WO2020088393A1 - 一种生产具有蛋白酶抗性的洗涤用酶的方法 - Google Patents

一种生产具有蛋白酶抗性的洗涤用酶的方法 Download PDF

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WO2020088393A1
WO2020088393A1 PCT/CN2019/113627 CN2019113627W WO2020088393A1 WO 2020088393 A1 WO2020088393 A1 WO 2020088393A1 CN 2019113627 W CN2019113627 W CN 2019113627W WO 2020088393 A1 WO2020088393 A1 WO 2020088393A1
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protease
cellulase
enzyme
washing
peptide
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PCT/CN2019/113627
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French (fr)
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张青
陈志兵
刘艳萍
田延军
许韡
管轶男
黄亦钧
吕家华
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青岛蔚蓝生物集团有限公司
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Priority to CN201980029782.2A priority Critical patent/CN112368388B/zh
Priority to US17/288,530 priority patent/US12018262B2/en
Priority to EP19877863.1A priority patent/EP3875591A4/en
Publication of WO2020088393A1 publication Critical patent/WO2020088393A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • the present invention relates to the technical field of genetic engineering, and in particular to a method for producing a protease-resistant washing enzyme.
  • the protease-resistant washing enzyme is obtained by fusing and expressing the protease-inhibiting peptide gene.
  • Bio enzymes are one of the major technological advances in the development of the synthetic detergent industry as a detergent aid. It promotes the sustainable development of the detergent industry; reducing the amount of surfactant and sodium tripolyphosphate in the detergent helps For the development of low-phosphorus or non-phosphorus detergents; it can improve the washing performance, but it is non-toxic and can be completely biodegraded, which can reduce the emission of pollutants; lower the washing temperature and reduce the number of rinsing, which is conducive to energy saving and water saving.
  • the biological enzyme itself is easy to degrade and the complexity of detergent formulation technology, such as surfactant, bleach, protease and other formulation components and the influence of pH, temperature and other formulation conditions, the stability of the enzyme has become the enzyme-added detergent formulation Outstanding issue.
  • Enzyme preparation companies and detergent developers use protein engineering technology, chemical modification technology, stabilizer addition and microencapsulation technology to improve the stability of biological enzymes in detergents and improve their effectiveness.
  • protease inhibitors added to detergents is much lower than that of borate protease inhibitors, as a compound containing aryl groups, its long-term use will cause certain adverse effects on the natural environment and animal health. Therefore, those skilled in the art are actively looking for environmentally friendly, non-toxic side effects of protease inhibitors or developing new methods to improve enzyme stability.
  • the present invention provides a method for producing a protease-resistant washing enzyme.
  • the method obtains the enzyme for washing which is resistant to protease by fusing and expressing the gene of the enzyme for washing and the gene of protease inhibitory peptide, which is beneficial to maintaining the stability of various enzyme components in the enzyme-added detergent and improving washing The effect of the agent.
  • the present invention provides a method for producing a protease-resistant washing enzyme, which includes the following steps:
  • the enzyme for washing and the protease inhibitory peptide are fused in a covalent manner.
  • the washing enzymes are cellulase, lipase, cutinase, amylase, carbohydrase, pectinase, mannanase, arabinase, galactanase, xylanase, laccase or Any one of peroxidases.
  • the enzyme for washing is preferably cellulase.
  • the enzyme for washing is preferably lipase.
  • protease inhibitory peptides are protease inhibitory peptides SSI derived from Streptomyces sp., Bowman-Birk protease inhibitory peptides, protease inhibitory peptides PCL derived from Barley, and protease inhibitors derived from Alteromonas Either one or two or three of the protein leader peptides derived from the peptide MST or Bacillus sp.
  • the host cells are Bacillus sp., Aspergillus sp., Trichoderma sp., Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli.
  • the host cell is preferably Trichoderma sp., And further preferably Trichoderma reesei.
  • the protease inhibitory peptide in the fusion protein may be fused at the N-terminus, C-terminus, or intermediate fusion of the enzyme for washing.
  • the present invention also provides a protease-resistant washing enzyme produced by the above method.
  • the invention also provides the application of the protease-resistant detergent enzyme in detergent.
  • the invention can significantly improve the resistance of protease by fusion expression of cellulase or lipase and protease inhibitory peptide, and effectively reduce the degradation effect of protease on cellulase or lipase.
  • the residual enzyme activity of cellulase SCD45 was 91.4% after 12 hours at room temperature, and the residual enzyme activity of cellulase SCD45-protease inhibitor peptide fusion protein was up to 99.7%; after 24 hours at 40 °C
  • the residual enzyme activity of cellulase SCD45 is only 8.1%, while the residual enzyme activity of cellulase SCD45-protease inhibitor peptide fusion protein is as high as 74.0%, which is a very significant improvement.
  • the cellulase with protease resistance obtained by the invention can be widely used in detergents, and can effectively improve the stability of enzymes in detergents. After being left at 37 ° C for 24h, the residual enzyme activity of the cellulase in the enzyme-added laundry detergent with cellulase SCD45 was 12.1%, while the cellulase in the enzyme-added laundry detergent with cellulase SCD45-protease inhibited peptide fusion protein.
  • Residual enzyme activity is as high as 59.5%; after being left at 37 ° C for 48h, the residual enzyme activity of the cellulase in the enzyme-added laundry liquid with the addition of cellulase SCD45 is 2.1%, while the residual cellulase in the enzyme-added laundry liquid with the addition of fusion protein The enzyme activity is as high as 28.9%; after being left at 37 ° C for 72h, the residual enzyme activity of the cellulase in the enzyme-added laundry detergent with cellulase SCD45 is 0.9%, while the residual enzyme of the cellulase in the enzyme-added laundry detergent with fusion protein added The activity is as high as 14.2%, which has been significantly improved and achieved unexpected technical effects.
  • Cellulase HGD45 and cellulase NT45 are also significantly increased in their protease resistance by fusion expression with protease inhibitory peptides.
  • the lipase TG-protease inhibitor peptide fusion protein was significantly improved after being placed at 37 ° C for 2d, 5d and 6d. After 6d, the residual enzyme activity of the fusion protein was as high as 64.6%, which was higher than fat TG increased by 86.7%, the effect is extremely significant.
  • the method of the present invention can also be widely applied to washing enzymes, such as cutinases, amylases, pectinases, mannanases, arabinase, galactanase, wood Glycanase, laccase, peroxidase, etc., can significantly improve the protease resistance of the enzyme, effectively reduce the degradation effect of the protease on the enzyme, and facilitate the stable coexistence of the enzyme and the protease in the detergent.
  • washing enzymes such as cutinases, amylases, pectinases, mannanases, arabinase, galactanase, wood Glycanase, laccase, peroxidase, etc.
  • Figure 1 is an SDS-PAGE electrophoresis detection diagram; among them, lane M is Marker, lanes 1 and 2 are T. reesei PS fermentation supernatant, and lanes 3 and 4 are T. reesei SCD45 fermentation supernatant.
  • the number range includes the numbers that define the range. Unless otherwise noted, nucleic acids are written from left to right in the 5 'to 3' direction; amino acid sequences are written from left to right in the direction from amino group to carboxyl group, respectively. In particular, practitioners may refer to Sambrook et al., 1989 and Ausubel FM et al., 1993 to understand the definitions and terms in the art. It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described, as these can be modified.
  • washing enzyme-protease inhibitor peptide fusion protein refers to at least one molecule of washing enzyme (or fragment or variant thereof) and at least one molecule of protease inhibitor peptide (or fragment or variant thereof) The protein formed by fusion.
  • the washing enzyme and protease inhibitory peptide fusion protein of the present invention comprises at least a fragment or variant of the enzyme for washing and at least a fragment or variant of the protease inhibitory peptide.
  • the polynucleotides encoding the whole or part of the washing enzyme are linked to each other in the same reading frame and the nucleic acid linked by the polynucleotide encoding the whole or part of the protease inhibitory peptide is translated).
  • the enzymes and protease inhibitory peptides for washing, once they become part of the fusion protein can be called “parts", “regions” or “modules” of the fusion protein.
  • the applicant obtained the cellulase SCD45-protease inhibitory peptide SSI fusion gene by fusing the cellulase SCD45 gene with the subtilisin inhibitory peptide SSI gene derived from Streptomyces sp.
  • amino acid sequence of the cellulase SCD45 is SEQ ID NO: 1, and the encoding nucleotide sequence is SEQ ID NO: 2;
  • amino acid sequence of the protease inhibitory peptide SSI is SEQ ID NO: 3, and its coding nucleotide sequence is SEQ ID NO: 4;
  • the above cellulase SCD45 and protease inhibitor peptide SSI were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. respectively.
  • primer 1 shown in SEQ ID NO: 7
  • primer 2 shown in SEQ ID NO: 8
  • subtilisin inhibitory peptide SSI gene As a template, using primer 3 (as shown in SEQ ID NO: 9) and primer 4 (as shown in SEQ ID NO: 10) to perform PCR amplification to obtain a gene fragment encoding the protease inhibitor peptide .
  • the purified cellulase SCD45 and the protease inhibitor peptide SSI U-Clone fragments are mixed in an equimolar ratio; using primer 1 and primer 4, respectively, and performing PCR with the above U-Clone fragment mixture as a template to obtain cellulase SCD45 -Protease inhibitor peptide SSI fusion gene, named SCD45-SSI, its nucleotide sequence is SEQ ID NO: 6, the encoded amino acid sequence is SEQ ID NO: 5, wherein the protease inhibitor peptide SSI is C with cellulase SCD45 End fusion.
  • the PCR reaction was performed for 30 cycles, each cycle being 94 ° C for 40 seconds, 62 ° C for 40 seconds, and 72 ° C for 1 minute. A final extension was performed at 72 ° C for 5 minutes, and the reaction was frozen to 16 ° C.
  • the fusion gene SCD45-SSI fragment obtained above was cloned into the Trichoderma expression plasmid pSC2G after double digestion with Xba I and Mlu I through the U-Clone kit, and the recombinant expression plasmid was constructed, named SCD45-SSI-pSC2G. To ensure accuracy, several clones were sequence verified. A recombinant expression plasmid carrying the cellulase SCD45 gene was constructed in the same manner as above, and named SCD45-pSC2G.
  • Trichoderma trichoderma (reechoderma reesei) SCHD4 Take the spore suspension of Trichoderma trichoderma (reechoderma reesei) SCHD4 and inoculate it on a PDA plate and incubate at 30 ° C for 6 days; after its spore production is rich, cut out a colony of about 1cm ⁇ 1cm and place it in 120mL containing YEG + U 0.5% yeast powder, 1% glucose, 0.1% uridine) liquid culture medium, shaking culture at 30 °C, 220 rpm for 14 to 16 hours;
  • Trichoderma PS Trichoderma PS
  • the applicant constructed the T. reesei engineering strain carrying the cellulase SCD45 gene according to the same operation as above, and named it Trichoderma reesei SCD45 (Trichoderma reesei SCD45).
  • the host bacteria Trichoderma reesei, the recombinant strains Trichoderma reesei SCD45 and Trichoderma reesei PS were inoculated on PDA plates and cultured at 30 ° C for 1 d.
  • the amount of enzyme required to degrade and release 1 ⁇ mol of the reducing end from a solution with a concentration of 1% CMC-Na per minute is 1 unit of enzyme activity.
  • Enzyme activity X A ⁇ 1 ⁇ 0.5 ⁇ n ⁇ 15
  • X enzyme activity unit, IU / g (mL);
  • the results of SDS-PAGE electrophoresis are shown in Figure 1.
  • the protein band indicated by the arrows in lanes 1 and 2 is the fusion protein recombinantly expressed by Trichoderma reesei PS, and its molecular weight is significantly higher than that indicated by the arrows in lanes 3 and 4.
  • the band of the cellulase SCD45 protein is large, which is basically consistent with the theoretical molecular weight of the fusion protein. This shows that the T.
  • reesei PS recombinant strain constructed by the present invention can effectively express the cellulase SCD45-protease inhibitory peptide SSI fusion protein, and further illustrates that the present invention is obtained by fusing the cellulase gene and the protease inhibitory peptide SSI gene
  • the fusion polynucleotide sequence, the cellulase and protease inhibitory peptides encoded by it are fused together in a covalent manner, and play its function as a complete protein.
  • the results of enzyme activity measurement showed that the enzyme activity of the host fermentation supernatant was only 4.5U / ml, while the recombinant strains T. reesei SCD45 and T. reesei PS fermentation supernatant had enzyme activities of 117U / mL and 109U / mL, respectively. . Therefore, the recombinant strain constructed by the present invention can efficiently express cellulase SCD45 and Trichoderma reesei PS can efficiently express the cellulase SCD45-protease inhibitor peptide SSI fusion protein without affecting the enzyme of cellulase Live level.
  • Trichoderma reesei SCD45 and Trichoderma reesei PS were inoculated on PDA plates and cultured at 30 ° C for 5 days; fresh spores grown on PDA plates were transferred to a liquid fermentation medium containing 1L (the liquid fermentation culture
  • the basic formula is: glucose 1%, corn steep liquor 1.5%, calcium chloride 0.05%, ammonium sulfate 0.9%, magnesium sulfate heptahydrate 0.2%, potassium dihydrogen phosphate 2%, Tween-80 0.02%, polypropylene glycol 0.02% , Inorganic salt solution 0.02%, pH5.5, where the inorganic salt solution consists of 5g / L FeSO 4 ⁇ 7H 2 O, 1.6g / L MnSO 4 ⁇ H 2 O, 1.2g / L ZnSO 4 ⁇ 7H 2 O) In a 3L shake flask, cultivate at 30 ° C and 200 rpm for 1 day in a shaker.
  • the cellulase enzyme activity of the fermentation supernatant was tested according to the method described in Example 1.4.
  • the results showed that the cellulase activity of the recombinant strains of Trichoderma reesei SCD45 and Trichoderma reesei PS fermentation supernatant were 1980U / mL and 1865U / mL, respectively. Therefore, it is further shown that the recombinant strain constructed by the present invention can efficiently express cellulase SCD45 and Trichoderma reesei PS can efficiently express the cellulase SCD45-protease inhibitor peptide SSI fusion protein.
  • the residual enzyme activity of cellulase SCD45 recombinantly expressed by Trichoderma reesei SCD45 was 91.4% in the presence of protease at room temperature for 12 hours, while that of Trichoderma reesei PS was recombinantly expressed.
  • the residual enzyme activity of the cellulase SCD45-protease inhibitor peptide SSI fusion protein is up to 99.7%; after 24 hours at 40 ° C, the residual enzyme activity of the cellulase SCD45 is only 8.1%, while the cellulase SCD45-protease inhibits peptide fusion
  • the residual enzyme activity of the protein is up to 74.0%, which is a very significant improvement.
  • the above results indicate that the present invention can significantly improve the protease resistance of cellulase by effectively expressing cellulase and protease inhibitory peptide SSI, effectively reduce the degradation effect of protease on cellulase, and obtain unexpected technical effects.
  • the cellulase with protease resistance provided by the present invention is more suitable for application in the field of washing.
  • the applicant obtained the cellulase HGD45-protease inhibitor peptide PCL fusion gene by fusing the cellulase HGD45 gene with the protease inhibitor peptide PCL gene derived from barley.
  • the amino acid sequence of the cellulase HGD45 is SEQ ID NO: 11, and the encoding nucleotide sequence is SEQ ID NO: 12.
  • amino acid sequence of the protease inhibitory peptide PCL derived from barley is SEQ ID NO: 13, and the encoding nucleotide sequence is SEQ ID NO: 14;
  • PCR amplification was performed using primer 5 (shown in SEQ ID NO: 17) and primer 6 (shown in SEQ ID NO: 18) to obtain a gene fragment encoding the cellulase HGD45.
  • primer 7 shown in SEQ ID NO: 19
  • primer 8 shown in SEQ ID NO: 20
  • HGD45-PCL Obtained cellulase HGD45-protease inhibitory peptide PCL fusion gene, named HGD45-PCL, its nucleotide sequence is SEQ ID NO: 16, its encoded amino acid sequence is SEQ ID NO: 15, wherein protease inhibitor peptide PCL is The C-terminal fusion of cellulase HGD45.
  • the PCR reaction was performed for 30 cycles, each cycle being 94 ° C for 40 seconds, 62 ° C for 40 seconds, and 72 ° C for 1 minute. A final extension was performed at 72 ° C for 5 minutes, and the reaction was frozen to 16 ° C.
  • the fusion gene HGD45-PCL fragment was cloned into the Trichoderma expression plasmid pSC2G after double digestion with Xba I and Mlu I through the U-Clone kit to construct a recombinant expression plasmid, named HGD45-PCL-pSC2G. To ensure accuracy, several clones were sequence verified.
  • the recombinant expression plasmid carrying cellulase HGD45 gene was constructed by the same method as above, and named as HGD45-pSC2G.
  • T. reesei engineering strain recombinantly expressing the cellulase HGD45-protease inhibitory peptide PCL fusion protein was constructed in the same manner as in Example 1.3, and named as Trichoderma reesei HGD45-PCL (Trichoderma reesei HGD45-PCL).
  • Trichoderma reesei HGD45 Trichoderma reesei HGD45
  • Example 1.4 The fermentation was carried out in the same manner as in Example 1.4 and Example 1.5, and the fermentation supernatant was subjected to cellulase enzyme activity measurement.
  • the fermentation supernatants of Trichoderma reesei HGD45 and Trichoderma reesei HGD45-PCL were diluted with pH7.5 acetate buffer to 1000U / ml; 2ml of the diluted fermentation supernatant, 0.06ml protease (pH 10. 5.
  • Protease enzyme activity (400,000U / ml), 18ml white cat laundry liquid base, after fully mixing the three, the enzyme-added laundry liquid can be obtained; after the enzyme-added laundry liquid is placed at 37 °C for 48h, 96h, 168h, respectively Perform cellulase enzyme activity detection. Taking the initial enzyme activity of cellulase in the enzyme-added laundry liquid as 100%, the residual enzyme activity of cellulase was calculated respectively. The results are shown in Table 3.
  • the applicant obtained the cellulase NT45-protease inhibitor peptide MST fusion gene by fusing the cellulase NT45 gene with the protease inhibitor peptide MST gene derived from Alteromonas.
  • the amino acid sequence of the cellulase NT45 is SEQ ID NO: 21, and the encoding nucleotide sequence is SEQ ID NO: 22.
  • amino acid sequence of the protease inhibitory peptide MST is SEQ ID NO: 23, and the encoding nucleotide sequence is SEQ ID NO: 24;
  • primer 11 shown in SEQ ID NO: 29
  • primer 12 shown in SEQ ID NO: 30
  • the cellulase NT45-protease inhibitor peptide MST fusion gene is named NT45-MST, its nucleotide sequence is SEQ ID NO: 26, and its encoded amino acid sequence is SEQ ID NO: 25, in which the protease inhibitor peptide MST is associated with fiber The C-terminal fusion of the enzyme NT45.
  • the PCR reaction was performed for 30 cycles, each cycle being 94 ° C for 40 seconds, 62 ° C for 40 seconds, and 72 ° C for 1 minute. A final extension was performed at 72 ° C for 5 minutes, and the reaction was frozen to 16 ° C.
  • NT45-MST-pSC2G The fusion gene NT45-MST fragment was cloned into the Trichoderma expression plasmid pSC2G after double digestion with Xba I and Mlu I through U-Clone kit to construct a recombinant expression plasmid, named NT45-MST-pSC2G. To ensure accuracy, several clones were sequence verified.
  • Recombinant expression plasmid carrying cellulase NT45 gene was constructed by the same method as above, named NT45-pSC2G.
  • the T. reesei engineering strain recombinantly expressing the cellulase NT45-protease inhibitory peptide MST fusion protein was constructed by the same method as in Example 1.3, and named as Trichoderma NT45-MST (Trichoderma NTreesei NT45-MST).
  • Trichoderma NT45 Trichoderma NTree
  • Example 1.4 The fermentation was carried out in the same manner as in Example 1.4 and Example 1.5, and the fermentation supernatant was subjected to cellulase enzyme activity measurement.
  • Control group mix 133ul of pH10.5 acetate buffer and 67ul of the above fermentation supernatant (cellulase activity is 100U / ml), mix well, and leave at 37 ° C for 3 days.
  • Cellulase Cellulase residual enzyme activity Cellulase NT45 77.3% Cellulase NT45-MST fusion protein 85.9%
  • amino acid sequence of the protein leader peptide PPS is SEQ ID NO: 31, and its encoding nucleotide sequence is SEQ ID NO: 32;
  • the gene sequence of protein leader peptide PPS was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • primer 13 shown in SEQ ID NO: 35
  • primer 14 shown in SEQ ID NO: 36
  • PCR amplification was performed using primer 15 (shown in SEQ ID NO: 37) and primer 16 (shown in SEQ ID NO: 38) to obtain a gene fragment encoding the cellulose.
  • the purified protein leader peptide PPS U-Clone fragment and the cellulase SCD45 U-Clone fragment are mixed in an equimolar ratio; using primer 13 and primer 16, respectively, using the above U-clone fragment mixture as a template for PCR, Obtained the protein leader peptide PPS-cellulase SCD45 fusion gene, named PPS-SCD45, its nucleotide sequence is SEQ ID NO: 34, and its encoded amino acid sequence is SEQ ID NO: 33, where the protein leader peptide PPS is The N-terminal fusion of cellulase SCD45.
  • the PCR reaction was performed for 30 cycles, each cycle being 94 ° C for 40 seconds, 62 ° C for 40 seconds, and 72 ° C for 1 minute. A final extension was performed at 72 ° C for 5 minutes, and the reaction was frozen to 16 ° C.
  • the fusion gene PPS-SCD45 fragment was cloned into the Trichoderma expression plasmid pSC2G after double digestion with Xba I and Mlu I through the U-Clone kit to construct a recombinant expression plasmid, named PPS-SCD45-pSC2G. To ensure accuracy, several clones were sequence verified.
  • T. reesei engineering strain which obtained the recombinant expression protein leader peptide PPS-cellulase SCD45 fusion protein was constructed by the same method as in Example 1.3, and named as Trichoderma reesei PPS-SCD45 (Trichoderma reesei PPS-SCD45).
  • the recombinant strain T. reesei SCD45 recombinantly expressing cellulase SCD45 constructed in Example 1.3 was used as a control.
  • Example 1.4 The fermentation was carried out in the same manner as in Example 1.4 and Example 1.5, and the fermentation supernatant was subjected to cellulase enzyme activity measurement.
  • Control group mix 133ul of pH10.5 acetate buffer and 67ul of the above-mentioned Trichoderma reesei fermentation supernatant (cellulase enzyme activity is 100U / ml), mix well, and leave at 37 ° C for 3 days.
  • Cellulase Cellulase residual enzyme activity Cellulase SCD45 79.4% PPS-cellulase SCD45 fusion protein 88.8%
  • the method provided by the present invention is also applicable to lipase, which can significantly improve the resistance of lipase to protease.
  • the applicant obtained the lipase TG-protease inhibitor peptide SSI fusion gene by fusing the lipase TG gene with the subtilisin inhibitor peptide SSI gene.
  • the amino acid sequence of the lipase TG is SEQ ID NO: 39, and the encoding nucleotide sequence is SEQ ID NO: 40.
  • the gene sequence of lipase TG was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • primer 17 shown in SEQ ID NO: 43
  • primer 18 shown in SEQ ID NO: 44
  • subtilisin inhibitory peptide SSI gene As a template, using primer 19 (as shown in SEQ ID NO: 45) and primer 4 as described in Example 1.1 (as shown in SEQ ID NO: 10), PCR amplification was performed to obtain the code Gene fragments of protease inhibitory peptides.
  • the PCR reaction was performed for 30 cycles, each cycle being 94 ° C for 40 seconds, 62 ° C for 40 seconds, and 72 ° C for 1 minute. A final extension was performed at 72 ° C for 5 minutes, and the reaction was frozen to 16 ° C.
  • the TL-SSI fragment of the fusion gene was cloned into the Trichoderma expression plasmid pSC2G after double digestion with Xba I and Mlu I through the U-Clone kit, and the recombinant expression plasmid was constructed and named TG-SSI-pSC2G. To ensure accuracy, several clones were sequence verified.
  • a recombinant expression plasmid carrying the lipase TG gene was constructed using the same method as above, and named TG-pSC2G.
  • T. reesei engineering strain recombinantly expressing the lipase TG-protease inhibitory peptide SSI fusion protein was constructed in the same manner as in Example 1.3, and it was named Trichoderma TGS (Trichoderma reesei TGS).
  • Trichoderma TG Trichoderma reeree TG
  • Example 1.4 The fermentation was carried out in the same manner as in Example 1.4 and Example 1.5, and the fermentation supernatant was subjected to lipase enzyme activity measurement.
  • Lipase 2d 5d 6d Lipase TG 78.5% 50.1% 34.6% Lipase TG-SSI fusion protein 105.7% 76.2% 64.6%
  • lipase TG-protease inhibitor peptide SSI fusion protein was placed at 37 °C for 2d, 5d and 6d, its residual enzyme activity Both have been significantly improved. Among them, after being placed for 6 days, the residual enzyme activity of the fusion protein was as high as 64.6%, which was 86.7% higher than that of lipase TG, and the effect was extremely significant.
  • the above results show that the present invention can significantly improve the resistance of lipase protease by fusion expression of lipase and protease inhibitory peptide, effectively reduce the degradation effect of protease on lipase, and achieve unexpected technical effects.
  • the lipase with protease resistance provided by the present invention is more suitable for application in the field of washing.
  • the method provided by the present invention is also applicable to other washing enzymes, such as cutinase, amylase, pectinase, mannanase, arabinase, galactanase, xylan Sugar enzymes, laccases, peroxidases, etc., can significantly increase the protease resistance of the enzyme, effectively reduce the degradation of the enzyme by the protease, and facilitate the stable coexistence of the enzyme and the protease in the detergent.
  • other washing enzymes such as cutinase, amylase, pectinase, mannanase, arabinase, galactanase, xylan Sugar enzymes, laccases, peroxidases, etc.
  • the method of the present invention is not limited to the fusion expression of washing enzymes with protease inhibitory peptide SSI, protease inhibitory peptide PCL, protease inhibitory peptide MST or protein leader peptide PPS, but can also be combined with Bowman-Birk protease inhibitory peptide or Bacillus-derived
  • the protein leader peptide is fused and expressed to improve the protease resistance of the washing enzyme.
  • the method of the present invention is not limited to the fusion expression of the washing enzyme with a single protease inhibitory peptide, but also the fusion of the washing enzyme with two or three kinds of protease inhibitory peptides to improve the protease of the washing enzyme Resistance.
  • the fusion is not limited to the fusion of the protease inhibitor peptide at the N-terminus or C-terminus of the washing enzyme, but can also be fused in the middle.

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Abstract

提供了一种生产具有蛋白酶抗性的洗涤用酶的方法。所述方法通过将洗涤用酶的基因与蛋白酶抑制肽的基因进行融合表达，获得对蛋白酶具有抗性的洗涤用酶，有利于维持加酶洗涤剂中多种酶组分的稳定性，改善洗涤剂的使用效果。

Description

一种生产具有蛋白酶抗性的洗涤用酶的方法
本申请要求于2018年10月31日提交中国专利局、申请号为2018112858609、发明名称为“一种生产具有蛋白酶抗性的洗涤用酶的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因工程技术领域，具体涉及一种生产具有蛋白酶抗性的洗涤用酶的方法，通过将洗涤用酶基因与蛋白酶抑制肽基因进行融合表达，获得具有蛋白酶抗性的洗涤用酶。
背景技术
早在1913年，Rohm就已将胰腺提取物用于洗涤剂的预浸泡组分Burnus，开创了生物酶在洗涤剂工业中应用的历史。国外已商品化的洗涤剂用酶主要为水解酶类，包括碱性蛋白酶、淀粉酶、碱性纤维素酶、脂肪酶以及它们的复合物；新开发的抗染料转移功能或消毒杀菌效用的氧化还原酶类(如过氧化氢酶)也具有较好的应用前景。
生物酶作为洗涤助剂是合成洗涤剂工业发展过程中的重大技术进步之一，它促进了洗涤剂工业的可持续发展；降低了洗涤剂中表面活性剂和三聚磷酸钠的用量，有助于低磷或无磷洗涤剂的开发；能够改善洗涤性能，但自身无毒并能完全生物降解，可以减少污染物质的排放；降低洗涤温度，减少漂洗次数，有利于节能节水。
但是生物酶自身易于降解以及洗涤剂配方技术的复杂性，如表面活性剂、漂白剂、蛋白酶等配方组分以及pH、温度等配方条件的影响，酶的稳定性成了加酶洗涤剂配方的突出问题。酶制剂公司和洗涤用品开发商通过综合运用蛋白质工程技术、化学修饰技术、稳定剂添加和微囊包载技术，来提高生物酶在洗涤剂中的稳定性，改善其使用效果。
在含有大量水分的液体洗涤剂中，酶通常是不稳定的。剂型的溶剂、缓冲剂以及带电荷的表面活性剂等组分可能促使贮存过程中酶的三级结构解析折叠，而且蛋白酶在水溶液中更容易降解其他生物酶。在液体洗涤 产品贮存过程中蛋白酶对自身的水解作用和蛋白酶对其他酶类的水解作用，造成酶的失活量随时间的延长而增加。因此添加适量稳定介质以抑制蛋白酶活性、调节电荷平衡、加强渗透保护，从而保障生物酶结构的刚性是解决液体洗涤剂用酶稳定性的主要技术方案，即稳定剂添加技术。目前该技术的重点是开发高效的蛋白酶抑制剂和适合配方的复合稳定体系。
长久以来人们都知道苯基硼酸和它的同分异构体对多种蛋白酶活有可逆的抑制作用。在液体洗涤剂贮存过程当中,通过添加硼酸盐类化合物能有效抑制蛋白酶的活力以防止蛋白酶对自身和其他酶的水解作用,从而避免了产品贮存过程中酶活的丧失；而在洗涤过程中，随着洗涤剂的稀释酶的活力又能快速有效地得到恢复。21世纪以来，诺维信公司和保洁公司都着重开发含芳基的有机硼酸盐作为蛋白酶抑制剂，然而动物实验结果将硼酸盐确定为第二类生殖毒性化合物。而后研究人员发现，在加入蛋白酶的液体洗涤剂中，α-羟基羧酸盐对酶的稳定性起到关键作用，特别是含芳基的羧酸盐衍生物更有效，成为新一代蛋白酶抑制剂的主流产品。诺维信公司开发了3-氯苯甲酸、4-氯苯甲酸、3-氯苯乙酸、3,5-二氯苯甲酸等多种带有芳基的羧酸盐衍生物作为枯草芽孢杆菌蛋白酶抑制剂，比硼酸盐具有更好的生物降解性。虽然这类蛋白酶抑制剂在洗涤剂中的添加量远低于硼酸盐类蛋白酶抑制剂，但作为含芳基的化合物，其长期使用对自然环境以及动物健康都会造成一定的不良影响。因此，本领域技术人员正在积极寻找对环境友好、无毒副作用的蛋白酶抑制剂或开发新的提高酶稳定性的方法。
发明内容
本发明为解决现有技术问题，提供了一种生产具有蛋白酶抗性的洗涤用酶的方法。所述方法通过将洗涤用酶的基因与蛋白酶抑制肽的基因进行融合表达，获得对蛋白酶具有抗性的洗涤用酶，有利于维持加酶洗涤剂中多种酶组分的稳定性，改善洗涤剂的使用效果。
本发明提供了一种生产具有蛋白酶抗性的洗涤用酶的方法，包括如下步骤：
(a)构建融合多核苷酸序列，该序列包含编码洗涤用酶的基因和编码蛋白酶抑制肽的基因；
(b)将序列引入宿主细胞；
(c)培养宿主细胞，其中细胞表达序列并产生洗涤用酶-蛋白酶抑制肽融合蛋白，所述融合蛋白即为具有蛋白酶抗性的洗涤用酶。
所述融合蛋白中洗涤用酶与蛋白酶抑制肽是以共价方式融合。
所述的洗涤用酶为纤维素酶、脂肪酶，角质酶，淀粉酶，糖酶，果胶酶，甘露聚糖酶，阿拉伯糖酶，半乳聚糖酶，木聚糖酶，漆酶或过氧化物酶中的任意一种。
所述的洗涤用酶优选纤维素酶。
所述的洗涤用酶优选脂肪酶。
所述的蛋白酶抑制肽为链霉菌(Streptomyces sp.)来源的蛋白酶抑制肽SSI、Bowman-Birk蛋白酶抑制肽、大麦(Barley)来源的蛋白酶抑制肽PCL、类单胞菌(Alteromonas)来源的蛋白酶抑制肽MST或芽孢杆菌(Bacillus sp.)来源的蛋白前导肽中的任意一种或两种或三种。
所述宿主细胞为芽孢杆菌(Bacillus sp.)，曲霉(Aspergillus sp.)，木霉(Trichoderma sp.)，毕赤酵母(Pichia pastoris)，酿酒酵母(Saccharomyces cerevisiae)或大肠杆菌(Escherichia coli)。
所述宿主细胞优选木霉(Trichoderma sp.)，进一步优选里氏木霉(Trichoderma reesei)。
所述融合蛋白中蛋白酶抑制肽可在洗涤用酶的N端融合、C端融合或中间融合。
本发明还提供了通过上述方法生产的具有蛋白酶抗性的洗涤用酶。
本发明还提供了所述具有蛋白酶抗性的洗涤用酶在洗涤剂中的应用。
本发明通过将纤维素酶或脂肪酶与蛋白酶抑制肽进行融合表达，能显著提高其蛋白酶抗性，有效降低蛋白酶对纤维素酶或脂肪酶的降解作用。其中，在蛋白酶存在的情况下，室温放置12h后，纤维素酶SCD45的残留酶活为91.4％，而纤维素酶SCD45-蛋白酶抑制肽融合蛋白的残留酶活高达99.7％；40℃放置24h后，纤维素酶SCD45的残留酶活仅为8.1％， 而纤维素酶SCD45-蛋白酶抑制肽融合蛋白的残留酶活高达74.0％，得到极显著的提高。
本发明获得的具有蛋白酶抗性的纤维素酶，可广泛应用于洗涤剂中，能有效提高洗涤剂中酶的稳定性。37℃放置24h后，添加纤维素酶SCD45的加酶洗衣液中纤维素酶的残留酶活为12.1％，而添加纤维素酶SCD45-蛋白酶抑制肽融合蛋白的加酶洗衣液中纤维素酶的残留酶活高达59.5％；37℃放置48h后，添加纤维素酶SCD45的加酶洗衣液中纤维素酶的残留酶活为2.1％，而添加融合蛋白的加酶洗衣液中纤维素酶的残留酶活高达28.9％；37℃放置72h后，添加纤维素酶SCD45的加酶洗衣液中纤维素酶的残留酶活为0.9％，而添加融合蛋白的加酶洗衣液中纤维素酶的残留酶活高达14.2％，得到极显著的提高，取得了意料不到的技术效果。纤维素酶HGD45、纤维素酶NT45通过与蛋白酶抑制肽融合表达，也显著提高了其蛋白酶抗性。
脂肪酶TG-蛋白酶抑制肽融合蛋白在37℃条件下放置2d、5d和6d后，其残留酶活均得到显著提高，其中，在放置6d后，融合蛋白的残留酶活高达64.6％，比脂肪TG提高了86.7％，效果极为显著。
除纤维素酶和脂肪酶外，本发明所述方法还可广泛应用于洗涤用酶，例如角质酶、淀粉酶、果胶酶、甘露聚糖酶、阿拉伯糖酶、半乳聚糖酶、木聚糖酶、漆酶、过氧化物酶等，均能显著提高所述酶的蛋白酶抗性，有效降低蛋白酶对酶的降解作用，有利于实现所述酶在洗涤剂中与蛋白酶的稳定共存。
附图说明
图1为SDS-PAGE电泳检测图；其中，泳道M为Marker，泳道1和2为里氏木霉PS发酵上清液，泳道3和4为里氏木霉SCD45发酵上清液。
具体实施方式
本发明现在将通过参考的方式、仅仅使用下面给出的定义和实施例进行详细描述。除非在此处另外定义，所有此处使用的技术和科学术语都和 本发明所属领域的普通技术人员所通常理解的术语意义相同。Singleton等所著的由纽约John Wiley and Sons于1994年出版的DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY第二版，以及由Hale和Marham所著，由纽约Harper Perennial于1991年出版的THE HARPER COLLINS DICTIONARY OF BIOLOGY，向技术人员提供用于本发明中的许多术语的综合词典。尽管与此处的描述相似或等价的任何材料和方法可以在本发明的实际应用和试验中使用，本发明还是描述了优选的方法和材料。数字范围包括了界定范围的数字。除非另外指出，分别地，核酸按照5′到3′的方向从左到右写出；氨基酸序列按照从氨基到羧基的方向从左到右写出。特别地，从业者可以参照Sambrook等，1989和Ausubel FM等，1993来理解本领域的定义和术语。应该理解，本发明不局限于所描述的具体方法学、方案和试剂，因为这些是可以改变的。
如本发明所使用，“洗涤用酶-蛋白酶抑制肽融合蛋白”指通过至少一个分子的洗涤用酶(或其片段或变体)与至少一个分子的蛋白酶抑制肽(或其片段或变体)融合而形成的蛋白质。本发明洗涤用酶与蛋白酶抑制肽融合蛋白包含至少洗涤用酶的片段或变体和至少蛋白酶抑制肽的片段或变体，它们通过诸如基因融合(即洗涤用酶与蛋白酶抑制肽融合蛋白是由其中编码完整或部分洗涤用酶的多核苷酸以相同读码框与编码完整或部分蛋白酶抑制肽的多核苷酸连接的核酸翻译生成的)彼此相互连接。洗涤用酶和蛋白酶抑制肽，一旦成为融合蛋白的一部分，可称为融合蛋白的“部份”、“区域”或“模块”。
下面结合实施例对本发明进行详细的描述。
实施例1纤维素酶SCD45与蛋白酶抑制肽SSI融合表达提高其蛋白酶抗性
1.1纤维素酶SCD45-蛋白酶抑制肽SSI融合基因的获得
申请人通过将纤维素酶SCD45基因与来源于链霉菌(Streptomyces sp.)的枯草杆菌蛋白酶抑制肽SSI基因进行融合，获得纤维素酶SCD45-蛋白酶抑制肽SSI融合基因。
所述纤维素酶SCD45的氨基酸序列为SEQ ID NO：1，其编码核苷酸序列为SEQ ID NO：2；
所述蛋白酶抑制肽SSI的氨基酸序列为SEQ ID NO：3，其编码核苷酸序列为SEQ ID NO：4；
由苏州金唯智生物科技有限公司分别合成上述纤维素酶SCD45和蛋白酶抑制肽SSI。
具体操作如下：
以纤维素酶SCD45基因为模板，利用引物1(如SEQ ID NO：7所示)和引物2(如SEQ ID NO：8所示)进行PCR扩增获得编码该纤维素酶的基因片段。
以枯草杆菌蛋白酶抑制肽SSI基因为模板，利用引物3(如SEQ ID NO：9所示)和引物4(如SEQ ID NO：10所示)进行PCR扩增获得编码该蛋白酶抑制肽的基因片段。
将纯化后的纤维素酶SCD45和蛋白酶抑制肽SSI的U-Clone片段按等摩尔比例混匀；分别利用引物1和引物4，以上述U-Clone片段混合物为模板进行PCR，获得纤维素酶SCD45-蛋白酶抑制肽SSI融合基因，命名为SCD45-SSI，其核苷酸序列为SEQ ID NO：6，编码的氨基酸序列为SEQ ID NO：5，其中蛋白酶抑制肽SSI是与纤维素酶SCD45的C端融合。PCR反应进行30个循环，每个循环为94℃40秒，62℃40秒，以及72℃1分钟。在72℃进行最后的延伸5分钟，将反应物冷冻至16℃。
1.2表达载体的构建
将上述获得的融合基因SCD45-SSI片段通过U-Clone试剂盒克隆至经Xba I和Mlu I双酶切后的木霉表达质粒pSC2G中，构建得到重组表达质粒，命名为SCD45-SSI-pSC2G。为确保准确，对若干克隆进行测序验证。用上述同样方法构建得到携带纤维素酶SCD45基因的重组表达质粒，命名为SCD45-pSC2G。
1.3里氏木霉工程菌的构建与筛选
(1)原生质体制备
取宿主菌里氏木霉(Trichoderma reesei)SCHD4孢子悬液，接种于 PDA平板上，30℃培养6天；待其产孢丰富后，切取约1cm×1cm的菌落置于含120mL YEG+U(0.5％酵母粉、1％葡萄糖、0.1％尿苷)的液体培养基中，30℃，220rpm振荡培养14～16h；
用无菌纱布过滤收集菌丝体，并用无菌水清洗一次；将菌丝体置于含有20mL 10mg/mL裂解酶液(Sigma L1412)的三角瓶中，30℃，90rpm作用1-2h；用显微镜观察检测原生质体转化进展；
将预冷的20mL 1.2M山梨醇(1.2M山梨醇，50mM Tris-Cl，50mM CaCl 2)加入上述三角瓶中，轻轻摇匀，用无菌Miracloth滤布过滤收集滤液，3000rpm，4℃离心10min；弃上清，加入预冷的5mL 1.2M山梨醇溶液悬浮菌体，3000rpm，4℃离心10min；弃上清，加入适量预冷的1.2M山梨醇悬浮分装(200μL/管，原生质体浓度为108个/mL)；
(2)表达载体转化与菌株验证
以下操作均在冰上进行，取10μg重组质粒SCD45-SSI-pSC2G加入到含有200μL原生质体溶液的7mL无菌离心管中，然后加入50μL 25％PEG(25％PEG，50mM Tris-Cl，50mM CaCl2)，轻弹管底混匀，冰上放置20min；加入2mL 25％PEG，混匀后室温放置5min；加入4mL 1.2M山梨醇，轻轻混匀后倒入熔化并保持在55℃的上层培养基中(0.1％MgSO 4，1％KH 2PO 4,0.6％(NH 4) 2SO 4,1％葡萄糖,18.3％山梨醇,0.35％琼脂糖)；轻轻混匀后铺在制备好的下层培养基平板上(2％葡萄糖，0.5％(NH 4) 2SO 4，1.5％KH 2PO 4，0.06％MgSO 4，0.06％CaCl 2，1.5％琼脂)，30℃培养5～7d至有转化子长出。
挑取转化子至下层培养基平板，30℃培养2d；取适量菌丝体置于2mL离心管中，加入100mg无菌石英砂和400μL抽提缓冲液(100mM Tris-HCl，100mM EDTA，250mM NaCl，1％SDS)；用珠打仪剧烈振荡2min；65℃水浴20min后，加入200μL 10M NH4AC，冰浴10min；13000rpm离心10min；取上清，加入2倍体积的无水乙醇，-20℃放置30min；13000rpm离心10min，弃上清；用70％乙醇洗涤2次；晾干，加水溶解，于-20℃保存。
以上述提取的转化子基因组DNA为模板，利用引物1和4进行PCR 扩增。PCR扩增条件为94℃4min；94℃40s；58℃40s，72℃1min，30个循环；72℃7min，16℃；利用凝胶回收试剂盒回收PCR扩增产物并进行测序分析。测序结果显示，扩增产物的核苷酸序列与SEQ ID NO：6一致。从而说明，本发明构建得到了携带纤维素酶SCD45-蛋白酶抑制肽SSI融合基因的里氏木霉工程菌，将其命名为里氏木霉PS(Trichoderma reesei PS)。
作为对照，申请人按照上述同样操作，构建得到携带纤维素酶SCD45基因的里氏木霉工程菌，命名为里氏木霉SCD45(Trichoderma reesei SCD45)。
1.4摇瓶发酵验证和酶活测定
将宿主菌里氏木霉，重组菌株里氏木霉SCD45和里氏木霉PS分别接种于PDA平板，30℃培养1d，待孢子丰富后，分别取两块直径1cm的菌丝块接种于含有50mL发酵培养基(1.5％葡萄糖，1.7％乳糖，2.5％玉米浆，0.44％(NH 4) 2SO 4，0.09％MgSO 4，2％KH 2PO 4，0.04％CaCl 2，0.018％吐温-80，0.018％微量元素)的250mL三角瓶中，30℃培养48小时，然后25℃培养48小时，取发酵上清液分别进行SDS-PAGE电泳检测和纤维素酶酶活测定。
(1)酶活测定方法
在50℃、pH值为6.0的条件下，每分钟从浓度为1％CMC-Na的溶液中降解释放1μmol还原末端所需要的酶量为1个酶活单位。
取试管各加入已稀释好的酶液0.5ml；同时放置50±0.1℃水浴中，预热2min；准确向样品试管中加入0.5mL底物溶液，准确即时15min，迅速向各管中加入碳酸钠溶液0.2ml，于空白管中加入底物溶液0.5ml，摇匀。以空白管调零，在分光光度计波长410nm下测量。
酶活X＝A×1÷0.5×n÷15
其中：X——酶活力单位，IU/g(mL)；
A——吸光度在标准曲线上算得的对硝基苯酚含量，μmol；
1/0.5——所加入的酶液体积；
15——待测液与底物的反应时间；
n——稀释倍数；
(2)实验结果
SDS-PAGE电泳检测结果如图1所示，泳道1和2中箭头所指处的蛋白条带即为里氏木霉PS重组表达的融合蛋白，其分子量明显比泳道3和4中箭头所指处的纤维素酶SCD45蛋白条带大，与融合蛋白的理论分子量大小基本一致。从而说明，本发明构建的里氏木霉PS重组菌株能有效表达纤维素酶SCD45-蛋白酶抑制肽SSI融合蛋白，也进一步说明，本发明通过将纤维素酶基因与蛋白酶抑制肽SSI基因进行融合获得的融合多聚核苷酸序列，其编码的纤维素酶和蛋白酶抑制肽是以共价方式融合在一起，作为一个完整的蛋白发挥其功能。
酶活测定结果显示：宿主菌发酵上清液酶活仅为4.5U/ml，而重组菌株里氏木霉SCD45和里氏木霉PS发酵上清液酶活分别达到117U/mL和109U/mL。从而说明，本发明构建的重组菌株里氏木霉SCD45能高效表达纤维素酶SCD45，里氏木霉PS能高效表达纤维素酶SCD45-蛋白酶抑制肽SSI融合蛋白，且不影响纤维素酶的酶活水平。
1.5发酵放大
将重组菌株里氏木霉SCD45和里氏木霉PS分别接种至PDA平板上，30℃培养5天；将PDA平板上长出的新鲜孢子转移至含有1L液体发酵培养基(所述液体发酵培养基的配方为：葡萄糖1％，玉米浆1.5％，氯化钙0.05％，硫酸铵0.9％，七水硫酸镁0.2％，磷酸二氢钾2％，吐温-80 0.02％，聚丙二醇0.02％，无机盐溶液0.02％，pH5.5，其中无机盐溶液由5g/L FeSO 4·7H 2O、1.6g/L MnSO 4·H 2O、1.2g/L ZnSO 4·7H 2O组成)的3L摇瓶中，30℃，200rpm摇床培养1天。
将摇瓶发酵液转移至装有10L上述发酵培养基(pH5.5)的20L发酵罐；温度控制在28±1℃，pH值5.0±0.2，发酵培养165h后，收集发酵液并过滤用于进一步的酶活检测分析。
参照实施例1.4所述方法对发酵上清液进行纤维素酶酶活检测。结果显示：重组菌株里氏木霉SCD45和里氏木霉PS发酵上清液中纤维素酶酶活分别为1980U/mL和1865U/mL。从而进一步说明，本发明构建的重 组菌株里氏木霉SCD45能高效表达纤维素酶SCD45，里氏木霉PS能高效表达纤维素酶SCD45-蛋白酶抑制肽SSI融合蛋白。
1.6纤维素酶的蛋白酶抗性实验
首先，将里氏木霉SCD45和里氏木霉PS的发酵上清液分别用pH7.5乙酸缓冲液稀释至600U/ml；然后分别取8ml稀释后的发酵上清液，1ml pH10.5、蛋白酶酶活为120000U/ml的蛋白酶，将两者充分混匀，得混合酶液；将混合酶液在室温下放置12h和40℃放置24h后，分别进行纤维素酶酶活检测。以混合酶液中纤维素酶的初始酶活计100％，分别计算纤维素酶的残留酶活，具体结果见表1。
表1纤维素酶残留酶活比较
纤维素酶 室温放置12h 40℃放置24h
纤维素酶SCD45 91.4％ 8.1％
纤维素酶SCD45-SSI融合蛋白 99.7％ 74.0％
从表1的数据可以看出，在蛋白酶存在的情况下，室温放置12h后，里氏木霉SCD45重组表达的纤维素酶SCD45的残留酶活为91.4％，而里氏木霉PS重组表达的纤维素酶SCD45-蛋白酶抑制肽SSI融合蛋白的残留酶活高达99.7％；40℃放置24h后，上述纤维素酶SCD45的残留酶活仅为8.1％，而上述纤维素酶SCD45-蛋白酶抑制肽融合蛋白的残留酶活高达74.0％，得到极显著的提高。
上述结果表明，本发明通过将纤维素酶与蛋白酶抑制肽SSI进行融合表达，能显著提高纤维素酶的蛋白酶抗性，有效降低蛋白酶对纤维素酶的降解作用，取得了意料不到的技术效果。本发明提供的具有蛋白酶抗性的纤维素酶更适合在洗涤领域中的应用。
1.7纤维素酶在洗衣液中的稳定性实验
首先，将里氏木霉SCD45和里氏木霉PS的发酵上清液分别用pH7.5乙酸缓冲液稀释至1000U/ml；分别取2ml稀释后的发酵上清液，0.06ml蛋白酶(pH10.5、蛋白酶酶活480000U/ml)，18ml白猫洗衣液基料，将三者充分混匀后，得加酶洗衣液；将加酶洗衣液在37℃下放置24h、48h、72h后，参照实施例3所述方法分别进行纤维素酶酶活检测。以加酶洗衣 液中纤维素酶的初始酶活计100％，分别计算纤维素酶的残留酶活，结果如表2所示。
表2加酶洗衣液中纤维素酶在37℃条件下的残留酶活
纤维素酶 24h 48h 72h
纤维素酶SCD45 12.1％ 2.1％ 0.9％
纤维素酶SCD45-SSI融合蛋白 59.5％ 28.9％ 14.2％
从表2的数据可以看出，添加纤维素酶SCD45-蛋白酶抑制肽融合蛋白的加酶洗衣液在37℃放置24h、48h和72h后，其中的纤维素酶残留酶活均显著高于添加纤维素酶SCD45的加酶洗衣液。从而表明，本发明通过将纤维素酶与蛋白酶抑制肽进行融合表达，获得具有蛋白酶抗性的纤维素酶，可广泛应用于洗涤剂中，能有效提高洗涤剂中纤维素酶的稳定性，取得了意料不到的技术效果。
实施例2纤维素酶HGD45与蛋白酶抑制肽PCL融合表达提高其蛋白酶抗性
2.1纤维素酶HGD45-蛋白酶抑制肽PCL融合基因的获得与表达
申请人通过将纤维素酶HGD45基因与大麦来源的蛋白酶抑制肽PCL基因进行融合，获得纤维素酶HGD45-蛋白酶抑制肽PCL融合基因。
所述纤维素酶HGD45的氨基酸序列为SEQ ID NO：11，其编码核苷酸序列为SEQ ID NO：12。
所述大麦来源的蛋白酶抑制肽PCL的氨基酸序列为SEQ ID NO：13，其编码核苷酸序列为SEQ ID NO：14；
由苏州金唯智生物科技有限公司分别合成上述纤维素酶HGD45和蛋白酶抑制肽PCL的基因。
具体操作如下：
以纤维素酶HGD45基因为模板，利用引物5(如SEQ ID NO：17所示)和引物6(如SEQ ID NO：18所示)进行PCR扩增获得编码该纤维素酶HGD45的基因片段。
以蛋白酶抑制肽PCL基因为模板，利用引物7(如SEQ ID NO：19 所示)和引物8(如SEQ ID NO：20所示)进行PCR扩增获得编码该蛋白酶抑制肽的基因片段。
将所述蛋白酶抑制肽PCL的U-Clone片段与纤维素酶HGD45的U-Clone片段按等摩尔比例混匀；分别利用引物5和引物8，以上述U-clone片段混合物为模板进行PCR，，获得纤维素酶HGD45-蛋白酶抑制肽PCL融合基因，命名为HGD45-PCL，其核苷酸序列为SEQ ID NO：16，其编码的氨基酸序列为SEQ ID NO：15，其中蛋白酶抑制肽PCL是与纤维素酶HGD45的C端融合。PCR反应进行30个循环，每个循环为94℃40秒，62℃40秒，以及72℃1分钟。在72℃进行最后的延伸5分钟，将反应物冷冻至16℃。
将融合基因HGD45-PCL片段通过U-Clone试剂盒克隆至经Xba I和Mlu I双酶切后的木霉表达质粒pSC2G中，构建得到重组表达质粒，命名为HGD45-PCL-pSC2G。为确保准确，对若干克隆进行测序验证。
采用上述同样方法构建得到携带纤维素酶HGD45基因的重组表达质粒，命名为HGD45-pSC2G。
采用与实施例1.3同样的方法构建得到重组表达纤维素酶HGD45-蛋白酶抑制肽PCL融合蛋白的里氏木霉工程菌，将其命名为里氏木霉HGD45-PCL(Trichoderma reesei HGD45-PCL)。
作为对照，同时构建得到重组表达纤维素酶HGD45的里氏木霉工程菌，命名为里氏木霉HGD45(Trichoderma reesei HGD45)。
采用与实施例1.4和实施例1.5同样的方法进行发酵，并将发酵上清液进行纤维素酶酶活测定。
2.2纤维素酶的蛋白酶抗性实验
将里氏木霉HGD45和里氏木霉HGD45-PCL的发酵上清液分别用pH7.5乙酸缓冲液稀释至1000U/ml；分别取2ml稀释后的发酵上清液，0.06ml蛋白酶(pH10.5、蛋白酶酶活400000U/ml)，18ml白猫洗衣液基料，将三者充分混匀后，得加酶洗衣液；将加酶洗衣液在37℃下放置48h、96h、168h后，分别进行纤维素酶酶活检测。以加酶洗衣液中纤维素酶的初始酶活计100％，分别计算纤维素酶的残留酶活，结果如表3所示。
表3加酶洗衣液中纤维素酶在37℃条件下的残留酶活
纤维素酶 48h 96h 168h
纤维素酶HGD45 45.9％ 28.8％ 20.6％
纤维素酶HGD45-PCL融合蛋白 74.9％ 65.2％ 49.0％
从表3的数据可以看出，添加纤维素酶HGD45-蛋白酶抑制肽PCL融合蛋白的加酶洗衣液在37℃放置48h、96h和168h后，其中的纤维素酶残留酶活均显著高于添加纤维素酶HGD45的加酶洗衣液。从而表明，本发明通过将纤维素酶HGD45与蛋白酶抑制肽PCL进行融合表达，能显著提高纤维素酶的蛋白酶抗性，有效提高洗涤剂中纤维素酶的稳定性，取得了意料不到的技术效果。
实施例3纤维素酶NT45与蛋白酶抑制肽MST融合表达提高其蛋白酶抗性
3.1纤维素酶NT45-蛋白酶抑制肽MST融合基因的获得与表达
申请人通过将纤维素酶NT45基因与类单胞菌(Alteromonas)来源的蛋白酶抑制肽MST基因进行融合，获得纤维素酶NT45-蛋白酶抑制肽MST融合基因。
所述纤维素酶NT45的氨基酸序列为SEQ ID NO：21，其编码核苷酸序列为SEQ ID NO：22。
所述蛋白酶抑制肽MST的氨基酸序列为SEQ ID NO：23，其编码核苷酸序列为SEQ ID NO：24；
由苏州金唯智生物科技有限公司分别合成上述纤维素酶NT45和蛋白酶抑制肽MST的基因。
具体操作如下：
以纤维素酶NT45基因为模板，利用引物9(如SEQ ID NO：27所示)和引物10(如SEQ ID NO：28所示)进行PCR扩增获得编码纤维素酶NT45的基因片段。
以蛋白酶抑制肽MST基因为模板，利用引物11(如SEQ ID NO：29 所示)和引物12(如SEQ ID NO：30所示)进行PCR扩增获得编码该蛋白酶抑制肽的基因片段。
将所述蛋白酶抑制肽MST的U-Clone片段与纤维素酶NT45的U-Clone片段按等摩尔比例混匀；分别利用引物9和引物12，以上述U-clone片段混合物为模板进行PCR，获得纤维素酶NT45-蛋白酶抑制肽MST融合基因，命名为NT45-MST，其核苷酸序列为SEQ ID NO：26，其编码的氨基酸序列为SEQ ID NO：25，其中蛋白酶抑制肽MST是与纤维素酶NT45的C端融合。PCR反应进行30个循环，每个循环为94℃40秒，62℃40秒，以及72℃1分钟。在72℃进行最后的延伸5分钟，将反应物冷冻至16℃。
将融合基因NT45-MST片段通过U-Clone试剂盒克隆至经Xba I和Mlu I双酶切后的木霉表达质粒pSC2G中，构建得到重组表达质粒，命名为NT45-MST-pSC2G。为确保准确，对若干克隆进行测序验证。
采用上述同样方法构建得到携带纤维素酶NT45基因的重组表达质粒，命名为NT45-pSC2G。
采用与实施例1.3同样的方法构建得到重组表达纤维素酶NT45-蛋白酶抑制肽MST融合蛋白的里氏木霉工程菌，将其命名为里氏木霉NT45-MST(Trichoderma reesei NT45-MST)。
作为对照，同时构建得到重组表达纤维素酶NT45的里氏木霉工程菌，命名为里氏木霉NT45(Trichoderma reesei NT45)。
采用与实施例1.4和实施例1.5同样的方法进行发酵，并将发酵上清液进行纤维素酶酶活测定。
3.2纤维素酶的蛋白酶抗性实验
实验组：用pH10.5乙酸缓冲液将蛋白酶稀释至3000U/ml，将133ul蛋白酶稀释液和67ul上述发酵上清液(纤维素酶酶活为100U/ml)，混匀后，37℃放置3天。
对照组：将133ul pH10.5乙酸缓冲液和67ul上述发酵上清液(纤维素酶酶活为100U/ml)，混匀后，37℃放置3天。
分别对实验组和对照组进行纤维素酶酶活检测。以对照组酶活计 100％，计算实验组纤维素酶的残留酶活，具体结果见表4。
表4纤维素酶在37℃条件下放置3天后的残留酶活
纤维素酶 纤维素酶残留酶活
纤维素酶NT45 77.3％
纤维素酶NT45-MST融合蛋白 85.9％
从表4的结果可以看出，37℃放置3天后，里氏木霉NT45-MST重组表达的纤维素酶NT45-蛋白酶抑制肽MST融合蛋白的残留酶活高达85.9％，显著高于纤维素酶NT45。从而说明，纤维素酶NT45-蛋白酶抑制肽MST融合蛋白的蛋白酶抗性得到明显提升。
实施例4纤维素酶SCD45与蛋白前导肽PPS融合表达提高其蛋白酶抗性
4.1蛋白前导肽PPS-纤维素酶SCD45融合基因的获得与表达
申请人通过纤维素酶SCD45与来源于解淀粉芽孢杆菌(Bacillus amyloliquefacien)的蛋白前导肽PPS基因进行融合，获得蛋白前导肽PPS-纤维素酶SCD45融合基因。
所述蛋白前导肽PPS的氨基酸序列为SEQ ID NO：31，其编码核苷酸序列为SEQ ID NO：32；
由苏州金唯智生物科技有限公司合成蛋白前导肽PPS的编码基因序列。
具体操作如下：
以蛋白前导肽PPS基因为模板，利用引物13(如SEQ ID NO：35所示)和引物14(如SEQ ID NO：36所示)进行PCR扩增获得编码该蛋白前导肽的基因片段。
以纤维素酶SCD45基因为模板，利用引物15(如SEQ ID NO：37所示)和引物16(如SEQ ID NO：38所示)进行PCR扩增获得编码该纤维素的基因片段。
将纯化后的蛋白前导肽PPS的U-Clone片段与纤维素酶SCD45的U-Clone片段按等摩尔比例混匀；分别利用引物13和引物16，以上述U-clone片段混合物为模板进行PCR，获得蛋白前导肽PPS-纤维素酶 SCD45融合基因，命名为PPS-SCD45，其核苷酸序列为SEQ ID NO：34，其编码的氨基酸序列为SEQ ID NO：33，其中蛋白前导肽PPS是与纤维素酶SCD45的N端融合。PCR反应进行30个循环，每个循环为94℃40秒，62℃40秒，以及72℃1分钟。在72℃进行最后的延伸5分钟，将反应物冷冻至16℃。
将融合基因PPS-SCD45片段通过U-Clone试剂盒克隆至经Xba I和Mlu I双酶切后的木霉表达质粒pSC2G中，构建得到重组表达质粒，命名为PPS-SCD45-pSC2G。为确保准确，对若干克隆进行测序验证。
采用与实施例1.3同样的方法构建得到重组表达蛋白前导肽PPS-纤维素酶SCD45融合蛋白的里氏木霉工程菌，将其命名为里氏木霉PPS-SCD45(Trichoderma reesei PPS-SCD45)。
以实施例1.3构建得到的重组表达纤维素酶SCD45的重组菌株里氏木霉SCD45为对照。
采用与实施例1.4和实施例1.5同样的方法进行发酵，并将发酵上清液进行纤维素酶酶活测定。
4.2纤维素酶的蛋白酶抗性实验
实验组：用pH10.5乙酸缓冲液将蛋白酶稀释至3000U/ml，将133ul蛋白酶稀释液和67ul上述里氏木霉发酵上清液(纤维素酶酶活为100U/ml)，混匀后，37℃放置3天。
对照组：将133ul pH10.5乙酸缓冲液和67ul上述里氏木霉发酵上清液(纤维素酶酶活为100U/ml)，混匀后，37℃放置3天。
分别对实验组和对照组进行纤维素酶酶活检测。以对照组酶活计100％，计算实验组纤维素酶的残留酶活。具体结果见表5。
表5纤维素酶在37℃条件下放置3天后的残留酶活
纤维素酶 纤维素酶残留酶活
纤维素酶SCD45 79.4％
PPS-纤维素酶SCD45融合蛋白 88.8％
从表5的结果可以看出，在蛋白酶存在的情况下，37℃放置3天后，里氏木霉PPS-SCD45重组表达的蛋白前导肽PPS-纤维素酶SCD45融合 蛋白的残留酶活高达88.8％，显著高于纤维素酶SCD45，其蛋白酶抗性得到明显提升。
实施例5脂肪酶与蛋白酶抑制肽SSI融合表达提高其蛋白酶抗性
除了纤维素酶外，本发明提供的方法同样适用于脂肪酶，可显著提高脂肪酶的蛋白酶抗性。
5.1脂肪酶TG-蛋白酶抑制肽SSI融合基因的获得与表达
申请人通过将脂肪酶TG的基因与枯草杆菌蛋白酶抑制肽SSI基因进行融合，获得脂肪酶TG-蛋白酶抑制肽SSI融合基因。
所述脂肪酶TG的氨基酸序列为SEQ ID NO：39，其编码核苷酸序列为SEQ ID NO：40。
由苏州金唯智生物科技有限公司合成脂肪酶TG的编码基因序列。
具体操作如下：
以脂肪酶TG基因为模板，利用引物17(如SEQ ID NO：43所示)和引物18(如SEQ ID NO：44所示)进行PCR扩增获得编码该脂肪酶的基因片段。
以枯草杆菌蛋白酶抑制肽SSI基因为模板，利用引物19(如SEQ ID NO：45所示)和实施例1.1所述的引物4(如SEQ ID NO：10所示)进行PCR扩增获得编码该蛋白酶抑制肽的基因片段。
将上述蛋白酶抑制肽SSI的U-Clone片段与脂肪酶TG的U-Clone片段按等摩尔比例混匀；分别利用引物17和实施例1.1所述的引物4，以上述U-clone片段混合物为模板进行PCR，获得脂肪酶TG-蛋白酶抑制肽SSI-融合基因，命名为TG-SSI，其核苷酸序列为SEQ ID NO：42，其编码的氨基酸序列为SEQ ID NO：41，其中蛋白酶抑制肽SSI是与脂肪酶TG的C端融合。PCR反应进行30个循环，每个循环为94℃40秒，62℃40秒，以及72℃1分钟。在72℃进行最后的延伸5分钟，将反应物冷冻至16℃。
将融合基因TL-SSI片段通过U-Clone试剂盒克隆至经Xba I和Mlu I双酶切后的木霉表达质粒pSC2G中，构建得到重组表达质粒，命名为 TG-SSI-pSC2G。为确保准确，对若干克隆进行测序验证。
采用上述同样方法构建得到携带脂肪酶TG基因的重组表达质粒，命名为TG-pSC2G。
采用与实施例1.3同样的方法构建得到重组表达脂肪酶TG-蛋白酶抑制肽SSI融合蛋白的里氏木霉工程菌，将其命名为里氏木霉TGS(Trichoderma reesei TGS)。
作为对照，同时构建得到重组表达脂肪酶TG的里氏木霉工程菌，命名为里氏木霉TG(Trichoderma reesei TG)
采用与实施例1.4和实施例1.5同样的方法进行发酵，并将发酵上清液进行脂肪酶酶活测定。
5.2脂肪酶的蛋白酶抗性实验
将里氏木霉TG和里氏木霉TGS的发酵上清液分别用pH7.5乙酸缓冲液稀释至3000U/ml；然后分别取9ml稀释后的发酵上清液，1ml pH10.5、蛋白酶酶活为5000U/ml的蛋白酶，将两者充分混匀，得混合酶液；将混合酶液在37℃放置一周，分别在第2d、5d、6d时取样进行脂肪酶酶活检测。以混合酶液中脂肪酶的初始酶活计100％，分别计算脂肪酶的残留酶活，具体结果见表6。
表6脂肪酶在37℃条件下的残留酶活
脂肪酶 2d 5d 6d
脂肪酶TG 78.5％ 50.1％ 34.6％
脂肪酶TG-SSI融合蛋白 105.7％ 76.2％ 64.6％
从表6的数据可以看出，与脂肪酶TG相比，在蛋白酶存在的情况下，脂肪酶TG-蛋白酶抑制肽SSI融合蛋白在37℃条件下放置2d、5d和6d后，其残留酶活均得到显著提高。其中，在放置6d后，融合蛋白的残留酶活高达64.6％，比脂肪酶TG提高了86.7％，效果极为显著。
上述结果表明，本发明通过将脂肪酶与蛋白酶抑制肽进行融合表达，能显著提高脂肪酶的蛋白酶抗性，有效降低蛋白酶对脂肪酶的降解作用，取得了意料不到的技术效果。本发明提供的具有蛋白酶抗性的脂肪酶更适合在洗涤领域中的应用。
除了纤维素酶和脂肪酶外，本发明提供的方法同样适用于其他洗涤用酶，例如角质酶、淀粉酶、果胶酶、甘露聚糖酶、阿拉伯糖酶、半乳聚糖酶、木聚糖酶、漆酶、过氧化物酶等，均能显著提高所述酶的蛋白酶抗性，有效降低蛋白酶对酶的降解作用，有利于实现所述酶在洗涤剂中与蛋白酶的稳定共存。
本发明所述方法并不仅限于将洗涤用酶与蛋白酶抑制肽SSI、蛋白酶抑制肽PCL、蛋白酶抑制肽MST或蛋白前导肽PPS进行融合表达，也可以与Bowman-Birk蛋白酶抑制肽或芽孢杆菌来源的蛋白前导肽进行融合表达，提高洗涤用酶的蛋白酶抗性。而且，本发明所述方法也不仅限于将洗涤用酶与单一的蛋白酶抑制肽进行融合表达，还可以将洗涤用酶同时与两种或三种蛋白酶抑制肽进行融合表达，提高洗涤用酶的蛋白酶抗性。所述融合也不仅限于蛋白酶抑制肽在洗涤用酶的N端融合或C端融合，还可以在中间融合。
以上对本发明所提供的一种生产具有蛋白酶抗性的洗涤用酶的方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种生产具有蛋白酶抗性的洗涤用酶的方法，其特征在于，所述的方法包括如下步骤：
    (a)构建融合多核苷酸序列，该序列包含编码洗涤用酶的基因和编码蛋白酶抑制肽的基因；
    (b)将序列引入宿主细胞；
    (c)培养宿主细胞，其中细胞表达序列并产生洗涤用酶-蛋白酶抑制肽融合蛋白，所述融合蛋白即为具有蛋白酶抗性的洗涤用酶。
  2. 如权利要求1所述的方法，其特征在于，所述的融合蛋白中洗涤用酶与蛋白酶抑制肽是以共价方式融合。
  3. 如权利要求1或2所述的方法，其特征在于，所述的洗涤用酶为纤维素酶、脂肪酶，角质酶，淀粉酶，糖酶，果胶酶，甘露聚糖酶，阿拉伯糖酶，半乳聚糖酶，木聚糖酶，漆酶或过氧化物酶中的任意一种。
  4. 如权利要求3所述的方法，其特征在于，所述的洗涤用酶为纤维素酶。
  5. 如权利要求3所述的方法，其特征在于，所述的洗涤用酶为脂肪酶。
  6. 如权利要求1-5任一所述的方法，其特征在于，所述的蛋白酶抑制肽为链霉菌(Streptomyces sp.)来源的蛋白酶抑制肽SSI、Bowman-Birk蛋白酶抑制肽、大麦(Barley)来源的蛋白酶抑制肽PCL、类单胞菌(Alteromonas)来源的蛋白酶抑制肽MST或芽孢杆菌(Bacillus sp.)来源的蛋白前导肽中的任意一种或两种或三种。
  7. 如权利要求1-6任一所述的方法，其特征在于，所述的蛋白酶抑制肽可在洗涤用酶的N端融合、C端融合或中间融合。
  8. 如权利要求1-7任一所述的方法，其特征在于，所述的宿主细胞为芽孢杆菌(Bacillus sp.)，曲霉(Aspergillus sp.)，木霉(Trichoderma sp.)，毕赤酵母(Pichia pastoris)，酿酒酵母(Saccharomyces cerevisiae)或大肠杆菌(Escherichia coli)中的任意一种。
  9. 如权利要求8所述的方法，其特征在于，所述的宿主细胞为里氏木霉(Trichoderma reesei)。
  10. 权利要求1-9任一所述的方法生产的具有蛋白酶抗性的洗涤用酶。
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