Patent Application: US-41234799-A

Abstract:
catalase enzymes derived from bacteria from the genera alcaligenes and microscilla are disclosed . the enzymes are produced from native or recombinant host cells and can be utilized to destroy or detect hydrogen peroxide , e . g ., in production of glyoxylic acid and in glucose sensors , and in processes where hydrogen peroxide is used as a bleaching or antibacterial agent , e . g . in contact lens cleaning , in bleaching steps in pulp and paper preparation and in the pasteurization of dairy products .

Description:
in order to facilitate understanding of the following description and examples which follow certain frequently occurring methods and / or terms will be described . the term “ isolated ” means altered “ by the hand of man ” from its natural state ; i . e ., if it occurs in nature , it has been changed or removed from its original environment , or both . for example , a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “ isolated ”, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “ isolated ”, as the term is employed herein . for example , with respect to polynucleotides , the term isolated means that it is separated from the nucleic acid and cell in which it naturally occurs . as part of or following isolation , such polynucleotides can be joined to other polynucleotides , such as dnas , for mutagenesis , to form fusion proteins , and for propagation or expression in a host , for instance . the isolated polynucleotides , alone or joined to other polynucleotides such as vectors , can be introduced into host cells , in culture or in whole organisms . introduced into host cells in culture or in whole organisms , such polynucleotides still would be isolated , as the term is used herein , because they would not be in their naturally occurring form or environment . similarly , the polynucleotides and polypeptides may occur in a composition , such as a media formulation ( solutions for introduction of polynucleotides or polypeptides , for example , into cells or compositions or solutions for chemical or enzymatic reactions which are not naturally occurring compositions ) and , therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein . the term “ ligation ” refers to the process of forming phosphodiester bonds between two or more polynucleotides , which most often are double stranded dnas . techniques for ligation are well known to the art and protocols for ligation are described in standard laboratory manuals and references , such as , for instance , sambrook et al ., molecular cloning , a laboratory manual , 2nd ed . ; cold spring harbor laboratory press , cold spring harbor , n . y . ( 1989 ). the term “ gene ” means the segment of dna involved in 4 producing a polypeptide chain ; it includes regions preceding and following the coding region ( leader and trailer ) as well as intervening sequences ( introns ) between individual coding segments ( exons ). a coding sequence is “ operably linked to ” another coding sequence when rna polymerase will transcribe the two coding sequences into a single mrna , which is then translated into a single polypeptide having amino acids derived from both coding sequences . the coding sequences need not be contiguous to one another so long as the expressed sequences ultimately process to produce the desired protein . “ recombinant ” enzymes refer to enzymes produced by recombinant dna techniques ; i . e ., produced from cells transformed by an exogenous dna construct encoding the desired enzyme . “ synthetic ” enzymes are those prepared by chemical synthesis . a dna “ coding sequence of ” or a “ nucleotide sequence encoding ” a particular enzyme , is a dna sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences . “ plasmids ” are designated by a lower case “ p ” preceded and / or followed by capital letters and / or numbers . the starting plasmids herein are either commercially available , publicly available on an unrestricted basis , or can be constructed from available plasmids in accord with published procedures . in addition , equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan . “ digestion ” of dna refers to catalytic cleavage of the dna with a restriction enzyme that acts only at certain sequences in the dna . the various restriction enzymes used herein are commercially available and their reaction conditions cofactors and other requirements were used as would be known to the ordinarily skilled artisan . for analytical purposes , typically 1 μg of plasmid or dna fragment is used with about 2 units of enzyme in about 20 μl of buffer solution . for the purpose of isolating dna fragments for plasmid construction , typically 5 to 50 μg of dna are digested with 20 to 250 units of enzyme in a larger volume . appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer . incubation times of about 1 hour at 37 ° c . are ordinarily used , but may vary in accordance with the supplier &# 39 ; s instructions . after digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment . size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by goeddel et al ., nucleic acids res ., 8 : 4057 ( 1980 ). “ oligonucleotides ” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized . such synthetic oligonucleotides have no 5 ′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an atp in the presence of a kinase . a synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated . “ ligation ” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments ( maniatis , t ., et al ., id ., p . 146 ). unless otherwise provided , ligation may be accomplished using known buffers and conditions with 10 units of t4 dna ligase (“ ligase ”) per 0 . 5 μg of approximately equimolar amounts of the dna fragments to be ligated . unless otherwise stated , transformation was performed as described in sambrook and maniatis , molecular cloning : a laboratory manual , cold spring harbor laboratory , 1989 . in accordance with an aspect of the present invention , there are provided isolated nucleic acids ( polynucleotides ) which encode for the mature enzyme having the deduced amino acid sequence of fig1 ( seq id no : 6 ). in accordance with another aspect of the present invention , there are provided isolated nucleic acids ( polynucleotides ) which encode for the mature enzyme having the deduced amino acid sequence of fig2 ( seq id no : 9 ). the polynucleotides of this invention were originally recovered from a genomic gene library derived from two sources . the first , alcaligenes ( delaya ) aquanarinus , is a β - proteobacteria . it is a gram - negative rod that grows optimally at 26 ° c . and ph 7 . 2 . the second , microscilla furvescens , is a cytophagales ( bacteria ) isolated from samoa . it is a gram - negative rod with gliding motility that grows optimally at 30 ° c . and ph 7 . 0 . with respect to alcaligenes ( delaya ) aquamarinus , the protein with the closest amino acid sequence identity of which the inventors are currently aware is the microscilla furvescens catalase ( 59 . 5 % protein identity ; 60 % dna identity ). the next closest is a mycobacterium tuberculosis catalase ( katg ), with a 54 % protein identity . with respect to microscilla furvescens , the protein with closest amino acid sequence identity of which the inventors are currently aware is catalase i of bacillus stearothermophilus , which has a 69 % amino acid identity . accordingly , the polynucleotides and enzymes encoded thereby are identified by the organism from which they were isolated . such are sometimes referred to below as “ 64ca2 ” ( fig1 and seq id nos : 5 and 6 ) and “ 53ca1 ” ( fig2 and seq id nos : 7 and 8 ). one means for isolating the nucleic acid molecules encoding the enzymes of the present invention is to probe a gene library with a natural or artificially designed probe using art recognized procedures ( see , for example : current protocols in molecular biology , ausubel f . m . et al . ( eds .) green publishing company assoc . and john wiley interscience , new york . 1989 , 1992 ). it is appreciated by one skilled in the art that the polynucleotides of ( seq id nos : 5 and 7 ), or fragments thereof ( comprising at least 12 contiguous nucleotides ), are particularly useful probes . other partjcularly , useful probes for this purpose are hybridizable fragments of the sequences of ( seq id nos : 5 and 7 ) ( i . e ., comprising at least 12 contiguous nucleotides ). with respect to nucleic acid sequences which hybridize to specific nucleic acid sequences disclosed herein , hybridization may be carried out under conditions of reduced stringency , medium stringency or even stringent conditions . as an example of oligonucleotide hybridization , a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45 ° c . in a solution consisting of 0 . 9 m nacl , 50 mm nah 2 po 4 , ph 7 . 0 , 5 . 0 mm na 2 edta , 0 . 5 % sds , 10ox denhardt &# 39 ; s , and 0 . 5 mg / ml polyriboadenylic acid . approximately 2 × 10 7 cpm ( specific activity 4 - 9 × 10 8 cpm / ug ) of 32 p end - labeled oligonucleotide probe are then added to the solution . after 12 - 16 hours of incubation , the membrane is washed for 30 minutes at room temperature in 1 × set ( 150 mm nacl , 20 mm tris hydrochloride , ph 7 . 8 , 1 mm na 2 edta ) containing 0 . 5 % sds , followed by a 30 minute wash in fresh 1 × set at ( tm less 10 ° c .) for the oligo - nucleotide probe . the membrane is then exposed to auto - radiographic film for detection of hybridization signals . stringent condition means hybridization will occur only if there is at least 90 % identity , preferably at least 95 % identity and most preferably at least 97 % identity between the sequences . further , it is understood that a section of a 100 bps sequence that is 95 bps in length has 95 % identity with the 1090 bps sequence from which it is obtained . see j . sambrook et al ., molecular cloning , a laboratory manual , 2d ed ., cold spring harbor laboratory ( 1989 ) which is hereby incorporated by reference in its entirety . also , it is understood that a fragment of a 100 bps sequence that is 95 bps in length has 95 % identity with the 100 bps sequence from which it is obtained . as used herein , a first dna ( rna ) sequence is at least 70 % and preferably at least 80 % identical to another dna ( rna ) sequence if there is at least 70 % and preferably at least a 80 % or 90 % identity , respectively , between the bases of the first sequence and the bases of the another sequence , when properly aligned with each other , for example when aligned by blastn . the present invention relates to polynucleotides which differ from the reference polynucleotide such that the differences are silent , for example , the amino acid sequence encoded by the polynucleotides is the same . the present invention also relates to nucleotide changes which result in amino acid substitutions , additions , deletions , fusions and truncations in the polypeptide encoded by the reference polynucleotide . in a preferred aspect of the invention these polypeptides retain the same biological action as the polypeptide encoded by the reference polynucleotide . the polynucleotides of this invention were recovered from genomic gene libraries from the organisms identified above . gene libraries were generated from a lambda zap ii cloning vector ( stratagene cloning systems ). mass excisions were performed on these libraries to generate libraries in the pbluescript phagemid . libraries were generated and excisions were performed according to the protocols / methods hereinafter described . the polynucleotides of the present invention may be in the form of rna or dna , which dna includes cdna , genomic dna , and synthetic dna . the dna may be double - stranded or single - stranded , and if single stranded may be the coding strand or non - coding ( anti - sense ) strand . the coding sequences which encodes the mature enzymes may be identical to the coding sequences shown in fig1 - 2 ( seq id nos : 5 and 7 ) or may be a different coding sequence which coding sequence , as a result of the redundancy or degeneracy of the genetic code , encodes the same mature enzymes as the dna of fig1 - 2 ( seq id nos : 6 & amp ; 8 ). the polynucleotide which encodes for the mature enzyme of fig1 - 2 ( seq id nos : 6 and 8 ) may include , but is not limited to : only the coding sequence for the mature enzyme ; the coding sequence for the mature enzyme and additional coding sequence such as a leader sequence or a proprotein sequence ; the coding sequence for the mature enzyme ( and optionally additional coding sequence ) and non - coding sequence , such as introns or non - coding sequence 5 ′ and / or 3 ′ of the coding sequence for the mature enzyme . thus , the term “ polynucleotide encoding an enzyme ( protein )” encompasses a polynucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and / or non - coding sequence . the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments , analogs and derivatives of the enzymes having the deduced amino acid sequences of fig1 - 2 ( seq id nos : 6 and 8 ). the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non - naturally occurring variant of the polynucleotide . thus , the present invention includes polynucleotides encoding the same mature enzymes as shown in fig1 - 2 ( seq id nos : 6 and 8 ) as well as variants of such polynucleotides which variants encode for a fragment , derivative or analog of the enzymes of fig1 - 2 ( seq id nos : 6 and 8 ). such nucleotide variants include deletion variants , substitution variants and addition or insertion variants . as hereinabove indicated , the polynucleotides may have a coding sequence which is a naturally occuring allelic variant of the coding sequences shown in fig1 - 2 ( seq id nos : 5 and 7 ). as known in the art , an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution , deletion or addition of one or more nucleotides , which does not substantially alter the function of the encoded enzyme . also , using directed and other evolution strategies , one may make very minor changes in dna sequence which can result in major changes in function . fragments of the full length gene of the present invention may be used as hybridization probes for a cdna or a genomic library to isolate the full length dna and to isolate other dnas which have a high sequence similarity to the gene or similar biological activity . probes of this type preferably have at least 10 , preferably at least 15 , and even more preferably at least 30 bases and may contain , for example , at least 50 or more bases . in fact , probes of this type having at least up to 150 bases or greater may be preferably utilized . the probe may also be used to identify a dna clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions , exons and introns . an example of a screen comprises isolating the coding region of the gene by using the known dna sequence to synthesize an oligonucleotide probe . labeled oligonucleotides having a sequence complementary or identical to that of the gene or portion of the gene sequences of the present invention are used to screen a library of genomic dna to determine which members of the library the probe hybridizes to . it is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe . useful reagents include but are not limited to radioactivity , fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product . the probes are thus useful to isolate complementary copies of dna from other sources or to screen such sources for related sequences . the present invention further relates to polynucleotides which hybridize to the hereinabove - described sequences if there is at least 70 %, preferably at least 90 %, and more preferably at least 95 % identity between the sequences . ( as indicated above , 70 % identity would include within such definition a 70 bps fragment taken from a 100 bp polynucleotide , for example .) the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove - described polynucleotides . as herein used , the term “ stringent conditions ” means hybridization will occur only if there is at least 95 % and preferably at least 97 % identity between the sequences . the polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode enzymes which either retain substantially the same biological function or activity as the mature enzyme encoded by the dna of fig1 - 2 ( seq id nos : 5 and 7 ). in referring to identity in the case of hybridization , as known in the art , such identity refers to the complementarity of two polynucleotide segments . alternatively , the polynucleotide may have at least 15 bases , preferably at least 30 bases , and more preferably at least 50 bases which hybridize to any part of a polynucleotide of the present invention and which has an identity thereto , as hereinabove described , and which may or may not retain activ polynucleotides may be employed as probes for the polynucleotides of ( seq id nos : 5 and 7 ), for example , for recovery of the polynucleotide or as a diagnostic probe or as a pcr primer . thus , the present invention is directed to polynucleotides having at least a 70 % identity , preferably at least 90 % identity and more preferably at least a 95 % identity to a polynucleotide which encodes the enzymes of ( seq id nos : 6 and 8 ) as well as fragments thereof , which fragments have at least 15 bases , preferably at least 30 bases , more preferably at least 50 bases and most preferably fragments having up to at least 150 bases or greater , which fragments are at least 90 % identical , preferably at least 95 % identical and most preferably at least 97 % identical to any portion of a polynucleotide of the present invention . the terms “ fragment ,” “ derivative ” and “ analog ” when referring to the enzymes of fig1 - 2 ( seq id nos : 6and 8 ) means enzymes which retain essentially the same biological function or activity as such enzymes . thus , an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature enzyme . the enzymes of the present invention may be a recombinant enzyme , a natural enzyme or a synthetic enzyme , preferably a recombinant enzyme . the fragment derivative or analog of the enzymes of fig1 - 2 ( seq id nos : 6 and 8 ) may be ( i ) one in which one or more of the amino acid residues are substituted with a conserved or non - conserved amino acid residue ( preferably a conserved amino acid residue ) and such substituted amino acid residue may or may not be one encoded by the genetic code , or ( ii ) one in which one or more of the amino acid residues includes a substituent group , or ( iii ) one in which the mature enzyme is fused with another compound , such as a compound to increase the half - life of the enzyme ( for example , polyethylene glycol ), or ( iv ) one in which the additional amino acids are fused to the mature enzyme , such as a leader or secretory sequence or a sequence which is employed for purification of the mature enzyme or a proprotein sequence . such fragments , derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein . the enzymes and polynucleotides of the present invention are preferably provided in an isolated form , and preferably are purified to homogeneity . the present invention also relates to vectors which include polynucleotides of the present invention , host cells which are genetically engineered with vectors of the invention and the production of enzymes of the invention by recombinant techniques . host cells are genetically engineered ( transduced or transformed or transfected ) with the vectors of this invention which may be , for example , a cloning vector such as an expression vector . the vector may be , for example , in the form of a plasmid , a phage , etc . the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters , selecting transformants or amplifying the genes of the present invention . the culture conditions , such as temperature , ph and the like , are those previously used with the host cell selected for expression , and will be apparent to the ordinarily skilled artisan . the polynucleotides of the present invention may be employed for producing enzymes by recombinant techniques . thus , for example , the polynucleotide may be included in any one of a variety of expression vectors for expressing an enzyme . such vectors include chromosomal , nonchromosomal and synthetic dna sequences , e . g ., derivatives of sv40 ; bacterial plasmids ; phage dna ; baculovirus ; yeast plasmids ; vectors derived from combinations of plasmids and phage dna , viral dna such as vaccinia , adenovirus , fowl pox virus , and pseudorabies . however , any other vector may be used as long as it is replicable and viable in the host . the appropriate dna sequence may be inserted into the vector by a variety of procedures . in general , the dna sequence is inserted into an appropriate restriction endonuclease site ( s ) by procedures known in the art . such procedures and others are deemed to be within the scope of those skilled in the art . the dna sequence in the expression vector is operatively linked to an appropriate expression control sequence ( s ) ( promoter ) to direct mrna synthesis . as representative examples of such promoters , there may be mentioned : ltr or sv40 promoter , the e . coli . lac or trp , the phage lambda p l promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses . the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator . the vector may also include appropriate sequences for amplifying expression . in addition , the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture , or such as tetracycline or ampicillin resistance in e . coli . the vector containing the appropriate dna sequence as hereinabove described , as well as an appropriate promoter or control sequence , may be employed to transform an appropriate host to permit the host to express the protein . as representative examples of appropriate hosts , there may be mentioned : bacterial cells , such as e . coli , streptomyces , bacillus subtilis ; fungal cells , such as yeast ; insect cells such as drosophila s2 and spodoptera sf9 ; animal cells such as cho , cos or bowes melanoma ; adenoviruses ; plant cells , etc . the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein . more particularly , the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above . the constructs comprise a vector , such as a plasmid or viral vector , into which a sequence of the invention has been inserted , in a forward or reverse orientation . in a preferred aspect of this embodiment , the construct further comprises regulatory sequences , including , for example , a promoter , operably linked to the sequence . large numbers of suitable vectors and promoters are known to those of skill in the art , and are commercially available . the following vectors are provided by way of example ; bacterial : pqe70 , pqe60 , pqe - 9 ( qiagen ), pbluescript ii ks ( stratagene ), ptrc99a , pkk223 - 3 , pdr540 , prit2t ( pharmacia ); eukaryotic : pxt1 , psg5 ( stratagene ) psvk3 , pbpv , pmsg , psvl sv40 ( pharmacia ). however , any other plasmid or vector may be used as long as they are replicable and viable in the host . promoter regions can be selected from any desired gene using cat ( chloramphenicol transferase ) vectors or other vectors with selectable markers . two appropriate vectors are pkk232 - 8 and pcm7 . particular named bacterial promoters include laci , lacz , t3 , t7 , gpt , lambda p r , p l and trp . eukaryotic promoters include cmv immediate early , hsv thymidine kinase , early and late sv40 , ltrs from retrovirus , and mouse metallothionein - i . selection of the appropriate vector and promoter is well within the level of ordinary skill in the art . in a further embodiment , the present invention relates to host cells containing the above - described constructs . the host cell can be a higher eukaryotic cell , such as a mammalian cell , or a lower eukaryotic cell , such as a yeast cell , or the host cell can be a prokaryotic cell , such as a bacterial cell . introduction of the construct into the host cell can be effected by calcium phosphate transfection , deae - dextran mediated transfection , or electroporation ( davis , l ., dibner , m ., battey , i ., basic methods in molecular biology , ( 1986 )). the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence . alternatively , the enzymes of the invention can be synthetically produced by conventional peptide synthesizers . mature proteins can be expressed in mammalian cells , yeast , bacteria , or other cells under the control of appropriate promoters . cell - free translation systems can also be employed to produce such proteins using rnas derived from the dna constructs of the present invention . appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by sambrook et al ., molecular cloning : a laboratory manual , second edition , cold spring harbor , n . y ., ( 1989 ), the disclosure of which is hereby incorporated by reference . transcription of the dna encoding the enzymes of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector . enhancers are cis - acting elements of dna , usually about from 10 to 300 bp that act on a promoter to increase its transcription . examples include the sv40 enhancer on the late side of the replication origin bp 100 to 270 , a cytomegalovirus early promoter enhancer , the polyoma enhancer on the late side of the replication origin , and adenovirus enhancers . generally , recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell , e . g ., the ampicillin resistance gene of e . coli and s . cerevisiae trp1 gene , and a promoter derived from a highly - expressed gene to direct transcription of a downstream structural sequence . such promoters can be derived from operons encoding glycolytic enzymes such as β - phosphoglycerate kinase ( pgk ), α - factor , acid phosphatase , or heat shock proteins , among others . the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences , and preferably , a leader sequence capable of directing secretion of translated enzyme . optionally , the heterologous sequence can encode a fusion enzyme including an n - terminal identification peptide imparting desired characteristics , e . g ., stabilization or simplified purification of expressed recombinant product . useful expression vectors for bacterial use are constructed by inserting a structural dna sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter . the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to , if desirable , provide amplification within the host . suitable prokaryotic hosts for transformation include e . coli , bacillus subtilis , salmonella typhimurium and various species within the genera pseudomonas , streptomyces , and staphylococcus , although others may also be employed as a matter of choice . as a representative but nonlimiting example , useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pbr322 ( atcc 37017 ). such commercial vectors include , for example , pkk223 - 3 ( pharmacia fine chemicals , uppsala , sweden ) and pgemi ( promega biotec , madison , wis ., usa ). these pbr322 “ backbone ” sections are combined with an appropriate promoter and the structural sequence to be expressed . following transformation of a suitable host strain and growth of the host strain to an appropriate cell density , the selected promoter is induced by appropriate means ( e . g ., temperature shift or chemical induction ) and cells are cultured for an additional period . cells are typically harvested by centrifugation , disrupted by physical or chemical means , and the resulting crude extract retained for further purification . microbial cells employed in expression of proteins can be disrupted by any convenient method , including freeze - thaw cycling , sonication , mechanical disruption , or use of cell lysing agents , such methods are well known to those skilled in the art . various mammalian cell culture systems can also be employed to express recombinant protein . examples of mammalian expression systems include the cos - 7 lines of monkey kidney fibroblasts , described by gluzman , cell , 23 : 175 ( 1981 ), and other cell lines capable of expressing a compatible vector , for example , the c127 , 3t3 , cho , hela and bhk cell lines . mammalian expression vectors will comprise an origin of replication , a suitable promoter and enhancer , and also any necessary ribosome binding sites , polyadenylation site , splice donor and acceptor sites , transcriptional termination sequences , and 5 ′ flanking nontranscribed sequences . dna sequences derived from the sv40 splice , and polyadenylation sites may be used to provide the required nontranscribed genetic elements . the enzyme can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation , acid extraction , anion or cation exchange chromatography , phosphocellulose chromatography , hydrophobic interaction chromatography , affinity chromatography , hydroxylapatite chromatography and lectin chromatography . protein refolding steps can be used , as necessary , in completing configuration of the mature protein . finally , high performance liquid chromatography ( hplc ) can be employed for final purification steps . the enzymes of the present invention may be a naturally purified product , or a product of chemical synthetic procedures , or produced by recombinant techniques from a prokaryotic or eukaryotic host ( for example , by bacterial , yeast , higher plant , insect and mammalian cells in culture ). depending upon the host employed in a recombinant production procedure , the enzymes of the present invention may be glycosylated or may be non - glycosylated . enzymes of the invention may or may not also include an initial methionine amino acid residue . antibodies generated against the enzymes corresponding to a sequence of the present invention can be obtained by direct injection of the enzymes into an animal or by administering the enzymes to an animal , preferably a nonhuman . the antibody so obtained will then bind the enzymes itself . in this manner , even a sequence encoding only a fragment of the enzymes can be used to generate antibodies binding the whole native enzymes . such antibodies can then be used to isolate the enzyme from cells expressing that enzyme . for preparation of monoclonal antibodies , any technique which provides antibodies produced by continuous cell line cultures can be used . examples include the hybridoma technique ( kohler and milstein , nature , 256 : 495 - 497 , 1975 ), the trioma technique , the human b - cell hybridoma technique ( kozbor et al ., immunology today 4 : 72 , 1983 ), and the ebv - hybridoma technique to produce human monoclonal antibodies ( cole et al ., in monoclonal antibodies and cancer therapy , alan r . liss , inc ., pp . 77 - 96 , 1985 ). techniques described for the production of single chain antibodies ( u . s . pat . no . 4 , 946 , 778 ) can be adapted to produce single chain antibodies to immunogenic enzyme products of this invention . also , transgenic mice may be used to express humanized antibodies to immunogenic enzyme products of this invention . antibodies generated against an enzyme of the present invention may be used in screening for similar enzymes from other organisms and samples . such screening techniques are known in the art , for example , one such screening assay is described in sambrook and maniatis , molecular cloning : a laboratory manual ( 2d ed . ), vol . 2 : section 8 . 49 , cold spring harbor laboratory , 1989 , which is hereby incorporated by reference in its entirety . the present invention will be further described with reference to the following examples ; however , it is to be understood that the present invention is not limited to such examples . all parts or amounts , unless otherwise specified , are by weight . an e . coli catalase negative host strain cat500 was infected with a phage solution containing sheared pieces of dna from alcaligenes ( deleya ) aquamanrinus in pbluescript plasmid and plated on agar containing lb with ampicillin ( 100 μg / ml ), methicillin ( 80 μg / ml ) and kanamycin ( 100 μg / ml ) according to the method of hay and short ( hay , b . and short , j ., j . strategies , 5 : 16 , 1992 ). the resulting colonies were picked with sterile toothpicks and used to singly inoculate each of the wells of 96 - well microtiter plates . the wells contained 250 μl of sob media with 100 μg / ml ampicillin , 80 μg / ml methicillin , and ( sob amp / meth / kan ). the cells were grown overnight at 37 ° c . without shaking . this constituted generation of the “ sourcegenebank ”; each well of the source genebank thus contained a stock culture of e . coli cells , each of which contained a pbluescript plasmid with a unique dna insert . same protocol was adapted for screening catalase from microscilla furvescens . the plates of the source genebank were used to multiply inoculate a single plate ( the “ condensed plate ”) containing in each well 200 μl of sob amp / meth / kan . this step was performed using the high density replicating tool ( hdrt ) of the beckman biomek with a 1 % bleach , water , isopropanol , air - dry sterilization cycle in between each inoculation . each well of the condensed plate thus contained 4 different pbluescript clones from each of the source library plates . nine such condensed plates were prepared and grown for 16 h at 37 ° c . one hundred ( 100 ) μl of the overnight culture was transferred to the white polyfiltronic assay plates containing 100 μl hepes / well . a 0 . 03 % solution of hydrogen peroxide was made in 5 % triton and 20 μl of this solution was added to each well . the plates were incubated at room temperature for one hour . after an hour , 50 μl of 120 mm 3 -( p - hydroxyphenyl )- propionic acid and 1 unit of horseradish peroxidase were added to each well and the plates were incubated at room temperature for 1 hour . to quench the reaction , 50 μl of 1 m tris - base was added to each well . the wells were excited on a fluorometer at 320 nm and read at 404 nm . a low value signified a positive catalase hit . in order to isolate the individual clone which carried the activity , the source genebank plates were thawed and the individual wells used to singly inoculate a new plate containing sob amp / meth / kan . as above the plate was incubated at 37 ° c . to grow the cells , and assayed for activity as described above . once the active well from the source plate was identified , the cells from the source plate were streaked on agar with lb / amp / meth / kan and grown overnight at 37 ° c . to obtain single colonies . eight single colonies were picked with a sterile toothpick and used to singly inoculate the wells of a 96 - well microtiter plate . the wells contained 250 μl of sob amp / meth / kan . the cells were grown overnight at 37 ° c . without shaking . a 100 μl aliquot was removed from each well and assayed as indicated above . the most active clone was identified and the remaining 150 μl of culture was used to streak an agar plate with lb / amp / meth / kan . eight single colonies were picked , grown and assayed as above . the most active clone was used to inoculate 3 ml cultures of lb / amp / meth / kan , which were grown overnight . the plasmid dna was isolated from the cultures and utilized for sequencing . dna encoding the enzymes of the present invention , ( seq id nos : 6 and 8 ), were initially amplified from a pbluescript vector containing the dna by the pcr technique using the primers noted herein . the amplified sequences were then inserted into the respective pqe vector listed beneath the primer sequences , and the enzyme was expressed according to the protocols set forth herein . the 5 ′ and 3 ′ oligonucleotide primer sequences used for subcloning and vectors for the respective genes are as follows : alcaligenes ( deleya ) aquamarinus catalse : ( pqet vector ) 5 ′ primer ccgagaattcattaaagaggagaaattaactatgaataacgcatccgctgac ecori ( seq id no : 1 ) 3 ′ primer cggaaagcitttacgacgcgacgtcgaaacg hindiii ( seq id no : 2 ) microscilla furvescens catalase : ( pqet vector ) 5 ′ primer ccgagaattcattaaagaggagaaattaactatggaaaatcacaaacactca ecori ( seq id no : 3 ) 3 ′ primer cgaaggtaccttatttcagatcaaaccggtc kpni ( seq id no : 4 ) the restriction enzyme sites indicated correspond to the restriction enzyme sites on the bacterial expression vector indicated for the respective gene ( qiagen , inc . chatsworth , calif .). the pqet vector encodes antibiotic resistance ( amp r ), a bacterial origin of replication ( ori ), an ipitg - regulatable promoter operator ( p / o ), a ribosome binding site ( rbs ), a 6 - his tag and restriction enzyme sites . the pqet vector was digested with the restriction enzymes indicated . the amplified sequences were ligated into the respective pqet vector and inserted in frame with the sequence encoding for the rbs . the native stop codon was incorporated so the genes were not fused to the his tag of the vector . the ligation mixture was then used to transform the e . coli strain um255 / prep4 ( qiagen , inc .) by electroporation . um255 / prep4 contains multiple copies of the plasmid prep4 , which expresses the lad repressor and also confers kanamycin resistance ( kan r ). transformants were identified by their ability to grow on lb plates and ampicillin / kanamycin resistant colonies were selected . plasmid dna was isolated and confirmed by restriction analysis . clones containing the desired constructs were grown overnight ( o / n ) in liquid culture in lb media supplemented with both amp ( 100 ug / ml ) and kan ( 25 ug / ml ). the o / n culture was used to inoculate a large culture at a ratio of 1 : 100 to 1 : 250 . the cells were grown to an optical density 600 ( o . d . 600 ) of between 0 . 4 and 0 . 6 . iptg (“ isopropyl - b - d - thiogalacto pyranoside ”) was then added to a final concentration of 1 mm . iptg induces by inactivating the laci repressor , clearing the p / o leading to increased gene expression . cells were grown an extra 3 to 4 hours . cells were then harvested by centrifugation . the primer sequences set out above may also be employed to isolate the target gene from the deposited 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