Abstract: Alkaline proteases that are stable in the presence of linear alkylbenzene sulfonates can be obtained from bacteria isolated from soil and by other means. The soils are collected from an alkaline environment which has been exposed to extremely high pH and/or detergent contamination. The proteases are at least twice as stable as Esperase.TM. when stored in commercial liquid detergents.

Abstract: A functional food which comprises a glucosyltransferase and/or a fructosyltransferase each having a water-soluble polysaccharide production ability, and a base therefor.

Abstract: There are described, normally sporulating mutant Bacillus strain(s) which produce no detectable proteolytic activity during any phase of its growth. The absence of detectable proteolytic activity is due to the deletion of one or more codons specifying the mature subtilisin protease and the mature neutral protease. Also described are methods for producing such normally sporulating, protease deficient Bacillus mutants.

Abstract: The present invention provides a method for redesigning proteins to increase the stability of the protein by altering amino acid residue(s) that are in close proximity to the protein's metal ion binding site(s). Further, the invention is directed to proteins that have been redesigned to have enhanced stability according to the methods of this invention.

Abstract: A highly purified alkaline protease preparation is produced by separating a mixture containing amorphous form and crystalline form of alkaline protease from ultrafiltrate containing impurities, through a method which includes the incubation of an aqueous alkaline protease concentrate with sodium chloride and carbohydrate hydrolyse enzymes at an elevated temperature greater than 20.degree. C. and a pH between pH 4.0 and pH 6.5 at constant agitation.

Abstract: Alkaline protease which has leucine or isoleucine in the place of valine at the amino acid number 40 of wild type alkaline protease, a gene encoding the amino acid sequence of alkaline protease which has the substitution as described above, a recombinant DNA comprising said gene, a method of producing the above described alkaline protease, and a DNA fragment used for the expression of a gene.

Abstract: The mutants of neutral protease (NP) described can retain their enzymatic activities at temperatures at which wild-type neutral protease becomes inactive, the mutants being characterised in that the Gly 189 and/or Gly 147 residues of the aminoacid sequence of NP are replaced by different residues selected from the natural aminoacids.Mutagenised genes of neutral protease which code for the mutants, recombinant plasmids containing the genes, and strains of Bacillus transformed by the plasmids are also described. The mutants of neutral protease having the characteristics described above are particularly useful in the food sector.

Abstract: The invention relates to a process for the production of optically active (-)-oxabicyclo[3.3.0]octanolones of formula (-)-I ##STR1## wherein R is defined herein, comprising subjecting a racemic mixture of oxabicyclo[3.3.0]octanolone acylate to stereospecific acylate hydrolysis and separating the resultant products.

Abstract: The invention relates to subtilisin enzymes which have been modified by mutating a nucleotide sequence (gene) coding for the subtilisin. The modified subtilisin enzymes have enhanced thermal stability.

Abstract: The subject invention describes the cloning and overexpression of leader peptidase genes. A method for isolating a leader peptidase gene is disclosed. Overexpression of the signal peptidase in a suitable host species leads to an enhanced rate of protein processing.

Abstract: A novel method for ester hydrolysis catalyzed by enzymes having an oxyanion hole wherein the amide hydrolysis reaction is minimized. Enzymes are selected or alternatively derived by replacement of amino acid residues, which have minimal hydrogen bonding at or within 15 angstroms of the oxyanion hole.

Abstract: Novel methods and novel industrial unicellular microorganism strains, particularly industrial Bacillus strains, are provided for enhanced production of endogenous and exogenous polypeptides. Cloning vehicles containing the gene expressing the polypeptide of interest are introduced into a compatible host. Transformed hosts harboring the introduced vehicle in a stable way by integration of the vehicle into the host cells chromosome are selected Efficient transfer of the vehicle containing the gene of interest is achieved, with the resulting industrial strain transformants being effective, stable producers of the desired polypeptide product.

Abstract: Novel carbonyl hydrolase mutants derived from the DNA sequences of naturally-occurring or recombinant non-human carbonyl hydrolases are disclosed. The mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to generate the substitution of one or more amino acid residues in the amino acid sequence of a precursor carbonyl hydrolase. Such mutant carbonyl hydrolases have properties which are different from those of the precursor hydrolase and are especially useful in detergent formulations. The substituted amino acid residues correspond to position +123 and/or +274 in Bacillus amyloliquefaciens subtilisin.

Abstract: Novel carbonyl hydrolase mutants derived from the DNA sequences of naturally-occurring or recombinant non-human carbonyl hydrolases. The mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution of an amino acid in the amino acid sequence of a precursor carbonyl hydrolase. Such mutants have properties which are different than the precursor hydrolase.

Abstract: A substantially pure keratinaceous material-degrading B. licheniformis PWD-1 enzyme is disclosed. The substantially pure enzyme is characterized by a molecular weight of 33 kiloDaltons, an isoelectric point of 7.25, an optimum pH of 7.5, an optimum temperature of 45.degree.-50.degree. C., and thermal stability at low temperatures.

Abstract: There are described certain subtilisins wherein the amino acid sequence of such subtilisins has been modified at a position equivalent to +225 in Bacillus amyloliquefaciens, such that an amino acid selected from the group consisting of alanine, leucine, methionine, glutamine, valine, and serine, has been substituted for the amino acid residue naturally occurring at such position.

Abstract: A biologically degradable film is prepared consisting of a synthetic polymer and a biologically degradable polymer. The biologically degradable polymer is divided into small particles by means of enzymes produced by microbes in the form of spores, which enzymes split and release small molecules from the surface of the biopolymer particles. After achieving desired particle size, an emulsion is formed with vegetable oil and particles coated with enzyme protein become coated with vegetable oil, which at the same time interrupts the degradation of the biopolymer particles by the enzyme. The coated particles with the oil is separated from the suspension to remove small molecules after which the particles are redried and then pulverized. The final film is prepared in a film extruder in which the biopolymer is mixed with the synthetic polymer and possibly with other additives that are generally used in forming polymer films.

Abstract: The methods of the invention can be used to identify sites of cleavage of an enzyme in the presence of hypochlorite. It has been found that such cleavage occurs at tryptophan. Chemical modifications or genetic manipulation to change or delete tryptophan can be done to produce a more stable enzyme which retains activity in the presence of hypochlorite. The invention is particularly applicable to alkaline proteases which are useful in detergent compositions.

Abstract: Novel circular vectors containing a recplicable DNA sequence and DNA sequences encoding all or part of at least two distinct parental polypeptides are disclosed. Such vectors are used in novel processes utilizing in vivo recombination to produce recombined circular vectors containing said replicable DNA sequences and hybrid DNA sequences comprising: (1) a first DNA sequence encoding the amino-terminal portion of a hybrid polypeptide corresponding to a first part of a first parental polypeptide sequence and (2) a second DNA sequence encoding a carboxy-terminal portion of said hybrid polypeptide corresponding to a first part of a second parental polypeptide sequence. The hybrid DNA sequences of such recombined circular vectors can exprress novel hybrid polypeptides such as hybrid enzymes in general and in particular hybrid amylases and proteases. Various other processes are disclosed to isolate the recombined circular vector containing said hybrid DNA sequences.

Abstract: The use of T7 bacteriophage to produce DNA length standards by enzymatically joining terminally repetitious, blunt-ended DNA has now been demonstrated. It is now possible to precisely control the formation of concatemeric DNAs thereby generating custom-made size-ranges of length standards. Furthermore, the standards thus produced are stable over time providing a highly reproducible and convenient product for the molecular biologist.

Abstract: The invention relates to subtilisin enzymes which have been modified by mutating a nucleotide sequence (gene) coding for the subtilisin. The modified subtilisin enzymes have enhanced thermal stability.

Abstract: The invention relates to modified subtilisin enzymes which have increased thermal stability. The modified subtilisin enzymes have at least two or more amino acid mutations which confer increased thermal stability. It has been discovered that combining individual stabilizing mutations in subtilisin frequently results in an additive increase in thermal stability. In addition, the invention pertains to cloned mutant genes coding for a subtilisin material having at least two amino acid substitution which has increased thermal stability.

Abstract: Addition of alkaline proteolytic enzymes derived from Bacillis licheniformis in the anaerobic stage of bacterial digestion processes to improve the settling properties of the bacterial biomass.

Abstract: Cloned DNA is mutated by creating a single-stranded target region in a cloned DNA segment, and introducing a mutation into the single-stranded target region by treating the target region with a chemical or biological mutagenizing agent capable of introducing mutations into single-stranded DNA. The mutated target region then is rendered double-stranded and a microorganism is transformed with the mutated double-stranded DNA present in an expression vector. The transformed microorganism is cultivated under conditions wherein the mutated DNA is expressed to form an expression product, and the expression product is screened to identify a desired mutation in the DNA segment. Mutant subtilisins of enhanced thermal stability are also disclosed.

Abstract: Cheese is prepared using rennin obtained from insoluble refractile bodies of a recombinant microbial host cell. The rennin is obtained by rupturing the recombinant hose cell, isolating and solubilizing the insoluble refractile bodies, and recovering active rennin. Recombinant techniques involve preparing cDNA corresponding to the coding sequence for calf rennin, introducing into an expression vector and expressing in a host cell. As much as 200 mg rennin per liter of culture may be recovered. Prorennin or preprorennin may be produced and rennin derived therefrom.

Abstract: A method of degrading keratinaceous material is disclosed. The method comprises the steps of combining the keratinaceous material with Bacillus licheniformis to form a fermentation media and then fermenting the media for a time sufficient to degrade the material. The method can be used to produce amino acids from keratinaceous material and to produce a hydrolyzed feather product useful as a feed additive from the keratinaceous material.A preferred keratinaceous material for carrying out the present invention is feather, and a preferred bacteria for carrying out the invention is Bacillus licheniformis PWD-1.

Abstract: Rennin for making cheese is obtained from insoluble refractile bodies of a recombinant microbial host cell. The rennin is obtained by rupturing the recombinant host cell, isolating and solubilizing the insoluble refractile bodies, and recovering active rennin. Recombinant techniques involve preparing cDNA corresponding to the coding sequence for calf rennin, introducing into an expression vector and expressing in a host cell. As much as 200 mg rennin per liter of culture may be recovered. Prorennin or preprorennin may be produced and rennin derived therefrom.

Abstract: Combinations of alkaline Bacillus protease with alkaline fungal or actinomycete protease show improved detergency. Proteases obtainable from Fusarium sp. or Paecilomyces sp. (fungal) and Nocardiopsis sp. (actinomycete) are preferred.

Abstract: Exoenzymes, such as proteases, xylanases and amylases, are obtained continuously by cultivation of exoenzyme-producing microorganisms in one step in a fermenter which is operated with continuous flow and in which a deficiency state corresponding to maximal enzyme productivity is effected. Optical density of the culture (as a measure of the biomass density) and exoenzyme concentration in culture can be monitored to control the timing and extent of the deficiency state. It is particularly advantageous to impose an oxygen limitation and to maintain the deficiency state continuously by exerting an effect on the oxygen input.

Abstract: A class of subtilisin analogs suitable for admixture to cleaning compositions and having improved stability over naturally occurring Bacillus subtilisins are prepared by expressing a modified gene encoding the subtilisin analog in Bacillus subtilis. The subtilisin analogs are characterized as having a modified calcium binding site to improve calcium binding and either an Asn or a Gly replaced in any Asn-Gly sequences present in the subtilisin.

Abstract: In a method of continuously producing ethanol from sugar-containing substrates by fermentation of sugars by means of a flocculating strain of Zymomonas mobilis under anaerobic conditions and at a pH of from 4.5 to 7 a substrate is led commonly with Zymomonas mobilis cells through at least three fermentation stages without preceding sterilization, a concentration of at least 4% by volume of ethanol is maintained in each fermentation stage, a residence time of the fermentation medium in the entire system of maximally 31/3 h corresponding to a dilution rate of the fermentation medium in the entire system of at least 0.3 h.sup.-1 is adjusted, the Zymomonas mobilis cells are separated by sedimentation after the final fermentation stage, the Zymomonas mobilis cells are recycled into the first fermentation stage, and the ethanol-containing substrate separated from the Zymomonas mobilis cells is drawn off.

Abstract: A process for preparing pectin which comprises subjecting a plant tissue containing pectic substances to the action of a microorganism which belongs to the genus Bacillus and possesses an activity liberating pectin from a plant tissue but substantially does not possess an activity of decomposing pectin, or a culture broth or processed material thereof to liberate pectin from said plant tissue and recovering the pectin, which allows to obtain readily a pectin of high molecular weight in high yield.

Abstract: Novel microorganisms are provided which belong to the genus Bacillus and have ability of producing novel alkaline proteases. The alkaline proteases have excellent stability in highly alkaline conditions when blended with detergents, and improve detergency of the detergents. A process for the production of such alkaline proteases is also provided wherein the novel microorganisms are cultured.

Abstract: The invention relates to a fermentation process. This process is characterized by the fact that recourse is had, as a proteinic nutrient substance introduced into the fermentation medium, to an effective amount of potato protein obtained by coagulation from red waters.

Abstract: A cloned subtilisin gene has been modified at specific sites to cause amino acid substitutions at certain spots in the enzyme. The modified enzyme, preferably produced by Bacillus, is useful in combination with detergents.

Abstract: The invention relates to a process for the separation of biotechnologically produced valuable materials form a cell suspension by crossflow microfiltration to obtain an high specific permeate flux while retention stays near 0% for long periods. To enable crossflow microfiltration to be carried out on an industrial scale in biotechnology in the separation of biotechnologically produced extracellular valuable materials from a cell suspension, particularly in the separation of alkaline protease for recovering enzyme, under economically acceptacle conditions, alkaline protease of relatively high molecular weight, more especially enzyme >20,000 daltons, is separated from a fermenter solution using polysulfone tubes having micropore diameters of from 0.3 to 0.5 .mu.

Abstract: Contact lens cleaning compositions and a method which comprises treating soft contact lenses with general purpose proteases in combination with endoproteinase lys-C are effective in dissolving away and hydrolyzing lysozyme, the major protein component of tears.

Abstract: A process for the recovery of enzymes from an aqueous system having at least two aqueous phases wherein the partition coefficient with respect to an enzyme may be improved through the addition of organo-compatible, compounds having anionic functionality, such as polyacrylic acid, polyvinyl sulfonate and sodium dodecylsulfate.

Abstract: A method is provided for increasing the solubility of an enzyme in an aqueous solution comprising providing a water-soluble anionic surfactant to the solution and further providing the solution with a pH above that which result in the formation of an enzyme/surfactant precipitate. The application is further directed to an aqueous solution containing an anionic surfactant and having an enzyme concentration contained therein which is higher than that possible in the absence of the surfactant.

Abstract: Extracellular enzymes can be recovered from a whole fermentation beer by adding to the whole beer a mixture of a polymer and an inorganic salt. This total mixture produces an enzyme-rich polymer phase and a cell debris-containing, enzyme-poor salt phase which can be separated to produce an enzyme-rich product from the polymer phase.

Abstract: Certain novel aryl sulfonyl fluorides, their preparation, and their use in inhibiting serine proteases with chymotrypsin-like and elastase-like specificity.

Abstract: This invention contemplates a novel process for the preparation of solution stable alpha-amylase obtained from Bacillus licheniformis, and to the high potency liquid enzyme product prepared by this process. Typically, the enzyme-containing solution from a fermentation is concentrated and starch is added to the concentrate. Alternatively, a precipitation agent such as salt is added to the solution, and a cake containing the enzyme precipitates. The cake is then contacted with an aqueous solution containing starch to extract the enzyme out of the cake to provide a stable liquid enzyme product.

Abstract: This invention relates to a novel process for the recovery of enzymes obtained from enzyme-producing microorganisms, and to the liquid enzyme product recovered by this process. Typically, the enzyme-containing filtrate from a fermentation of an enzyme-secreting microorganism is concentrated and a precipitation agent such as a salt or an organic solvent is added to the concentrate, thereby forming a cake. Then, a polyol solvent is circulated through the cake to solubilize the enzyme or enzyme complex from the cake and provide a liquid enzyme product. Particularly effective is propylene glycol as the polyol solvent. The liquid enzyme product may be shipped as is or subjected to further treatment to remove the solvent and create an essentially solvent-free enzyme product. The process is especially effective for the recovery of alkaline protease or alpa amylase.

Abstract: This invention relates to a novel process for the recovery of enzyme crystals. The enzymes may be obtained from any enzyme-producing microorganisms such as bacteria, fungi, and yeasts. The invention contemplates supersaturation and/or crystallization to obtain enzymes in the crystalline form, and is particularly effective for the recovery of heat stable alpha-amylase in a crystal form.

Abstract: Functional protein having reduced nucleic acid content is produced without initial denaturation of the protein by contacting undenatured yeast cells with an alkaline protease at a temperature of about 20.degree. C. to 40.degree. C. for about 2 minutes to 2 hours at a pH of about 8 to 11. The yeast cells are preferably Pichia pastoris and the alkaline protease is preferably from Bacillus lichenformis.

Abstract: A storage stable liquid enzyme concentrate of Subtilisin Carlsberg containing 0.5-6.5 Anson Units of proteinase per gram of concentrate and method for preparing same. Solid form proteinase is extracted with 70-100 parts by volume propylene glycol, 30-0 parts by volume of water, then adjusted as necessary to 60-85% by wt. of the glycol. Stabilizing agents in the concentrate are 0.1-1 mol/Kg of a member selected from the group consisting of Na, K, and Ca glutamates, and glycinates, and acetamide, and also a calcium ion content of 0.04-0.5% by wt., pH range is 5-8.

Abstract: A stable, cooperative enzyme system which is stable under use conditions is disclosed which comprises at least two enzymes having activity towards a relatively complex substrate with at least partial activity over the same pH range, wherein their combined activities are greater than the sum of their individual activities as determined by the formula: ##EQU1## wherein E.sub.1 and E.sub.2 are said enzymes. No additional chemical stabilizers, modifiers or activators are added to the enzymes of this invention.Particularly preferred enzymes in this invention are proteases having optimal activity in acidic, neutral or alkaline media, and mixtures of the same.A method of making this cooperative enzyme system is also disclosed.The enzyme systems of this invention have a wide variety of uses in cleaning and other applications.

Abstract: Alkaline protease API-21 having the following physicochemical properties:(1) Activity: Hydrolyzes casein under alkaline conditions, optimum pH range is 10 to 11, and has optimum temperature range for activity of 45.degree. C. to 50.degree. C.;(2) Stability: No decrease in enzymatic activity observed up to 40.degree. C. upon heating without substrated for 10 minutes at a pH of 10, but 90% or more decrease of enzymatic activity observed at 50.degree. C. for 10 minutes at a pH of 10. Deactivation occurs at 50.degree. C. for 10 minutes at a pH of 8 and complete deactivation substantially occurs at 60.degree. C. for 10 minutes at a pH of 8. The protease is stable under alkaline conditions, especially within a pH range of 7 to 11.5, and is stable against freezing and freeze-drying; and(3) Enzyme molecule: The protease is protein having a molecular weight of about 22000 as estimated by a gel filtration method, an isoelectric point of 7.

Abstract: Bacillus serine proteases are acylated with an acyl radical of a monocarboxylic or dicarboxylic acid of about 1 to 6 carbon atoms to thermally destabilize the proteases at least about 3.degree. C. The acylated proteases have at least about 50% of their activity before acylation, and are advantageous in processes where it is desired to inactivate the proteases at a certain point in the process.

Abstract: A novel protease product suitable for admixture to washing compositions and exhibiting substantially attenuated allergenic properties is prepared by cultivating strains of Bacillus licheniformis which have been mutated to block their synthesis of protease other than Subtilopeptidase A (subtilisin). The commercial Bacillus licheniformis derived protease products are mixtures of subtilisin and a non-serine protease of lower stability and greater allergenicity than subtilisin.