AN ENZYME EXHIBITING CELLULASE ACTIVITY

Abstract: An enzyme which exhibits cellulase activity, which enzyme is producible by a strain of Bacillus spp., NCIMB 40250, or a related Bacillus spp. strain, or a derivative of said cellulase. In particular, the enzyme is an endoglucanase with an apparent molecular weight of 75, 56 or 45 kD or a cleavage product thereof with endoglucanase activity. An enzyme which comprises a core region derived from an endoglucanase combined with a cellulose-binding domain derived from another cellulase enzyme, or which comprises a core region derived from another cellulase enzyme combined with a cellulose-binding domain derived from an endoclucanase.

Description: AN ENZYME EXHIBITING CELLULASE ACTIVITY FIELD OF THE INVENTION The present invention relates to an enzyme exhibiting cellulase activity, a DNA construct encoding the enzyme, a cellulolytic agent comprising the enzyme and a detergent composition containing the enzyme. BACKGROUND OF THE INVENTION Biomass which largely consists of cellulose, hemicellulose and lignin has attracted increasing attention as an important renewable source of energy (including nutritional energy) . The amount of carbon fixed by photosynthesis has been estimated to be 100x10° tons per year worldwide, and half of that is contained in cellulose. If this material, or at least a significant part of it, could be converted into liquid fuel, gas and feed protein, this would constitute a significant contribution to solving the problem of recycling and conservation of resources. However, it has been found difficult to develop an economically viable process of converting cellulosic material into fermentable sugars. The currently most promising of the suggested processes involves the use of enzymes which are able to degrade cellulose. These enzymes which are collectively known as cellulases are produced by a number of microorganisms, including fungi (e.g. Trichoderma reseei. Humicola insolens, Fusarium oxysporum. etc.) and bacteria (e.g. Clostridium thermocellu . Cellulomonas spp., Thermonospora spp. , Bacterioides spp. , Microbispora bispora. etc.) . The economics of the production of fermentable sugars from biomass by means of such enzymes is not yet competitive with, for instance, the production of glucose from starch by means of α-amylase due to the ineffeciency of the cellulase enzymes. The most significant problems connected with the use of cellulases is their low specific activity and the high cost of their production. Therefore, there is a need to develop cellulases which are more efficient in degrading cellulosic materials into fermentable sugars. Apart from their utility for the degradation of biomass, cellulases have also been suggested for use in detergent compositions for the treatment of cotton-containing fabrics which largely consist of cellulose. It is well known that repeated washing of cotton-containing fabrics generally causes a pronounced, unpleasant harshness in the fabric due to the presence of amorphous regions in the cellulose fibres, which regions form protruding parts on the otherwise smooth fibres. Several methods for overcoming this problem have previously been suggested. For example, US 1.368.599 of Unilever Ltd. teaches the use of cellulases for reducing the harshness of cotton- containing fabrics. Also, US 4.435.307 (of Novo Industri A/S) teaches the use of a cellulytic enzyme derived from Humicola insolens as well as a fraction thereof as a harshness reducing detergent additive. Other uses of cellulases mentioned in the art include * soil removal from and colour clarification of fabric (cf. for instance EP 220 016) . Although the use of cellulase enzymes for harshness reduction of cotton-containing fabrics was suggested and demonstrated nearly 20 years ago the mechanism of this process has not been elucidated and is still not known in detail. Among other things, this is due to the multiplicity of the enzymes and the enzyme- catalyzed reactions involved. As a matter of fact, cellulases generated in nature e.g. by microbial species are indeed complex mixtures of cellulases. Accordingly, the conversion of natural¬ ly occurring materials, like cotton, catalyzed by cellulases is exceedingly difficult to analyze in detail. Due to these circumstances, the practical exploitation of cellulases for harshness reduction and prevention as well as colour clarification, however desirable, has not become widespread and of great practical utility: it is difficult to optimise production of multiple enzyme systems and thus to implement industrial cost-effective production of cellulase en¬ zymes, and their actual use has been hampered by difficulties arising from the need to employ rather large quantities of the cellulases to achieve the desired reduction and prevention of the harshness of cotton fabrics: for instance, addition of large quantities of the enzymes to detergent compositions is not com¬ patible with the optimal function of other ingredients in the detergent formulation nor is the addition of very large quantities of enzymes to the detergent composition in the interests of, e.g., consumer safety. The object of the present invention is therefore to provide cellulase enzymes with a high specific activity. SUMMARY OF THE INVENTION The present invention relates to an enzyme which exhibits cellulase activity, which enzyme is producible by a strain of Bacillus spp., NCIMB 40250, or by a related Bacillus spp. strain, or a derivative of said cellulase. The strain NCIMB 40250 was deposited on 18 January, 1990, in the National Collection of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen, Scotland, UK, in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. In the present context, the expression "enzyme exhibiting cellulase activity" is meant to be understood as an enzyme which is involved in the process of cellulose degradation. There are three different types of cellulases which act synergistically to produce soluble sugars: endoglucanases which show affinity for cellulose and which attack amorphous regions of low crystallinity in the cellulose fibre resulting in the formation of free ends; exoglucanases which initiate degradation from the non-reducing chain ends by removing cellobiose units; and β- glucosidases which hydrolyse cellobiose to glucose. The expression "related Bacillus spp. strain" is intended to indicate a strain belonging to the same Bacillus species as the strain NCIMB 40250 or a strain of a closely related species. The species to which the strain NCIMB 40250 belongs has been identified as Bacillus lautus. The scope of the present invention is also intended at least to include cellulase enzymes producible by other Bacillus lautus strains than NCIMB 40250. The term "derivative" is intended to indicate a protein which is derived from the native protein by addition of one or more amino acids to either or both the C- and N-terminal end of the the native protein, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or more sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence. Although the enzyme of the invention may be produced by cultivating the Bacillus spp. strain NCIMB 40250 or a related strain and isolating the enzyme from the culture, it will generally be more advantageous to produce the enzyme by recombinant DNA techniques which make it possible to optimize the yield of the enzyme produced. Furthermore, cloned genes encoding the enzymes may be modified in order to provide enzymes with improved properties. Thus, in another aspect, the present invention relates to a DNA construct which comprises a DNA sequence encoding an enzyme exhibiting cellulase activity, which enzyme is derivable from a strain of Bacillus spp. , NCIMB 40250, or a related Bacillus spp. strain, or a derivative of said cellulase. The invention further relates to an expression vector which carries an inserted DNA construct as indicated above, as well as to a cell transformed with the DNA construct or with the vector. In a still further aspect, the invention relates to a cellulolytic agent capable of degrading amorphous regions of cellulose fibres, the agent comprising an enzyme exhibiting cellulase activity as defined above. The invention also relates to a detergent composition comprising the cellulolytic agent. The cellulase enzyme of the invention has surprisingly been found to be more stable during washing (for 60 minutes at 40 β C) in the presence of conventional detergents than a commerci •al cellulase preparati•on (CelluzymeTM, a cellulase preparation isolated from Humicola insolens. available from Novo Nordisk, A/S) . The cause of the increased stability may reside in the alkalophilic nature of the enzyme (see example 5 below) . It is further speculated that it may also be ascribed to stability towards oxidation or towards the proteases commonly included in detergents. If so, the celluase enzyme of the invention may also show increased storage stability in liquid detergents containing proteases. DETAILED DISCLOSURE OF THE INVENTION The cellulase enzyme of the present invention is preferably one which exhibits endoglucanase activity (referred to in the following as an endoglucanase) , in particular one which exhibits an endoglucanase activity of at least about 10, more preferably at least about 20, most preferably at least about 25, such as about 30, CMC-endoase units per mg of total protein under alkaline conditions. The endoglucanase activity is determined as the viscosity decrease of a solution of carboxymethyl cellulose (CMC) after incubation with the enzyme of the present invention under the following conditions: A substrate solution is prepared, containing 35 g/1 CMC (Her¬ cules 7 LFD) in 0.1 M tris buffer at pH 9.0. The enzyme sample to be analyzed is dissolved in the same buffer. 10 ml substrate solution and 0.5 ml enzyme solution are mixed and transferred to a viscosimeter (e.g. Haake VT 181, NV sensor, 181 rpm) , thermostated at 40°C. Viscosity readings are taken as soon as possible after mixing and again 30 minutes later. The amount of enzyme that reduces the viscosity by one half under these conditions is defined as 1 CMC-endoase unit. It should be noted that the endoglucanase of the invention is one which is active (in terms of CMC-endoase activity) under alkaline conditions. More specifically, the endoglucanase is one which has a pH optimum at a pH of about 7.5-10.5. Contrary to several known cellulases which are active at an acid pH and relatively inactive at alkaline pH values, this characteristic makes the endoglucanase of the invention particularly useful for washing purposes, in particular as an ingredient of a detergent composition, as washing of clothes is typically conducted under alkaline conditions due to the alkalinity of most washing detergents. Alkalophilic cellulases are known, e.g. from EP 271 004, but they are not indicated to have a high affinity for cellulose, which is the case with the cellulase enzyme of the present invention which also exhibits a higher specific activity. The enzyme of the present invention is preferably one which is active at the temperatures at which clothes are typically washed, which is usually a temperature of up to about 60°C. Thus, the native enzyme isolated from strain NCIMB 40250 is active at temperatures between about 45 and 65°C. This, however, does not preclude the possibility that the enzyme may, under certain conditions, be active at temperatures above 65"C. One enzyme according to the invention is an endoglucanase with an apparent molecular weight of 75 kD or a cleavage product thereof exhibiting endoglucanase activity. The term "cleavage product" is intended to indicate a shorter form of the enzyme resulting from, for instance, chemical or enzymatic cleavage (e.g. by means of a suitable protease) after recovery of the enzyme or from posttranslational processing by the organism producing the enzyme, e.g. N- and/or C-terminal processing, which may give rise to a mature form of the enzyme. A specific example of a cleavage product of the ~75 kD enzyme which is of interest for the present purpose is a product of approximately 58 kD produced on cultivating a host organism transformed with DNA encoding the ~75 kD enzyme. The ~75 kD enzyme (and its ~58 kD cleavage product) are referred to in the following examples as Endol. The enzyme of the invention may be an endoglucanase encoded by the DNA sequence shown in the appended Sequence Listing ID#1 (showing the sequence of the -75 kD enzyme) , or a modification thereof encoding a derivative of said endoglucanase. Endoglucanase derivatives may conveniently be provided by suitably modifying the DNA sequence coding for the native endoglucanase. Examples of suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid sequence of the endoglucanase, but which may correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different polypeptide structure without, however, impairing the properties of the endoglucanase. Other examples of possible modifications are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence and deletion of one or more nucleotides at either end of or within the sequence. Such modifications of DNA coding for native proteins are well known and widely practised in the art. Another enzyme according to the invention is an endoglucanase with an apparent molecular weight of 56 kD or a cleavage product thereof (as defined above) exhibiting endoglucanase activity. In example 2 below, this enzyme is referred to as Endo2. The enzyme of the invention may be an endoglucanase encoded by the DNA sequence shown in the appended Sequence Listing ID#3 (showing the sequence of the -56 kD enzyme) , or a modification thereof (as defined above) encoding a derivative (as defined above) of said endoglucanase. A further enzyme according to the invention is an endoglucanase with an apparent molecular weight of 45 kD or a cleavage product thereof (as defined above) exhibiting endoglucanase activity. A specific example of such a cleavage product is a protein of approximately 30 kD produced on cultivating a host organism transformed with DNA encoding the -45 kD enzyme. In example 3 below, the -45 kD enzyme is referred to as Endo3A. The enzyme of the invention may be an endoglucanase encoded by the DNA sequence shown in the appended Sequence Listing ID#5 (showing the DNA sequence encoding the -45 kD product) , or a modification thereof (as defined above) encoding a derivative (as defined above) of said endoglucanase. Other enzymes exhibiting endoglucanase activity produced from endoglucanase clone 3 (cf. example 3 below) are proteins of approximately 60 and 56 kD, referred to as Endo3B and Endo3C. respectively. A still further enzyme according to the invention is an endoglucanase with an apparent molecular weight of 92 kD or a cleavage product thereof (as defined above) exhibiting endoglucanase activity. In example 4 below, this enzyme is referred to as Endo4. Other enzymes exhibiting endoglucanase activity produced from endoglucanase clone 4 (cf. example 4 below) are proteins of approximately 74 and 71 kD. Either or both of these may be individual enzymes or cleavage products of the -92 kD enzyme. It has been found that enzymes of the invention, e.g. Endol and Endo3A. are composed of a core region comprising the catalytically active domain and a region comprising a domain whose function is to mediate binding to cellulosic substrates (i.e. the cellulose-binding domain; this corresponds to a similar domain in an endocellulase from Bacillus subtilis (Nakamura et al., 1987). For example, the full-length -75 kD form of Endol comprises a core region and a C-terminal cellulose-binding domain which, in some cases, may be cleaved off proteolytically leaving a core region of -58 kD. The presence of the cellulose-binding domain has been found to be important for obtaining a colour clarification effect in prewashed textiles (cf. example 6 below) . Based on this finding, it may be possible to generate novel derivatives of cellulase enzymes which, for instance, combine a core region derived from an endoglucanase of the present invention with a cellulose-binding domain derived from another cellulase enzyme (e.g. one derived from a Cellulomonas fimi cellulase) . Alternatively, it may be possible to combine a core region derived from another cellulase enzyme with a cellulose- binding domain derived from an endoglucanase of the present invention. In a particular embodiment, the core region may be derived from a cellulase enzyme which does not, in nature, comprise a cellulose-binding domain, and which is C-terminally extended with a cellulose-binding domain derived from an endoglucanase of the present invention. In this way, it may be possible to construct cellulase enzymes with improved binding properties. The DNA construct of the invention may be one which comprises a DNA sequence encoding any one of the enzymes described above, or derivatives of these enzymes as defined above. The DNA construct may be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library of an appropriate Bacillus spp. strain (e.g. strain NCIMB 40250 or a related strain) and screening for DNA sequences coding for all or part of the appropriate cellulase by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Maniatis et al., 1982). The expression vector of the invention carrying the inserted DNA construct encoding the enzyme of the invention may be any vector which is capable of replicating autonomously in a given host organism, typically a plasmid or bacteriophage. In the vector, the DNA sequence encoding the enzyme of the invention should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host organism. The promoter is preferably derived from a gene encoding a protein homologous to the host organism. Examples of suitable promoters are lac of E.coli. dagA of Streptomyces coelicolor and amvL of Bacillus lichenifor is. The expression vector of the invention further comprises a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO and pIJ702. The expression vector may further comprise a DNA sequence coding for a signal peptide in order to provide extracellular expression of the enzyme. The DNA sequence may, for instance, be the signal sequence from the α-amylase gene of B___ licheniformis. The vector may also comprise a selectable marker, e.g. a gene whose product confers antibiotic resistance, such as ampicillin, chloramphenicol or tetracycline resistance, or the dal genes from B.subtilis or B.licheniformis. The procedures used to ligate the DNA sequences coding for the enzyme of the invention and the promoter, respectively, and to insert them into suitable vectors containing the information necessary for replication in the host cell, are well known to persons skilled in the art (cf. , for instance, Maniatis et al., op.cit.) . The host cell of the present invention may be transformed with the DNA construct of the invention encoding the cellulase enzyme described above. In this case, the DNA construct may conveniently be integrated in the host chromosome which may be an advantage as the DNA sequence coding for the cellulase is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous recombination. Alternatively, the host cell may be transformed with an expression vector as described above. The host cell used in the process of the invention may be any suitable bacterium which, on cultivation, produces large amounts of the enzyme of the invention. Examples of suitable bacteria may be grampositive bacteria such as bacteria belonging to the genus Bacillus, e.g. Bacillus subtilis. Bacillus licheniformis. Bacillus lentus. Bacillus brevis. Bacillus stearothermophilus. Bacillus alkalophilus. Bacillus amyloliquefaciens. Bacillus coagulans. Bacillus circulans or Bacillus lautus. or gramnegative bacteria such as Escherichia coli. The transformation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se. In a yet further aspect, the present invention relates to a method of producing a cellulase enzyme of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the cellulase or derivative thereof and recovering the cellulase or derivative thereof from the cells and/or culture medium. The medium used to cultivate the cells may be any conventional medium suitable for growing bacteria. The cellulase may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary after disruption of the cells, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like. In a particular embodiment of the method of the invention, the cellulase is recovered in mature form, either as a result of posttranslational processing of a proenzyme as explained above or as a result of appropriate modifications of the DNA sequence encoding the enzyme in the form of deletions of DNA corresponding to truncations in the N- and/or C-terminal sequences of the enzyme. There is reason to believe that different cellulases may exert a synergistic effect with respect to the degradation of cellulose. The cellulolytic agent of the invention may therefore advantageously comprise a combination of two or more cellulase enzymes of the invention or a combination of one or more cellulase enzymes of the invention with one or more other enzymes exhibiting cellulase activity. Such cellulases may be endocellulases or exocellulases dependent on the intended use of the cellulolytic agent (e.g. the degree of cellulose degradation aimed at) . The other cellulases may be selected from those which may be isolated from species of Humicola such as Humicola insolens (e.g. strain DSM 1800) , Fusarium such as Fusarium oxysporum (e.g. strain DSM 2672) , Myceliopthora such as Mvceliopthora thermophile. Erwinia such as Erwinia chrysanthermis (cf. M.H. Boyer et al., Eur. J. Biochem. 162. 1987, pp. 311-316), Trichoderma such as Trichoderma reseei, Microbispora such as Microbispora bispora. Neocallimastix such as Neocallimastix frontalis. Piromonas such as Piromonas communis f Robillarda spp. , Cellulomonas such as Cellulomonas fimi, Clostridium such as Clostridium thermocellum. Pseudomonas spp. , Ther onospora spp. , Bacterioides spp. or Ru inococcus spp. The cellulolytic agent of the invention may suitably be in the form of a non-dusting granulate, stabilized liquid or protected enzyme. Non-dusting granulates may be produced e.g. according to US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method disclosed in EP 238,216. The cellulolytic agent may suitably exhibit an endoglucanase activity of 500-10,000 CMC-endoase units (as defined above) per gram of the agent. The cellulolytic agent is suitably a detergent additive which may comprise one or more other enzymes, such as a protease, lipase and/or amylase, conventionally included in detergent additives. The detergent composition of the invention comprising the cellulolytic agent described above additionally comprises one or more surfactants which may be of the anionic, non-ionic, cationic, amphoteric, or zwitterionic type as well as mixtures of these surfactant classes. Typical examples of anionic surfactants are linear alkyl benzene sulfonates (LAS) , alpha olefin sulfonates (AOS) , alcohol ethoxy sulfates (AES) and alkali metal salts of natural fatty acids. The detergent composition of the invention may contain other detergent ingredients known in the art as e.g. builders, bleaching agents, bleach activators, anti-corrosion agents, sequestering agents, anti soil-redeposition agents, perfumes, enzyme stabilizers, etc. The detergent composition of the invention may be formulated in any convenient form, e.g. as a powder or liquid. The enzyme may, if required, be stabilized in a liquid detergent by inclusion of enzyme stabilizers as indicated above. Usually, the pH of a solution of the detergent composition of the invention will be 7-12 and in some instances 7.0-10.5. Other detergent enzymes such as proteases, lipases or amylases may be included in the detergent composition of the invention, either separately or in a combined additive as described above. The softening, soil removal and colour clarification effects obtainable by means of the cellulase enzyme of the invention generally require a concentration of the cellulase in the washing solution corresponding to an endoglucanase activity of 5 - 200 CMC-endoase units per liter. The detergent composition of the invention is typically employed in concentrations of 0.5 - 20 g/1 in the washing solution. Consequently, the cellulase concentration of the detergent composition of the invention is about 0.3 - 400 CMC-endoase units per gram. In general, it is most convenient to add the detergent additive in amounts of 0.1 - 5 % w/w * or, preferably, in amounts of 0.2 - 2 % of the detergent composition. For special applications, however, for instance when the detergent composition is to be used for colour clarification or harsness reduction of fabric which has been damaged by repeated washing, it may be convenient to include a much larger amount of the cellulolytic agent, such as about 20% w/w. In a still further aspect, the present invention relates to a method of reducing the rate at which cellulose-containing fabrics become harsh or of reducing the harshness of cellulose- containing fabrics, the method comprising treating a cellulose- containing fabric with a cellulolytic agent as described above. The method of the invention may be carried out by treating cellulose-containing fabrics during washing. The cellulolytic agent may either be added as such in the amount required to obtained the desired effect, or it may be added as an ingredient of a detergent composition as described above. However, if desired, treatment of the fabrics may also be carried out during soaking or rinsing or simply by adding the cellulolytic agent to water in which the fabrics are or will be immersed. The cellulolytic agent of the invention may also be employed to obtain colour clarification of cellulose-containing fabrics. After repeated washing, such fabrics often develop a grayish appearance. This effect is particularly evident with coloured fabrics, especially dark fabrics, and may probably be ascribed to undyed parts of the cellulose fibres becoming apparent when the cellulose fibres of which the fabric is composed are damaged by mechanical forces. The damaged parts of the fibres are assumed to be more amorphous than intact cellulose fibres and therefore more susceptible to the action of the cellulases of the present invention. The colour clarification effect is more pronounced when the cellulolytic agent contains an endoglucanase which comprises a cellulose-binding domain (cf. example 6 below) . Accordingly " , the present invention further relates to a method of treating a coloured, cellulose-containing fabric in order to provide colour clarification, the method comprising treating the cellulose-containing fabric with a cellulolytic agent according to the invention. The method of the invention may be carried out by treating cellulose-containing fabrics in an aqueous medium during washing. The cellulolytic agent may either be added as such in the amount required to obtained the desired effect, or it may be added as an ingredient of a detergent composition as described above. However, if desired, treatment of the fabrics may also be carried out during soaking or rinsing or simply by adding the cellulolytic agent to water in which the fabrics are or will be immersed. For colour clarification purposes, the aqueous medium may suitably exhibit an endoglucanase activity of more than about 250 CMC-endoase units per liter of the aqueous medium. It may furthermore be possible to employ a cellulolytic agent according to the invention to provide a localized variation in the colour of a fabric to impart a "stone-washed" appearance to the fabrics (for the use generally of cellulase enzymes for this purpose, see for instance EP 307 564) . The cellulolytic agent of the invention is also contemplated to be useful in the field of paper pulp processing, e.g. pulp drainage (for the use generally of cellulase enzymes for this purpose, see for instance EP 262 040) , as well as for deinking of paper intended for recycling (for the use generally of cellulase enzymes for this purpose, see for instance JP 59-9299 or JP 63-59494) . The invention is further illustrated by the following examples which are not in any way intended to limit the scope of the invention, with reference to the appended drawings, wherein Fig. 1 Restriction maps of the Bacillus spp. DNA insert contained on the plasmids in the 4 endoglucanase- p'ositive E.coli clones. The position and direction of transcription of endoglucanase 1 (Endol. pPL517) and endoglucanase 2 (Endo2, pPL382) is indicated. Restric¬ tion enzyme sites are indicated as follows: Pstl (P) , Hindlll (H) , Smal. (S) , Sail (Sa) , BamHI (Ba) , B lll (B) , Sphl (Sp) , EcoRI (E) . ____WΛ indicates pBR322 DNA. Fig. 2 Restriction maps of different plasmids carrying the Endol gene fl ?) . The /S-lactamase promoter of pBR322 is indicated by the arrow (Pb) and the direction of the transcription of the Endol gene is indicated by the arrow (Pb) . (MM): pBR322, (■■ ) : "tail" of pBR322 encoded amino acids. Restriction enzyme sites are indicated as follows: Pstl (P) , BallI (B) , EcoRI. (E) , Clal (C) . The endglucanase activity in extracts of E.coli MC1000 containing the indicated plasmids is shown to the right as (A) cellulase units/ml culture medium. SUBSTITUTESHEET Fig. 3 Effect of temperature on the cellulase (Endol) i extracts of E.coli MClOOO (pPL517) and on the multipl cellulase activities in the supernatant of Bacillu spp. PL236. The activity was measured at the tempera ture indicated after an incubation period of 30 min See Materials and Methods. The activity at the dif ferent temperatures is presented as cellulase units/m of the original culture volume. Fig. 4 Relevant restriction sites and sequencing strategy o the endoglucanase Endol indicated as endocellulase in the figure. (J ■ > ): extent and direction o sequence reactions. Abbreviations: EcoRI (E) , Pst (P) , BallI (Bg) , BstNI (Bs) , Aval (A) , Clal (C) , BaittH (Ba). Fig. 5 is a restriction map of plasmid pDN 2801. Restrictio enzyme sites are indicated as follows: EcoRI(E), Bgll {Bg) , Hindlll (H) , Smal (Sm) , Sail (Sa) , Sphl (Sp) Pstl (P) , Eagl (Ea) , Clal (C) , BamHI (B) . CAT indicate the gene mediating chloramphenicol resistence. P indicates the Bacillus maltogenic α-amylase promoter Fig. 6 Restriction maps of different plasmids (B.subtili replication origin) carrying the Endol gene. Th maltogenic alpha-amylase promoter is indicated by th arrow (P ffl ) , which also indicates the direction of th transcription of the Endol gene; (■-■-■) : pDN2801, (M) : tail of PDN2801 encoded sequences. Restrictio enzyme sites are indicated as follows: Eagl (Ea) , Bgll (Bg) , Pstl (P) , BamHI (B) .The endocellulase activit in extracts of B.subtilis DN 1815 containing th indicated plasmids is shown to the right (A) a endoclucanase units/ml culture medium. SUBSTITUTE SHEET Fig. 7a Construction of pTL05 and pLA03. (| ): represents the and b C-terminal "tails" encoded by vector sequences. (^>) : indicates the expected C-terminal cleavage site. Restriction sites are abbreviated as follows: Pstl (P) , Pvu (Pv) , BamHI (B) , Bαlll (Bg) , Sail (S) . Fig. 8a Construction of pCH57. (JSSSJ) 1 Signal sequence of the and b alpha-amylase. ( μιιi|iιιi) : signal sequence of the Endol glucanase ( m ) : "direct repeat" e.g. start of the mature Endol gene. (• ) ribosome binding site of the alpha-amylase. (O) ribosome binding site of the Endol glucanase (M^) : alpha-amylase promoter. Restriction sites are abbreviated as follows: Pstl (P) ; Sail (S) ; Konl (K) ; Eaαl (Ea) ; Bσlll (Bg) . Fig. 9 Relevant restriction sites and sequencing strategy of the endoglucanase Endo2 (indicated as endocellulase 2 in the figure) . ( \ ...)■ ) : extent and direction of sequence reactions. Abbreviations: Seal (Sc) , Sacl (Sa) , Xmnl (X) , Hindi! (He) , Hindlll (H) , Pstl (P) . Fig. 10 Zymogram showing the molecular weight of the active proteins resulting from different plasmids carrying the original endoglucanase 3 clone (pPL591) as well as deletions in the original insert. Fig. 11 Restriction maps of the endoglucanase 3 clone (in¬ dicated in the figure as endocellulase 3) (pPL591) and plasmids containing deletions in the original insert. The position of the endoglucanase genes Endo3A, Endo3B and Endo3C. on the insert in plasmid pPL591 predicted from the data shown in the zymogram (Fig. 10) is shown on the restriction map. Restriction enzyme sites are indicated as follows: Hindlll (H) , Smal (S) , EcoRI (E) . SUBSTITUTE SHEET EXAMPLES MATERIALS AND METHODS a) Bacterial strains and plasmids The donor strain. Bacillus spp.. strain PL236, was isolated from a compost sample from Lyngby, Denmark, on the basis of its high cellulolytic activity. A sample of this strain was deposited on 18th January, 1990 in the National Collection of Industrial and Marine Bacteria, 23 St. Machar Drive, Aberdeen, Scotland, with the accession No. NCIMB 40250. The following E. coli strains and plasmid were used: MC 1000 faraD139) . (ara, leu)7697. lacX74. qalU. σalK. rpsL) (Casaba- dan et al., 1980); CSR603 (F-, thr-1. leuB6. proA2. prh-1. RecAl f argE3_, thi-1. uyrA6. ara-14. lacYl, galK2, xyl-5 f mtl- 1, rpsL31. tsx-33. -, SUPE44) (Sancar et al., 1979); PL248 is MClOOO containing the plasmid pNF2690 which contains the replication origin and the kanamycin resistance gene from pACYC177 (Chang and Cohen, 1978) and the cI857 repressor gene from the coliphage lambda; pBR322 (Bolivar, 1977) ; pUNl21 (Nilsson et al., 1983) pUClδ (Yanisch-Perron et al., 1985); pPLc28 (Remaut et al., 1981); pPL170 (Jørgensen, P.L. , 1983); For the experiments with J3. subtilis. the following B. subtilis strains and plasmids were used: DN1885 (amyE. amyR, spo*, pro * ) (Diderichsen, Novo Industri A/S) is a derivative of B. subtilis 168; PL1801 is a derivative of DN1885 lacking the two main exoproteases (apr " , npr " ) ; pDN2801 has the origin of replication from pUBHO (Keggins et al., 1987), the Cat gene of pC194 (Horinouchi and eisblum, 1982) and the maltogenic alpha- amylase promotor (Pm) from B. stearothermophilus (Diderichsen and Christiansen, 1988) followed by a polylinker; The B. subtil- is/E.coli shuttle vector pJKK3-l is described by Kreft et al. (1983) ; pPL1759 has the origin and kanamycin resistance gene of pUBHO and the promotor, ribosome binding site and signal sequence from the alpha-amylase gene from B.licheniformis (Stephens et al., 1984). b) Media Phosphoric acid swollen cellulose (ASC) was prepared from chromatography cellulose (MN 300, Machery, Nagel) as described by Walseth (1952) with the exception that the cellulose powder was suspended in acetone before treatment with phosphoric acid. The medium for detection of cellulase activity was prepared as standard m)-medium (Maniatis et al., 1982) containing 0.2% ASC or microcrystalline cellulose (Avicel, Merck). Bacillus spp.. B.subtilis and E.coli cells were grown in NY medium (von Meyenburg et al., 1982) , LB medium (Maniatis et al., 1982) , or BPX medium (100 g/1 potato starch, 50 g/1 barley flour, 0.1 g/1 BAN 5000 SKB, 10 g/1 sodium caseinate, 20 g/1 soybean meal; 9 g/1 Na 2 HP0 4 , 12H 2 0, 0.1 g/1 Pluronic) . The media were solidified by the addition of agar (20 g/1) . Tetracycline (20μg/ml) , or kanamycin (10 μg/ml) were added as required. c) Isolation of DNA To isolate the chromosomal DNA from Bacillus spp. PL236, cells from 250 ml overnight culture were resuspended in 10 ml (50 πM Tris-HCl, pH 8.0, 100 mM EDTA) , and incubated with 25 mg lysozyme for 20 min. at 37*C. To the mixture was added 2 ml of 10% (w/v) SDS, mixed and put on ice for 10 min. To the solution was then added 15 ml of phenol saturated with TE-buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), heated to 65"C, mixed gently and cooled on ice. After centrifugation for 30 min. at 40000 g the aqueous phase was ether extracted, ethanol precipitated and the pellet was resuspended in TE-buffer. The DNA was further purified by banding in a CsCl density gradient (Maniatis et al. , 1982) . E.coli plasmid DNA was prepared by the SDS lysis method (Maniatis et al., 1982); minipreparations of plasmid DNA for restriction enzyme analysis and transformations were prepared according to Holmes and Quigley (1981) . B.subtilis plasmid DNA was prepared by the alkaline lysis method. (Maniatis et al., 1982). d) Cloning of chromosomal DNA from Bacillus spp. PL236 into E. coli Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs and used as described by the manufacturers. After digestion with £stl, EcoRI or Hindlll restriction enzyme the DNA was heated to 65 * C for 10 min. and ethanol precipita¬ ted. 10 μ of linearized pBR322, pJKK3-l or pUN121 and 20 g of fragmented B_\ spp. chromosomal DNA were ligated with 5 units of DNA ligase in a final volume of 100 μl (16 h, 15"C) . The ligated DNA was used to transform competent E.coli MClOOO to tetracycline resistance essentially as described by Mandel and Higa (1970). (pBR322: 20 μg/ml, pUN121: 7 μg/ l and pJKK3-l: 10 μg/ml) . e) Detection of cellulase-positive E.coli clones The detection of cellulase activity on plates was performed using a modification of the technique of Teather and Wood (1982) . E.coli clones were grown overnight on solid NY medium at 37*C. Cells were lysed by overlaying the colonies by topagar containing phosphate buffer (100 mM, pH 7.0), agar (0.7%) CMC (0.2%), SDS (0.25 mg/ml) , and chloramphenicol (200 μ/ml) and subsequent incubation overnight at 37'C. Plates were then flooded with an aqueous solution of Congo red (1 mg/ml) for 15 min. and subsequently washed with 1 M NaCl. Cellulase-positive colonies were surrounded by a yellow halo on a red background. f) Maxicells Plasmid-encoded proteins were analysed using the maxicell method of Sancar et al. (1979) with the following modification. After UV irradiation, the surviving cells were killed by incubating the cells with D-cycloserine (150 μg/ml) for 48 h at 37 β C. g) Gel electrophoresis ( 3S S)-L.methionine labelled maxicell proteins and other proteins were analysed by electrophoresis on 15% (0.075 per cent bisacrylamide) SDS-polyacrylamide gels (Laemmli, 1970) . Proteins were visualized either by staining with Coomassie Blue G 250 or by autoradiography. Analysis of DNA was done by electrophoresis on agarose gels with the buffer described by Loening (1967) . h) Detection of cellulase activity in polyacrylamide gels Detection of. cellulase activity in protein bands separated by SDS-polyacrylamide gel electrophoresis was done by a modified zymogram technique described by Beguin (1983) . Protein prepara¬ tions were electrophoresed on a SDS-polyacrylamide gel as described above and the gel was washed 3 times 30 min. in phosphate buffer (100 mM, pH 7.0) layered on to a thin (0.8 mm) agarose gel (agarose, (1.8 per cent), CMC, (0.2 per cent), phosphate buffer, (100 mM, pH 7.0)), and incubated for 4 h at 42"C. Cellulase activity was visualized by staining the agarose gel for 30 min. in an aqueous solution of Congo red (1 mg/ml) followed by washing the gel in 1 M NaCl. i) Colorimetric cellulase assay Cellulase activity in cell extracts was analysed by measuring the increase in reducing groups released by the hydrolysis of CMC (Miller, 1959) . An appropriate amount of enzyme was in- cubated with 1.5 ml of 1 per cent CMC in phosphate buffer (100 mM, pH 7.0). After 30 min. of incubation at 55*-C, 1.5 ml of dinitrosalicyclic acid reagent was added and the samples were boiled for 5 min. The absorbance was read at 550 nm against blanks containing equivalent amounts of extract from the E.coli recipient strain. One unit of cellulase released 1 nM of glucose equivalents per second by reference to a standard curve. ) DNA-Sequencing Single end labelled DNA fragments were isolated and sequenced by the chemical modification method (Maxam and Gilbert, 1980) . The cleavage products were separated on 8% or 20% polyacryl-amide gels and thereafter autoradiographed at -70"C using intensi¬ fying screens. The dideoxyribonucleotide method of Sanger et al., (1977) was used for the sequencing of Endo3A using derivatives of pUClδ (Yanisch-Perron, 1985) . k) Southern*•analysis Chromosomal DNA from Bacillus spp. (PL236) was digested with restriction enzymes as required and fractionated on 1% (w/v) agarose gels. DNA was then blotted onto nitrocellulose filters. 32 -labelled DNA probes (recombinant plasmids) were prepared by nick translation (Rigby et al., 1977) using 32 ^ p (Amersham) and hybridization was carried out as described by Southern (1975) . Autoradiography was performed at -70 β C using intensi¬ fying screens. 1) Transformation of competent B. subtilis cells A modified version of Dubnau and Davidoff-Adelson's (1971) procedure for preparing competent cells of B. subtilis is used. 10 ml of LB-medium is inoculated with the strain in the morning. 7 hours later sequential dilutions in KM-1-medium are made and incubated overnight at 37 β C. The following morning, the second- most diluted and growing culture is diluted ten times in KM-2- medium. The cells are harvested after 45-60 minutes of incubation by centrifuging for 3 minutes at 7K. They are resuspended in 1/10 volume of the supernatant and 1/50 volume of 86% glycerol is added. 0.1 ml amounts are frozen on liquid nitrogen and stored at -80 β C. In order to transform these competent cells, the method of Ehrlich (1986) is used, with some modifications. BTF is prepared and preheated to 42 β C. 0.01 ml of DNA is placed in a reaction vessel, and the competent cells are thawed at 42°C. BTF is added to the cells at a ratio of 1:1, and 0.1 ml of the mixture is added to the DNA. The cells and DNA incubate with shaking for 20 minutes at 37 β C. A further 30 minutes of gene expression with 0.1 ml of NY-medium is needed, if kanamycin resistance is desired. The cells are finally spread on relevant plates. Recipes for stock-solutions are as follows: Stock solutions for making B.subtilis competent cells Salt mix: 10 mM CaCl 2 , ImM FeCl 3 and l M MnCl 2 . 10 X MM: 20 g (NH 4 ) 2 S0 4 , 60 g KH 2 P0 4 , 140 g K 2 HP0 4 β 3H 2 0 in 1L. KM-stock: 100 ml 10 X MM, 10 ml 10% Na- citrate, 2 ml 1M MgS0 4 in 1L. KMl-stock: 960 ml KM-stock, 20 ml 20% glucose, 1 ml 20% casamino acids, 20 ml 5% yeast extract, 30 1 ImM MnCl 2 in 1L. KM2-stock: 960 ml KM-stock, 20 ml 20% glucose, 1 ml 20% casamino acids, 20 ml 5% yeast extract, 1 ml salt mix, 1 ml 0.5 M CaCl 2 , and 0.8 ml 1M MgCl 2 in 1L. BCG: 100 ml 10 x MM, 10 ml Na-citrate, 2 ml 1M Mgso 4 , 1 ml saltmix, 20 ml 20% glucose in 1L. BTF: 800 ml BCG, 100 ml lOmM EGTA, 100 mM MgCl 2 in 1L. (Prepared fresh before use) . ISOLATION AND CHARACTERIZATION OF CELLULOLYTIC STRAINS In order to clone genes coding for cellulose-degrading enzymes the following screening programme was set up to find suitable cellulolytic donor strains. Various compost samples were used as source of cellulolytic microorganisms. Serial dilutions of compost samples were plated out on ASC agar medium and cellulolytic activity was detected through the formation of clearing zones around the colonies. Several cellulolytic bacteria were isolated. One of the most active of these isolates which was identified as Bacillus spp. PL236 (NCIMB 40250) was selected as the donor strain for the cloning experiments. The strain rapidly degraded both acid swollen cellulose and microcrystalline cellulose (Avicel, Merck) on agar medium. At the optimum temperature of growth (42*C) the clearing zones appeared in 2-3 days. Adding small amounts of Bacillus spp. PL236 culture to suspen¬ sions of microcrystalline cellulose makes the cellulose crystals lump together tightly and sediment. This suggests that either the Bacillus spp. PL236 cells or the extracellular cellulase enzymes have a strong affinity for the cellulose substrate and tightly bind the cellulose crystals together. SCREENING ASSAY FOR RECOMBINANT CLONES The first attempts to clone cellulases from Bacillus spp. PL236 were directed towards the endocellulases genes from Bacillus spp. PL236. To facilitate the screening on plates of endocellu¬ lase positive clones, an assay using the dye Congo red (Teather and Wood, 1982) was adapted to E.coli. In order to detect cellulase activity trapped inside the recombinant E.coli cells the cells were lysed by adding SDS to the top agar. This modification of the top agar had no measure- able effect on the CMC-degrading enzymes of Bacillus spp.PL236 f when the modified assay was used on this organism. MOLECULAR CLONING OF ENDOCELLULASES Several endocellulase genes were cloned from the cellulolytic Bacillus spp. strain (PL236) . An endocellulase 1 clone (PL517) was made by ligating Pstl partially digested PL236 chromosomal DNA with Pstl cleaved pBR322 and subsequently transforming competent E.coli cells. An endocellulase 2 clone (pPL382) was made by ligating Hindlll partially digested PL236 chromosomal DNA with Hindlll cleaved pJKK3-l (an E.coli/B.subtilis shuttle-vector) and subsequently transforming competent E.coli cells. An endocellulase 3 clone (pPL591) was made by ligating EcoRI partially digested PL236 DNA with EcoRI cleaved pUN121 and subsequently transforming competent E.coli cells. An endocellulase 4 clone (pPL592) was made by ligating Hindlll partially digested PL236 DNA with Hindlll cleaved pUN121 and subsequently transforming competent E.coli cells. Transformants derived from the use of both the pBR322 and the pUN121 plasmid vector were screened by their tetracycline resistance (pBR322: 20 g/ml, pUN121: 7 μg/ml and pJKK3-l: 10 μg/ml) . The transformants were replicated to another set of plates and overlayed by the modified CMC top agar. The plates were in¬ cubated overnight and stained with Congo red as described above. Under the reisolation procedure it was observed that it was possible to detect positive clones without adding the cell lyzing agent SDS to the topagar, although the diameter of the halo was considerably smaller. EXPRESSION AND CHARACTERIZATION OF THE CLONED CELLULASES Plasmids from the cellulase-positive transformants were isolated and analyzed with restriction enzymes. Restriction enzyme maps of representative plasmids from all four cloning experiments are shown in Fig. 1. To determine the molecular weight of the cloned endoglucanases a zymogram technique (Beguin, 1983) was used. Total protein preparations from representative endoglucanase clones were separated on a SDS-polyacrylamide gel. The proteins in the gel were then renatured by washing out the SDS and replicated onto an agarose gel containing CMC. Renatured proteins diffuse to this activity gel and proteins representing endoglucanase activity hydrolyse the CMC in the gel. The endoglucanase bands were then visualised by staining the activity gel with Congo red as described above. As appears from Fig. 1, the restriction maps of the cloned DNA (as well as the molecular weight of the endoglucanases encoded by the cloned DNA) are different in the four different clones. This indicates that these four clones represent at least four different endocellulase genes from Bacillus spp. (PL236) . The detailed analysis of the different endocellulases represen- ted by these four clones is described in the following. Example 1 Endocellulase 1 (Endol) Physical mapping of endocellulase gene 1 In the cloning experiment described above several cellulase- positive E.coli clones were obtained, which contained different fragments of Pstl partially cleaved Bacillus spp. PL236 DNA. Fig. 2 shows the restriction maps of some of these clones. The plasmids invariably contained two Pstl fragments (1000 and 1350 bp.) indicating that both were necessary for synthesis of a polypeptide with cellulase activity: Nucleotide sequencing has later shown that the 500 bp. Pstl fragment present the on pPL217 and pPL517 (Fig. 2) contains the C-terminal part of the complete cellulase gene (data are presented in the following) . The structure of this part of the B.spp. chromosome was confirmed by Southern analysis (data are presented in the following) . Activity measurements Extracts from the E.coli clones containing pPL212, pPL216, pPL517 and pBR322 were prepared from overnight cultures grown in NY medium supplemented with tetracycline. Cells were concentrated 10-fold in 100 mM phosphate buffer, pH 7.0. DNase was added and the cells were ruptured by passing them twice through a French Press (12000 lb/in) . The extracts were centrifuged for 60 min. at 40000 X g and the supernatants were assayed for cellulase activity. The cell-free extract of E.coli MClOOO(pPL212) , E.coli MClOOO(pPL517) and E.coli MClOOO(pPL216) contained 8 units, 7.2 units and 0.2 units respectively of cellulase/ml of original culture volume. (Fig. 2) . 1/7 of the total cellulase activity in these cultures was found in the supernatant. The E.coli MClOOO(pBR322) clone showed no cellulase activity. The high level of cellulase activity in extracts of strains carrying the plasmids pPL212 and pPL517 is most likely due to an increased transcription of the cellulase gene originating from the /9-lactamase promoter on pBR322. It was concluded that the cellulase is expressed in the direction shown in Fig. 2. Measurements of the viscosity of a CMC solution and of the release of reducing sugars indicated that the cloned cellulase is an endo-(l,4)-3-glucanase. Maxicell and zymogram analysis The molecular weight of the endoglucanase protein was analysed by the maxicell technique. The plasmids pPL212, pPL216 and pBR322 were transformed into the maxicell strain CSR603 (Sancar et al., 1979) to analyse for plasmid-encoded proteins. The plasmid pPL212 gave rise to three polypeptides of 75000 D, 65000 D and 58000 D in addition to the proteins encoded by pBR322. Apart from the pBR322 proteins, no proteins encoded by the plasmid pPL216 could be detected, due to the low expression of the cellulase gene. The three polypeptides from pPL212 were tested for cellulase activity using a gel replica technique, (Beguin, 1983). A comparison of the cellulase activity bands and the bands on the autoradiogram from the polyacrylamide gel showed that the 75000 D and at least one of the 58000 and 65000 D protein bands had cellulase activity. It was also found the supernatant of the Bacillus spp. PL236 culture contained at least three different proteins with cellulase activity. One of these proteins comigra- ted with the 58000 D protein synthesized in the maxicell. Cell extracts from E.coli MClOOO (pPL212) only revealed one active band comigrating with the 58000 protein from the maxicell E.coli CSR603 (pPL212) . As mentioned previously, the following sequence data showed that the plasmids pPL212 and pPL216 did not contain the entire Endol endoglucanase gene. The 75000 D active protein seen in the maxicell E.coli CSR603 (pPL212) is thus a fusion protein, where 105 C-terminal amino acids are encoded by pBR322 sequences (Fig. 2) . This fusion protein is apparently posttrans- lationally processed, ending up with the 58000 D mature endoglu¬ canase. Cell extracts from E.coli MClOOO (pPL217) and E.coli MClOOO (pPL517) , which contain the complete endoglucanase gene, also gave activity bands of M r approx 58000 D and 75000 D. The 75000 D active,protein from the plasmids pPL217 and pPL517, thus represents the "genuine" initial translation product from the Endol glucanase gene, which apparently is processed more slowly than the fusion protein from pPL212. However, both the 75000 D fusion protein synthesized from pPL212 and the "genuine" 75000 D protein synthesized from pPL517 and pPL217 are processed down to a 58000 D protein with high cellulase activity. From the sequence data it can also be predicted that the endoglucanase expressed from pPL216 is synthesized a fusion protein, where the 26 C-terminal amino acids are encoded by pBR322 sequences (Fig. 2) . This protein was not detected by the maxicell technique and zymograms using extract from PL216 only revealed one active band of 58000 D, which most likely repre¬ sents the processed protein. The processing in E.coli of the two fusion proteins from pPL212 and pPL216, which represent two different lengths of the C-terminal "tail" thus results, in both case, in an active protein of approx. 58000 D. This indicates that the endocellulase is processed from the C-terminal, because N-terminal processing would result in two proteins with a difference in M r of approx. 9000 D, which would easily have been detected on the zympgrams. It is therefore most likely that endoglucanase 1 (Endol) is synthesized as a proenzyme which at least in E.coli (and possibly also in B.spp.) is modified by stepwise removal of approx. 150 C-terminal amino acid residues and approx. 30 N- terminal amino acid residues, corresponding to the removal of the signal peptide. The endoglucanase seems to be modified correctly as indicated by the fact that the final processing, product, the 58000 D activity band present in E.coli MClOOO (pPL212) extract, apparently comigrate with one of the endoglu¬ canases present in the supernatant of cultures of Bacillus spp. PL236. Temperature optimum and stability The cellulase activity of the extracts was measured at dif¬ ferent temperatures and the highest activity of Endol produced in E.coli was found at 60"C (Fig. 3). The heat stability of the endoglucanase was tested by incubating the extracts at 50 β C, 55 " C and 60 β C for varying periods and the residual activity was measured as outlined in Materials and Methods. Although the highest activity was observed at 60 β C with a fixed incubation time of 30 min. , the enzyme is inactivated at this temperature with a t^ of 1.2 h. At 50"C and 55 β C no inactivation was observed after 5 h of incubation. DNA-sequence The nucleotide sequence of the endocellulase gene 1 (Endol) was deduced from the plasmid pPL517 which contains approx. 2850 bp Of Bacillus spp. PL236 DNA. The sequence wasa determined by the chemical modification method (Maxam and Gilbert, 1980) using the partial restriction map and the sequencing strategy outlined in Fig. 4. The complete nucleotide sequence is shown in Sequence listing ID#1. A computer analysis of this sequence revealed only one open reading frame long enough to encode the approx. 75000 D protein detected by the maxicell and zymogram analysis of extracts from the cellulase-positive E.coli MClOOO(pPL517) . This sequence which begins at nucleotide 677 and ends at nucleotide 2776, encodes an enzyme of 700 amino acids. The M r calculated from the DNA sequence was 77006 D. Within the open reading frame there were three potential initia¬ tion codons (ATG at positions 677, 737 and 749) , but only the ATG codon at position 677 was preceeded by a ribosome binding site (AAGGAGG (Mclaughlin et al., 1981). It was therefore concluded that the ATG codon at position 677 was the correct initiation codon. The initiation codon is followed by an amino acid sequence which resembles signal sequences found in gram-positive organisms. Such sequences consist of a relatively short hydrophilic region at the N-terminal followed by a longer sequence of hydrophobic residues. By using the signal sequence cleavage model proposed by Heijne (1983) the cleavage site can be predicted to be between the two first alanine residues in the sequence Asn-Ala-Ala-Ala. The signal sequence is thus 31 amino acids long. The upstream and downstream regions contained no significant homology to the consensus sequence of the sigma 43 promoter of B. subtilis and no terminator-like sequences. Southern analysis The Bacillus spp. (PL236) chomosomal DNA was digested with Hindlll. Pstl, EcoRI and Xhol and plasmids pPL212 and pPL509 were used as probes for the hybridization. Plasmid pPL212 contains two Pstl fragments (1350 bp. and 1000 bp.) and plasmid pPL509 contains only the 500 bp. Pstl fragment of the entire Endol gene, represented by the plasmid pPL517, which contains three Pstl fragments (1350 bp., 1000 bp. and 500 bp.) of Bacillus spp. PL236 DNA. The pPL212 probe recognized the expected two Pstl fragments (1350 bp. , 1000 bp.) and the pPL509 the 500 bp. Pstl fragment in the Bacillus spp. PL236 Pstl digest. Both the pPL212 and the pPL509 probe also recognized the same overlapping EcoRI fragment and the same overlapping Hindlll fragment in Bacillus spp. PL236, EcoRI and Hindlll digest. These results indicate that the Bacillus spp. PL236 DNA insert in pPL517 was cloned in a non-deleted form and that the three Pstl fragments in pPL517 are continuous on the Bacillus spp. PL236 chromosome. Expression of the Endol gene in B.subtilis For the cloning experiments in B.subtilis. pPL517 was used as the donor of the Endol gene and pDN2801, carrying a strong Bacillus promoter P m , was used as the Bacillus vector (Fig. 5) . The Endol gene-containing Eagl fragment was ligated to Eagl cleaved pDN2801 and by subsequent transformation to compentent B.subtilis cells (DN1885) , strain CH7 was obtained. To test whether the processed C-terminal part was necessary for the expression of the Endol gene in B.subtilis cells, a construc¬ tion was made where the Endol gene was fused to vector sequen- ces in the internal BollI site. This fusion replaces the coding region for the C-terminal 94 amino acids with 55 "random" amino acids encoded by vector sequences. Similar constructions made in E.coli vectors, though fused to different vector sequences, resulted in an active periplasmic endoglucanase in E.coli. which was processed in the "correct" manner. Part of the Endol gene contained in the Bglll fragment from pPL517 was ligated with BamHI cleaved pDN2801 and sub¬ sequent transformation to competent B. subtilis cells (DN1885) resulted in strain CH14. Transformants were in both cases screened for their chloramphe- nicol resistance, and the desired plasmid constructions in the strains CH7 and CH14 by restriction analysis of their plasmids. The two versions of the Endol gene on the plasmids pCH7 and pCH14 are thus transcribed from the same promoter P m . A restriction map of the plasmids is shown in Fig. 6. The B.subtilis DN1885 used for these experiments produces an endoglucanase of its own, which of course gave some background activity. The Endol gene product was exported to the culture medium from the recombinant strain CH7, and the activity measured in the culture supernatant was approximately 20 times higher than the background activity (Fig. 6) . No extracellular activity above the background level was detected from the recombinant strain CH14 which contains the Endol gene with the substituted C-terminal. The culture supernatants from the strains CH7 and CH14 and cell extract from CH7 cells were analysed by the zymogram technique. The zymogram revealed active protein bands of approx. 75000 D and 58000 D from the CH7 cell extract and only one active protein band of approx. 58000.D from the CH7 culture superna¬ tant. These bands correspond to those observed in E.coli and the processing of approx. 90 amino acids from the C-terminal appears to take place in B.subtilis too. The plasmid pCH7 was transformed to PL1801, which is a der¬ ivative of DN1885 lacking the two main exoproteases (apr ~ ,npr " ) , resulting in the strain CH14. The Endol cellulase as produced from CH14 was processed "normally" indicating that the two main exoproteases from B.subtilis are not responsible for the C- terminal processing of the Endol cellulase. A very weak active band of approx. 58000 D was detected from the CH14 culture supernatant, indicating that the manipulated gene is expressed and processed in at least almost the same way as the native gene product. Among other things, the very low expression from pCH14 and the fact that the two genes are expressed from the same expression signals may indicate that the approx. 90 C-terminal amino acids are necessary for the export of the Endol gene product from B.subtilis. Optimization of expression of the Endol gene in E. coli In order to optimize the expression of Endol. the Endol gene was combined with the strong E.coli promoters P R and P L originating from phage lambda (Remaut et al., 1981). Both promoters are repressible by the lambda cI857 repressor, which is heat labile, thus rendering the P R and P L promoters heat inducible, in cells producing the lambda CI857 gene product. (Ptashne et al. , 1982) . The P R promoter is contained on the expression plasmid pPL170 together with the lambda cI857 gene. (Fig. 7; Jørgensen, 1983). The P R promoter was placed upstream of the Endol gene by ligating the P R containing Pvul - Sail fragment from pPL170, to the Endol gene containing Pvul - Sail fragment. Transformation to competent MClOOO cells resulted in the strain TL05 containing the plasmid pTL05. In the plasmid pTL05 the 0-lactamase promoter is deleted, thus bringing the Endol gene under transcriptional control of the P R promoter. At this point, the Endol gene was believed to be contained within the Bglll fragment from pPL2129. The Endol gene fusion to vector sequences on pPL212 was therefore transferred to pTL05, resulting in a fusion protein where 105 C-terminal amino acids are encoded by vector sequences. This fusion protein is however processed correctly as shown earlier with the strain PL212. The cellulase production from TL05 is completely repressed at 28 ° C and induced at 42°C. The P L promoter provided on the plasmid pPLc28 (Remaut et al., 1981) was combined with the Endol gene by ligating the Bglll fragment from pPL212 to BamHI cleaved pPLc28. Transforming to competent PL248 cells, which are harbouring the lambda cI857 gene on a compatible pACYC177 based plasmid (pNF2690) , resulted in the strain LA03, containing the plasmid pLA03 (Fig. 7). The Endol gene is thus fused to vector sequences, but due to unspecified DNA sequences in pPLc28 the length and nature of the resulting fusion protein is unknown. The cellulase production from LA03 was completely repressed at 28 β C and induced at 40.5°C. The cellulase production from LA03 and TL05 was evaluated at different temperatures. LA03, TL05 and PL212 were grown overnight at 28 β C in NY medium supplemented with the appropriate antibiotics (AMP + KAN, KAN and TET, respectively) . For each strain the overnight cultures were diluted 100 fold in NY medium (AMP + KAN, KAN and TET, respectively) , and the diluted cultures were grown at different temperatures between 28°C and 42 β C. Cells from each culture were harvested at OD 450 = 1 and lysed on a French Press, and the activity in the extracts was determined as described earlier. LA03 which exhibited the highest cellulase production was unable to grow at temperatures above 40.5°C. The experiment was repeated without antibiotic selection pressure in the diluted cultures. Similar results were obtained, but LA03 grew very slowly at temperatures above 40.5°C. However, this growth was followd by a significant loss of the plasmid pLA03. No signifi¬ cant loss of pLA03 at temperatures up to 40.5 β C or pPL212 and pTL05 at any temperature, was observed. Optimization of expression of the Endol gene in B.subtilis In order to optimize the expression of the Endol gene in B.subtilis. the Endol gene was fused to the expression-signals (promoter, ribosome binding site and signal sequence) from the alpha-amylase gene from B.licheniformis. which is expressed in high amounts in B.subtilis. PPL1759 contains the promoter, ribosome binding site and most of the signal peptide of the B.licheniformis alpha-amylase (Stephens et aJL., 1984) . The downstream side of this region ends with a Pstl site, which again is followed by a polylinker (Fig. 8) . Between the Pstl and the Sail site in the polylinker of pPL1759, a synthetic DNA fragment consisting of two comple¬ menting oligonucleotides creating Pstl and Sail "sticky" ends was inserted. In the resulting plasmid pCH52 the synthetic linker reconstitutes the missing part of the signal peptide of the alpha-amylase and further encodes the first 14 N-terminal amino acids of the mature Endol cellulase (Fig. 8) . The linker thus creates a hybrid signal peptide cleavage site between the alpha-amylase and the cellulase. The expected cleavage site is shown in Fig. 8. From pPL517 the Endol gene was excised without promoter on an Eagl fragment and inserted into the unique Eagl site in pCH52. The plasmid in which the Endol gene was inserted in the correct orientation was named pCH54. pCH54 contains two direct repeated sequences of 45 bp (e.g. the 45 N-terminal base pairs of the mature Endol gene) which may recombine, deleting the region between them (Ehrlich et al., 1986) . This recombination event, however, occurs with a very low frequency when the repeat is as small as 45 bp. In order to enrich the amount of plasmid that has recombined, a plasmid preparation of pCH54 was cut with the enrichment restriction enzyme Kpnl. pCH54 contains a unique Kpnl site between the two direct repeats and only non-recombinant plasmids are cut with Kpnl. while recombinant plasmids stay circular. When B.subtilis (DN 1885) was retransformed with this mixture, transformants were mostly (90%) containing recombinant plasmids, since B.subtilis competent cells are not transformed with linerized plasmid DNA. The recombinant plasmid was called pCH57 and is contained in the strain CH57. The structure was confirmed by restriction analysis, but the gene fusion was not confirmed by DNA sequencing. In this construction pCH57 the Endol gene is thus perfectly fused to the alpha-amylase expression signals. The endoglucanase is produced extracellularly from the B. subtilis strain CH57, indicating that the hybrid signal cleavage site is functioning. The secreted Endol endoglucanase is processed to the expected M r , namely 58000 D. The production of the Endol cellulase from CH57 was evaluated in two different media, NY (overnight at 37 β C) and BPX (7 days at 37 β C) . The BPX medium is a very rich medium in which the nutrients are slowly released, thus keeping the cells in an early stationary fase for several days during fermentation. The alpha-amylase expression signals function particularly well in this medium. The results appear from Table 1 below. Table 1 STRAIN PLASMID U/ml U/ml NY BPX Analysis of the culture supernatant (BPX-medium) on PAGE revealed a dominant (90%) endoglucanase band corresponding to a concentration of endoglucanase in the supernatant of approx. 0.5 g/L. Example 2 Endocellulase 2 (Endo 2) DNA-sequence The nucleotide sequence of endocellulase 2 (Endo2) was deduced from the plasmid pPL382 which is described above. The plasmid contains approx. 2500 bp. of Bacillus spp. PL236 DNA. The sequence was determined by the chemical modification method (Maxam and Gilbert, 1980) using the partial restriction map and the sequencing strategy outlined in Fig. 9. The complete nucleotide sequence is shown in Sequence Listing ID#3. A computer analysis of this sequence revealed only one open reading frame long enough to encode for the approx. 56000 D protein detected in the zymogram analysis of the extract from the cellulase positive clone E.coli MClOOO(pPL382) . This sequence begins at position 172 and ends at 1869 and encodes an enzyme consisting of 566 amino acids. The calculated M r is 62551 D which is slightly higher than the M r of 56000 D determined by zymogram analysis. This difference could be due to inaccuracy in the zymogram analysis or to post-translational processing beyond the expected processing of the signal peptide. The ATG initiation codon in position 172 was selected because it was the only initiation codon within the open reading frame, which was proceeded by a ribosome binding site AAGGAGG (Mclaughlin et al., 1981) . This initiation codon was followed by a signal sequence-like sequence, and by use of the signal sequence cleavage model proposed by Heijne (1983) , the cleavage site could be predicted to be between the two alanine residues in the middle of the sequence Leu-Ala-Ala-Ala. The signal sequence of the Endo12 is thus 30 amino acids long. The region upstream of the open reading frame contained a sequence homologous with the sigma 43 type promoters of B.subt- ilis (Johnson et al., 1983) at position 46-75. This sequence consists of TTTACA as the -35 region and TATTAT as the -10 region; the two are separated by 18 nucleotides. A palindromic repeat sequence of 13 bp. was found downstream of the termination codon at position 1956-1981, which seems to resemble a rho-independent terminator (Rosenberg and Court, 1979) . Southern analysis The Bacillus spp. (PL236) chromosomal DNA was digested with Hindlll, Pstl, EcoRI and Xhol and the plasmid pPL382 was used as a probe for the hybridization. The hybridization pattern obtained confirmed that the Bacillus spp. PL236 DNA was cloned in non-deleted form, that the two Hindlll fragments from pPL382 was continuous on the Bacillus spp. PL236 chromosome and that the Endo2 gene was different from the other cloned endoglucana¬ ses. Expression of Endo2 in B.subtilis Plasmid pPL382 was transformed to B.subtilis DN1885 to achieve secretion of the mature Endo2 product. B. subtilis DN1885 (pPL382) was grown aerobically in 640 ml LB-medium containing 10 μg/ml tetracycline for 30 hours. The supernatant was concentrated by precipitation for 24 hours with (NH 4 ) 2 S0 4 at 70% saturation. After 5 hours of dialysis against 100 mM Tris-HCl pH 7, the concentrated supernatant was heated to 55°C for 15 minutes. Denatured protein was removed by centrifugation and the soluble proteins were subsequently precipitated with (NH) 2 S0 4 at 70% saturation. The resolubilized proteins were dialyzed against 100 mM Tris-HCl pH 7 with a final volume of 2 ml and were applied to a 80 cm x 1 cm gel filtration column containing Ultrogel AcA 44 (LKB) . Active fractions were pooled, concentrated and applied to a SDS containing polyacryla ide gel, where the endoglucanase appeared as a single band at 56 kDa. The activity yield of the method was approximately 10%. The endoglucanase comigrates with the endoglucanase obtained from extracts of MClOOO (pPL382) as detected by Zymogram analysis in the supernatant of DN1885 (pPL382) . The endogluca- nase activity of DN1885 (pPL382) is about 25 times that of strain DN1885 when grown in NY medium. EXAMPLE 3 Endocellulase 3 Physical mapping of the endocellulase clone 3 The endocellulase clone 3 is represented by the plasmid pPL591 which contains a 11000 bp. EcoRI fragment of Bacillus spp. PL236 DNA. A partial restriction map of this plasmid is shown in Fig. 10. Zymogram analysis of extracts from E.coli MClOOO(pPL591) shows that the insert gives rise to four proteins with cellulase activity. The approx. M r of these proteins were 60000 D, 56000 D, 45000 D and 30000 D (Fig. 10). Southern analysis showed that the EcoRI fragment from pPL591 was cloned in a non-deleted form from the Bacillus spp. PL236 chromosome, and indicates that the DNA did not contain the DNA- sequences encoding the Endol and Endo2 genes. To analyse whether these proteins represented post-trans- lational processing products from one or several cellulase genes, deletion plasmids were made using the restriction enzymes Hindlll and Smal. Deletion of the 4800 bp. Smal fragment, resulting in the plasmid pPL538, did not eliminate any of the four cellulase bands on the zymogram. The B.spp. DNA insert on pPL591 contained 5 Hindlll sites. Digestion of pPL591 with Hindlll thus gave 6 fragments of 6600 bp. (vector fragment) , 3200 bp., 1700 bp. , 1550 bp. , 1350 bp. and 900 bp., where the 1700 bp. fragment had originated from the Hindlll site within the pUN121 vector plasmid (Fig. 11) . Elimination of all of the Hindlll fragments except the 1700 bp. fragment (reinserted in the opposite direction) resulted in the plasmid pPL540. Removal of the rest of the Bacillus spp. PL236 DNA by eliminating the EcoRI fragment from pPL540 resulted in the plasmid pPL587. Both the E.coli MClOOO(pPL540) and the E.coli MClOOO(pPL587) were cellulase-positive and zymogram analysis of extracts from these clones revealed only the 45000 D and 30000 D proteins. The protein-coding capacity of the Bacillus spp. PL236 DNA (approx. 1500 bp.) is approx. 55000 D, which is too small to contain two endoclucanase genes of 45000 D and 30000 D. The 30000 D protein on the zymogram is thus most likely a result of immature post-translational processing of the 45000 D protein. The cellulase gene encoding the 45000 D protein was designated Endo3A. The Endo3A gene was cloned in both direc¬ tions on the plasmids pPL587 and pPL538 giving rise to the same two proteins, thus eliminating the chance of the protein being a fusion protein. Elimination of the 1550 bp. , the 1350 bp. and the 900 bp. fragment resulted in the plasmid pPL542. Extracts from E.coli MClOOO(pPL542) revealed cellulase positive proteins of approx. 30000 D, 45000 D, 49000 D and 56000 D (Fig. 10) . From these preliminary results, the existence of two additional endogluca- nase genes within the original insert on pPL591 and pPL538 are postulated. The additional endoglucanase genes are designated Endo3B and Endo3C. Their postulated position on the Bacillus spp. PL236 DNA is shown in Fig. 11. The postulated model is based on the assumption that the 60000 D protein made from pPL538 and pPL591 is converted to a truncated fusion protein of 49000 D made from pPL542 where the Hindlll fragment of 1350 bp. is deleted. DNA-seguence of Endo3A The DNA sequence of the Endo3A gene was deduced from the plasmid pPL540 containing approx. 1500 bp. of Bacillus spp. PL236 DNA using the dideoxy chain termination method. The gene was placed in pUC18 in both orientations, and a number of deletions were constructed. Standard primers were used except for one synthe- tic oligonucleotide that was used for sequencing a region with no practical restriction sites. The C-terminal part of the gene was deduced from the plasmid pPL538. The partial DNA-sequence is shown in Sequence Listing ID#6. The sequence revealed an open reading frame coding for a protein with a M r of about 62000 D which is in agreement with the observed protein of 60kD in the zymograms. The ATG start codon (position 30) is preceded by a typical ribosome binding site (Mclaughlin et al., 1981). The initiation codon is followed by a typical gram-positive signal sequence and by using the signal sequence cleavage model (Heijne, 1983) a signal sequence of 36 amino acids is revealed. EXAMPLE 4 Endocellulase 4 Zymogram analysis The endocellulase clone No. 4 is represented by the plasmid pPL592, which contains approx. 14000 bp. of Bacillus spp. PL236 DNA. A partial restriction map is shown in Fig. 1. A zymogram analysis of extracts from E.coli MClOOO(pPL592= revealed three cellulase active proteins with M r values of approx. 92000 D, 74000 D and 71000 D. Further analysis is necessary to determine whether these proteins are encoded by one or several cellulase genes. Southern analysis confirmed the origin of the cloned DNA on the Bacillus spp. PL236 chromosome, and indicates that the cloned DNA is not represented on the other endoglucanase clones. EXAMPLE 5 A. Endol cloned and expressed in Bacillus subtilis An agar slant was inoculated with _____ subtilis strain CH 57 and incubated for 20 hours at 37 β C. 10 ml of a 0.9 % NaCl-solution was added to the test tube which was shaken to suspend the cells. The cell suspension was used to inoculate a 2 1 fermentor. The following parameters were used to run the fermentation: Temperature: 37"C. Aeration: 1,1 1/minute. Stirring: 1100 rpm. Fer entor: A 2 1 model with a working volume of 1,5 1. The pH was maintained between 6.2 and 7.2 for the first 40 hours of fermentation. After that the pH was maintained between 6.7 and 7.2. The pH was maintained within this range by dosing with NH 3 and H 3 P0 4 . Dosing of a glucose solution was initiated after 40 hours at a flow rate of 3.7 ml/hour. Substrate Potato starch degraded with Termamyl* 50 g Soybean meal 110 g Corn steep Liquor 16.5 g Alburex (potato protein) 27.5 g (NH 4 ) 2 S04 2.2 g KH 2 P0 4 1,2 g Na 2 HP0 4 .2H 2 0 5,9 g Water added up to 1100 ml. *Termamyl is a commercial B_ t _ licheniformis α-amylase avalable from Novo Nordisk A/S. Glucose solution Glucose.H 2 0 600 g Citric acid 0,6 g Water added up to 1000 ml The fermentation was stopped after 166 hours of fermentation at aann OODD 665500 vvaalluuee ooff 112222.. AAtt tthhaatt ttiinme there were 1200 ml of fermentation broth in the fermentor. The fermentation broth was centrifuged, and the extracellular volume was 400 ml containing 40 CMC-endoase units per ml (16.000 CMC-endoase units in all) . The culture medium was further processed by filtration and dilution followed by concentration on an Amicon ultrafiltration module with a cut-off at 10.000 MW. The concentrated enzyme solution was frozen. Half of the frozen liquid was thawed and diluted with deionized water and then concentrated once more on an Amicon ultrafiltration module. The total yield was 4471 CMC-endoase units (from 8000 CMC-endoase units) . The total volume of 1050 ml was subjected to ion exchange chromatography at pH 7. The enzyme was bound to a DEAE- Sephacryl anion exchange column (300 ml volume) at pH 7 (50 mM tris-HCl) . The Endol enzyme was eluted at pH 7 with 0.3 M NaCl. The purified enzyme has a molecular weight of 58,000 D on SDS- PAGE. The pi is 4.0. Its activity is 30 CMC-endoase units per mg protein. The protein determination is based on the amino acid composition of the enzyme deduced from the DNA sequence: 13 tryptophan, 30 tyrosine and a molecular weight of 57,566 D. The extinction coefficient is calculated by means of the following formula: ( 13 X 5559 + 30 X 1197 ) / 57566 = 1.88 The purified enzyme has an endoglucanase activity of 57 CMC- endoase units-per ml and an absorbance at 280nm of 3.6. Thus, (57 x 1.88)/3.6 = 30 CMC-endoase units per mg protein. B. Stability of Endol in detergents The following 4 detergent compositions were used: 1. USA liquid detergent: 2 gram per liter of 6" hardness water ( 1 part tap water to 2 parts deionized water) . The pH was measured to 7,29. 2. USA Heavy Duty Powder detergent: 0,9 gram per liter of 6° hardness water. The pH was measured to 9,2. 3. Heavy Duty Powder detergent (2) with bleach and activator: 0.12 gram/1 Na-perborate tetrahydrate and 0,088 gram/1 NOBS. The pH was measured to 9,2. 4. European Heavy Duty Powder detergent with bleach and activator (Batch DR 8806 Europe). 5 gram per liter in 9° hardness water. Celluzyme™ (batch CAX 007 crude enzyme with cellulase and other enzymes) 2353 CMC-endoase units per gram was compared with Endol 30000 CMC-endoase units per gram. The enzymes were diluted to 3 CMC-endoase units per ml in all 4 detergent solutions: The endoglucanase activity after dilution was measured as described above (by determining the decrease in the viscosity of CMC) . The endoglucanase activity after 60 min. incubation at 40 °C was measured and compared with the initial activity. The following results were obtained: Detergent solution 1 2 3 4 Celluzyme™ • 89% 75% 66% 75% Endol 106% 90% 97% 98% The standard deviation is 10%, It appears from the table that Endol is more stable at a pH of 9-10 compared wi •th CelluzymeTM i•n these detergents. EXAMPLE 6 Preparation of full-length (-75 kD) Endol in B. subtilis 10 1 of LB medium containing 1 mM CuCl 2 and 10 μg/ml chloramphenicol was inoculated with 10 ml of an overnight culture of B___ subtilis DN969 (B. Diderichsen et al., J.Bacteriol. 172(8). 1990, pp. 4315-4321) containing the plasmid pCH7 (described above in example 1) , divided among 10 sterile 2 1 flasks and incubated with vigorous shaking for 36 hours at 37°C. The culture was centrifuged for 10 minutes at lOOOOxg and 4 β C after which EDTA, pH 8, was added to the supernatant to a final concentration of 5 mM. 25 g of Avicel PH-105 which had been hydrated in ethanol and washed with destilled water was added to the supernatant which was left standing with gentle stirring for 2 hours at 4°C. The supernatant/Avicel mixture was centrifuged for 10 minutes at lOOOOxg and 4 β C. The cleared supernatant was decanted off immediately after the rotor had stopped. The Avicel/enzyme cake was resuspended and washed in 200 ml of ImM EDTA, and the mixture was centrifuged for 1 minute at lOOOOxg. This procedure was repeated twice. The Avicel/enzyme cake was then resuspended in 150 ml (1% triethylamine and l mM EDTA) and was -left standing with vigorous stirring for 1 hour at 4°C. The mixture was centrifuged for 1 minute at lOOOOxg and 4"C. The supernatant was retained. This procedure was repeated twice. The solution of enzyme and triethylamine (about 300 ml) was was evaporated jLn vacuo to 100 ml. The temperature of the solution was not allowed to exceed 10 ° C. The pH was adjusted to 7 by adding 1M HC1, and the solution was frozen at -70°C. The amount of protein in the 100 ml enzyme solution was determined to be 40 mg by means of a Bradford reagent (available from BioRad) using bovine serum albumin as the standard. B. Characterisation of the -75 kD Endol The enzyme obtained above had a purity of about 90%. The enzyme was found to have a molecular weight of 75 kD on SDS-PAGE. In immunoprecipitation experiments (carried out by rocket immunoelectrophoresis in agarose gel as described by N. Axelsen et al. , Chapter 2 in A Manual of Quantitative Immunoelectrophoresis. Blackwell Scientific Publ. 1973), ~75 kD Endol was immunoreactive with a monospecific rabbit antibody raised against the core region (-58 kD form) of Endol. The -75 kD and -58 kD (processed) forms of Endol were tested for their ability to bind to cellulose (Avicel) . 10 μg of each of -75 kD Endol and -58 kD Endol were added to 50 μl of a cellulose suspension (0.1% w/w Avicel, 5 mM EDTA, pH 8.0) . The suspensions were shaken for 30 minutes, and the Avicel was harvested at 10000 rpm for 2 minutes. The amount of Endol cellulase remaining in the supernatant was analysed by SDS-PAGE. More than 95% of the -75 kD Endol was bound to the cellulose, while less than 5% of the -58 kD Endol was similarly bound. This shows that the C- terminal part of the -75 kD Endol cellulase comprises a cellulose-binding domain. Extensive amino acid sequence homology was found between this region (from amino acid 554 to 700) of the Endol cellulase and other cellulases, e.g. an endocellulase from Bacillus subtilis (Nakamura et al., 1987), the middle part of the bifunctional cellulase from Caldocellum saccharolvticum (D.J. Saul et al., Nucl.Acids Res. 17. 1988, p. 439), two endocellulases from Clostridium stercorarium (W. Schwarz et al., Biotech.Let . 11. 1989, pp. 461-466) . C. Colour clarification effect of -75 kD Endol The colour clarification effect of -75 kD Endol was determined by exposing a prewashed worn textile surface to the enzyme and then measuring the clarity of the surface colour compared to the clarity of the surface colour of textiles which had not been treated with the enzyme. Black 100% cotton swatches (15 x 10 cm) were prewashed and tumble-dried under the following conditions Detergent: Keminus (available from Irma A/S) , 1.5g/l Temperature: 70 β C Washing time: 60 minutes Drying time: 30 minutes No. prewashing/ drying treatments:15 The swatches were prewashed in a conventional washing machine (Miele Deluxe Electronic W761) . After each wash, the swatches were dried in a tumble-drier. The visual effect of the prewashing/drying was that the surface colour turned greyish due to the presence of damaged cellulose fibres causing the worn look. After prewashing, the swatches were washed in a Terg-O-Tometer (toploaded mini washing machine) under the following conditions No. of treatments: 3 Standard detergent: LAS NANSA 1169/P: 10% AE Berol 160: 15% Ethanol, 96%: 10% TEA: 5% Water: 60% After each wash, the swatches were rinsed in tap water and dried at room temperature. The surface colour of the swatches was analysed by measuring reflected light. White light was projected onto the surface, and the reflection/remission was measured at 16 wavelengths (400 n - 700 nm) . The results from the measurements were processed (by means of an "Elrepho 2000" apparatus available from Datacolor, Switzerland) into Hunter coordinates of which the L-coordinate represents the grey scale values. Each swatch was analysed twice on each side, and the results shown below are are a total average from the measurements of the two swatches from the same treatment. In the table, white is L = 100, and black is L = 0. Dosage 0 CMC endoase/160 CMC endoase/1 L 16.78 15.50 S.D. 0.08 0.03 Delta L - 1.28 Comparable results were obtained with Celluzyme™ (batch PPC 2174 containing a mixture of enzymes from Humicola insolens. DSM 1800) Dosage (CMC endoase/1) 0 15 30 60 Delta L - 1. 02 1. 48 1. 90 REFERENCES Beguin, P.: Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas. Anal. Biochem. 131 (1983) 333-336. Bolivar, F. , Rodriguez, R.L., Green, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W. Crosa, J.H. , and Falkow, S.: Construction and characterization of new cloning vehicles II. A multipurpose cloning system. Gene 2 (1977) 95-113. Casadaban, J.M. and Cohen, S.N.: Analysis of gene control signals by DNA fusion and cloning in Esc erichia coli. J. Mol. Biol. 138 (1980) 179-207. Chang, A.C.Y. and Cohen, S.N. J. Bacteriol., 134, 1141. (1978). Diderichsen, B. and Christiansen, L. Cloning of a maltogenic alpha-amylase from Bacillus stearothermophilus. FEMS Microbiol. lett. 56, 53-60. (1988). Dubnau, D. and Davidoff-Adelson, R. Fate of transforming DNA following uptake by competent B.subtilis 1. Formation and properties of the donor recipient complex. J. Mol. Biol. 56, 209-221. (1971). Ehrlich, S.D., Riele, H. , Petit, M.A. , Janniere, L. , Noirot, P. and Michael, B. DNA recombination in plasmids and the chromosome of B.subtilis. Bacillus Mol. Genet, and Biotech.Appl. 27-33. (1986) . Heijne, G. : Patterns of amino acids near signal-sequence cleaage sites. Eur. J. Biochem. 133, 17-21 (1983). Holmes, D.S. and Quigley, M. : A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114 (1981) 193-197. Horinouchi, S. and Weisblum, B. : Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bact. 150, 2, 815-825. Johnson, W.C., Moran, C.P. Jr. and Losich, R. : (1983) Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gen. Jørgensen, P.L. : Construction of vectors for cloning of pro- motorer. M.Sc. Thesis, The Technical University of Denmark (1983) . Keggins, K.M. r Lovell, P.S. and Duvall, E.J. Molecular cloning of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUBHO. Proc.Natl.Acad.Sci.USA.75, 3, 1423-1427. (1978) . Kreft, J., Burger, J.K. and Goebel, W. : Expression of antibi- otic resistance genes from E.coli in Bacillus subtilis. Mol.Gen.Genet. 190: 384-389 (1983). Laemmli, U.K.: Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227 (1970) 680- 685. Loening, U.E. : The fractionation of high-molecular-weight ribonucleic acid polyacrylamide gel electrophoresis. Biochem. J. 102 (1967) 251-257. Mandel, M. and Higa, A.: Calcium dependent bacteriophage DNA infection, J. Mol. Biol. 53 (1970) 154-162. Maniatis, T. , Fritsch, E.F., and Sambrook, J. : Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Maxam, A.M. and Gilbert, W. : Sequencing end-labelled DNA with base-specific chemical cleavage. In: Grossman, L. and Moldave, K. (Eds., Methods in Enzymology, Vol. 65, Academic Press, New York, 1980, pp 499-560. Mclaughlin, J.R., Murray, C.L. and Babinowitz, C.J. 1981. Unique features in the ribosome binding site sequence of the gram- positive Staphy1ococuccus aureus B-laclamase gene. J. Biol. Chem. 256: 11283-11291. von Meyenburg, K. , Jørgensen, B.B., Nielsen, J. , and Hansen, F.G.: Promoters of the atp operon coding for the membrane bound ATP synthase of Escherichia coli mapped by TnlO insertion mutations. Mol. Gen. Genet. 188 (1982) 240-248. Miller, G.L.: Use of dinitrosalicyclic reagent for determina¬ tion of reducing sugars, Anal. Chem. 31. (1959) 426-428. Nakamura, A., Uozumi, T. , Beppu, T. (1987). Nucleotide sequence of a cellulase gene of Bacillus subtilis. Eur. J. Biochem. 164, 317-320. Nilsson, B. , Uhlen, M. , Josephson, S., Gatenbeck, S. and Philipson, L. : An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nucleic Acids Res. 11: 8019-8030. Ptashne, M. , Johnson, A.D. and Pabo, CO.: A genetic switch in a bacterial virus. Scientific American, November (1982) . Remaut et al., Gene 15. 1981, pp. 81-93. Remaut, E. , Stanssens, P. and Fiirs, W. : Plasmid vectors for high-efficiency expression controlled by the P L promoter of coliphage lambda. Gene, 15, 81-93. (1981). Rigby, P.W.J., Dieckmann, M. , Rhodes, C. and Berg, P. (1977): Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. I. Mol. Biol. 113, 237-251. Rosenberg, M. and Court, D. : Regulatory sequences involved in the promotion and termination of RNA transcription. Ann. Rev. Genet. 1979 13: 319-53. Sancar, A., Hack, A.M., and Rupp, W.P.: Simpel methods for identifications of plasmid-coded proteins, J. Bacteriol. 137 (1979) 692-693. Sanger, F. , Nichlen, S. and Coulson, A.R. (1977). Proc. Natl. Acad. Sci. USA 74., 5463-5467. Southern, E.: Petection of specific sequences among PNA fragments separated by gel electrophoresis. I. Mol. Biol. 98, 503-517 (1975) . Stephens, M.A. , Ortlepp, S.A., Ollington, J.F. and McConnel, D.J. J.bact. Vol.158, No 1, p 369-372. (1984). Teather, R.M. and Wood, P.J. : Use of Congo red-polysaccharide interactions in enumeration and characterization of celluloly¬ tic bacteria from the bovine rumen. Appl. Environ. Microbiol. -3 (1982) 777-780. Walseth, C.S.: Occurrence of cellulases in enzyme preparations from microorganisms. Tappi, Vol. 35, No. 5 (1952) 228-233. Yanisch-Perron, C. , Vieira, J. and Messing, J. Gene, 33, 103- 119. (1985). SEQUENCE LISTING ( 1 ) GENERAL INFORMATION : (i) APPLICANT: Novo Nordisk A/S (ii) TITLE OF INVENTION: An Enzyme Exhibiting Cellulase Activity (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Novo Nordisk A/S, Patent Department (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: DENMARK (F) ZIP: DK-2880 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: (B) COMPUTER: (C) OPERATING SYSTEM: (D) SOFTWARE: (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: DK 164/90 (B) FILING DATE: 19-JAN-1990 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Thalsø-Madsen, Kine Birgit (B) REGISTRATION NUMBER: (C) REFERENCE/DOCKET NUMBER: 3425.204-WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: +4544448888 (B) TELEFAX: +4544493256 (C) TELEX: 37304 (2) INFORMATION FOR SEQ ID N0:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2977 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus lautus (B) STRAIN: NCIMB 40250 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 677..2776 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: ATCTTGAAGC TTCGAGGTTC GCGTAACCAG GTCGATCGGC TGACTTTCCT CGCCAAGCTG 60 CACCATGGCT TCATAGATTA ATTGGTGAGG AGGATCATAG AAATCTTCCG TCCGCACCGC 120 TCCATGGCGG TAATCAGCGC TTCCGACTGC AGTACAGAGC GGCCGATACA AATGCAGCCG 180 ACAACCAGAT CAAGCCGTCC TTCAACATCA AAAACAACGG TACTTCGGCT GTTGATTTAA 240 GCACGCTCAA AATCCGCTAC TACTTCACCA AGGATGGTTC TGCGGCGGTG AACGGCTGGA 300 TCGACTGGGC GCAGCTCGGC GGCAGCAACA TTCAGATCTC GTTTGGCAAC CATACTGGCA 360 CGAATTCGGA TACGXACGTG GAGCTGAGCT TCTCGTCCGA GGCAGGCTCG ATTGCGGCGG 420 GCGGCCAATC CGGTGAAATC CAGCTGCGCA TGTCCAAGAC GGACTGGTCG AACTTTAACG 480 AGGCGAACGA CTACTCGTTC GATGGGACGA AGACGGCCTT TGCTGACTGG GATCGGGTCG 540 TATTGTACCA GAACGGCCAA ATAGTGTGGG GAACTGCTCC ATAAACCGAT ACAGGGGAAT 600 GTGCCGGAAC CGCTCTTTTG CAGGGCAGAC TGGCGGTATC CCTTGCTGAA ATGACTATTC 660 CTGGGAGGGA TCAAAA ATG AAG ACA AGA CAA AGA AAG CGG CTG TTC GTC 709 Met Lys Thr Arg Gin Arg Lys Arg Leu Phe Val 1 5 10 AGT GCG GCG CTG GCA GTA TCC TTG ACA ATG ACC GTA CCG ATG CCC GCT 757 Ser Ala Ala Leu Ala Val Ser Leu Thr Met Thr Val Pro Met Pro Ala 15 20 25 TCT GTA AAT GCA GCT GCG AGT GAT GTC ACT TTC ACG ATT AAT ACG CAG 805 Ser Val Asn Ala Ala Ala Ser Asp Val Thr Phe Thr He Asn Thr Gin 30 35 40 TCG GAA CGT GCA GCG ATC AGC CCC AAT ATT TAC GGA ACC AAT CAG GAT 853 Ser Glu Arg Ala Ala He Ser Pro Asn He Tyr Gly Thr Asn Gin Asp 45 50 55 CTG AGC GGG ACG GAG AAC TGG TCA TCC CGC AGG CTC GGA GGC AAC CGG 901 Leu Ser Gly Thr Glu Asn Trp Ser Ser Arg Arg Leu Gly Gly Asn Arg 60 65 70 75 CTG ACG GGT TAC AAC TGG GAG AAC AAC GCA TCC AGC GCC GGA AGG GAC 949 Leu Thr Gly Tyr Asn Trp Glu Asn Asn Ala Ser Ser Ala Gly Arg Asp 80 85 90 TGG CTT CAT TAC AGC GAT GAT TTT CTC TGC GGC AAC GGT GGT GTT CCA 997 Trp Leu His Tyr Ser Asp Asp Phe Leu Cys Gly Asn Gly Gly Val Pro 95 100 105 GAC ACC GAC TGC GAC AAG CCG GGG GCG GTT GTT ACC GCT TTT CAC GAT 1045 Asp Thr Asp Cys Asp Lys Pro Gly Ala Val Val Thr Ala Phe His Asp 110 115 120 AAA TCT TTG GAG AAT GGA GCT TAC TCC ATT GTA ACG CTG CAA ATG GCG 1093 Lys Ser Leu Glu Asn Gly Ala Tyr Ser He Val Thr Leu Gin Met Ala 125 130 135 GGT TAT GTG TCC CGG GAT AAG AAC GGT CCA GTT GAC GAG AGT GAG ACG 1141 Gly Tyr Val Ser Arg Asp Lys Asn Gly Pro Val Asp Glu Ser Glu Thr 140 145 150 155 GCT CCG TCA CCG CGT TGG GAT AAG GTC GAG TTT GCC AAA AAT GCG CCG 1189 Ala Pro Ser Pro Arg Trp Asp Lys Val Glu Phe Ala Lys Asn Ala Pro 160 165 170 TTC TCC CTT CAG CCT GAT CTG AAC GAC GGA CAA GTG TAT ATG GAT GAA 1237 Phe Ser Leu Gin Pro Asp Leu Asn Asp Gly Gin Val Tyr Met Asp Glu 175 180 185 GAA GTT AAC TTC CTG GTC AAC CGG TAT GGA AAC GCT TCA ACG TCA ACG 1285 Glu Val Asn Phe Leu Val Asn Arg Tyr Gly Asn Ala Ser Thr Ser Thr 190 195 200 GGC ATC AAA GCG TAT TCG CTG GAT AAC GAG CCG GCG CTG TGG TCT GAG 1333 Gly He Lys Ala Tyr Ser Leu Asp Asn Glu Pro Ala Leu Trp Ser Glu 205 210 215 ACG CAT CCA AGG ATT CAT CCG GAG CAG TTA CAA GCG GCA GAA CTC GTC 1381 Thr His Pro Arg He His Pro Glu Gin Leu Gin Ala Ala Glu Leu Val 220 225 230 235 GCT AAG AGC ATC GAC TTG TCA AAG GCG GTG AAG AAC GTC GAT CCG CAT 1429 Ala Lys Ser He Asp Leu Ser Lys Ala Val Lys Asn Val Asp Pro His 240 245 250 GCC GAA ATA TTC GGT CCT GCC CTT TAC GGT TTC GGC GCA TAT TTG TCT 1477 Ala Glu He Phe Gly Pro Ala Leu Tyr Gly Phe Gly Ala Tyr Leu Ser 255 260 265 CTG CAG GAC GCA CCG GAT TGG CCG AGT TTG CAA GGC AAC TAC AGC TGG 1525 Leu Gin Asp Ala Pro Asp Trp Pro Ser Leu Gin Gly Asn Tyr Ser Trp 270 275 280 TTT ATC GAT TAC TAT CTG GAT CAG ATG AAG AAT GCT CAT ACG CAG AAC 1573 Phe He Asp Tyr Tyr Leu Asp Gin Met Lys Asn Ala His Thr Gin Asn 285 290 295 GGC AAA AGA TTG CTC GAT GTG CTG GAC GTC CAC TGG TAT CCG GAA GCA 1621 Gly Lys Arg Leu Leu Asp Val Leu Asp Val His Trp Tyr Pro Glu Ala 300 305 310 315 CAG GGC GGA GGC CAG CGA ATC GTC TTT GGC GGG GCG GGC AAT ATC GAT 1669 Gin Gly Gly Gly Gin Arg He Val Phe Gly Gly Ala Gly Asn He Asp 320 325 330 ACG CAG AAG GCT CGC GTA CAA GCG CCA AGA TCG CTA TGG GAT CCG GCT 1717 Thr Gin Lys Ala Arg Val Gin Ala Pro Arg Ser Leu Trp Asp Pro Ala 335 340 345 TAC CAG GAA GAC AGC TGG ATC GGC ACA TGG TTT TCA AGC TAC TTG CCC 1765 Tyr Gin Glu Asp Ser Trp He Gly Thr Trp Phe Ser Ser Tyr Leu Pro 350 355 360 TTA ATT CCG AAG CTG CAA TCT TCG ATT CAG ACG TAT TAT CCG GGT ACG 1813 Leu He Pro Lys Leu Gin Ser Ser He Gin Thr Tyr Tyr Pro Gly Thr 365 . 370 375 AAG CTG GCG ATC ACA GAG TTC AGC TAC GGC GGA GAC AAT CAC ATT TCG 1861 Lys Leu Ala He Thr Glu Phe Ser Tyr Gly Gly Asp Asn His He Ser 380 385 390 395 GGA GGC ATA GCT ACC GCG GAC GCG CTC GGC ATT TTT GGA AAA TAT GGC 1909 Gly Gly He Ala Thr Ala Asp Ala Leu Gly He Phe Gly Lys Tyr Gly 400 405 410 GTT TAT GCC GCG AAT TAC TGG CAG ACG GAG GAC AAT ACC GAT TAT ACC 1957 Val Tyr Ala Ala Asn Tyr Trp Gin Thr Glu Asp Asn Thr Asp Tyr Thr 415 420 425 AGC GCT GCT TAC AAG CTG TAT CGC AAC TAC GAC GGC AAT AAA TCG GGG 2005 Ser Ala Ala Tyr Lys Leu Tyr Arg Asn Tyr Asp Gly Asn Lys Ser Gly 430 435 440 TTC GGC TCG ATC AAA GTG GAC GCC GCT ACG TCC GAT ACG GAG AAC AGC 2053 Phe Gly Ser He Lys Val Asp Ala Ala Thr Ser Asp Thr Glu Asn Ser 445 450 455 TCG GTA TAC GCT TCG GTA ACT GAC GAG GAG AAT TCC GAA CTC CAC CTG 2101 Ser Val Tyr Ala Ser Val Thr Asp Glu Glu Asn Ser Glu Leu His Leu 460 465 470 475 ATC GTG CTG AAT AAA AAT TTC GAC GAT CCG ATC AAC GCT ACT TTC CAG 2149 He Val Leu Asn Lys Asn Phe Asp Asp Pro He Asn Ala Thr Phe Gin 480 485 490 CTG TCT GGT GAT AAA ACC TAC ACA TCC GGG AGA GTA TGG GGC TTC GAC 2197 Leu Ser Gly Asp Lys Thr Tyr Thr Ser Gly Arg Val Trp Gly Phe Asp 495 500 505 CAA ACC GGA TCC GAC ATT ACG GAA CAA GCA GCT ATA ACG AAT ATT AAC 2245 Gin Thr Gly Ser Asp He Thr Glu Gin Ala Ala He Thr Asn He Asn 510 515 520 AAC AAT CAA TTC ACG TAT ACG CTT CCT CCA TTG TCG GCT TAC CAC ATT 2293 Asn Asn Gin Phe Thr Tyr Thr Leu Pro Pro Leu Ser Ala Tyr His He 525 530 535 GTT CTG AAA GCG GAT AGC ACC GAA CCG GTC AAC TCC GAT CTC GTC GTG 2341 Val Leu Lys Ala Asp Ser Thr Glu Pro Val Asn Ser Asp Leu Val Val 540 545 550 555 CAG TAT AAG GAC GGT GAT CGC AAC AAT GCA ACC GAC AAT CAG ATC AAG 2389 Gin Tyr Lys Asp Gly Asp Arg Asn Asn Ala Thr Asp Asn Gin He Lys 560 565 570 CCG CAT TTC AAT ATT CAA AAT AAA GGG ACC AGC CCG GTA GAT CTG AGT 2437 Pro His Phe Asn He Gin Asn Lys Gly Thr Ser Pro Val Asp Leu Ser 575. 580 585 TCC CTT ACC CTG CGC TAC TAT TTT ACC AAA GAC AGC TCT GCA GCG ATG 2485 Ser Leu Thr Leu Arg Tyr Tyr Phe Thr Lys Asp Ser Ser Ala Ala Met 590 595 600 AAC GGC TGG ATC GAT TGG GCG AAG CTC GGC GGC AGC AAC ATT CAG ATT 2533 Asn Gly Trp He Asp Trp Ala Lys Leu Gly Gly Ser Asn He Gin He 605 610 615 TCG TTC GGT AAT CAT AAT GGC GCG GAT TCG GAT ACG TAC GCG GAG CTG 2581 Ser Phe Gly Asn His Asn Gly Ala Asp Ser Asp Thr Tyr Ala Glu Leu 620 625 630 635 GGC TTC TCG TCC GGC GCA GGC TCG ATT GCG GAG GGC GGT CAA TCC GGC 2629 Gly Phe Ser Ser Gly Ala Gly Ser He Ala Glu Gly Gly Gin Ser Gly 640 645 650 GAA ATC CAG CTG CGC ATG TCG AAG GCG GAC TGG TCG AAC TTC AAC GAG 2677 Glu He Gin Leu Arg Met Ser Lys Ala Asp Trp Ser Asn Phe Asn Glu 655 660 665 GCG AAC GAC TAC TCG TTC GAT GGG GCG AAG ACG GCC TAT ATA GAT TGG 2725 Ala Asn Asp Tyr Ser Phe Asp Gly Ala Lys Thr Ala Tyr He Asp Trp 670 675 680 GAT CGC GTG ACG CTA TAC CAA GAC GGA CAA CTC GTA TGG GGA ATC GAG 2773 Asp Arg Val Thr Leu Tyr Gin Asp Gly Gin Leu Val Trp Gly He Glu 685 690 695 CCG TAGAAGATGA CTAGACAACA TTAGTGATGA GACGCGGCCG GCCATAACGG 2826 Pro 700 CTGTCTTGAC TCTGATTCGA TCAAAAAATC AAAGCAAAGG GGATGAAAGT AATGAATGTT 2886 GCGATTCAAA AGAGAATCGG ATCAATATTG ATGATTGCCT CACTAATTAT TAGCTTATTG 2946 CCGTTAGGGA GCAGCAGAAC GTATGCTGCA G 2977 (2) INFORMATION FOR SEQ ID N0:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 700 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: Met Lys Thr Arg Qln Arg Lys Arg Leu Phe Val Ser Ala Ala Leu Ala 1 5 10 15 Val Ser Leu Thr Met Thr Val Pro Met Pro Ala Ser Val Asn Ala Ala 20 25 30 Al a Ser Asp Val Thr Phe Thr He Asn Thr Gin Ser Gl u Arg Al a Al a 35 40 45 He Ser Pro Asn He Tyr Gly Thr Asn Gin Asp Leu Ser Gly Thr Glu 50 55 60 Asn Trp Ser Ser Arg Arg Leu Gly Gly Asn Arg Leu Thr Gly Tyr Asn 65 70 75 80 Trp Glu Asn Asn Ala Ser Ser Ala Gly Arg Asp Trp Leu His Tyr Ser 85 90 95 Asp Asp Phe Leu Cys Gly Asn Gly Gly Val Pro Asp Thr Asp Cys Asp 100 105 110 Lys Pro Gly Ala Val Val Thr Ala Phe His Asp Lys Ser Leu Glu Asn 115 120 125 Gly Ala Tyr Ser He Val Thr Leu Gin Met Ala Gly Tyr Val Ser Arg 130 135 140 Asp Lys Asn Gly Pro Val Asp Glu Ser Glu Thr Ala Pro Ser Pro Arg 145 150 155 160 Trp Asp Lys Val Glu Phe Ala Lys Asn Ala Pro Phe Ser Leu Gin Pro 165 170 175 Asp Leu Asn Asp Gly Gin Val Tyr Met Asp Glu Glu Val Asn Phe Leu 180 185 190 Val Asn Arg Tyr Gly Asn Ala Ser Thr Ser Thr Gly He Lys Ala Tyr 195 200 205 Ser Leu Asp Asn Glu Pro Ala Leu Trp Ser Glu Thr His Pro Arg He 210 215 220 His Pro Glu Gin Leu Gin Ala Ala Glu Leu Val Ala Lys Ser He Asp 225 230 235 240 Leu Ser Lys Ala Val Lys Asn Val Asp Pro His Ala Glu He Phe Gly 245 250 255 Pro Ala Leu Tyr Gly Phe Gly Ala Tyr Leu Ser Leu Gin Asp Ala Pro 260 265 270 Asp Trp Pro Ser Leu Gin Gly Asn Tyr Ser Trp Phe He Asp Tyr Tyr 275 . 280 285 Leu Asp Gin Met Lys Asn Ala His Thr Gin Asn Gly Lys Arg Leu Leu 290 295 300 Asp Val Leu Asp Val His Trp Tyr Pro Glu Ala Gin Gly Gly Gly Gin 305 310 315 320 Arg He Val Phe Gly Gly Ala Gly Asn He Asp Thr Gin Lys Ala Arg 325 330 335 Val Gin Ala Pro Arg Ser Leu Trp Asp Pro Ala Tyr Gin Glu Asp Ser 340 345 350 Trp He Gly Thr Trp Phe Ser Ser Tyr Leu Pro Leu He Pro Lys Leu 355 360 365 Gin Ser Ser He Gin Thr Tyr Tyr Pro Gly Thr Lys Leu Ala He Thr 370 375 380 Glu Phe Ser Tyr Gly Gly Asp Asn His He Ser Gly Gly He Ala Thr 385 390 395 400 Ala Asp Ala Leu Gly He Phe Gly Lys Tyr Gly Val Tyr Ala Ala Asn 405 410 415 Tyr Trp Gin Thr Glu Asp Asn Thr Asp Tyr Thr Ser Ala Ala Tyr Lys 420 425 430 Leu Tyr Arg Asn Tyr Asp Gly Asn Lys Ser Gly Phe Gly Ser He Lys 435 440 445 Val Asp Ala Ala Thr Ser Asp Thr Glu Asn Ser Ser Val Tyr Ala Ser 450 455 460 Val Thr Asp Glu Glu Asn Ser Glu Leu His Leu He Val Leu Asn Lys 465 470 475 480 Asn Phe Asp Asp Pro He Asn Ala Thr Phe Gin Leu Ser Gly Asp Lys 485 490 495 Thr Tyr Thr Ser Gly Arg Val Trp Gly Phe Asp Gin Thr Gly Ser Asp 500 505 510 He Thr Glu Gin Ala Ala He Thr Asn He Asn Asn Asn Gin Phe Thr 515 520 525 Tyr Thr Leu Pro Pro Leu Ser Ala Tyr His He Val Leu Lys Ala Asp 530 535 540 Ser Thr Glu Pro Val Asn Ser Asp Leu Val Val Gin Tyr Lys Asp Gly 545 550 555 560 Asp Arg Asn Asn Ala Thr Asp Asn Gin He Lys Pro His Phe Asn He 565 570 575 Gin Asn Lys Gly Thr Ser Pro Val Asp Leu Ser Ser Leu Thr Leu Arg 580 585 590 Tyr Tyr Phe Thr Lys Asp Ser Ser Ala Ala Met Asn Gly Trp He Asp 595 600 605 Trp Ala Lys Leu Gly Gly Ser Asn He Gin He Ser Phe Gly Asn His 610 615 620 Asn Gly Ala Asp Ser Asp Thr Tyr Ala Glu Leu Gly Phe Ser Ser Gly 625 630 635 640 Al a Gly Ser He Al a Gl u Gly Gly Gin Ser Gly Gl u He Gin Leu Arg 645 650 655 Met Ser Lys Al a Asp Trp Ser Asn Phe Asn Gl u Al a Asn Asp Tyr Ser 660 665 670 Phe Asp Gly Al a Lys Thr Al a Tyr He Asp Trp Asp Arg Val Thr Leu 675 680 685 Tyr Gin Asp Gly Gi n Leu Val Trp Gly He Gl u Pro 690 695 700 (2) INFORMATION FOR SEQ ID N0:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2323 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus lautus (B) STRAIN: NCIMB 40250 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 172..1869 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: GGGGTTTTGT TCATTTCAAG AAAGAGCACG AATAAACATC GCTAATTTAC ACATGATTTA 60 TTCTCTAATT ATTATGGTGC ATGCCAATTG GAAAATGTAG TAGATTAGTC ATCGTAACCT 120 GCTTTCATGC CAAGATATGT AATTTTTAAA AAAGAGTAAA GGAGAATTCA G ATG AAA 177 Met Lys 1 AAA CGT AGA AGC AGT AAA GTT ATT CTT TCG TTG GCC ATC GTT GTT GCA 225 Lys Arg Arg Ser Ser Lys Val He Leu Ser Leu Ala He Val Val Ala 5 10 15 TTA TTG GCA GCC GTC GAA CCT AAT GCC GCT TTA GCA GCG GCT CCA CCA 273 Leu Leu Ala Ala Val Glu Pro Asn Ala Ala Leu Ala Ala Ala Pro Pro 20 25 30 AGT GCC ATG CAG TCC TAT GTT GAA GCG ATG CAG CCT GGC TGG AAC CTT 321 Ser Ala Met Gin Ser Tyr Val Glu Ala Met Gin Pro Gly Trp Asn Leu 35 40 45 50 GGC AAT TCT CTG GAT GCT GTC GGT GCG GAT GAG ACG CTG GCA CGG GGC 369 Gly Asn Ser Leu Asp Ala Val Gly Ala Asp Glu Thr Leu Ala Arg Gly 55 60 65 AAT CCG CGG ATC ACG AAA GAG CTC ATT CAG AAC ATC GCT GCG CAA GGC 417 Asn Pro Arg He Thr Lys Glu Leu He Gin Asn He Ala Ala Gin Gly 70 75 80 TAT AAG AGC ATA CGG ATT CCT GTT ACC TGG GAT TCC CAT ATC GGC GCG 465 Tyr Lys Ser He Arg He Pro Val Thr Trp Asp Ser His He Gly Ala 85 90 95 GCC CCA AAT TAT CAA ATT GAA GCT GCG TAC CTC AAT CGA GTG CAG GAG 513 Ala Pro Asn Tyr Gin He Glu Ala Ala Tyr Leu Asn Arg Val Gin Glu 100 105 110 GTC GTA CAG TGG GCT TTG GAC GCG AAC CTC TAT GTG ATG ATT AAT GTC 561 Val Val Gin Trp Ala Leu Asp Ala Asn Leu Tyr Val Met He Asn Val 115 120 125 130 CAT CAT GAT TCC TGG CTA TGG ATC AGC AAA ATG GAG TCG CAG CAC GAT 609 His His Asp Ser Trp Leu Trp He Ser Lys Met Glu Ser Gin His Asp 135 140 145 CAA GTA CTG GCC CGT TAT AAT GCG ATT TGG ACG CAA ATT GCC AAC AAG 657 Gin Val Leu Ala Arg Tyr Asn Ala He Trp Thr Gin He Ala Asn Lys 150 155 160 TTC AAG AAC AGC CCG AGC AAG CTG ATG TTC GAG AGC GTG AAT GAG CCT 705 Phe Lys Asn Ser Pro Ser Lys Leu Met Phe Glu Ser Val Asn Glu Pro 165 170 175 CGC TTT ACG GAT GGC GGA ACT ACG GAT GAA GCC AAG CAG CAA AAA ATG 753 Arg Phe Thr Asp ly Gly Thr Thr Asp Glu Ala Lys Gin Gin Lys Met 180 185 190 CTG GAC GAG CTG AAC GTA TCC TTT TTC AAC ATC GTC AGA AAT TCC GGC 801 Leu Asp Glu Leu Asn Val Ser Phe Phe Asn He Val Arg Asn Ser Gly 195 200 205 210 GGC CAG AAC GCG ACT CGC CCG CTA GTT CTT TCT ACG TTG GAG GCC TCT 849 Gly Gin Asn Ala Thr Arg Pro Leu Val Leu Ser Thr Leu Glu Ala Ser 215 220 225 CCC ACC CAA GAG AGA ATG ACG GCG CTT TAT AAT ACG ATG ACC AAA CTG 897 Pro Thr Gin Glu Arg Met Thr Ala Leu Tyr Asn Thr Met Thr Lys Leu 230 235 240 AAC GAC AAG AAT CTG ATC GCA ACC GTT CAT TTT TAT GGA TTC TGG CCG 945 Asn Asp Lys Asn Leu He Ala Thr Val His Phe Tyr Gly Phe Trp Pro 245 250 255 TTT AGC GTA AAT ATC GCA GGA TAT ACG AAA TTT GAT GCG GAG ACG CAA 993 Phe Ser Val Asn He Ala Gly Tyr Thr Lys Phe Asp Ala Glu Thr Gin 260 265 270 AAT GAT ATT ATA ACG ACC TTC GAT AAC GTG TAT AAC ACA TTT GTA GCA 1041 Asn Asp He He Thr Thr Phe Asp Asn Val Tyr Asn Thr Phe Val Ala 275 280 285 290 AAG GGA ATC CCG GTG GTA GTC GGC GAA TAT GGC CTT CTT GGA TTC GAT 1089 Lys Gly He Pro Val Val Val Gly Glu Tyr Gly Leu Leu Gly Phe Asp 295 300 305 AAG AAT ACC GGC GTC ATT GAA CAG GGT GAG AAA TTG AAA TTT TTC GAG 1137 Lys Asn Thr Gly Val He Glu Gin Gly Glu Lys Leu Lys Phe Phe Glu 310 315 320 TTT TTT GCC CAG TAT GTG AAG CAA AAA AGC ATT TCC ACT ATG CTA TGG 1185 Phe Phe Ala Gin Tyr Val Lys Gin Lys Ser He Ser Thr Met Leu Trp 325 330 335 GAT AAC GGA CAG CAC TTC AAC CGC ACG AGC TTC AAG TGG TCT GAC CCG 1233 Asp Asn Gly Gin His Phe Asn Arg Thr Ser Phe Lys Trp Ser Asp Pro 340 345 350 GAT TTA TTC AAT ATG ATC AAG GCC AGT TGG ACC GGA CGT TCA TCC ACG 1281 Asp Leu Phe Asn Met He Lys Ala Ser Trp Thr Gly Arg Ser Ser Thr 355 360 365 370 GCT TCC AGC GAC CTG ATC CAT GTC AAG CAG GGC ACG GCG GTA AAA GAT 1329 Ala Ser Ser Asp Leu He His Val Lys Gin Gly Thr Ala Val Lys Asp 375 380 385 ACT TCG GTT CAG CTC AAT CTT AAC GGG AAT ACG CTA ACT TCC CTT TCC 1377 Thr Ser Val Gin Leu Asn Leu Asn Gly Asn Thr Leu Thr Ser Leu Ser 390 395 400 GTA AAT GGA ACG ACA CTG AAA TCA GGC ACA GAT TAC ACT TTA AAC AGC 1425 Val Asn Gly Thr Thr Leu Lys Ser Gly Thr Asp Tyr Thr Leu Asn Ser 405 410 415 AGC AGA TTA ACT TTT AAA GCG AGC CAG TTG ACC AAG CTG ACC TCC TTG 1473 Ser Arg Leu Thr Phe Lys Ala Ser Gin Leu Thr Lys Leu Thr Ser Leu 420 425 430 GGC AAA TTG GGG GTC AAC GCG ACG ATC GTG ACT AAA TTC AAT AGA GGC 1521 Gly Lys Leu Gly Val Asn Ala Thr He Val Thr Lys Phe Asn Arg Gly 435 440 445 450 GCC GAC TGG AAG TTC AAC GTA GTC CTG TAC AAT ACG CCT AAG CTT AGC 1569 Ala Asp Trp Lys Phe Asn Val Val Leu Tyr Asn Thr Pro Lys Leu Ser 455 460 465 AGT ACG ACG GGG ACT ACT TCT TCC TTT GCG ATT CCA ACG GCT TTC AAC 1617 Ser Thr Thr Gly Thr Thr Ser Ser Phe Ala He Pro Thr Ala Phe Asn 470 475 480 GGG GAT CAG CTT GCT ACG ATG GAA GCG GTC TAT GTA AAC GGC GGC AAT 1665 Gly Asp Gin Leu Ala Thr Met Glu Ala Val Tyr Val Asn Gly Gly Asn 485 490 495 GCC GGT CCG CAT AAC TGG ACT TCC TTT AAG GAA TTC GAA ACG ACG TTC 1713 Ala Gly Pro His Asn Trp Thr Ser Phe Lys Glu Phe Glu Thr Thr Phe 500 505 510 AGC CCC GCT TAT AGC GAG GGG AAA ATC AAA CTG CAG CAG GCG TTC TTT 1761 Ser Pro Ala Tyr Ser Glu Gly Lys He Lys Leu Gin Gin Ala Phe Phe 515 520 525 530 AAT GAA GTG AAT GAT ACC ACA GTC ACG CTC AAG TTC CAA TTC TGG AGC 1809 Asn Glu Val Asn Asp Thr Thr Val Thr Leu Lys Phe Gin Phe Trp Ser 535 540 545 GGG GAG ATC GTC AAC TAC ACG ATT AAA AAG AGC GGT TCG ACG GTG ACG 1857 Gly Glu He Val Asn Tyr Thr He Lys Lys Ser Gly Ser Thr Val Thr 550 555 560 GGT ACG GCT TCA TAAGCGAGTT TGGCAAAAAA GGACCGATAT ACTGCCTAAT 1909 Gly Thr Ala Ser 565 TTGGTATTGC CTTAGTTGAA AGCAATTGCT CCGAATAAAC AGAATGAAGC CCCGGCCAGC 1969 TGGCCGGGAC TTATGCGTTT AGGAAGTATA AACGAATCAT CAGCAATTTA TTTAGCTCGT 2029 CTCAGTTCAG CAATATCGGC TTCATGTGAA ACGGAGCGGA TGAACAATCT TTCGAGCAAT 2089 TTCTCATGCT CCTGCfGGGT TTGGAGAACG GTTTGCTGAT TAGTTTTAAG TACAGATATA 2149 TCCTCACGGA CTTGATTGAT TCATGTGGTC CGTTAGTTCT TCTACCTTTG TATTTGTGGC 2209 AGCAACGATA TGAATTAATT GTTGAATGTG CCCGCCATGA CTGTTTAGCT GCTCATTGTG 2269 GCTTTGTAAC TGTTCTCGGA TTTCTTTGAA TTCTTGGTCG TGCTCATTAA GCTT 2323 ( 2) INFORMATION FOR SEQ ID NO : 4 : (i ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 566 amino acids (B) TYPE: amino acid (D) TOPOLOGY : l inear ( i i ) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID N0:4 : Met Lys Lys Arg Arg Ser Ser Lys Val H e Leu Ser Leu Al a H e Val 1 5 10 15 Val Al a Leu Leu Al a Al a Val Gl u Pro Asn Al a Al a Leu Al a Al a Al a 20 25 30 Pro Pro Ser Ala Met Gin Ser Tyr Val Glu Ala Met Gin Pro Gly Trp 35 40 45 Asn Leu Gly Asn Ser Leu Asp Ala Val Gly Ala Asp Glu Thr Leu Ala 50 55 60 Arg Gly Asn Pro Arg He Thr Lys Glu Leu He Gin Asn He Ala Ala 65 70 75 80 Gin Gly Tyr Lys Ser He Arg He Pro Val Thr Trp Asp Ser His He 85 90 95 Gly Ala Ala Pro Asn Tyr Gin He Glu Ala Ala Tyr Leu Asn Arg Val 100 105 110 Gin Glu Val Val Gin Trp Ala Leu Asp Ala Asn Leu Tyr Val Met He 115 120 125 Asn Val His His Asp Ser Trp Leu Trp He Ser Lys Met Glu Ser Gin 130 135 140 His Asp Gin Val Leu Ala Arg Tyr Asn Ala He Trp Thr Gin He Ala 145 150 155 160 Asn Lys Phe Lys Asn Ser Pro Ser Lys Leu Met Phe Glu Ser Val Asn J65 170 175 Glu Pro Arg Phe Thr Asp Gly Gly Thr Thr Asp Glu Ala Lys Gin Gin 180 185 190 Lys Met Leu Asp Glu Leu Asn Val Ser Phe Phe Asn He Val Arg Asn 195 200 205 Ser Gly Gly Gin Asn Ala Thr Arg Pro Leu Val Leu Ser Thr Leu Glu 210 215 220 Ala Ser Pro Thr Gin Glu Arg Met Thr Ala Leu Tyr Asn Thr Met Thr 225 230 235 240 Lys Leu Asn Asp Lys Asn Leu He Ala Thr Val His Phe Tyr Gly Phe 245 250 255 Trp Pro Phe Ser Val Asn He Ala Gly Tyr Thr Lys Phe Asp Ala Glu 260 265 270 Thr Gin Asn Asp He He Thr Thr Phe Asp Asn Val Tyr Asn Thr Phe 275 280 285 Val Ala Lys Gly He Pro Val Val Val Gly Glu Tyr Gly Leu Leu Gly 290 295 300 Phe Asp Lys Asn Thr Gly Val He Glu Gin Gly Glu Lys Leu Lys Phe 305 310 315 320 Phe Glu Phe Phe Ala Gin Tyr Val Lys Gin Lys Ser He Ser Thr Met 325 330 335 Leu Trp Asp Asn Gly Gin His Phe Asn Arg Thr Ser Phe Lys Trp Ser 340 345 350 Asp Pro Asp Leu Phe Asn Met He Lys Ala Ser Trp Thr Gly Arg Ser 355 360 365 Ser Thr Ala Ser Ser Asp Leu He His Val Lys Gin Gly Thr Ala Val 370 375 380 Lys Asp Thr Ser Val Gin Leu Asn Leu Asn Gly Asn Thr Leu Thr Ser 385 390 395 400 Leu Ser Val Asn Gly Thr Thr Leu Lys Ser Gly Thr Asp Tyr Thr Leu 405 410 415 Asn Ser Ser Arg Leu Thr Phe Lys Ala Ser Gin Leu Thr Lys Leu Thr 420 425 430 Ser Leu Gly Lys Leu Gly Val Asn Ala Thr He Val Thr Lys Phe Asn 435 440 445 Arg Gly Ala Asp Trp Lys Phe Asn Val Val Leu Tyr Asn Thr Pro Lys 450 455 460 Leu Ser Ser Thr Thr Gly Thr Thr Ser Ser Phe Ala He Pro Thr Ala 465 470 475 480 Phe Asn Gly Asp Gin Leu Ala Thr Met Glu Ala Val Tyr Val Asn Gly 485 490 495 Gly Asn Ala Gly Pro His Asn Trp Thr Ser Phe Lys Glu Phe Glu Thr 500 505 510 Thr Phe Ser Pro Ala Tyr Ser Glu Gly Lys He Lys Leu Gin Gin Ala 515 520 525 Phe Phe Asn Glu Val Asn Asp Thr Thr Val Thr Leu Lys Phe Gin Phe 530 535 540 Trp Ser Gly Glu He Val Asn Tyr Thr He Lys Lys Ser Gly Ser Thr 545 550 555 560 Val Thr Gly Thr Ala Ser 565 (2) INFORMATION FOR SEQ ID NO: 5 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 1775 base pai rs (B) TYPE: nucleic acid (C) STRANDEDNESS : doubl e (D) TOPOLOGY : l inear (ii) MOLECULE TYPE: DNA (genomic) (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus lautus (B) STRAIN: NCIMB 40250 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 30..(1625.1775) (C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /partial /evidence= EXPERIMENTAL /transl_except= (pos: 1446..1458, aa: OTR) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TTGAAGCGCT GAATTCAGGA GGTTAAATAA TGCGTATTCA TGCAATTCGG CAATCTTGCC 60 GTTTGGTATT GACGATGGTT TTGATGCTTG GCTTATTGCT GCCTGTGGGC GCCCCCAAAG 120 GCTATGCCGC TCCGGCTGTT CCTTTTGGCC AATTAAAAGT TCAGGGCAAT CAATTGGTAG 180 GACAGTCCGG GCAAGCTGTT CAACTGGTTG GCATGAGCTC ACATGGATTG CAGTGGTATG 240 GCAATTTCGT CAACAAATCG TCGTTGCAGT GGATGAGAGA TAACTGGGGC ATCAACGTCT 300 TCCGTGCCGC TATGTATACT TCCGAAGATG GTTACATTAC GGATCCTTCC GTCAAGAACA 360 AGGTGAAGGA GGCGGTTCAG GCATCCATCG ATCTGGCCTT GTACGTTATT ATTGACTGGC 420 ATATCTTGTC TGATGGGAAT CCGAATACGT ACAAAGCGCA ATCGAAAGCG TTCTTCCAAG 480 AGATGGCCAC ATTGTACGGC AACACGCCGA ATGTAATCTA TGAAATCGCG ACGAGCCCAA 540 CGGAATGTGT CCTGGGCAGA TGTCAGTCGT CGGAAGAAGT GATCACGGCC ATTCGTTCGA 600 TTGACCCCGA CGGAGTGGTT ATCGTTGGCA GTCCAACCTG GAGCCAGGAT ATTCATCTGG 660 CGGCCGATAA CCCGGTATCA CATAGCAATG TCATGTATGC GCTTCATTTC TATTCAGGCA 720 CGCATGGACA GTTTTTGAGA GACCGAATTA CCTATGCGAT GAACAAAGGA GCAGCGATCT 780 TCGTTACCGA GTGGGGCACC AGTGATGCAT CCGGGAACGG CGGGCCGTAT TTGCCTCAGT 840 CCAAAGAGTG GATCGATTTC TTGAATGCTC GCAAGATCAG CTGGGTGAAC TGGTCGCTCG 900 CTGATAAAGT AGAAACGTCT GCTGCTCTTA TGCCAGGTGC ATCGCCTACC GGCGCTGGAC 960 CGATGCCCAA TTGTCGAATG GGCAAATCGG GTTCGCGATC AAATCCGGCA AGCAACTGGA 1020 GGCGGCAGGG CAATCCAACT GCACCGGCTG CCCCTACTAA CCTCTCGGCA AACGGCGGCA 1080 ACGCCCAGGT ATCATTAACC TGGAACGCAG TTAGCGGGGC GACGAGCTAT ACCGTAAAGC 1140 GAGCAACGAC GAGCGGCGGT CCGTACACGA ATGTGGACCG GGGTGTCACG GCGACGAGCT 1200 ACACGAACAC CGGGCTGACG AATGGCACGA CGTATTATTA TGTCGTGAGG GCATCCAATA 1260 GCGCGGGCAG CAGCGCGAAC TCCGCGCAAG CGAGCGCAAC GCCGGCTAGC GGCGGCGCCA 1320 GTACGGGGAA CCTTGTTGTC CAATACAAAG TTGGCGACAC TAGCGCCACG GATAACCAAA 1380 TGAAGCCTTC CTTTAACATC AAGAACAACG GTACAACCCC TGTTAACCTG AGCGGCCTCA 1440 AGCTTNNNNN NNNNNNNNAA AAAGACGGAC CTGCGGATAT GAGCTGCTCG ATCGACTGGG 1500 CGCAAATCGG CCGAACGAAT GTTCTGCTGG CATTCGCTAA CTTTACCGGG AGTAATACGG 1560 ATACTTACTG TTGTGAGCTA AGCTTTAGCT GCACTGCAGG TTCGTATCCC GGCTATGCGT 1620 GGACNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1680 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1740 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNN 1775 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1609 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (v1) ORIGINAL SOURCE: (A) ORGANISM: Baci l l us l autus (B) STRAIN: NCIMB 40250 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 30..1607 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6: TTGAAGCGCT GAATTCAGGA GGTTAAATA ATG CGT ATT CAT GCA ATT CGG CAA 53 Met Arg He His Ala He Arg Gin 1 5 TCT TGC CGT TTG GTA TTG ACG ATG GTT TTG ATG CTT GGC TTA TTG CTG 101 Ser Cys Arg Leu Val Leu Thr Met Val Leu Met Leu Gly Leu Leu Leu 10 15 20 CCT GTG GGC GCC CCC AAA GGC TAT GCC GCT CCG GCT GTT CCT TTT GGC 149 Pro Val Gly Ala Pro Lys Gly Tyr Ala Ala Pro Ala Val Pro Phe Gly 25 30 35 40 CAA TTA AAA GTT CAG GGC AAT CAA TTG GTA GGA CAG TCC GGG CAA GCT 197 Gin Leu Lys Val Gin Gly Asn Gin Leu Val Gly Gin Ser Gly Gin Ala 45 50 55 GTT CAA CTG GTT GGC ATG AGC TCA CAT GGA TTG CAG TGG TAT GGC AAT 245 Val Gin Leu Val Gly Met Ser Ser His Gly Leu Gin Trp Tyr Gly Asn 60 65 70 πc GTC AAC AAA TCG TCG TTG CAG TGG ATG AGA GAT AAC TGG GGC ATC 293 Phe Val Asn Lys Ser Ser Leu Gin Trp Met Arg Asp Asn Trp Gly He 75 80 85 AAC GTC TTC CGT GCC GCT ATG TAT ACT TCC GAA GAT GGT TAC ATT ACG 341 Asn Val Phe Arg Ala Ala Met Tyr Thr Ser Glu Asp Gly Tyr He Thr 90 95 100 GAT CCT TCC GTC AAG AAC AAG GTG AAG GAG GCG GTT CAG GCA TCC ATC 389 Asp Pro Ser Val Lys Asn Lys Val Lys Glu Ala Val Gin Ala Ser He 105 110 115 120 GAT CTG GCC TTG TAC GTT ATT ATT GAC TGG CAT ATC TTG TCT GAT GGG 437 Asp Leu Ala Leu Tyr Val He He Asp Trp His He Leu Ser Asp Gly 125 130 135 AAT CCG AAT ACG TAC AAA GCG CAA TCG AAA GCG TTC TTC CAA GAG ATG 485 Asn Pro Asn Thr Tyr Lys Ala Gin Ser Lys Ala Phe Phe Gin Glu Met 140 145 150 GCC ACA TTG TAC GGC AAC ACG CCG AAT GTA ATC TAT GAA ATC GCG ACG 533 Ala Thr Leu Tyr Gly Asn Thr Pro Asn Val He Tyr Glu He Ala Thr 155 160 165 AGC CCA ACG GAA TGT GTC CTG GGC AGA TGT CAG TCG TCG GAA GAA GTG 581 Ser Pro Thr Glu Cys Val Leu Gly Arg Cys Gin Ser Ser Glu Glu Val 170 175 180 ATC ACG GCC ATT CGT TCG ATT GAC CCC GAC GGA GTG GTT ATC GTT GGC 629 He Thr Ala He Arg Ser He Asp Pro Asp Gly Val Val He Val Gly 185 190 195 200 AGT CCA ACC TGG AGC CAG GAT ATT CAT CTG GCG GCC GAT AAC CCG GTA 677 Ser Pro Thr Trp Ser Gin Asp He His Leu Ala Ala Asp Asn Pro Val 205 210 215 TCA CAT AGC AAT GTC ATG TAT GCG CTT CAT TTC TAT TCA GGC ACG CAT 725 Ser His Ser Asn Val Met Tyr Ala Leu His Phe Tyr Ser Gly Thr His 220 225 230 GGA CAG TTT TTG AGA GAC CGA ATT ACC TAT GCG ATG AAC AAA GGA GCA 773 Gly Gin Phe Leu Arg Asp Arg He Thr Tyr Ala Met Asn Lys Gly Ala 235 240 245 GCG ATC TTC GTT ACC GAG TGG GGC ACC AGT GAT GCA TCC GGG AAC GGC 821 Ala He Phe Val Thr Glu Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly 250 255 260 GGG CCG TAT TTG CCT CAG TCC AAA GAG TGG ATC GAT TTC TTG AAT GCT 869 Gly Pro Tyr Leu Pro Gin Ser Lys Glu Trp He Asp Phe Leu Asn Ala 265 270 275 280 CGC AAG ATC AGC TGG GTG AAC TGG TCG CTC GCT GAT AAA GTA GAA ACG 917 Arg Lys He Ser Trp Val Asn Trp Ser Leu Ala Asp Lys Val Glu Thr 285 290 295 TCT GCT GCT CTT ATG CCA GGT GCA TCG CCT ACC GGC GCT GGA CCG ATG 965 Ser Ala Ala Leu Met Pro Gly Ala Ser Pro Thr Gly Ala Gly Pro Met 300 305 310 CCC AAT TGT CGA ATG GGC AAA TCG GGT TCG CGA TCA AAT CCG GCA AGC 1013 Pro Asn Cys Arg Met Gly Lys Ser Gly Ser Arg Ser Asn Pro Ala Ser 315 320 325 AAC TGG AGG CGG CAG GGC AAT CCA ACT GCA CCG GCT GCC CCT ACT AAC 1061 Asn Trp Arg Arg Gin Gly Asn Pro Thr Ala Pro Ala Ala Pro Thr Asn 330 335 340 CTC TCG GCA AAC GGC GGC AAC GCC CAG GTA TCA TTA ACC TGG AAC GCA 1109 Leu Ser Ala Asn Gly Gly Asn Ala Gin Val Ser Leu Thr Trp Asn Ala 345 350 355 360 GTT AGC GGG GCG ACG AGC TAT ACC GTA AAG CGA GCA ACG ACG AGC GGC 1157 Val Ser Gly Ala Thr Ser Tyr Thr Val Lys Arg Ala Thr Thr Ser Gly 365 370 375 GGT CCG TAC ACG AAT GTG GAC CGG GGT GTC ACG GCG ACG AGC TAC ACG 1205 Gly Pro Tyr Thr Asn Val Asp Arg Gly Val Thr Ala Thr Ser Tyr Thr 380 385 390 AAC ACC GGG CTG ACG AAT GGC ACG ACG TAT TAT TAT GTC GTG AGG GCA 1253 Asn Thr Gly Leu Thr Asn Gly Thr Thr Tyr Tyr Tyr Val Val Arg Ala 395 400 405 TCC AAT AGC GCG GGC AGC AGC GCG AAC TCC GCG CAA GCG AGC GCA ACG 1301 Ser Asn Ser Ala Gly Ser Ser Ala Asn Ser Ala Gin Ala Ser Ala Thr 410 415 420 CCG GCT AGC GGC GGC GCC AGT ACG GGG AAC CTT GTT GTC CAA TAC AAA 1349 Pro Ala Ser Gly Gly Ala Ser Thr Gly Asn Leu Val Val Gin Tyr Lys 425 430 435 440 GTT GGC GAC ACT AGC GCC ACG GAT AAC CAA ATG AAG CCT TCC TTT AAC 1397 Val Gly Asp Thr Ser Ala Thr Asp Asn Gin Met Lys Pro Ser Phe Asn 445 450 455 ATC AAG AAC AAC GGT ACA ACC CCT GTT AAC CTG AGC GGC CTC AAG CTT 1445 He Lys Asn Asn Gly Thr Thr Pro Val Asn Leu Ser Gly Leu Lys Leu 460 465 470 NNN NNN NNN NNN NAA AAA GAC GGA CCT GCG GAT ATG AGC TGC TCG ATC 1493 Xaa Xaa Xaa Xaa Xaa Lys Asp Gly Pro Ala Asp Met Ser Cys Ser He 475 480 485 GAC TGG GCG CAA ATC GGC CGA ACG AAT GTT CTG CTG GCA TTC GCT AAC . 1541 Asp Trp Ala Gin He Gly Arg Thr Asn Val Leu Leu Ala Phe Ala Asn 490 495 500 TTT ACC GGG AGT AAT ACG GAT ACT TAC TGT TGT GAG CTA AGC TTT AGC 1589 Phe Thr Gly Ser Asn Thr Asp Thr Tyr Cys Cys Glu Leu Ser Phe Ser 505 510 515 520 TGC ACT GCA GGT TCG TAT CCC GGC TAT GCG TGG AC 1624 Cys Thr Ala Gly Ser Tyr Pro Gly Tyr Ala Trp 525 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 526 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7: Met Arg He His Ala He Arg Gin Ser Cys Arg Leu Val Leu Thr Met 1 5 10 15 Val Leu Met Leu Gly Leu Leu Leu Pro Val Gly Ala Pro Lys Gly Tyr 20 25 30 Ala Ala Pro Ala Val Pro Phe Gly Gin Leu Lys Val Gin Gly Asn Gin 35 40 45 Leu Val Gly Gin Ser Gly Gin Ala Val Gin Leu Val Gly Met Ser Ser 50 55 60 His Gly Leu Gin Trp Tyr Gly Asn Phe Val Asn Lys Ser Ser Leu Gin 65 70 75 80 Trp Met Arg Asp Asn Trp Gly He Asn Val Phe Arg Ala Ala Met Tyr 85 90 95 Thr Ser Glu Asp Gly Tyr He Thr Asp Pro Ser Val Lys Asn Lys Val 100 105 110 Lys Glu Ala Val Gin Ala Ser He Asp Leu Ala Leu Tyr Val He He 115 120 125 Asp Trp His He Leu Ser Asp Gly Asn Pro Asn Thr Tyr Lys Ala Gin 130 135 140 Ser Lys Ala Phe Phe Gin Glu Met Ala Thr Leu Tyr Gly Asn Thr Pro 145 150 155 160 Asn Val He Tyr Glu He Ala Thr Ser Pro Thr Glu Cys Val Leu Gly 165 170 175 Arg Cys Gin Ser Ser Glu Glu Val He Thr Ala He Arg Ser He Asp 180 185 190 Pro Asp Gly Val Val He Val Gly Ser Pro Thr Trp Ser ' Gln Asp He 195 200 205 His Leu Ala Ala Asp Asn Pro Val Ser His Ser Asn Val Met Tyr Ala 210 215 220 Leu His Phe Tyr Ser Gly Thr His Gly Gin Phe Leu Arg Asp Arg He 225 230 235 240 Thr Tyr Ala Met Asn Lys Gly Ala Ala He Phe Val Thr Glu Trp Gly 245 250 255 Thr Ser Asp Ala Ser Gly Asn Gly Gly Pro Tyr Leu Pro Gin Ser Lys 260 265 270 Glu Trp He Asp Phe Leu Asn Ala Arg Lys He Ser Trp Val Asn Trp 275 280 285 Ser Leu Ala Asp Lys Val Glu Thr Ser Ala Ala Leu Met Pro Gly Ala 290 295 300 Ser Pro Thr Gly Ala Gly Pro Met Pro Asn Cys Arg Met Gly Lys Ser 305 310 315 320 Gly Ser Arg Ser Asn Pro Ala Ser Asn Trp Arg Arg Gin Gly Asn Pro 325 330 335 Thr Ala Pro Ala Ala Pro Thr Asn Leu Ser Ala Asn Gly Gly Asn Ala 340 345 350 Gin Val Ser Leu Thr Trp Asn Ala Val Ser Gly Ala Thr Ser Tyr Thr 355 360 365 Val Lys Arg Ala Thr Thr Ser Gly Gly Pro Tyr Thr Asn Val Asp Arg 370 375 380 Gly Val Thr Ala Thr Ser Tyr Thr Asn Thr Gly Leu Thr Asn Gly Thr 385 390 395 400 Thr Tyr Tyr Tyr Val Val Arg Ala Ser Asn Ser Ala Gly Ser Ser Ala 405 410 415 Asn Ser Ala Gin Ala Ser Ala Thr Pro Ala Ser Gly Gly Ala Ser Thr 420 425 430 Gly Asn Leu Val Val Gin Tyr Lys Val Gly Asp Thr Ser Ala Thr Asp 435 440 445 Asn Gin Met Lys Pro Ser Phe Asn He Lys Asn Asn Gly Thr Thr Pro 450 455 460 Val Asn Leu Ser Gly Leu Lys Leu Xaa Xaa Xaa Xaa Xaa Lys Asp Gly 465 470 475 480 Pro Ala Asp Met Ser Cys Ser He Asp Trp Ala Gin He Gly Arg Thr 485 490 495 Asn Val Leu Leu Ala Phe Ala Asn Phe Thr Gly Ser Asn Thr Asp Thr 500 505 510 Tyr Cys Cys Glu Leu Ser Phe Ser Cys Thr Ala Gly Ser Tyr Pro Gly 515 520 525 Tyr Ala Trp 530 Intemtt-OM- AppJicrton No: PCT/ MICROORGANISMS _. 3 20 , iMimneA-πoM or oapoarr • D 1 NATIONAL COLLECTION OF INDUSTRIAL & MARINE BACTERIA LTD. Torry Research Station, P.O. Box 31, 23 St Machar Drive, Aberdeen AB9 8DG, Scotland o— i *»»*__»» 18 January 1990 NCIMB 40250 **. AODmONAL INDtCATtOIIS ' (IMM MM* M MI —*-fk\*). Th-a 1-mrWM to ottMnutt M ■ tmH r i ahaat Q In respect of those designations in which a European patent is sought, a sample of the deposited micro¬ organism will be made available only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC) until the publication of the mention of the grant of the Euro¬ pean patent or until the date on which the appli¬ cation has been refused or is deemed to be withdrawn. C. DMIM ATIO STAfM POH WHtCM INDICATIONS AM HAM • 01 *• HHIC * NHM . Ml Itf _- . MPARATI rUMNIVMIN* Of INDICATIONS • (tan MMk M Ml M»»eMK> Tha HMWMIwi NMM *_**_ *-** ■ * ha MtmKM M UM Iw-Hrmmml Iwii Mar * <lpatH> IM f**Hwn*l MM ≠ MM - ACCaMfeA WyniMf a« 0*MM ** ) I. K] TMi ilmt *»•• racau-aa * axHli the MtamrtaMl _ pU___m_m ahan Ma* (I* M cMcka* k IM MCHilm OMca) Q Tha MM •< racaipt (tnm lha βMHcM!) ky the Venn PCT/M IM WM OT IW)

Claims: CLAIMS 1. An enzyme which exhibits cellulase activity, which enzyme is producible by a strain of Bacillus spp., NCIMB 40250, or a related Bacillus spp. strain, or a derivative of said cellulase. 2. An enzyme according to claim 1, which exhibits an endoglucanase activity of at least about 10, more preferably at least about 20, most preferably at least about 25, such as about 30, CMC-endoase units (as defined herein) per mg of total protein under alkaline conditions. 3. An enzyme according to claim 1, which is active at a temperature of up to about 65 β C. 4. An enzyme according to any of claims 1-3, which is an endoglucanase with an apparent molecular weight of 75 kD or a cleavage product thereof exhibiting endoglucanase activity. 5. An enzyme according to claim 4, which is an endoglucanase encoded by the DNA sequence shown in the appended Sequence Listing ID#1, or a modification thereof encoding a derivative of said endoglucanase. 6. An enzyme according to any of claims 1-3, which is an endoglucanase with an apparent molecular weight of 56 kD or a cleavage product thereof exhibiting endoglucanase activity. 7. An enzyme according to claim 6, which is an endoglucanase encoded by the DNA sequence shown in the appended Sequence Listing ID##, or a modification thereof encoding a derivative of said endoglucanase. 8. An enzyme according to any of claims 1-3, which is an endoglucanase with an apparent molecular weight of 45 kD or a cleavage product thereof exhibiting endoglucanase activity. 9. An enzyme according to claim 8, which is an endoglucanase encoded by the DNA sequence shown in the appended Sequence Listing ID#6, or a modification thereof encoding a derivative of said endoglucanase. 10. An enzyme according to any of claims 1-3, which is an endoglucanase with an apparent molecular weight of 60 kD or a cleavage product thereof exhibiting endoglucanase activity, or which is an endoglucanase with an apparent molecular weight of 56 kD or a cleavage product thereof exhibiting endoglucanase activity. 11. An enzyme according to any of claims 1-3, which is an endoglucanase with an apparent molecular weight of 92 kD or a cleavage product thereof exhibiting endoglucanase activity. 12. An enzyme which comprises a core region derived from an endoglucanase according to any of claims 1-11 combined with a cellulose-binding domain derived from another cellulase enzyme, or a core region derived from another cellulase enzyme combined with a cellulose-binding domain derived from an endoglucanase according to any of claims 1-11. 13. An enzyme according to claim 12, wherein the core region is derived from a cellulase enzyme which does not, in nature, comprise a cellulose-binding domain. 14. A DNA construct which comprises a DNA sequence encoding an enzyme exhibiting cellulase activity, which enzyme is derivable from a strain of Bacillus spp., NCIMB 40250, or a related Bacillus spp. strain, or a derivative of said cellulase. 15. A DNA construct according to claim 14, wherein the DNA sequence encodes an endoglucanase with an apparent molecular weight of 75 kD. 16. A DNA construct according to claim 15, wherein the DNA sequence is the one shown in the appended Sequence Listing ID#1, or a modification thereof encoding a derivative of said endoglucanase. 17. A DNA construct according to claim 14, wherein the DNA sequence encodes an endoglucanase with an apparent molecular weight of 56 kD. 18. A DNA construct according to claim 17, wherein the DNA sequence is the one shown in the appended Sequence Listing ID#3, or a modification thereof encoding a derivative of said endoglucanase. 19. A DNA construct according to claim 14, wherein the DNA sequence encodes an endoglucanase with an apparent molecular weight of 45 kD. 20. A DNA construct according to claim 19, wherein the DNA sequence is the one shown in the appended Sequence Listing ID#6, or a modification thereof encoding a derivative of said endoglucanase. 21. A DNA construct according to claim 14, wherein the DNA sequence encodes an endoglucanase with an apparent molecular weight of 92 kD. 22. An expression vector which carries an inserted DNA construct according to any of claims 14-21. 23. A cell which is transformed with a DNA construct according to any of claims 14-21 or with an expression vector according to claim 22. 24. A cell according to claim 23, which is a bacterium. 25. A cell according to claim 24, which is a grampositive bacterium. 26. A cell according to claim 25, wherein the grampositive bacterium is selected from the group consisting of Bacillus subtilis. Bacillus licheniformis. Bacillus lentus. Bacillus brevis. Bacillus stearothermophilus. Bacillus alkalophilus. Bacillus amyloliquefaciens. Bacillus coaqulans. Bacillus circulans or Bacillus lautus. 27. A cell according to claim 24, which is a gra negative bacterium, e.g. Escherichia coli. 28. A method of producing an enzyme according to any of claims 1-13, wherein a host cell according to any of claims 23-27 is cultured under conditions conducive to the production of the endoglucanase or a derivative thereof, and the endoglucanase or derivative thereof is subsequently recovered from the culture. 29. A method according to claim 28, wherein the endoglucanase is recovered in mature form. 30. A cellulolytic agent capable of degrading amorphous regions of cellulose fibres, the agent comprising an enzyme according to any of claims 1-13. 31. An agent according to claim 30, which comprises a combination of two or more cellulases recited in any of claims 1-13, or a combination of one or more cellulases recited in any of claims 1-13 with one or more other enzymes with cellulase.activity. 32. An agent according to claim 30 or 31, which is in the form of a non- dusting granulate, stabilized liquid or protected enzyme. 33. An agent according to any of claims 30-32, which exhibits an endoglucanase activity of 500-10,000 CMC-endoase units per gram of the agent. 34. An agent according to any of claims 30-33, which is a detergent additive. 35. An agent according to claim 34, which additionally comprises another enzyme such a a protease, lipase and/or amylase. 36. A detergent composition comprising a cellulolytic agent according to any of claims 30-35. 37. A detergent composition according to claim 36, which exhibits an endoglucanase activity of 0.3-400 CMC-endoase units per gram of detergent. 38. A method of reducing the rate at which cellulose-containing fabrics become harsh or of reducing the harshness of cellulose-containing fabrics, the method comprising treating cellulose-containing fabrics with a cellulolytic agent according to any of claims 30-35. 39. A method according to claim 38, wherein the treatment of the fabrics with the cellulolytic agent is conducted during soaking, washing or rinsing of the fabrics. 40. A method of treating a coloured, cellulose-containing fabric in order to provide colour clarification, the method comprising treating the cellulose- containing fabric with a cellulolytic agent according to any of claims 30- 35. 41. A method according to claim 40, wherein the treatment of the fabric with the cellulolytic agent is conducted in an aqueous medium during soaking, washing or rinsing of the fabric. 42. A method according to claim 41, wherein the aqueous medium exhibits an endoglucanase activity of more than about 250 CMC-endoase units per liter of the aqueous medium.

Inventors: HANSEN CHRISTIAN, JOERGENSEN PER LINAA, SCHUELEIN MARTIN, HANSEN, CHRISTIAN, JOERGENSEN, PER, LINAA, SCHUELEIN, MARTIN
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1991
Main classification: C12N15
IPC codes: C11D3, C12N1, C12N9, C12N15, C12R1, C12S11, D06M16