92 results about "Keratinase" patented technology

The invention discloses keratinase Phyto-Keratinase and application of the keratinase Phyto-Keratinase. The keratinase Phyto-Keratinase comprises the following components: (1) protease which is coded by a nucleotide sequence as shown in SEQ ID NO.1; and / or (2) protease which is derived from bacillus subtilis, is coded by a nucleic acid sequence with more than 98% of identity with the SEQ ID NO.1 nucleotide sequence and has the characteristic of hydrolyzing keratin. According to the plant keratinase Phyto-Keratinase disclosed by the invention, the Phyto-Keratinase (plant keratinase) produced by utilizing microbial fermentation is a serine proteolytic enzyme, is also a biocompatible cutinase, and is obtained by fermenting saccharomyces cerevisiae. Phyto-Keratinase provides a nitrogen source for the growth of plants (beans), and contains various amino acids (functional peptides) and special proteolytic enzymes derived from the plants at the same time. The Phyto-keratinase can provide the effects of resisting inflammation, removing cutin and providing nutrition for skin regeneration at the same time, can relieve various inflammations by inhibiting PAR-2 receptors, and has potential value in the fields of cosmetics, beauty medicine and environment.

The invention relates to an enzyme and bacterium combined feed additive for pre-digestion of feed raw materials as well as preparation and application of the enzyme and bacterium combined feed additive. The feed additive comprises: (a) an enzyme preparation, which is composed of protease, cellulase, xylanase, mannase, phytase, pectinase and keratinase, and has a total enzyme activity of 42500-45000 U / g; the probiotics are composed of bacillus subtilis, lactobacillus plantarum and saccharomyces cerevisiae, and the viable count is larger than or equal to 107 CFU / g; and (c) the mass ratio of enzyme to bacteria is (1-2): 1. According to the additive, the enzyme preparation and the probiotics are jointly used, and the raw materials are effectively pretreated, so that the nutritional value and the digestibility of the feed are improved. The feed additive disclosed by the invention comprises a specific enzyme preparation and probiotic combination, and predigestion treatment on the feed raw materials is realized through an optimized preparation process and fermentation process, so that the nutritional value and the utilization value of the feed raw materials are improved. The additive is simple in preparation method, low in cost and environment-friendly, and has a wide application prospect.

The invention discloses an eggshell membrane peptide for enhancing bone mineral density as well as a preparation method and application thereof, belongs to the technical field of egg by-product processing, and particularly relates to a preparation method of the eggshell membrane peptide for enhancing bone mineral density, which comprises the following steps: carrying out vacuum freeze drying and crushing on an eggshell membrane to obtain eggshell membrane powder; mixing the eggshell membrane powder with deionized water to obtain an eggshell membrane solution; adding keratinase, performing enzymolysis, and taking supernate; adding collagenase, carrying out enzymolysis, and centrifuging to take supernate, so as to obtain primary enzymatic hydrolysate; adding alkaline phosphatase, performing enzymolysis and centrifugation, and taking supernate to obtain enzymatic hydrolysate; and performing ultrafiltration and drying to obtain the eggshell membrane peptide. According to the eggshell membrane peptide for enhancing the bone mineral density, keratin cross-linking is destroyed through enzymolysis of keratinase, active peptide capable of stimulating osteoblast proliferation is released through enzymolysis of collagenase, phosphopeptide calcium binding sites are further activated through enzymolysis of alkaline phosphatase, calcium salt deposition is promoted, and the eggshell membrane peptide for enhancing the bone mineral density is provided.

Engineered, modified, or mutated Bacillus strains that produce keratinases or serine proteases (KerS) that biologically degrade keratin-containing materials, such as feathers; compositions containing these Bacillus strains or the keratinases they produce, and methods for biologically degrading keratin-containing materials using these strains or keratinases.

The present invention relates to the technical field of bacterial enzyme fermentation, and provides a bacterial enzyme synergistic fermentation method for removing antinutritional factors in soybean meal, the method comprises the following steps: S1, soybean meal pretreatment: soybean meal, water and an auxiliary agent are mixed and then subjected to steam explosion treatment, and the auxiliary agent comprises citric acid, a hydrogen peroxide solution and polyphenol; s2, bacterial enzyme fermentation: mixing the pretreated soybean meal with molasses and water to obtain a raw material, adding a complex microbial inoculant and a complex enzyme, uniformly stirring, and fermenting; the complex microbial inoculants comprise saccharomycetes, bacillus subtilis, lactobacillus rhamnosus and pediococcus pentosaceus; the compound enzyme comprises acid protease, pectinase, phytase, glucanase, alpha-galactosidase and keratinase; and S3, after fermentation is finished, drying the materials to obtain the fermented soybean meal. According to the fermentation method, the glycinin degradation rate can reach 79% or above, the acid soluble protein content is increased to 26% or above, the anti-nutritional factor content is remarkably reduced, and the nutritional value is greatly increased.

The invention provides a pet hair decomposer with synergistic effect of bacillus subtilis and keratinase, the pet hair decomposer comprises bacillus subtilis ZYpet-015 and keratinase, the preservation number of the bacillus subtilis ZYpet-015 is CGMCC (China General Microbiological Culture Collection Center) No.34790, and the preservation date is June 6, 2025; the protease activity of the bacillus subtilis is greater than or equal to 129.92 U, the survival rate of the bacillus subtilis in simulated gastric juice within 3 hours is greater than 50%, and the tolerance rate of the bacillus subtilis in intestinal juice within 4 hours is greater than 90%. According to the pet hair decomposing agent, hair keratin is synergistically degraded through protease secreted by the bacillus subtilis ZYpet-015 and keratinase, and the hair decomposing efficiency reaches 28.07% + / -2.79% and is remarkably higher than that of a single treatment group. Meanwhile, experiments prove that the pet hair decomposer can effectively destroy the hair cuticle structure, is suitable for a pet hair-melting nutritional supplement and can relieve the hair ball symptom.

The invention discloses freeze-dried powder for promoting alimentary canal hair discharge of pet cats and a preparation method thereof.The freeze-dried powder is prepared from, by mass, 80%-85% of chicken breast, 8%-10% of cellulose, 4%-6% of whole goat milk powder, 2.5%-3.5% of barley seedlings, 0.25%-0.35% of keratinase, 0.08%-0.12% of vitamin E and 0.08%-0.12% of rosemary extract.The freeze-dried powder not only has good palatability but also is good in hair discharge effect; in addition, diarrhea or constipation cannot be caused.

The invention belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to a preparation method of an acid-resistant high-activity keratinase preparation and application of the acid-resistant high-activity keratinase preparation in feather meal fermented feed. According to the invention, acid resistance design is carried out based on cotG-KERQ7, and the acid stability and activity of the keratinase mutant are both improved through a combined mutation strategy of three rounds of base catalytic residue replacement in a catalytic center, surface unstable positive charge amino acid replacement and flexible region introduction. By making the zymoprotein surface of serine keratinase more suitable for an acidic environment, the volume of a substrate binding cavity of protease in the acidic environment is increased, and the substrate can be better accepted and catalyzed, so that the acid stability and activity are effectively improved; finally, the acid-resistant and high-activity keratinase preparation B.subtilis cotG-KERQ7-M2 is obtained, and reference is provided for subsequent industrialized and fermented feed preparation for green conversion of feather waste into acid-soluble protein.

The present invention discloses a keratinase variant that improves hydrolysis ability by fusing a substrate binding domain, and belongs to the field of genetic engineering and enzyme engineering technology. The present invention uses PCR and Gibson connection technology to successfully fuse and express a substrate binding domain from Trichoderma reesei at the C-terminus of keratinase KerZ1 through a connecting peptide to obtain a keratinase variant KerZ1 / CBM‑L8. The enzyme activity assay results show that the enzyme activity of the keratinase variant KerZ1 / CBM‑L8 is only 41.5% of that of KerZ1, but the feather hydrolysis experiment results show that the average enzyme activity of the keratinase variant KerZ1 / CBM‑L8 is 2.71 times that of KerZ1, which has more application value and potential.

The invention belongs to the technical field of polyester surface modification, and relates to a method for promoting cutinase to hydrolyze polyester by using triton. The method comprises the following steps: adding triton into a Tris-HCl buffer solution, uniformly mixing, and adjusting the pH value with hydrochloric acid to obtain a triton buffer solution; and immersing the refined terylene into a triton buffer solution, adding a cutinase preparation for hydrolysis reaction, inactivating cutinase, taking out the terylene, washing and drying. Triton is used as an additive to improve the activity and stability of cutinase, promote the hydrolysis effect of cutinase on polyester, and increase the hydrolysis release product by 229.8%, so that cutinase can hydrolyze polyester with higher crystallinity.

The invention discloses candida utilis engineering bacteria and a method for producing single-cell protein by fermenting corn thin pulp, and belongs to the technical field of biology. According to the candida utilis engineering bacteria disclosed by the invention, the candida utilis is subjected to gene modification, a chitin synthase gene in the candida utilis is knocked out, and an exogenous keratinase gene is integrated, so that the candida utilis engineering bacteria can utilize corn thin pulp as a substrate for fermentation. In the fermentation process, carbon and nitrogen sources in the thin corn pulp can be effectively utilized for growth, macromolecular protein in the thin corn pulp can be hydrolyzed and converted into single-cell protein, the content of the single-cell protein in final fermentation liquor is increased, the protein content of the final fermentation liquor can reach 60% or above, and the proportion of the single-cell protein can reach 30% or above; the corn thin pulp can be efficiently utilized, and meanwhile, single-cell protein in the fermentation liquor can be used for adding animal feed so as to meet the requirement for animal protein feed in the market.

The present invention relates to a pharmaceutical composition for treating peptic gastric ulcer and a preparation method thereof, belonging to the field of biotechnology. The pharmaceutical composition contains chicken gizzard-membrane peptide, pumpkin seed extract, complex bacteria, white hyacinth bean flower extract and bletilla striata extract; the chicken gizzard-membrane peptide contains the products obtained by successively subjecting chicken gizzard-membrane to fractional enzymolysis with pepsin, chymotrypsin and keratinase; the pumpkin seed extract contains the products obtained by subjecting pumpkin seeds to co-enzymolysis with cellulase and pectinase and then successively to fractional enzymolysis with pepsin, nattokinase and aminopeptidase; the complex bacteria contain the co-fermentation bacteria of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus casei; the white hyacinth bean flower extract is an ethanol percolation extract; the bletilla striata extract contains the products obtained by subjecting bletilla striata tubers to co-enzymolysis with cellulase and pectinase and then successively to fractional enzymolysis with pepsin, bromelain and serrapeptase. The various components are used in combination, having a good therapeutic effect on gastric ulcer, quickly and efficiently repairing gastric mucosa, and being safe and free of side effects.

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of tumor infiltrating lymphocytes. Keratinase modified by myristoyl coenzyme A cooperates with fibronectin and elastase, so that tumor cells quickly fall off from tumor blocks, and loose tissues are subjected to low-temperature enzymolysis by protease-K, so that the phenotype and activity of tumor infiltrating lymphocytes can be protected; an eicosapentaenoic acid solution is mixed with polysorbate, ultrapure water and a culture medium matrix to obtain an emulsion mixed solution, the emulsion mixed solution is subjected to rotary evaporation to remove a solvent, and the amplification-promoting nanoparticles obtained after freeze drying can make tumor infiltrating lymphocytes ingest effective components more quickly; the tumor infiltrating lymphocytes extracted by the invention are high in activity and rapid in proliferation and growth, still have high cell activity after cryopreservation, and have good anti-breast tumor activity.

This invention discloses an acidic serine keratinase gene and its applications, belonging to the field of genetic engineering technology. This invention screened a novel serine keratinase from the acidic hot spring environment of Niujie, Yunnan. This keratinase naturally possesses characteristics of acid resistance, good thermal stability, and high enzyme activity, allowing it to adapt well to the acidic microenvironment of the skin and temperature fluctuations throughout the product's production, storage, and transportation processes. Furthermore, it eliminates the need for complex stabilization treatments, simplifying the production process, reducing production costs, and extending the product's shelf life. This achieves green and efficient preparation, fundamentally solving key problems of existing keratinases in skin applications, such as irritation, poor stability, and difficulty in storage. It is particularly suitable for the gentle exfoliation of human keratin plugs, demonstrating outstanding application value and broad prospects in the daily chemical skincare field.

The present invention provides a feed additive containing a keratinase mutant and bile acid and its application. The present invention uses a saturation mutation method to Bacillus licheniformis The keratinase gene of the source is mutated to obtain keratinase mutants KS01, KS02, KS03, and KS04. Compared with the original keratinase, the optimal reaction temperature of the mutants is reduced from 55°C to 38°C, 45°C, 46°C, and 50°C, respectively, and the residual enzyme activity of the mutant KS01 is above 65% under the reaction conditions of 25°C to 35°C. The present invention also uses the enzyme preparation prepared by fermentation, filtration and drying of the engineered bacteria containing the mutants and compounding with bile acid, which not only has a good effect of enzymolysis of soybean meal antigen protein, but also can be used in poultry and livestock breeding, can obviously play an anti-diarrhea role, and protect the gastrointestinal tract of animals, and has good industrial value and market application prospects.

A method for decomposition of keratin material, e. g. mixture of feathers, is disclosed. The method comprises treating the keratin material with Bacillus cereus KB043 strain, subsequently with sodium hydroxide and potassium hydroxide, and finally with neutralizing acid. The end product is intended for use as an amino acid fertilizer.

The invention discloses preparation and application of bacillus licheniformis for decomposing pigeon feathers, and relates to the field of environmental resource application in the breeding industry, and the preparation method comprises the following steps: step 1, strain activation: firstly resuscitating a freeze-dried strain, and then culturing and activating by adopting a gradient activation mode; 2, preparing a seed solution, namely culturing the strain activated in the step 1 in a seed culture medium to obtain the seed solution; according to the method, the components of the pre-activation culture medium comprise 1% of corn flour, 0.5% of soybean meal and 0.1% of dipotassium phosphate, the production cost is low, and the preparation feasibility and economic benefits of the bacillus licheniformis are improved; the feather decomposition efficiency is improved by 2-3 times through substrate pretreatment, the residual feather amount is reduced, meanwhile, the fermentation period is shortened to 40-50 hours, and the feather decomposition efficiency of the pigeons is improved; in the fermentation tank, the activity of the keratinase can reach 600U / mL, so that the decomposition efficiency can be improved, subsequent reuse is facilitated, and the utilization rate is increased.

The invention relates to the technical field of parasite prevention and control, and provides a probiotic fungus capable of promoting parasite egg death and a preparation method thereof, the strain is recombinant aspergillus niger, and an exogenous chitinase gene (SEQ ID NO: 1) and a keratinase gene (SEQ ID NO: 2) are integrated in a genome of the strain. Aspergillus niger is transformed through genetic engineering, multi-enzyme synergistic degradation of the horny cortex of the worm eggs of the echinococcosis is achieved, the survival rate of the worm eggs is remarkably reduced, meanwhile, environmental safety and long-acting prevention and control capacity are both considered, and an innovative solution is provided for comprehensive prevention and control of echinococcosis.

The invention belongs to the technical field of pet food and biological medicine, and discloses a composite probiotic composition with a hair ball biodegradation effect and application thereof. The composition comprises Witzia ciliaris, Bacillus subtilis and a camellia seed extract. According to the present invention, the camellia seed extract with the specific source is adopted as the biological surfactant, such that the oil seal sebum layer on the pet hair surface is emulsified and cracked, the hair surface tension is significantly reduced, the keratinase secreted by the bacillus subtilis is induced to efficiently wet and adsorb on the hair fiber so as to achieve the deep biodegradation of the keratin; meanwhile, the lactic acid generated by the Witzia clostridium is combined to promote intestinal peristalsis, and the decomposition and excretion of hair balls are synergistically realized. Experimental results show that the dry weight loss rate of the composition to cat hair reaches 65-78%, the vomiting frequency can be effectively reduced, and the composition has excellent acid-resistant stability, palatability and safety.

The invention discloses heat-resistant keratinase, the amino acid sequence of the heat-resistant keratinase is shown as SEQ ID NO: 1, the gene is obtained from Brevibacillus gelatini LD5, heterologous expression, purification and characterization are carried out on the keratinase through a genetic engineering means, experimental data show that the keratinase has keratinase activity at 30-90 DEG C, the reaction temperature of the keratinase is as high as 90 DEG C, and the keratinase can be used for preparing the heat-resistant keratinase. High thermal stability is realized at 60 DEG C; after the keratinase BrgM4 is used for carrying out enzymolysis on chicken feathers for 48 hours at the temperature of 50 DEG C and the pH value of 6.5, the soluble keratin content reaches 8.87 mg / mL; the keratinase disclosed by the invention is beneficial to degradation of keratin waste at high temperature.

The invention provides a mild multi-effect anti-acne enzyme composition for teenagers and application of the mild multi-effect anti-acne enzyme composition. The composition comprises a fig extract, a roselle extract and keratinase. The composition provided by the invention is safe and non-irritant in components, the fig extract and the keratinase play a cutin renewal role of biological enzyme, and the roselle extract fundamentally promotes the generation of skin renewal related enzymes and assists in accelerating the decomposition of keratosuppository. The three components are synergistically compatible, pores are dredged, the skin problems such as teenager closed acne and blackhead are solved, and efficient and mild acne resistance is achieved.

The invention discloses skin mucosa maintenance liquid capable of killing fungi and HPV (human papillomavirus) as well as a preparation method and application of the skin mucosa maintenance liquid, and belongs to the technical field of sterilization and virus removal. The skin mucosa maintenance liquid is prepared from octanol ether, benzalkonium bromide, phenoxyethanol, yermate, parachlorometaxylenol, taetinic acid, chitosan, keratinase, fluconazole, glycerol, borneol, menthol, absolute ethyl alcohol and tween-40. Wherein the skin mucosa maintenance liquid has an effect of regulating apoptosis induction on abnormal cells and an effect on vaginal flora, and has an obvious inhibition effect on cervicitis and colpitis mycotica, namely an anti-inflammatory effect; meanwhile, apoptosis of cells containing HPV16 and HPV18 genes can be induced, shedding of abnormal cells is promoted, diseased cells are killed, and vaginal tissues with pathological changes are removed.

The application provides a method for promoting degradation of waste feather by feather powder. The method is characterized in that feather powder is added to an inorganic salt culture medium containing waste feather as the only carbon and nitrogen source, and then microorganisms with the function of degrading keratin are inoculated to degrade the waste feather. The method has the advantages of short degradation time of waste feather, significantly improved degradation rate and keratinase activity, low cost and easy operation. The application can accelerate the green transformation and high-value application of waste feather, and can screen and enrich germplasm resources for degrading keratin, thereby helping to realize the green and circular high-quality development of the livestock and poultry breeding industry.

The invention relates to the technical field of cosmetics, in particular to a mark fading and repairing composition as well as a preparation method and application thereof. The invention relates to a mark fading and repairing composition which comprises the following components in percentage by weight: 0.1%-5% of dragon's blood extract, 0.01%-0.5% of keratinase and 1%-5% of nicotinamide, the preparation method comprises the following steps: preparing the eutectic solvent; performing ultrasonic-assisted extraction; separation and post-treatment; the invention has the following beneficial effects: the resina draconis extract can inhibit continuous pigmentation caused by acne inflammation residue, promote healing of skin minimal wounds and reduce dermis layer injury of acne mark parts; keratinase specifically hydrolyzes stratum corneum aged keratin, cutin metabolism of acne mark parts is accelerated, and accumulation of pigments in stratum corneum is avoided; nicotinamide inhibits melanocytes from transferring melanin to keratinocytes; through the synergistic effect of the three components, a closed-loop printing fading mechanism of anti-inflammatory repair-keratin metabolism-pigment inhibition is formed, the printing fading effect is more comprehensive and more efficient, and skin repair and barrier protection can be both considered.

The application discloses a method for hydrophilic modification of polyester by DES ultrasonic pretreatment combined with enzymes, and belongs to the technical field of surface modification. The polyester hydrophilic modification method is specifically as follows: after refining, polyester fabric is put into a DES solvent for ultrasonic treatment, and after the treatment, washing and drying are performed; the dried polyester fabric is put into a 5mmol / L Tris-Hcl buffer solution with pH 8, and Hic keratinase enzyme liquid is added for hydrolysis reaction; after the reaction, the reaction residue is inactivated, and the fabric is washed and dried; the hydrogen bond donor of the DES solvent is an alcohol or an amide monomer, and the hydrogen bond acceptor is one or two of choline chloride, betaine and dihydrocholine citrate; compared with a traditional chemical modification method, the method is environmentally friendly, has no pollution, has a small influence on mechanical properties, and is mild and efficient in modification effect.

The invention belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to a preparation method and application of a keratinase mutant for efficiently degrading feather feed in an acidic gastric juice environment. The method comprises the following steps: replacing amino acid Arg247 with positive charges on the zymoprotein surface based on cotG-KERQ7 in an environment with the pH value of 4, then carrying out rational design on the chemical property of a KERQ7 oxygen negative ion cave, and directly introducing acidic amino acid, so that a mechanism that the serine protease oxygen negative ion cave actively dynamically adjusts the charge density through pH-dependent protonation is introduced, and the pH-dependent protonation of the serine protease oxygen negative ion cave is changed into the pH-dependent protonation of the serine protease oxygen negative ion cave. According to the method, the keratinase is used as a raw material, the oxygen anion hole adaptive low-pH environment mutant is obtained, the acid stability and activity of the keratinase mutant are both improved, an efficient mutant enzyme preparation with activity and acid resistance remarkably improved in the low-pH environment is finally obtained, reference is provided for subsequent improvement of the application value of the keratinase in the feed industry, and the method has a wide application prospect. Wide market application prospects are realized.

The present application relates to a kind of multifunctional pharmaceutical composition of dental comprehensive repair and its preparation method, belong to biopharmaceutical technical field, and pharmaceutical composition includes keratin peptide, coral fungus extract, eggshell derived nano calcium phosphate, catechin zinc complex, hydroxyapatite, modified chitosan-PEG gel and other components.Keratin peptide is the product between 3kDa-10kDa obtained by keratinase enzymolysis to white goose velvet;Coral fungus extract is the product obtained by cellulase, subtilisin and bromelain enzymolysis to coral fungus, and purified by G-25 dextran gel column;Modified chitosan-PEG gel is the product obtained by sodium cyanoborohydride stabilization and glycerol plasticization to the reactant of chitosan and dialdehyde polyethylene glycol.Integrated multiple functions such as antibacterial, mineralization and tissue repair, through optimizing component and dosage form, realize efficient, minimally invasive dental damage repair, and good biocompatibility and long-acting.

The invention belongs to the technical field of microorganism application, and particularly relates to bacillus pacificus YM01 capable of degrading feathers and application of the bacillus pacificus YM01. The bacillus pacificus YM01 is preserved in the China Center for Type Culture Collection on June 23, 2025, and the preservation number of the bacillus pacificus YM01 is CCTCC (China Center for Type Culture Collection) M 20251388. The strain can rapidly grow by taking feather as a unique carbon and nitrogen source and degrade and convert the feather into small molecular compounds such as peptide and amino acid, a culture method of the bacillus pacificus YM01 is optimized, conditions for degrading the feather by fermentation liquor are studied, keratinase BPK1 and chitinase BPC1 are screened out, and the application prospect is wide. The invention further establishes a method for degrading feathers by using the strain and fermentation broth thereof, namely extracellular crude enzyme, and provides a feasible technology for biotransformation and utilization of feather resources.

This invention discloses a BhrPETase keratinase mutant and its application in the degradation of polyethylene terephthalate (PET), belonging to the fields of environmental science and enzyme engineering. The site numbers are based on the amino acid sequence shown in SEQ ID NO:1. The mutant contains substitutions of S14G, H78Y, and S184G relative to SEQ ID NO:1, with the preferred amino acid sequence shown in SEQ ID NO:9. Under conditions of 1 mg enzyme protein / g substrate, 65°C, and 96 h, the preferred mutant exhibits a degradation rate of 90.4% for PET membranes and a half-life of 150 h at 60°C, making it suitable for enzymatic degradation and depolymerization of PET.

The application belongs to the technical field of genetic engineering. The application provides application of a thiol-oxidoreductase in promoting keratin enzymolysis and a method for promoting keratin enzymolysis. The thiol-oxidoreductase is applied to a keratin enzymolysis system, and the enzymolysis effect is obviously improved. Through testing, the enzymolysis effect of the thiol-oxidoreductase combined with a protease on keratin is not only obviously higher than the enzymolysis effect of a single protease, but also is better than the hydrolysis of keratin by a chemical reducing agent with the same concentration. The application retains the advantages of enzyme method "green chemistry" and is friendly to the environment.