PROTEASE ENZYME MANUFACTURE USING NON-PROTEIN PROTEASE INHIBITORS

Abstract: Methods for manufacturing protease enzymes comprising the use of certain non-protein, dissolved protease inhibitor material having Ki of less than about 1x10<-4> during the manufacturing process.

Description: Protease enzyme manufacture using non-protein protease i nhibi tors The present invention relates to methods for manufacturing protease enzymes comprising the use of certain non-protein, dissolved protease inhibitor material having Ki of less than about 1X10~4 during the manufacturing process. BACKGROUND OF THE INVENTION Protease-containing detergent compositions are well known. It is also known that a problem with such compositions is the degradation by protease enzymes of second enzymes present in these compositions, such as lipases, cellulases, amylases, etc., and possibly also some of the protease itself. Solving this problem in detergent compositions has been approached in a variety of ways including the use of protease inhibitors in such compositions. Cleaning compositions are describe, for example, in U.S. Patent 4,566,985, issued January 28, 1986, to Bruno et al (liquid cleaning compositions containing benzamidine hydrohalide); European Patent Publication No. 376,705 published July 4, 1990 by Cardinali et al (using lower aliphatic alcohol and a salt of a lower carboxylic acid and a surfactant system which is predominantly nonionic); European Patent Publication No. 381,262 published August 8, 1990 by Aronson et al (using a mixture of a polyol and boron compound which are capable of reacting); WO 92/03529, published March 5, 1992 by Novo Nordisk A/S (using reversible protease inhibitors of the peptide or protein type); WO 92/05239, published April 2, 1992 by Novo Nordisk A/S (also using reversible protease inhibitors of the peptide or protein type); WO 92/19707 published October 30, 1992 (liquid laundry detergents containing meta substituted boronic acids); U.S. Patent 4,908,150, issued March 13, 1990 to Hessel et al (liquid detergent compositions containing particular nonionic ethylene glycol-containg copolymers); European Patent Application Serial No. 90/870212, published November 14, 1990 (liquid detergent compositions containing certain bacterial serine proteases and lipases); US Patent 5,178,789, issued January 12, 1993 to Estell (liquid detergents using an inhibitor that binds to the enzyme); and US Patent 5,039,446, issued August 13, 1991 to Estell (liquid detergents using an inhibitor having a certain dissociation constant). The manufacture of protease enzymes, including those useful in detergent compositions, have been described in the literature. Examples of such proteases and their manufacturing process, which typically involve fermentation of bacteria which produce these proteases, include European Patent Publication No. 199,404 published October 29, 1986 by Venegas, and WO 93/13125, published July 8, 1993 by Novo Nordisk A/S. The latter is directed to a process for the microbial production of a protein susceptable to inactivation in a fluid production medium by continuously and reversibly protecting said protein against inactivation during the production stage, separating the protein from the production medium, deprotecting the protein, and recovering the protein product. The process is further said to be useful for obtaining increased yields of the protein by continuously precipitating the protein. At one point (at page 12) it is stated: "In connection with the subtilisin proteases mentioned below it is in this embodiment preferred to use a protease inhibitor, such as the CI-1, CI-2, PSI, Eglin C, Eglin B, TSI-1, SSI, or VSI inhibitor, variants thereof, or a mixture of any of these to protect the subtilisin protease" "In the above embodiment the de-protection of the protein in question, such as a protease, is usually performed through the dissolution of the complex when using the protease in e.g. a detergent composition that is dissolved when making up the wash liquor. " German Patent Specification 2, 131,451, filed by Nagase & Co., published December 30, 1971, discusses a process for the production of alkaline protease. This process is said to require the addition of 0.1 to 12% of water soluble borate. Therein it is stated (as translated to English): "The growth of bacteria producing protease and the productivity of protease are generally accelerated by the addition of phosphate. However, viscous compounds of high molecular weight, which make it extremely difficult to filter and perform other operating processes, are produced in this case as by-products. However, it was now found that with the addition of water soluble borates, such as borax or boric acid, in small amounts to the culture, the formation of viscous compounds can be greatly restrained, and the production of protease is favored at the same time. The filtering activity is greatly improved by doing this in the production of alkaline protease, and, accordingly, the enzyme yield with the filtration can be greatly increased. Thus, the expression jorate' used here also includes boric acid - as shown above. The mechanism of action of borate or boric acid has not been completely explained; however, it is certain that borate or boric acid inhibits the formation of viscous compound of high molecular weight without having a disadvantageous effect on the production of alkaline protease." Therein, there is no discussion or indication whether the water soluble borates have any inhibiting effect on the protease per se. It is indicated, however, that certain amounts of the borate can actually retard the production of the enzyme. In addition, Japanese patent publication J01-296987, published November 30, 1989, describes a method comprising culturing a microorganism capable of producing protease in a culture medium containing a substance showing affinity to protease (e.g., cinnamic acid; cinnamic acid ester; bacitracin; phenylbutylamine; phenylboric acid; aminophenylboric acid; phenylalanine; N-carbobenzoxy- phenylalanine; tyrosine). Preferred methods are said to comprise culturing the microorganism in a culture medium containing an adsorbent on which the substance showing affinity to the protease is adsorbed (e.g., silica; alumina; Sepharose; crosslinked polymethacrylate), separating the adsorbent and recovering the protease from the adsorbent. It is indicated that preparation of protease can be practiced while suppressing the autolysis of formed protease in the presence of a substance showing affinity for protease and that by using the adsorbent, formed protease can be easily isolated and refined. The inhibition constant (Ki) is ordinarily used as a measure of capacity to inhibit enzyme activity, with a low Ki indicating a more potent inhibitor. A discussion of the inhibition of one proteolytic enzyme, subtilisin, is provided in Philipp, M. and Bender, M.L., "Kinetics of Subtilisin and Thiolsubtilisin", Molecular & Cellular Biochemistry, 51. pp. 5-32 (1983). Peptide alkylboronic acid is discussed as an inhibitor of trypsin-like serine proteases such as thrombin, plasma kallikrein and plasmin, especially in pharmaceuticals, in European Patent Application 293,881, Kettner et al, published December 7, 1988. It has been surprisingly discovered by the present invention that the inclusion of non-protein, dissolved protease inhibitors having Ki values of less than about 1X10" in the culture medium of the fermentation step of manufacturing protease enzymes from bacteria provides for improved manufacturing processes. Surprisingly, such non-protein, dissolved inhibitors apparently do not interfere with bacteria's ability to produce the protease. However, while not to be limited by theory, it appears that these inhibitors limit the autolysis (self destruction) of the protease, and/or possibly also the destruction and/or other influence of the protease on the bacteria per se. It is therefore an object of the present invention to provide methods for manufacturing protease enzymes. It is a further object to provide a method of manufacturing protease enzyme by including dissolved protease enzyme inhibitor(s) in the culture medium for such methods. An additional object is to provide a method for manufacturing protease enzymes resulting in higher yields and/or higher purity and/or easier purification and/or more stable protease enzymes and/or higher activity protease enzymes. An object is to also manufacture protease enzymes stabilized by protease enzyme inhibitors. These and other objects of the present invention will be readily apparent from the detailed description provided herewith. All percentages are by weight, and all measurements are made at 25°C, unless otherwise indicated. All documents cited are incorporated herein by reference in their entirety. SUMMARY OF THE INVENTION The present invention relates to a method for manufacturing protease enzymes comprising including one or more non-protein, dissolved protease enzyme inhibitors having Ki value of less than about 1X10"^ in the manufacturing process. Preferred are methods which provide dissolved protease enzyme inhibitor to protease enzyme at molar ratio greater than about 1: 1. Preferred methods utilize fermentation broth comprising Bacillus subtilis and/or Bacillus licheniformis bacteria. DETAILED DESCRIPTION OF THE INVENTION (a) Protease Enzymes: Protease enzymes prepared by the present invention process can be of animal, vegetable or microorganism (preferred) origin. Preferred are the serine proteolytic enzymes. Particularly preferred is bacterial serine protease enzyme obtained from Bacillus subtilis and/or Bacillus licheniformis. Non limiting examples of protease enzymes include Novo Industri A/S Alcalase®, Esperase®, Savinase ® (Copenhagen, Denmark), Gist-brocades' Maxatase®, Maxacal®, and Maxapem 15® (protein engineered Maxacal®)(Delft, Netherlands), and subtilisin BPN and BPN' which are commercially available from Sigma Chemical Company. Preferred protease enzymes are also modified bacterial serine proteases, such as those made by Genencor International., Inc, (South San Francisco, California) which are described in European Patent Application 251,446, Wells et al., published January 7, 1988, and which is called herein "Protease B"; U.S. Patent 5,030,378, Venegas, issued July 9, 1991, which refers to a modified bacterial serine proteolytic enzyme which is called "Protease A" herein; and the protease enzymes described in U.S. Application Serial Number 08/136,797 filed October 14, 1993, by Baeck et al., entitled "Protease-Containing Cleaning Compositions" (this application is incorporated by reference herein in its entirety). Preferred protease enzymes are selected from the group consisting of Savinase ®, Maxacal®, Alcalase®, BPN', Protease A and Protease B, the proteases described in USSN 08/136,797, and mixtures thereof. (b) Protease Enzyme Inhibitors: The present invention method also requires the presence of one or more non- protein, dissolved protease enzyme inhibitors having Ki values less than about 1X10" 4 in the fermentation broth (aka, culture medium) to provide dissolved protease enzyme inhibitor. Preferred is dissolved protease enzyme inhibitor to protease enzyme molar ratio greater than about 1: 1, more preferably greater than about 2: 1, and most preferably within the range of from about 2:1 to about 10:1 (with this range being based on the quantity of protease enzyme present at the end of the fermentation step). The term "non-protein, dissolved protease enzyme inhibitor", as used herein, means any non-protein reversible inhibitor of the proteolytic activity of the protease enzyme being manufactured capable of being dissolved (in whole or in part) in the fermentation broth, and for peptide-derived inhibitors, no such inhibitors comprise more than about 10 amino acid units. Preferred inhibitors are those having Ki of less than about 1X10"^ M, preferably less than about 1X10"- 5 M, and most preferably less than about 1X10" 6 M. Ki values are determined by known procedures. For example, potential inhibitors of the protease subtilisin BPN' are evaluated on the basis of their ability to inhibit the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as described by DelMar for chymotrypsin, a protease similar to subtilisin BPN [DelMar et al., Analytical Biochemistry (1979) 99, 316-320]. Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and the inhibitor are added to a buffered aqueous solution (pH 8.6) containing 17% by volume of dimethyl sulfoxide. The rate is initiated by the introduction of a known amount of subtilisin BPN' and monitored spectrophotometrically at 25 C. The rates, which obey Michaelis Menten conditions, are analyzed by the method of Lineweaver and Burk to determine the inhibitor binding constants, Ki. [Biochemistry Third Edition (1988) by Lubert Stryer; W.H. Freeman and Company, pp 187-195]. The term "reversible inhibitor", as used herein, means those protease inhibitors which are capable of being released from the protease enzyme to allow restored proteolytic activity, including but not limited to competitive, noncompetitive and uncompetitive inhibitors as described for example in detail in Mahler et al., "Biological Chemistry, Second Edition" (published 1971 by Harper & Row) at pages 295-299, the disclosures of which are incorporated herein by reference in its entirety. Depending on the protease enzyme which is being manufactured, protease enzyme inhibitors can be selected which are more effective than others for reversibly inhibiting said protease. Existing proteases can be divided into trypsin, subtilisin, chymotrypsin and elastase type proteases, and the protease enzyme inhibitor(s) to be used in the manufacture of each such enzymes can be selected as appropriate for each of these type of enzyme being produced. Preferred protease enzyme inhibitors for use herein include boronic acid compounds, peptide aldehyde compounds, peptide boronic acid compounds, peptide trifluoromethylketone compounds, and mixtures thereof. Preferred are peptide boronic acid compounds, peptide aldehyde compounds, and mixtures thereof, preferably comprising from 2 to about 10, more preferably from 2 to about 5, amino acids and/or amino acid derivatives (e.g., the carboxylic acid terminus derivatized into an aldehyde moiety in the peptide aldehyde compounds, into a trifluoromethylketone moiety in the peptide trifluoromethylketone compounds, and into a boronic acid-containing moiety in the peptide boronic acid compounds). These compounds are described, for example, in PCT Patent Publications Numbers WO 94/0465 IE; WO 94/04652E; WO 94/04653; WO 94/04542; WO 94/04543; WO 94/04654, all published by The Procter & Gamble Company March 3, 1994, and U.S. Patent Application Serial No. 08/149,171, filed November 5, 1993 by Panandiker and Bjorkquist, the disclosures of all being herein by reference in their entirety. As used herein, the term "peptide aldehydes" means compounds comprising a peptidic chain wherein the C-terminal end of said chain is converted from a carboxylic group to an aldehyde group. Peptide aldehydes have been described, as well as processes for their manufacture, for example, in PCT Patent Publication No. WO 94/0465 IE, published by The Procter & Gamble Company March 3, 1994, the disclosures being incorporated herein by reference in their entirety. Preferred peptide aldehydes for use herein comprise from 2 to 6 amino acids (including the aldehyde derivatized unit), most preferably 3 or 4. Peptide aldehydes include, for example, Lys-Ala-LysH, Ile-Phe-LysH, Phe- Pro-ArgH, Phe-Val-ArgH, Lys-AJa-AlaH, Ala-Ala-ProH, Gly-Ala-LeuH, Gly-Ala- PheH, Phe-Gly-Ala-PheH, Phe-Gly-AJa-LeuH, Leu-Leu-PheH, Ala-Ala-PheH, Leu- Leu-TyrH, Val-Pro-ValH and Ala-Val-LeuH. Preferred peptide aldehydes for use herein are Lys-Ala-AlaH, Ala-Ala-ProH, Gly-Ala-LeuH, Gly-Ala-PheH, Phe-Gly- AJa-PheH, and Phe-Gly-Ala-LeuH. Particularly preferred is Phe-Gly-Aia-LeuH. All peptide aldehydes herein are preferably used in their methyl carbamate or methyl urea N-terminal protected form. As used herein, the term "peptide boronic acid" means compounds comprising a peptidic chain wherein the C-terminal end of said chain is converted from a carboxylic group to a boronic acid-containing group. Peptide boronic acid compounds have been described, as well as processes for their manufacture, for example in PCT Patent Publications Numbers WO 94/04542, WO 94/04543, WO 94/04654, and WO 94/04653, all published by The Procter & Gamble Company March 3, 1994, the disclosures of all being incorporated herein by reference in their entirety. Preferred peptide boronic acid compounds for use herein comprise from 2 to 6 amino acids units (including the boronic acid derivatized unit), most preferably 3 or 4. As used herein, the term "peptide trifluoromethylketone" means compounds comprising a peptidic chain wherein the C-terminal end of said chain is converted from a carboxylic group to a trifluoromethylketone group. Peptide trifluoromethylketone compounds have been described, as well as processes for their manufacture, for example in PCT Patent Publication Number WO 94/04652E, published by The Procter & Gamble Company March 3, 1994, the disclosures of which being incorporated herein by reference in their entirety. Preferred peptide trifluoromethylketone compounds for use herein comprise from 2 to 6 amino acids units (including the trifluoromethylketone derivatized unit), most preferably 3 or 4. The N-terminal end of said peptidic chain in the peptide aldehydes, peptide trifluoromethylketone, and peptide boronic acid compounds may be protected by appropriate protecting groups which are known in the art. However, preferred compounds have the N-terminal end of the peptidic chain protected by a methyl carbamate (CH3O-(O)C-) or methyl urea (CH3N-(O)C-) group. Examples include, but are not limited to, the following (having the indicated Ki values for subtilisin BPN'): Compound Kj (M) 1) Boronic Acid Compounds: 3.6X10" 5 2) Peptide Boronic Acid Compounds: 2.64X10" 7 3.6X10- 7 3) Peptide Aldehyde Compounds: 7.6X10- 7 (c) Method of Manufacturing: The present invention method comprises manufacturing protease enzymes by a process comprising including one or more non-protein, dissolved protease enzyme inhibitors having Ki values of less than about 1X10"4 in the manufacturing process. Methods for manufacturing protease enzymes, to which such protease enzyme inhibitors may be added according to the present invention, typically utilize a fermentation broth to produce the protease enzyme, followed by one or more purification steps to isolate the protease enzyme. Preferred methods herein utilize the fermentation of Bacillus subtilis and/or Bacillus licheniformis bacteria in a nutrient medium, and also comprise the non- protein, dissolved protease enzyme inhibitor(s) having Ki values of less than about IXIO' . Also preferred is the inclusion of the protease inhibitor(s) both in the fermentation broth and thereafter (by carry over from the fermentation broth and/or by separate addition(s)) in the enzyme purification process step(s). However, the present invention methods may utilize the protease enzyme inhibitor(s) during only the fermentation step, or the fermentation step plus all of the other manufacturing steps. The present invention methods for manufacturing protease enzymes preferably comprises: (a) growing protease enzymes in a fermentation broth comprising one or more non-protein, dissolved protease enzyme inhibitors having a Ki of less than about lXlO- 4 M; and (b) purifying said protease enzyme by one or more steps to concentrate the protease enzyme produced in step (a), said purification steps optionally comprising further addition of a non-protein, dissolved protease enzyme inhibitor having Ki values of less than about 1X10"4. More preferred processes are those wherein one or more dissolved protease enzyme inhibitors are added to the process at step (a), or steps (a) and (b) at a molar ratio of protease enzyme inhibitor to protease enzyme of at least about 1:1. Preferred methods use Bacillus subtilis bacteria to produce the protease enzyme in the fermentation broth of step (a). Preferred methods also comprise including the protease enzyme inhibitor in the fermentation broth. In general, it is desired that the protease enzyme inhibitor(s) concentration during the fermentation process, prior to concentrating the protease enzyme (when the inhibitor concentration would be increased), be less than about 0.1 mM, preferably less than about 0.05 mM. It is also generally desirable to utilize protease enzyme inhibitors) in a molar ratio relative to the final protease enzyme yield of at least about 1:1, preferably at least about 2:1, and most preferably within the range of from about 2: 1 to about 10:1 (inhibitoπenzyme). Also preferred is that protease enzyme inhibitors be present in the purification step(s), typically at a concentration of less than about 0.1 mM (more preferably less than about 0.05 mM) prior to concentrating the protease enzyme. Also preferred is the production of the protease enzymes in dry or concentrated liquid form prepared by the process of the present invention comprising the protease enzyme inhibitor, (d) Detergent Compositions: An effective amount of one or more protease enzymes produced by the present invention are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal. Such cleaning compositions include detergent compositions for cleaning hard surfaces, unlimited in form (e.g., liquid and granular); detergent compositions for cleaning fabrics, unlimited in form (e.g., granular, liquid and bar formulations); dishwashing compositions (unlimited in form); oral cleaning compositions, unlimited in form (e.g., dentifrice, toothpaste and mouthwash formulations); denture cleaning compositions, unlimited in form (e.g., liquid, tablet); and contact lens cleaning compositions, unlimited in form (e.g., liquid, tablet). As used herein, "effective amount of protease enzyme" refers to the quantity of protease enzyme necessary to achieve the enzymatic activity necessary in the specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like. Preferably the cleaning compositions of the present invention comprise from about 0.0001% to about 10% of one or more protease enzyme, more preferably from about 0.001% to about 1%, more preferably still from about 0.01% to about 0.1%. Several examples of various cleaning compositions wherein the protease enzyme prepared by the present invention may be employed are discussed in further detail below. All parts, percentages and ratios used herein are by weight unless otherwise specified. As used herein, "non-fabric cleaning compositions" include hard surface cleaning compositions, dishwashing compositions, oral cleaning compositions, denture cleaning compositions and contact lens cleaning compositions. A. Cleaning Compositions for Hard Surfaces. Dishes and Fabrics The protease enzymes prepared by the present invention can be used in many detergent compositions. Thus the protease enzymes prepared by the present invention can be used with various conventional ingredients to provide fully- formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions and the like. Such compositions can be in the form of liquids, granules, bars and the like. Such compositions can be formulated as modern "concentrated" detergents which contain as much as 30%-60% by weight of surfactants. The cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surfactants are typically present at levels of from about 5% to about 35% of the compositions. Nonlimiting examples of surfactants useful herein include the conventional C 11 -C i alkyl benzene sulfonates and primary and random alkyl sulfates, the C\Q- C 18 secondary (2,3) alkyl sulfates of the formulas CH3(CH 2 )x(CHOSO3)-M + )CH 3 and CH3 (CH2MCHOSO3 "M+) CH2CH3 wherein x and (y+ 1 ) are integers of at least about 7, preferably at least about 9, and M is a water-solubilizing cation, especially sodium, the Cio-Ci g alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), Cι n-Cι g alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the Ci()-Cι g alkyl polyglycosides, and their corresponding sulfated polyglycosides, Ci2-Cι g alpha-sulfonated fatty acid esters, Ci2-Cι g alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), l2" i8 betaines and sulfobetaines ("sultaines"), Cjo-Cig amine oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. (Use of such surfactants in combination with the aforesaid amine oxide and/or betaine or sultaine surfactants is also preferred, depending on the desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-C1 g N-methyl glucamides disclosed in US Patent 5, 194,639, Connor et al., issued March 16, 1993, incorporated herein by reference. A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C10- 16 alkolamides can be incorporated into the compositions, typically at about 1% to about 10% levels. The C10- 14 monoethanol and diethanol amides illustrate a typical class of such suds boosters. Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous. If desired, soluble magnesium salts such as MgCl2, MgSO4, an d the like, can be added at levels of, typically, from about 0.1% to about 2%, to provide additionally sudsing. The liquid detergent compositions herein can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable. Monohydric alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1,3-propanediol, ethylene glycol, glycerine, and 1,2- propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers. The detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11.0. Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art. When formulating the hard surface cleaning compositions and fabric cleaning compositions, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like. Other conventional builders are listed in standard formularies. Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases not prepared by the present invention process in such compositions, typically at levels of from about 0.001% to about 1% by weight. Various detersive and fabric care enzymes are well-known in the laundry detergent art. Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1% to about 15% by weight. If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1% to about 10% by weight. Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials. Enzyme stabilizers may also be used in the cleaning compositions of the present invention. Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1% to about 1%). 1. Hard surface cleaning compositions As used herein "hard surface cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like. Hard surface cleaning compositions may comprise an effective amount of one or more protease enzymes prepared by the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.01% to about 5%, more preferably still from about 0.05% to about 1% by weight of active enzyme of the composition. In addition to comprising one or more protease enzymes prepared by the present invention, such hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering builder. In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy/streaky residue on the glass surface. The surfactant component, when present, may comprise as little as 0.1% of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%, more preferably from about 1% to about 5% of surfactant. Typically the compositions will contain from about 0.5% to about 50% of a detergency builder, preferably from about 1% to about 10%. Preferably the pH should be in the range of about 8 to 12. Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary. Solvents may be included in the compositions. Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-trimethyl-l,3-pentanediol and 2-ethyl-l,3-hexanediol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, preferably from about 3% to about 11%. Additionally, highly volatile solvents such as isopropanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after "full strength" application of the composition to the surface. When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions. 2. Dishwashing Compositions Dishwashing compositions may comprise one or more protease enzymes prepared by the present invention. As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to, granular and liquid forms. 3. Fabric cleaning compositions Fabric cleaning compositions may comprise one or more protease enzymes prepared by the present invention. As used herein, "fabric cleaning composition" refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. a. Granular fabric cleaning compositions The granular fabric cleaning compositions may contain an effective amount of one or more protease enzymes prepared by the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1% by weight of active enzyme of the composition. In addition to one or more protease enzymes, the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent. b. Liquid fabric cleaning compositions Liquid fabric cleaning compositions may comprise an effective amount of one or more protease enzyme prepared by the present invention, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1%, by weight of active enzyme of the composition. Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water. c. Bar fabric cleaning compositions Bar fabric cleaning compositions suitable for hand-washing soiled fabrics contain an effective amount of one or more protease enzymes prepared by the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.01% to about 1% by weight of the composition. The protease enzymes are preferably included in an amount sufficient to provide an activity of from about 0.005 to about 0.1, more preferably from about 0.012 to about 0.04, Anson units per gram of composition. The following examples are illustrative and are not intended to limit the invention since one skilled in the art would be able to substitute protease enzymes and protease enzyme inhibitors based on the disclosure herein without undue experimentation or deviation from the spirit or scope of the claims. EXAMPLE 1 The method of the present invention is used to produce an unstable subtilisin BPN' variant as follows. Fifty ml of 2xYTc* y r media (as described in Sambrook et al., "Molecular cloning: A Laboratory Manual", published 1989 by Cold Spring Harbor Laboratory Press, incorporated herein by reference in its entirety) is inoculated with the unstable subtilisin BPN' variant in Bacillus subtilis strain BG2036 (as described in Yang et al., "Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation.", J. Bacteriol.. 160:15-21 (1984), incorporated herein by reference in its entirety). The culture is split into two 25 ml cultures, and to one is added 90 ul of a 10 mM solution of the methyl carbamate peptide aldehyde Phe-Gly-Ala-LeuH inhibitor (Ki = 7.5x10" 9 ) in 100% ethanol. (The expected expression levels of enzyme for this type of culture is 0.1 mg/ml or 3.6X10-6M; the inhibitor level used therefore gives about a 10 fold excess of inhibitor to enzyme.) Both cultures are grown at 37°C at 250 φm shaking for 24 hours. The results are: A^oo Reading pNA assay (of 1:10 dilution) 1 ) Control 1.74 0.0029 Inhibitor 1.77 0.0243 1) pNA assay buffer as described in DelMar et al., Anal. Biochem.. 99. 316-320 (1979). Use of this method results in about a 10 fold increase in the level of enzyme. EXAMPLE 2 The method of the present invention is used for purification of an unstable subtilisin BPN 1 variant as follows. A 25 ml culture of the unstable subtilisin BPN variant in 2xYTcMP media with 10 mM of the methyl carbamate peptide aldehyde Phe-Gly-Ala-LeuH inhibitor (about 10 fold excess) is grown overnight (18 hours) at 37°C and 250 φm shaking. The bacterial cells are then spun down (5K; 15 min.), and the supematent, cleared fermentation broth ("CFB") is transferred to a new tube. The CFB is diluted (1:10) and a pNA assay is conducted. A PD-10 column (Sephadex G-25M, Pharmacia) with 10 mM MOPS buffer pH 7.0 is equilibrated, and then 1 ml of CFB is applied to. the column and allowed to run in (eluate discarded), followed by 2 ml of buffer (elute discarded) and than an additional 2 ml of buffer (eluate is collected as a batch; this is diluted 1:10 and a pNA assay conducted). The eluate is added to a 0.5 ml column of S-Sepharose Fast Flow (Pharmacia) equilibrated with the above buffer. This is washed with 3 ml of buffer, and then 1.5 ml of 10 mM MOPS pH 7.0, 0.3 M NaCl is applied to the column and collected as a batch (this is diluted 1:10 and a pNA assay conducted). The results are: Volume pNA Assay Yield CFB 1 ml 0.0757 100% PD-10 2 ml 0.0305 81% S-Sepharose 1.5 ml 0.0129 31% Since the unstable subtilisin BPN' variant without inhibitor is lost by the PD- 10 stage, the present method results in a significant increase in yield.

Claims: What is claimed is: 1. A method for manufacturing protease enzymes comprising including one or more non-protein, dissolved protease enzyme inhibitors having Ki value of less than 1X10"4 in the manufacturing process. 2. The method of Claim 1 comprising the steps of: a) growing protease enzymes in a fermentation broth; and b) purifying said protease enzyme by one or more steps to concentrate the protease enzyme produced in step (a); and wherein one or more non-protein, dissolved protease enzyme inhibitors having Ki value of less than 1X10"4 are added to the process at step (a), step (b), or steps (a) and (b). 3. A method for manufacturing protease enzymes comprising the steps of (a) growing protease enzymes in a fermentation broth comprising one or more non-protein, dissolved protease enzyme inhibitors having a Ki of less than 1X10"4 M; and (b) purifying said protease enzyme by one or more steps to concentrate the protease enzyme produced in step (a), said purification steps optionally comprising further addition of a non-protein, dissolved protease enzyme inhibitor having Ki values of less than 1X10-4. 4. The method according to any of Claims 1-3 wherein the non-protein, dissolved protease enzyme inhibitor is selected from the group consisting of boronic acid compounds, peptide aldehyde compounds, peptide boronic acid compounds, peptide trifluoromethylketone compounds, and mixtures thereof. 5. The method according to any of Claims 1-4 wherein the protease enzyme inhibitor has a Ki value of less than 1X10-5 M. 6. The method according to any of Claims 1-5 wherein the protease enzyme inhibitor is present in the fermentation step at a concentration of less than 0.1 mM. 7. The method according to any of Claims 1-6 wherein the protease enzyme inhibitor is present in the purification medium at a concentration of less than 0.1 mM prior to concentrating the protease enzyme. 8. The method according to any of Claims 1-7 wherein the protease enzyme inhibitor has a Ki value of less than 1X10" * *', and the molar ratio of inhibitor to protease enzyme is at least 1:1. 9. A protease enzyme in dry or concentrated liquid form prepared by the method according to any of Claims 1-8 and comprising one or more of the non-protein, dissolved protease enzyme inhibitors utilized in the manufacturing process. 10. A detergent composition comprising one or more protease enzyme according to Claim 9.

Inventors: BARNETT BOBBY LEE, VENEGAS MANUEL GARCIA, BARNETT, BOBBY, LEE, VENEGAS, MANUEL, GARCIA
Applicants: PROCTER & GAMBLE, THE PROCTER & GAMBLE COMPANY
Application year: 1995
Main classification: C12N9
IPC codes: C11D3, C12N9