WO2020238339A1 - 一种脂肪酶突变体及其在去污方面的应用 - Google Patents

一种脂肪酶突变体及其在去污方面的应用 Download PDF

Info

Publication number
WO2020238339A1
WO2020238339A1 PCT/CN2020/079438 CN2020079438W WO2020238339A1 WO 2020238339 A1 WO2020238339 A1 WO 2020238339A1 CN 2020079438 W CN2020079438 W CN 2020079438W WO 2020238339 A1 WO2020238339 A1 WO 2020238339A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipase
seq
mutant
amino acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/079438
Other languages
English (en)
French (fr)
Inventor
王睿
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Publication of WO2020238339A1 publication Critical patent/WO2020238339A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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)

Definitions

  • the invention relates to a lipase mutant and its application in decontamination, and belongs to the technical field of enzyme engineering.
  • Lipase (Lipase, EC 3.1.1.3, glyceride hydrolase) is a general term for a class of enzymes that hydrolyze fat. In some reaction systems, lipase can catalyze hydrolysis, alcoholysis, transesterification, hydrogenolysis, and indirect redox transesterification. In addition, lipase also has the ability to hydrolyze racemic mixtures and synthesize esters and peptide bonds. Therefore, lipase has important applications in many fields, such as:
  • Lipase can be used in oil processing. Lipase hydrolysis of oil can be carried out under normal temperature and pressure, so it will not denature the highly unsaturated fatty acids and tocopherols in the oil;
  • Lipase can be used in dairy processing.
  • the application of lipase to hydrolyze milk fat in dairy products can enhance the flavor of cheese, milk powder and cream, promote the maturation of cheese, and improve the quality of dairy products;
  • Lipase can be used in the processing of pasta. Adding lipase to the pasta can improve the elasticity of the product to varying degrees. The dough is thin and transparent but not easy to break, improving the taste. In addition, adding lipase to the pasta, It can release monoglycerides, delay spoilage, and improve the fresh-keeping ability of pasta;
  • Lipase can be used in meat processing. Adding lipase to meat can effectively degrade fat in meat and make fat-free meat;
  • Lipase can be used in drug synthesis. Microbial lipase is used to enrich polyunsaturated fatty acids from animals and plants. Free polyunsaturated fatty acids and their mono- and double-glycerides are used to produce various drugs. Lipase is used in the synthesis of drugs such as diazepam, naproxen, and captopril;
  • Lipase can be used in diesel synthesis. Enzymatic synthesis of diesel uses lipase to catalyze the transesterification reaction between animal and vegetable oils and low-carbon alcohols to generate corresponding fatty acid esters. Compared with traditional chemically catalyzed diesel synthesis, this method It has the advantages of simple purification process, less investment equipment, low energy consumption, low pollution, and low requirements for oil and fat raw materials, which has attracted people's general attention.
  • Lipase can be used in polymer synthesis. At present, artificially synthesized polymer materials cause serious environmental pollution. Therefore, the application of enzyme-catalyzed synthesis of this molecular material that can be degraded by microorganisms has attracted more and more attention;
  • Lipase can be used in the synthesis of chiral compounds. Lipase can work in non-hydrolyzed substances. Its enzymatic reaction in non-hydrolyzed substances can synthesize optically active alcohols, fatty acids, lactones and other pharmaceuticals and pesticides. body.
  • Lipase can be used in leather production. In the leather processing process, an important step is to remove the residual fat and protein closely connected with the leather or fur. Among them, the intradermal fat is protected by the fat film. It is difficult to be removed by chemical treatment methods such as lime treatment. Using the decomposition effect of lipase or mixed use with protease, it can easily enter the fat and emulsify it, which obviously improves the quality of leather;
  • Lipase can be used in the preparation of detergents.
  • the application of genetic engineering to develop detergent lipase is one of the most successful applications of modern biotechnology in large-scale industrialization;
  • Lipase can be used in paper synthesis.
  • "Qiazhi” is the general term for all hydrophobic components in wood, including triglycerides and wax fats. It will deposit on the drying column and affect the quality and production capacity of paper. Enzymes can remove the "fat” in the pulp and improve the quality of the paper.
  • lipase has certain requirements on the stability of lipase.
  • higher temperature is often used, which has higher requirements for the thermal stability of lipase.
  • the leather processing and papermaking process need to use strong alkali, which has higher requirements for the pH stability of lipase under alkaline conditions, and most of the existing lipases have poor stability, for example, from The lipase RAL of Rhizopus arrhizus has an optimal temperature of only 35°C. After heat treatment at 50°C for 10 minutes, only 40% of the enzyme activity remains. After 40 minutes of treatment, the enzyme activity will be completely lost. Under the condition of pH greater than 9.5, it will lose more than 70% of its activity, which undoubtedly greatly limits the further development of lipase in the above-mentioned applications.
  • the technical problem to be solved by the present invention is to provide a lipase (Lipase, EC 3.1.1.3) with stability, especially thermal stability and strong pH stability under alkaline conditions.
  • the present invention provides a lipase mutant, the lipase mutant is obtained by modifying the starting amino acid sequence of the lipase at positions 151, 235, and 171 of SEQ ID NO.1
  • the amino acids at position, 343, 275, 332, 310 or 180 are mutated;
  • the enzyme mutant is obtained by simultaneously mutating the 316th and 340th amino acids of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1;
  • the enzyme mutant is obtained by simultaneously mutating the 178th and 238th amino acids of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1;
  • the enzyme mutant is obtained by simultaneously mutating amino acids at positions 316, 340, 178, and 238 of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1;
  • the enzyme mutant is obtained by simultaneously mutating amino acids 235, 343, 332, and 310 of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1;
  • the enzyme mutant is obtained by simultaneously mutating amino acids 151, 171, 275, and 180 of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1;
  • the enzyme mutant is obtained by changing the starting amino acid sequence of the lipase shown in SEQ ID NO. 1 at positions 235, 343, 332, 310, 151, 171, and The 275th and 180th amino acids were simultaneously mutated;
  • the enzyme mutant is obtained by changing the starting amino acid sequence of the lipase shown in SEQ ID NO. 1 at positions 235, 343, 332, 310, 151, 171, and The 275th, 180th, 316th, 340th, 178th and 238th amino acids were simultaneously mutated.
  • the lipase mutant is obtained by mutating serine at position 151 of the lipase shown in SEQ ID NO. 1 to asparagine (S151N), named Is L1;
  • the lipase mutant is obtained by mutating the 235th serine of the lipase whose starting amino acid sequence is as shown in SEQ ID NO. 1 to alanine (S235A), which is named L2;
  • the lipase mutant is obtained by mutating lysine at position 171 of the lipase whose starting amino acid sequence is as shown in SEQ ID NO. 1 to arginine (K171R), and is named L3;
  • the lipase mutant is obtained by mutating serine 343 of the lipase whose starting amino acid sequence is as shown in SEQ ID NO. 1 to tyrosine (S343Y), and named L4;
  • the lipase mutant is obtained by mutating the glycine at position 275 of the lipase shown in SEQ ID NO. 1 to alanine (G275A), which is named L5;
  • the lipase mutant is obtained by mutating glutamine at position 332 of the lipase whose starting amino acid sequence is as shown in SEQ ID NO. 1 to phenylalanine (Q332F), and is named L6;
  • the lipase mutant is obtained by mutating the 310th aspartic acid of the lipase whose starting amino acid sequence is as shown in SEQ ID NO. 1 to valine (D310V), which is named L7;
  • the lipase mutant is obtained by mutating leucine at position 180 of the lipase whose starting amino acid sequence is as shown in SEQ ID NO. 1 to histidine (L180H), and is named L8;
  • the lipase mutant is made by mutating the 316th phenylalanine to cysteine and the 340th glycine to cysteine of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1
  • the obtained (F316C ⁇ G340C) is named L9;
  • the lipase mutant is obtained by mutating the 178th serine to cysteine and the 238th glutamine to cysteine of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1 (S178C ⁇ Q238C), named L10;
  • the lipase mutant is made by mutating the 316th phenylalanine to cysteine and the 340th glycine to cysteine of the lipase whose starting amino acid sequence is as shown in SEQ ID NO.1 ,
  • the mutation of serine at position 178 to cysteine and the mutation of glutamine at position 238 to cysteine (F316C ⁇ G340C ⁇ S178C ⁇ Q238C), named L11;
  • the lipase mutant is made by mutating the 235th serine to alanine, the 343th serine to tyrosine, and the 332th lipase whose starting amino acid sequence is as shown in SEQ ID NO.1.
  • the aminoamide was mutated to phenylalanine and the 310th aspartic acid was mutated to valine (S235A ⁇ S343Y ⁇ Q332F ⁇ D310V), named L12;
  • the lipase mutant is made by mutating the 151st serine to asparagine, the 171st lysine to arginine, and the 275th lipase whose starting amino acid sequence is shown in SEQ ID NO.1. Mutation of position glycine to alanine and mutation of position 180 to histidine (S151N ⁇ K171R ⁇ G275A ⁇ L180H), named L13;
  • the lipase mutant is made by mutating the 235th serine to alanine, the 343th serine to tyrosine, and the 332th lipase whose starting amino acid sequence is as shown in SEQ ID NO.1.
  • Aminoamide was mutated to phenylalanine, aspartic acid at position 310 was mutated to valine, serine at position 151 was mutated to asparagine, lysine at position 171 was mutated to arginine, and glycine at position 275 was mutated to Alanine and leucine at position 180 were mutated to histidine (S235A ⁇ S343Y ⁇ Q332F ⁇ D310V ⁇ S151N ⁇ K171R ⁇ G275A ⁇ L180H), named L14;
  • the lipase mutant is made by mutating the 235th serine to alanine, the 343th serine to tyrosine, and the 332th lipase whose starting amino acid sequence is as shown in SEQ ID NO.1.
  • Aminoamide was mutated to phenylalanine, aspartic acid at position 310 was mutated to valine, serine at position 151 was mutated to asparagine, lysine at position 171 was mutated to arginine, and glycine at position 275 was mutated to Alanine, leucine at position 180 to histidine, phenylalanine at position 316 to cysteine, glycine at position 340 to cysteine, serine at position 178 to cysteine Acid and glutamine at position 238 were mutated to cysteine (S235A ⁇ S343Y ⁇ Q332F ⁇ D310V ⁇ S151N ⁇ K171R ⁇ G275A ⁇ L180H ⁇ F316C ⁇ G340C ⁇ S178C ⁇ Q238C), named L15.
  • the present invention also provides a gene encoding the above-mentioned lipase mutant.
  • the present invention also provides a recombinant plasmid which carries the above-mentioned gene.
  • the expression vector of the recombinant plasmid is pPIC9K vector, pPIC3.5K vector, pPICZ ⁇ vector or pPICZ vector.
  • the present invention also provides a host cell which carries the aforementioned gene or the aforementioned recombinant plasmid.
  • the host cell is a fungus or bacteria.
  • the host cell is Pichia pastoris.
  • the present invention also provides a detergent, which contains the above-mentioned lipase mutant.
  • the detergent contains the aforementioned lipase mutant, a surfactant, and a buffer.
  • the surfactant may be sodium dodecyl sulfate (SDS), sodium ethoxylated alkyl sulfate (AES), fatty alcohol polyoxyethylene ether (AEO-9) And/or disodium lauryl sulfosuccinate monoester (DLS).
  • SDS sodium dodecyl sulfate
  • AES sodium ethoxylated alkyl sulfate
  • AEO-9 fatty alcohol polyoxyethylene ether
  • DLS disodium lauryl sulfosuccinate monoester
  • the buffer may be phosphate buffer, Tris buffer, citrate buffer or sodium carbonate buffer.
  • the concentration of the above-mentioned lipase mutant in the detergent is 10-400 U/L.
  • the present invention also provides a decontamination method, the method is to add the above-mentioned lipase mutant to the article to be decontaminated; or, the method is to add the above-mentioned detergent to the article to be decontaminated.
  • the present invention also provides the above-mentioned lipase mutant or the above-mentioned gene or the above-mentioned recombinant plasmid or the above-mentioned host cell in oil processing, dairy processing, pasta processing, meat processing, drug synthesis, diesel synthesis, polymer synthesis, chiral compound synthesis, Leather production, detergent preparation or papermaking applications.
  • the present invention also provides the application of the aforementioned lipase mutant or the aforementioned detergent or the aforementioned decontamination method in decontamination.
  • the thermal stability of the lipase mutant of the present invention is significantly improved compared with the wild type.
  • the optimal temperature of L11 can reach 45°C, which is 5°C higher than that of the wild type, and the T m value of L11 can reach 49.2 °C, 4.2°C higher than the wild type.
  • the half-life of L11 at 65°C is 3.9min, which is 4.59 times that of the wild type; the optimum temperature of L12 is 45°C, which is 5°C higher than the wild type, and the T m value can reach 50.3 °C, 5.3°C higher than the wild type, the half-life of L12 at 65°C is 4.9min, which is 5.76 times that of the wild type; the optimum temperature of L13 is 43°C, which is 3°C higher than the wild type, and the T m value can reach 53.8 °C, 8.8°C higher than the wild type.
  • the half-life of L13 at 65°C is 7.6min, which is 8.94 times that of the wild type; the optimum temperature of L14 is 48°C, which is 8°C higher than the wild type, and the T m value can reach 60.8 °C, 15.8°C higher than the wild type, the half-life of L14 at 65°C is 19.8min, which is 23.29 times that of the wild type; the optimum temperature of L15 can reach 50°C, which is 10°C higher than the wild type, and the T m value can reach At 68.8°C, it is 23.8°C higher than the wild type.
  • the half-life of L15 at 65°C is 56.2min, which is 66.11 times that of the wild type.
  • the thermal stability of L1 ⁇ L10 is also improved compared with the wild type;
  • the pH stability of the lipase mutant of the present invention has been significantly improved compared with the wild type.
  • the optimal pH of L9 is increased from 8.0 of the wild type to 8.5;
  • the optimal pH of L11 is increased from 8.0 of the wild type to 8.0 9.0,
  • the enzyme activity of L11 under the condition of pH 10 can reach 80.5% of the enzyme activity under the optimum pH condition, which is 10.73 times higher than that of the wild type.
  • the residual enzyme activity of L11 after 30 minutes of treatment at pH 10 It can reach 90.8%, which is 2.20 times higher than that of the wild type; the optimum pH of L15 is increased from 8.0 of the wild type to 9.0; the enzyme activity of L15 under the condition of pH 10 can reach 75.2 of the enzyme activity under the condition of optimum pH %, which is 10.02 times higher than the wild type.
  • the residual enzyme activity of L15 treated at pH 10 for 30 minutes can reach 100%, which is 2.43 times higher than the wild type;
  • the lipase mutant of the present invention has good washing performance.
  • the oil removal rate of using L9 to wash oily soiled cloth can reach 92.8%; the oil removal rate of using L11 to wash oily soiled cloth can reach 93.5%; The oil removal rate can reach 93.6%;
  • the lipase mutant of the present invention has good thermal stability, good pH stability, and good washing performance. Therefore, the lipase mutant of the present invention is used in decontamination, oil processing, dairy processing, pasta processing, meat processing, It has important applications in drug synthesis, diesel synthesis, polymer synthesis, chiral compound synthesis, leather production, detergent preparation and papermaking.
  • Figure 1 PCR amplification electrophoresis diagram of the whole plasmid containing recombinant plasmids encoding different mutant genes
  • M is the DL10000 DNA molecular weight standard
  • 1 is the result of PCR amplification and electrophoresis of the whole plasmid of the recombinant plasmid containing the gene encoding mutant S151N
  • 2 is the result of PCR amplification and electrophoresis of the whole plasmid containing the recombinant plasmid encoding the gene of S235A
  • 3 is the result of PCR amplification and electrophoresis of the whole plasmid of the recombinant plasmid containing the gene encoding the mutation K171R.
  • FIG. 2 SDS-PAGE electrophoresis diagram of protein purification of the fermentation supernatant of Pichia pastoris engineered bacteria containing genes encoding different mutants;
  • M is the DL10000 DNA molecular weight standard
  • 1 is the result of protein purification SDS-PAGE electrophoresis of the fermentation supernatant of the engineered Pichia strain containing the gene encoding the mutant S151N
  • 2 is the Pichia pastoris containing the gene encoding the mutant S235A
  • 3 is the SDS-PAGE electrophoresis results of the protein purification of the fermentation supernatant of the engineered Pichia pastoris containing the gene encoding mutation K171R.
  • FIG. 3 Relative enzyme activity of wild type, L1, L2, L3, L4, L5, L6, L7, L8, L9, L10 at different temperatures.
  • Figure 4 Relative enzyme activity of wild type, L11, L12, L13, L14, L15, L16 and L17 at different temperatures.
  • Figure 5 Relative enzyme activity of wild type, L1, L2, L3, L4, L5, L6, L7, L8, L9, L10 at different pH.
  • Figure 6 Relative enzyme activity of wild type, L11, L12, L13, L14, L15, L16 and L17 at different pH.
  • the E. coli JM109 involved in the following examples was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; the Pichia pastoris GS115 involved in the following examples was purchased from Invitrogen; the pPIC9K vector involved in the following examples was purchased from Invitrogen .
  • lipase enzyme activity is defined as: reaction under the conditions of pH 8.0 and 40°C, the amount of enzyme that produces 1 ⁇ mol p-nitrophenol per minute is one enzyme activity unit (1U).
  • LB liquid medium m/m: peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0.
  • YPD liquid medium yeast extract 1%, tryptone 2%, glucose 2%, autoclaved at 121°C for 20 min.
  • YPD plate yeast extract 1%, tryptone 2%, glucose 2%, agar 2%, autoclave at 121°C for 20 min.
  • MD plate (m/m): Amino-free yeast nitrogen source 1.34%, biotin 4 ⁇ 10 -5 %, glucose 2%, agar 2%.
  • MM plate (m/m): Amino-free yeast nitrogen source 1.34%, biotin 4 ⁇ 10 -5 %, methanol 0.5%, agar 2%.
  • BMGY medium yeast extract 1%, tryptone 2%.
  • the gene encoding the lipase whose amino acid sequence is shown in SEQ ID NO.1 was amplified from Rhizopus chinonsis; the obtained gene proRCL was digested with Not I and EcoR I and connected with pPIC9K vector to obtain the recombinant Plasmid pPIC9K-proRCL; the recombinant plasmid pPIC9K-proRCL was transformed into E. coli JM109 to obtain E. coli JM109/pPIC9K-proRCL; E.
  • coli JM109/pPIC9K-proRCL was spread on the LB plate (containing 100mg/ L Kanamycin), after culturing at 37°C for 10 hours, the positive clones were screened and confirmed by sequencing to obtain the correct recombinant plasmid pPIC9K-proRCL; after linearization of the recombinant plasmid pPIC9K-proRCL with Sal I, it was electrotransformed into Pichi Pichia GS115/pPIC9K-proRCL was obtained from yeast GS115; Pichia GS115/pPIC9K-proRCL was coated on MD plate (containing 100mg/L kanamycin) and cultured at 37°C After 10 hours, the genome was extracted and the positive clones were identified by PCR, and positive transformants were obtained; the positive transformants were inoculated into a 250 mL shake flask containing 25 mL BMGY medium, and cultured at 30°C and 250 r/min to
  • the amplification primers are as follows:
  • Rm 5'-AATTCCAGTGCGGCCGCTTAATGATGATGATGATGATGAGAAGAACCCAAACAGCTTCCTTCGTTAATACC-3' (SEQ ID NO. 3).
  • mutants S151N, S235A, K171R, S343Y, G275A, Q332F, D310V, L180H, F316C ⁇ G340C, S178C ⁇ Q238C, F316C ⁇ G340C ⁇ S178C ⁇ Q238C, S235A ⁇ S343Y ⁇ Q332F ⁇ D310V, S151N ⁇ K171R ⁇ G275A ⁇ L180H, S235A ⁇ S343Y ⁇ Q332F ⁇ D310V ⁇ S151N ⁇ K171R ⁇ G275A ⁇ L180H, S235A ⁇ S343Y ⁇ Q332FN ⁇ D310V ⁇ D310V K171R ⁇ G275A ⁇ L180H ⁇ F316C ⁇ G340C ⁇ S178C ⁇ Q238C, D159R and T319V;
  • mutant primers are:
  • S151N-Fm 5'-CTGCTTACTGTCGT AAC GTCGTTCCAGGTACC-3' (SEQ ID NO. 4);
  • S151N-Rm 5'-GGTACCTGGAACGAC GTT ACGACAGTAAGCAG-3' (SEQ ID NO. 5);
  • S235A-Fm 5'-GTTTCCTTTCC GCA TACAACCAAGTTGTCAAAG-3' (SEQ ID NO.6);
  • S235A-Rm 5'-CTTTGACAACTTGGTTGTA TGC GGAAAGGAAAC-3' (SEQ ID NO. 7);
  • K171R-Fm 5'-GTCTCAAGTATGTTCCTGATGGT AGG CTTATCAAGAC-3' (SEQ ID NO. 8);
  • K171R-Rm 5'-GTCTTGATAAG CCT ACCATCAGGAACATACTTGAGAC-3' (SEQ ID NO. 9);
  • S343Y-Fm 5'-CCCGGTGTCGAA TAT TGGATCAAGGAAGAC-3' (SEQ ID NO. 10);
  • S343Y-Rm 5'-GGGGTGAAGATAACCGGCGGCTTGAGGAGG-3' (SEQ ID NO.11);
  • G275A-Fm 5'-GCCTTGCTCGCT GCC ATGGATCTCTACCAACGTG-3' (SEQ ID NO.12);
  • G275A-Rm 5'-CACGTTGGTAGAGATCCAT GGC AGCGAGCAAGGC-3' (SEQ ID NO. 13);
  • Q332F-Fm 5'-CCCTCATGTTCCTCCT TTC GCCTTCGGTTATCTTC-3' (SEQ ID NO.14);
  • Q332F-Rm 5'-GAAGATAACCGAAGGC GAA AGGAGGAACATGAGGG-3' (SEQ ID NO. 15);
  • D310V-Fm 5'-CATTCGCTTACTACGTC GTC AGCACCGGAATTCCC-3' (SEQ ID NO. 16);
  • D310V-Rm 5'-GGGAATTCCGGTGCTGAC GAC GTAGTAAGCGAATG-3' (SEQ ID NO. 17);
  • L180H-Fm 5'-GACCTTCACTTCTCTT CAC ACTGATACCAATGGC-3' (SEQ ID NO.18);
  • L180H-Rm 5'-GCCATTGGTATCAGT GTG AAGGCAAGTGAAGGTC-3' (SEQ ID NO.19);
  • F316C-Fm 5'-CACCGGAATTCCC TGC CACCGTACCGTTCAC-3' (SEQ ID NO.20);
  • F316C-Rm 5'-GTGAACGGTACGGTG GCA GGGAATTCCGGTG-3' (SEQ ID NO.21);
  • G340C-Fm 5'-GTTATCTTCACCCC TGC GTCGAATCTTGGATC-3' (SEQ ID NO. 22);
  • G340C-Rm 5'-GATCCAAGATTCGAC GCA GGGGTGAAGATAAC-3' (SEQ ID NO.23);
  • S178C-Fm 5'-GACCTTCACT TGC CTTCTCACTGATACCAATG-3' (SEQ ID NO. 24);
  • S178C-Rm 5'-CATTGGTATCAGTGAGAAG GCA AGTGAAGGTC-3' (SEQ ID NO. 25);
  • Q238C-Fm 5'-CCTTTCCTCATACAAC TGC GTTGTCAAAGACTACTTCCCCG-3' (SEQ ID NO.26);
  • Q238C-Rm 5'-CGGGGAAGTAGTCTTTGACA ACG CAGTTGTATGAGGAAAGG-3' (SEQ ID NO.27);
  • D159R-Fm 5'-GGTACCAAGTGG CGA TGTAAGCAATGTCTC-3' (SEQ ID NO. 28);
  • D159R-Rm 5'-GAGACATTGCTTACA TCG CCACTTGGTACC-3' (SEQ ID NO. 29);
  • T319V-Fm 5'-CCCTTCCACCGT GTC GTTCACAAGCGTG-3' (SEQ ID NO.30);
  • T319V-Rm 5'-CACGCTTGTGAAC GAC ACGGTGGAAGGG-3' (SEQ ID NO.31);
  • the PCR system is: 10 ⁇ M forward primer and reverse primer 1 ⁇ L each, dNTPMix 2 ⁇ L, 5 ⁇ PS Buffer 2.5 ⁇ L, PrimeStar GXL polymerase 0.5 ⁇ L, template 0.5 ⁇ L, add double distilled water to make up 25 ⁇ L;
  • the PCR conditions were: 95°C 2min; 95°C 20s, 55-63°C 1min, 68°C 11min, total 18 cycles; 68°C 5min.
  • the PCR product was detected by 1% agarose gel electrophoresis, the PCR product after the detection was digested with Dpn I, and then transformed into E. coli JM109 to obtain the E. coli engineered bacteria containing the gene encoding the mutant;
  • the engineered bacteria were spread on LB plates (containing 100mg/L kanamycin), and after culturing at 37°C for 10 hours, positive clones were screened and sequenced for confirmation, and the correct recombinant plasmids (containing S151N, S235A, K171R encoding mutants) were obtained.
  • the full plasmid PCR amplification electrophoresis diagram of the recombinant plasmid of the gene is shown in Figure 1); After linearizing the recombinant plasmid with Sal I, it is electrotransformed into Pichia pastoris GS115 to obtain the Pichia pastoris engineered bacteria containing the gene encoding the mutant; Pichia pastoris engineered bacteria were spread on MD plates (containing 100mg/L kanamycin), and after culturing at 37°C for 10 hours, the genome was extracted and the positive clones were identified by PCR to obtain positive transformants; the positive transformants were inoculated on In a 250 mL shake flask containing 25 mL of BMGY medium, culture at 30°C and 250 r/min to an OD 600 of 4.0 to obtain a bacterial solution; centrifuge the bacterial solution at 8 000 r/min for 5 minutes, collect the bacterial cells, and re-weight with 100 mL of BMMY medium Suspend the bacteria to an
  • Example 3 Study on the enzymatic properties of different lipases
  • the optimal temperature of the wild type is 40°C; the optimal temperature of L1, L2, L3, L4, L5, L6, L8, L9, L10, L16 and L17 is also 40°C, and Compared with wild type, there is no increase; but the optimum temperature of L7 is increased to 45°C; the optimum temperature of L13 is increased to 43°C; the optimum temperature of L11 and L12 is increased to 45°C; the optimum temperature of L14 is increased to 48°C; The optimal temperature of L15 increased the most, which was 50°C, which was 10°C higher than that of the wild type.
  • Example 2 The wild type obtained in Example 1 and the L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, and L17 obtained in Example 2 were respectively before Treat in a constant temperature water bath at 40°C ⁇ 70°C for 30min, then ice bath for 20min, and finally measure the lipase activity at 40°C.
  • the heat treatment temperature is the enzyme denaturation temperature (See Table 1 for test results).
  • the denaturation temperature of L1 ⁇ L15 has been improved to different degrees, among which the higher degree of improvement is L11 ⁇ L15, and the denaturation temperature is 49.2°C, 50.3°C, 53.8°C, 60.8. °C and 68.8°C, which are 4.2°C, 5.3°C, 8.8°C, 15.8°C and 23.8°C respectively higher than the wild type; while the denaturation temperature of L16 is 40.1°C, and the denaturation temperature of L17 is 38.5°C, which are respectively lower than the wild type by 4.9 °C and 6.5°C.
  • T m Numbering Denaturation temperature
  • Example 2 Place the wild type obtained in Example 1 and L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16 and L17 obtained in Example 2 respectively In a constant temperature water bath at 65°C, samples were taken at regular intervals to measure the residual enzyme activity and compare its thermal stability (see Table 2 for the test results).
  • the half-lives of L1 ⁇ L15 at 65°C have different degrees of prolongation.
  • the longer ones are L11 ⁇ L15, and the half-lives are 3.9min, 4.9min, 7.6min, 19.8min and 56.2min, respectively 4.59 times, 5.76 times, 8.94 times, 23.29 times and 66.11 times longer than the wild type; while the half-life of L16 is 0.75min, and the half-life of L17 is 0.5min, which are 11.76% lower than the wild type respectively And 41.18%.
  • citric acid buffer (pH 6) with a concentration of 0.05mol/L
  • phosphate buffer (pH 6-8) with a concentration of 0.05mol/L
  • Tris-HCl buffer (pH 6) with a concentration of 0.05mol/L.
  • Figure 5-6 shows that the optimal pH of the wild type is 8.0; the optimal pH of L1, L2, L3, L4, L5, L6, L7, L8, L10, L12, L13, L14, L16 and L17 is also 8.0 Compared with the wild type, the optimal pH of L9 both rises to 8.5, which is 0.5 higher than that of the wild type; the optimal pH of both L11 and L15 rises to 9.0, which is 1 higher than the wild type, and prefers alkaline conditions .
  • Table 3 The ratio of the enzyme activity of different lipases under the condition of pH 10 to the enzyme activity under the optimal pH condition
  • L1 8.5% L2 16.5% L3 17.6% L4 21.0% L5 24.5% L6 9.9% L7 17.8% L8 15.6% L9 90.6% L10 13.4% L11 80.5% L12 5.7% L13 30.3% L14 25.6% L15 75.2% L16 7.2% L17 6.9%
  • the residual enzyme activity of L5 treated at pH 10 for 30 minutes is 65.0%, which is 57.3% higher than the wild type; the residual enzyme activity of L7 treated at pH 10 for 30 minutes is 55.3% Compared with the wild type, the residual enzyme activity of L9 was 87.4% after 30min treatment at pH 10, which was 112% higher than that of the wild type; L11 was the residual enzyme activity after 30min treatment at pH 10 The activity is 98.8%, which is 139.8% higher than that of the wild type; the residual enzyme activity of L12 treated at pH 10 for 30 min is 55.8%, which is 35.4% higher than that of the wild type; L13 and L14 are at pH 10 After 30 minutes of treatment, the residual enzyme activity was 65.2% and 65.8%, which were 58.3% and 59.7% higher than the wild type respectively; the enzyme activity of L15 was treated at pH 10 for 30 minutes without loss, and remained 100%, which was higher than the wild type.
  • a phosphate buffer (pH 9.0) with a concentration of 0.1 mol/L
  • a Tris buffer (pH 10.0, 10.5, 11.0) with a concentration of 0.1 mol/L
  • an SDS solution with a concentration of 0.5% (m/m) and the examples 1
  • the obtained wild-type and the L9, L11, L15, L16 and L17 obtained in Example 2 are formulated into a washing liquid.
  • the final volume of the washing liquid is 100 mL.
  • the formula is as follows:
  • Detergent 1 buffer 50mL, deionized water 50mL;
  • Detergent 2-7 buffer 50mL, wild-type obtained in Example 1 and L9, L11, L15, L16 or L17100U obtained in Example 2, add deionized water to a total volume of 100mL;
  • Detergent 8 buffer 50mL, SDS solution 25mL, add deionized water to a total volume of 100mL;
  • Detergent 9-14 buffer 50mL, wild-type obtained in Example 1 and L9, L11, L15, L16 or L17100U obtained in Example 2, SDS solution 25mL, and deionized water added to a total volume of 100mL.
  • the oil removal rate is measured by the weight of the oil before and after washing:
  • Oil removal rate (%) [(oil weight before washing-oil weight after washing)/oil weight before washing] ⁇ 100;
  • Table 5 shows the washing effect of different lipases on oil-stained cloths.
  • the oil removal rate After adding L9 under the four conditions of pH 9.0 and 10.0, 10.5 and 11.0, the oil removal rate is nearly doubled than when washing with buffer only.
  • the detergent contains SDS
  • the oil removal rate can be From 79.9%, 78.6%, 75.8% and 76.1% to 88.1%, 90.5%, 92.8% and 85.5%, respectively, the increase rate is 10.3%, 15.3%, 22.4% and 12.4%; at pH 9.0 and 10.0, 10.5
  • the oil removal rate is doubled compared with the buffer washing only.
  • the oil removal rate can be increased from 79.9%, 78.6%, 75.8% And 76.1% rose to 87.5%, 90.4%, 93.5% and 86.3%, respectively, the increase rate was 9.9%, 15.0%, 23.4% and 13.4%; L15 was added under the four conditions of pH 9.0 and 10.0, 10.5 and 11.0 After that, the oil removal rate is doubled compared with the buffer washing only.
  • adding L15 will increase the oil removal rate from 79.9%, 78.6%, 75.8% and 76.1% to 87.1%, respectively. 89.1%, 93.6% and 86.5%, the increase rate is 9.0%, 13.4%, 23.4% and 13.7%, indicating that L9, L11, L15 have very good washing effects and broad application prospects;

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了多种热稳定性好、pH稳定性好、洗涤性能好的脂肪酶突变体，可用于去污、油脂加工、乳品加工、面食加工、肉类加工、药物合成、柴油合成、高分子合成、手性化合物合成、皮革生产、制备洗涤剂和造纸等方面，属于酶工程技术领域。

Description

一种脂肪酶突变体及其在去污方面的应用 技术领域
本发明涉及一种脂肪酶突变体及其在去污方面的应用，属于酶工程技术领域。
背景技术
脂肪酶(Lipase，EC 3.1.1.3，甘油酯水解酶)是水解脂肪的一类酶的总称。在有些反应系统中，脂肪酶可以催化水解、醇解、转酯、氢解、间接氧化还原转酯化作用，此外，脂肪酶还具有水解外消旋混合物以及酯合成和合成肽键的能力，因此，脂肪酶在很多领域均具有重要的应用，例如：
1、脂肪酶可在油脂加工中应用，油脂的脂肪酶水解可在常温常压下进行，因此，不会使得油脂中的高度不饱和脂肪酸和生育酚等生物物质变性；
2、脂肪酶可在乳品加工中应用，应用脂肪酶在乳品中进行乳脂水解，可增强干酪、奶粉、奶油的风味，促进干酪的成熟，改善乳制品的品质；
3、脂肪酶可在面食加工中应用，在面食中加入脂肪酶，可使产品的弹性得到不同程度的提高，面皮薄而透明但不易破裂，改善食感，另外，在面食中加入脂肪酶，可释放出单酸甘油酯，延缓腐败，提高面食的保鲜能力；
4、脂肪酶可在肉类加工中应用，在肉类中加入脂肪酶，可有效降解肉类中的脂类，制作无脂肪肉；
5、脂肪酶可在药物合成中应用，微生物脂肪酶被用来从动物和植物中富集多不饱和脂肪酸，游离的多不饱和脂肪酸和它们的单双甘酯被用来生产各种药物，如硫氮草酮、萘普生、卡托普利等药物的合成中都要用到脂肪酶；
6、脂肪酶可在柴油合成中应用，酶法合成柴油就是利用脂肪酶催化动植物油脂与低碳醇间的酯交换反应生成相应的脂肪酸酯，和传统化学催化合成柴油相比，该法具有提纯工艺简单、投资设备少、耗能低、污染小、对油脂原料要求低等优点，已日益引起人们的普遍关注；
7、脂肪酶可在高分子合成中应用，当前，人工合成的高分子材料造成了严重的环境污染，因此，应用酶催化法合成可以被微生物降解的该分子材料越来越受到人们的重视；
8、脂肪酶可在手性化合物合成中应用，脂肪酶可以在非水解质中起作用，其在非水解质中的酶催化反应可合成具有光学活性的醇、脂肪酸、内酯等医药农药中间体。
9、脂肪酶可在皮革生产中应用，在皮革的加工过程中，一个重要的步骤是脱除与皮革或皮毛紧密相连的残余脂肪和蛋白质，其中，皮内脂肪由于受到脂肪膜的保护，很难被石灰处 理等化学处理方法去除，利用脂肪酶的分解作用或与蛋白酶混合使用，能够轻易进入脂肪内部将其乳化出来，明显提高了皮革质量；
10、脂肪酶可在制备洗涤剂中应用，应用遗传工程开发出洗涤剂用脂肪酶是现代生物技术大规模工业化最成功的的应用之一；
11、脂肪酶可在造纸合成中应用，“洽脂”是木材中所有疏水性成分的总称，包括三甘酯和蜡脂，它会在干燥柱上沉积而影响纸的质量和生产能力，脂肪酶可除去纸浆中的“洽脂”，改善纸的质量。
然而，脂肪酶的大多数应用都对脂肪酶的稳定性有一定的要求，例如，药物合成的过程中，常需使用较高的温度，这对脂肪酶的热稳定性有了更高的要求；皮革加工和造纸过程中需使用强碱，这对脂肪酶在碱性条件下的pH稳定性有了更高的要求，而现有的大多数脂肪酶稳定性都比较差，例如，来源于少根根霉(Rhizopus arrhizus)的脂肪酶RAL，其最适温度仅有35℃，在50℃下热处理10min后，只能残余40％的酶活，处理到40min后，酶活会完全丧失，其在pH大于9.5的条件下，会损失70％以上活性，这无疑大大限制了脂肪酶在上述应用中的进一步发展。
因此，急需提供一种稳定性，尤其是热稳定性和在碱性条件下pH稳定性强的脂肪酶。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种稳定性，尤其是热稳定性和在碱性条件下pH稳定性强的脂肪酶(Lipase，EC 3.1.1.3)。
[技术方案]
为解决上述问题，本发明提供了一种脂肪酶突变体，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位、第235位、第171位、第343位、第275位、第332位、第310位或第180位氨基酸进行突变得到的；
或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位以及第340位氨基酸同时进行突变得到的；
或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第178位以及第238位氨基酸同时进行突变得到的；
或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位、第340位、第178位以及第238位氨基酸同时进行突变得到的；
或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位、第343位、第332位以及第310位氨基酸同时进行突变得到的；
或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位、第171位、第275位以及第180位氨基酸同时进行突变得到的；
或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位、第343位、第332位、第310位、第151位、第171位、第275位以及第180位氨基酸同时进行突变得到的；
或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位、第343位、第332位、第310位、第151位、第171位、第275位、第180位、第316位、第340位、第178位以及第238位氨基酸同时进行突变得到的。
在本发明的一种实施方式中，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位丝氨酸突变为天冬酰胺得到的(S151N)，命名为L1；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸得到的(S235A)，命名为L2；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第171位赖氨酸突变为精氨酸得到的(K171R)，命名为L3；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第343位丝氨酸突变为酪氨酸得到的(S343Y)，命名为L4；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第275位甘氨酸突变为丙氨酸得到的(G275A)，命名为L5；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第332位谷氨酰胺突变为苯丙氨酸得到的(Q332F)，命名为L6；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第310位天冬氨酸突变为缬氨酸得到的(D310V)，命名为L7；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第180位亮氨酸突变为组氨酸得到的(L180H)，命名为L8；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位苯丙氨酸突变为半胱氨酸以及第340位甘氨酸突变为半胱氨酸得到的(F316C\G340C)，命名为L9；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第178位丝氨酸突变为半胱氨酸以及第238位谷氨酰胺突变为半胱氨酸得到的(S178C\Q238C)，命名为L10；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位苯丙氨酸突变为半胱氨酸、第340位甘氨酸突变为半胱氨酸、第178位丝氨酸突变为半胱氨酸以及第238位谷氨酰胺突变为半胱氨酸得到的(F316C\G340C\S178C\Q238C)，命名为L11；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸、第343位丝氨酸突变为酪氨酸、第332位谷氨酰胺突变为苯丙氨酸以及第310位天冬氨酸突变为缬氨酸得到的(S235A\S343Y\Q332F\D310V)，命名为L12；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位丝氨酸突变为天冬酰胺、第171位赖氨酸突变为精氨酸、第275位甘氨酸突变为丙氨酸以及第180位亮氨酸突变为组氨酸得到的(S151N\K171R\G275A\L180H)，命名为L13；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸、第343位丝氨酸突变为酪氨酸、第332位谷氨酰胺突变为苯丙氨酸、第310位天冬氨酸突变为缬氨酸、第151位丝氨酸突变为天冬酰胺、第171位赖氨酸突变为精氨酸、第275位甘氨酸突变为丙氨酸以及第180位亮氨酸突变为组氨酸得到的(S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H)，命名为L14；
或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸、第343位丝氨酸突变为酪氨酸、第332位谷氨酰胺突变为苯丙氨酸、第310位天冬氨酸突变为缬氨酸、第151位丝氨酸突变为天冬酰胺、第171位赖氨酸突变为精氨酸、第275位甘氨酸突变为丙氨酸、第180位亮氨酸突变为组氨酸、第316位苯丙氨酸突变为半胱氨酸、第340位甘氨酸突变为半胱氨酸、第178位丝氨酸突变为半胱氨酸以及第238位谷氨酰胺突变为半胱氨酸得到的(S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H\F316C\G340C\S178C\Q238C)，命名为L15。
本发明还提供了一种基因，所述基因编码上述脂肪酶突变体。
本发明还提供了一种重组质粒，所述重组质粒携带上述基因。
在本发明的一种实施方式中，所述重组质粒的表达载体为pPIC9K载体、pPIC3.5K载体、pPICZα载体或pPICZ载体。
本发明还提供了一种宿主细胞，所述宿主细胞携带上述基因或上述重组质粒。
在本发明的一种实施方式中，所述宿主细胞为真菌或细菌。
在本发明的一种实施方式中，所述宿主细胞为毕赤酵母。
本发明还提供了一种洗涤剂，所述洗涤剂含有上述脂肪酶突变体。
在本发明的一种实施方式中，其特征在于，所述洗涤剂含有上述脂肪酶突变体、表面活性剂以及缓冲液。
在本发明的一种实施方式中，所述表面活性剂可为十二烷基硫酸钠(SDS)、乙氧基化烷基硫酸钠(AES)、脂肪醇聚氧乙烯醚(AEO-9)和/或月桂基磺化琥珀酸单酯二钠(DLS)。
在本发明的一种实施方式中，所述缓冲液可为磷酸缓冲液、Tris缓冲液、柠檬酸盐缓冲液或碳酸钠盐缓冲液。
在本发明的一种实施方式中，所述洗涤剂中上述脂肪酶突变体的浓度为10～400U/L。
本发明还提供了一种去污方法，所述方法为在需去污物品中添加上述脂肪酶突变体；或者，所述方法为在需去污物品中添加上述洗涤剂。
本发明还提供了上述脂肪酶突变体或上述基因或上述重组质粒或上述宿主细胞在油脂加工、乳品加工、面食加工、肉类加工、药物合成、柴油合成、高分子合成、手性化合物合成、皮革生产、制备洗涤剂或造纸方面的应用。
本发明还提供了上述脂肪酶突变体或上述洗涤剂或上述去污方法在去污方面的应用。
[有益效果]
(1)本发明脂肪酶突变体的热稳定性较野生型有了明显的改善，其中，L11的最适温度可达45℃，较野生型提高了5℃，L11的T m值可达49.2℃，较野生型提高4.2℃，L11的在65℃的半衰期为3.9min，是野生型的4.59倍；L12的最适温度为45℃，较野生型提高了5℃，T m值可达50.3℃，较野生型提高5.3℃，L12的在65℃的半衰期为4.9min，是野生型的5.76倍；L13的最适温度为43℃，较野生型提高了3℃，T m值可达53.8℃，较野生型提高8.8℃，L13的在65℃的半衰期为7.6min，是野生型的8.94倍；L14的最适温度为48℃，较野生型提高了8℃，T m值可达60.8℃，较野生型提高15.8℃，L14的在65℃的半衰期为19.8min，是野生型的23.29倍；L15的最适温度可达50℃，较野生型提高了10℃，T m值可达68.8℃，较野生型提高23.8℃，L15的在65℃的半衰期为56.2min，是野生型的66.11倍；L1～L10的热稳定性也相比于野生型有了一定的改善；
(2)本发明脂肪酶突变体的pH稳定性较野生型有了明显的改善，其中，L9的最适pH由野生型的8.0提升至8.5；L11的最适pH由野生型的8.0提升至9.0，L11在pH为10的条 件下的酶活可达最适pH条件下的酶活的80.5％，较野生型提高了10.73倍，L11在pH为10的条件下处理30min后的残余酶活可达90.8％，较野生型提高了2.20倍；L15的最适pH由野生型的8.0提升至9.0；L15在pH为10的条件下的酶活可达最适pH条件下的酶活的75.2％，较野生型提高了10.02倍，L15在pH为10的条件下处理30min后的残余酶活可达100％，较野生型提高了2.43倍；
(3)本发明脂肪酶突变体的洗涤性能好，其中，使用L9洗涤油污布的去油率可达92.8％；使用L11洗涤油污布的去油率可达93.5％；使用L15洗涤油污布的去油率可达93.6％；
(4)本发明脂肪酶突变体的热稳定性好、pH稳定性好、洗涤性能好，因此，本发明的脂肪酶突变体在去污、油脂加工、乳品加工、面食加工、肉类加工、药物合成、柴油合成、高分子合成、手性化合物合成、皮革生产、制备洗涤剂和造纸等方面均有很重要的应用。
附图说明
图1：含有编码不同突变体基因的重组质粒的全质粒PCR扩增电泳图；
其中，M为DL10000DNA分子量标准，1为含有编码突变体S151N的基因的重组质粒的全质粒PCR扩增电泳结果，2为含有编码突变体S235A的基因的重组质粒的全质粒PCR扩增电泳结果，3为含有编码突变K171R的基因的重组质粒的全质粒PCR扩增电泳结果。
图2：含有编码不同突变体基因的毕赤酵母工程菌的发酵上清液的蛋白纯化SDS-PAGE电泳图；
其中，M为DL10000DNA分子量标准，1为含有编码突变体S151N的基因的毕赤酵母工程菌的发酵上清液的蛋白纯化SDS-PAGE电泳结果，2为含有编码突变体S235A的基因的毕赤酵母工程菌的发酵上清液的蛋白纯化SDS-PAGE电泳结果，3为含有编码突变K171R的基因的毕赤酵母工程菌的发酵上清液的蛋白纯化SDS-PAGE电泳结果。
图3：野生型、L1、L2、L3、L4、L5、L6、L7、L8、L9、L10在不同温度下的相对酶活。
图4：野生型、L11、L12、L13、L14、L15、L16以及L17在不同温度下的相对酶活。
图5：野生型、L1、L2、L3、L4、L5、L6、L7、L8、L9、L10在不同pH下的相对酶活。
图6：野生型、L11、L12、L13、L14、L15、L16以及L17在不同pH下的相对酶活。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中涉及的大肠杆菌JM109购自生工生物工程(上海)有限公司；下述实施例中涉及的毕赤酵母GS115购自Invitrogen公司；下述实施例中涉及的pPIC9K载体购自Invitrogen公司。
下述实施例中涉及的检测方法如下：
脂肪酶酶活的检测方法：
pNPP法，具体参考文献：Pencreach G et al.Enzyme and Microbial Technol.1996,18:417-422.。
其中，脂肪酶酶活的定义为：在pH 8.0，40℃的条件下反应，每分钟产生1μmol对硝基苯酚的酶量为一个酶活力单位(1U)。
下述实施例中涉及的培养基如下：
LB液体培养基(m/m)：蛋白胨1％、酵母提取物0.5％、NaCl 1％，pH7.0。
YPD液体培养基(m/m)：酵母提取物1％、胰蛋白胨2％、葡萄糖2％，121℃高压灭菌20min。
YPD平板(m/m)：酵母提取物1％、胰蛋白胨2％、葡萄糖2％、琼脂2％，121℃高压灭菌20min。
MD平板(m/m)：无氨基酵母氮源1.34％、生物素4×10 -5％、葡萄糖2％、琼脂2％。
MM平板(m/m)：无氨基酵母氮源1.34％、生物素4×10 -5％、甲醇0.5％、琼脂2％。
BMGY培养基(m/m)：酵母提取物1％、胰蛋白胨2％。
实施例1：野生型脂肪酶的表达
具体步骤如下：
从华根霉(Rhizopus chinonsis)中扩增得到编码氨基酸序列如SEQ ID NO.1所示的脂肪酶的基因；将获得的基因proRCL通过Not I和EcoR I双酶切与pPIC9K载体连接，得到重组质粒pPIC9K-proRCL；将重组质粒pPIC9K-proRCL转化至大肠杆菌JM109中，得到大肠杆菌工程菌E.coli JM109/pPIC9K-proRCL；将E.coli JM109/pPIC9K-proRCL涂布于LB平板(含100mg/L卡那霉素)上，于37℃培养10h后，筛选阳性克隆并测序确认，得到验证正确的重组质粒pPIC9K-proRCL；将重组质粒pPIC9K-proRCL用Sal I线性化后，电转化入毕赤酵母GS115中，得到毕赤酵母工程菌Pichia GS115/pPIC9K-proRCL；将毕赤酵母工程菌Pichia GS115/pPIC9K-proRCL涂布于MD平板(含100mg/L卡那霉素)上，于37℃培养10h后，提取基因组、PCR鉴定确定阳性克隆，得到阳性转化子；将阳性转化子接种于含25mL BMGY培养基的250mL摇瓶中，30℃、250r/min培养至OD 600为4.0，得到菌液；将菌液于8 000r/min离心5min后收集菌体，用100mL的BMMY培养基重悬菌体至OD 600为1.0，得到重悬液；将重悬液于30℃、250r/min继续培养，每24h补加0.5％(体积比)甲醇诱导表达，96h后收集菌液，将菌液于8 000r/min离心5min，得到含有野生型酶的上清液；将上清液利用镍 柱进行亲和层析，在250mmol/L咪唑浓度时洗脱目的蛋白，得到纯化后的野生型酶，命名为野生型；
其中，扩增引物如下：
Fm：5’-TCAAGATCCCTAGGGTTCCTGTTGCTGGTCATAAAGGTTC-3’(SEQ ID NO.2)；
Rm：5’-AATTCCAGTGCGGCCGCTTAATGATGATGATGATGATGAGAAGAACCCAAACAGCTTCCTTCGTTAATACC-3’(SEQ ID NO.3)。
实施例2：脂肪酶突变体的制备及表达
具体步骤如下：
利用全质粒PCR技术，以实施例1获得的重组质粒pPIC9K-proRCL为模板进行定点突变，获得突变体S151N、S235A、K171R、S343Y、G275A、Q332F、D310V、L180H、F316C\G340C、S178C\Q238C、F316C\G340C\S178C\Q238C、S235A\S343Y\Q332F\D310V、S151N\K171R\G275A\L180H、S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H、S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H\F316C\G340C\S178C\Q238C、D159R以及T319V；
其中，突变引物为：
S151N：
S151N-Fm：5’-CTGCTTACTGTCGT AACGTCGTTCCAGGTACC-3’(SEQ ID NO.4)；
S151N-Rm：5’-GGTACCTGGAACGAC GTTACGACAGTAAGCAG-3’(SEQ ID NO.5)；
S235A：
S235A-Fm：5’-GTTTCCTTTCC GCATACAACCAAGTTGTCAAAG-3’(SEQ ID NO.6)；
S235A-Rm：5’-CTTTGACAACTTGGTTGTA TGCGGAAAGGAAAC-3’(SEQ ID NO.7)；
K171R：
K171R-Fm：5’-GTCTCAAGTATGTTCCTGATGGT AGGCTTATCAAGAC-3’(SEQ ID NO.8)；
K171R-Rm：5’-GTCTTGATAAG CCTACCATCAGGAACATACTTGAGAC-3’(SEQ ID NO.9)；
S343Y：
S343Y-Fm：5’-CCCGGTGTCGAA TATTGGATCAAGGAAGAC-3’(SEQ ID NO.10)；
S343Y-Rm：5’-GGGGTGAAGATAACCGGCGGCTTGAGGAGG-3’(SEQ ID NO.11)；
G275A：
G275A-Fm：5’-GCCTTGCTCGCT GCCATGGATCTCTACCAACGTG-3’(SEQ ID NO.12)；
G275A-Rm：5’-CACGTTGGTAGAGATCCAT GGCAGCGAGCAAGGC-3’(SEQ ID NO.13)；
Q332F：
Q332F-Fm：5’-CCCTCATGTTCCTCCT TTCGCCTTCGGTTATCTTC-3’(SEQ ID NO.14)；
Q332F-Rm：5’-GAAGATAACCGAAGGC GAAAGGAGGAACATGAGGG-3’(SEQ ID NO.15)；
D310V：
D310V-Fm：5’-CATTCGCTTACTACGTC GTCAGCACCGGAATTCCC-3’(SEQ ID NO.16)；
D310V-Rm：5’-GGGAATTCCGGTGCTGAC GACGTAGTAAGCGAATG-3’(SEQ ID NO.17)；
L180H：
L180H-Fm：5’-GACCTTCACTTCTCTT CACACTGATACCAATGGC-3’(SEQ ID NO.18)；
L180H-Rm：5’-GCCATTGGTATCAGT GTGAAGGCAAGTGAAGGTC-3’(SEQ ID NO.19)；
F316C\G340C：
F316C-Fm：5’-CACCGGAATTCCC TGCCACCGTACCGTTCAC-3’(SEQ ID NO.20)；
F316C-Rm：5’-GTGAACGGTACGGTG GCAGGGAATTCCGGTG-3’(SEQ ID NO.21)；
G340C-Fm：5’-GTTATCTTCACCCC TGCGTCGAATCTTGGATC-3’(SEQ ID NO.22)；
G340C-Rm：5’-GATCCAAGATTCGAC GCAGGGGTGAAGATAAC-3’(SEQ ID NO.23)；
S178C\Q238C：
S178C-Fm：5’-GACCTTCACT TGCCTTCTCACTGATACCAATG-3’(SEQ ID NO.24)；
S178C-Rm：5’-CATTGGTATCAGTGAGAAG GCAAGTGAAGGTC-3’(SEQ ID NO.25)；
Q238C-Fm：5’-CCTTTCCTCATACAAC TGCGTTGTCAAAGACTACTTCCCCG-3’(SEQ ID NO.26)；
Q238C-Rm：5’-CGGGGAAGTAGTCTTTGACA ACGCAGTTGTATGAGGAAAGG-3’ (SEQ ID NO.27)；
F316C\G340C\S178C\Q238C、S235A\S343Y\Q332F\D310V、S151N\K171R\G275A\L180H、S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H以及S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H\F316C\G340C\S178C\Q238C：同上；
D159R；
D159R-Fm：5’-GGTACCAAGTGG CGATGTAAGCAATGTCTC-3’(SEQ ID NO.28)；
D159R-Rm：5’-GAGACATTGCTTACA TCGCCACTTGGTACC-3’(SEQ ID NO.29)；
T319V：
T319V-Fm：5’-CCCTTCCACCGT GTCGTTCACAAGCGTG-3’(SEQ ID NO.30)；
T319V-Rm：5’-CACGCTTGTGAAC GACACGGTGGAAGGG-3’(SEQ ID NO.31)；
PCR体系为：10μM正向引物和反向引物各1μL，dNTPMix 2μL，5×PS Buffer 2.5μL，PrimeStar GXL polymerase0.5μL，模板0.5μL，加双蒸水补齐25μL；
PCR条件为：95℃2min；95℃20s，55～63℃1min，68℃11min，共18个循环；68℃5min。
PCR产物用1％琼脂糖凝胶电泳进行检测，将检测后正确的PCR产物用Dpn I进行消化，后转化至大肠杆菌JM109中，得到含有编码突变体的基因的大肠杆菌工程菌；将大肠杆菌工程菌涂布于LB平板(含100mg/L卡那霉素)上，于37℃培养10h后，筛选阳性克隆并测序确认，得到验证正确的重组质粒(含有编码突变体S151N、S235A、K171R的基因的重组质粒的全质粒PCR扩增电泳图见图1)；将重组质粒用Sal I线性化后，电转化入毕赤酵母GS115中，得到含有编码突变体的基因的毕赤酵母工程菌；将毕赤酵母工程菌涂布于MD平板(含100mg/L卡那霉素)上，于37℃培养10h后，提取基因组、PCR鉴定确定阳性克隆，得到阳性转化子；将阳性转化子接种于含25mL BMGY培养基的250mL摇瓶中，30℃、250r/min培养至OD 600为4.0，得到菌液；将菌液于8 000r/min离心5min后收集菌体，用100mL的BMMY培养基重悬菌体至OD 600为1.0，得到重悬液；将重悬液于30℃、250r/min继续培养，每24h补加0.5％(体积比)甲醇诱导表达，96h后收集菌液，将菌液于8 000r/min离心5min，得到分别含有不同突变体的上清液(含有编码突变体S151N、S235A、K171R的基因的毕赤酵母工程菌的发酵上清液的蛋白纯化SDS-PAGE电泳图见图2)；将上清液利用镍柱进行亲和层析，在250mmol/L咪唑浓度时洗脱目的蛋白，得到纯化后的突变体S151N、S235A、K171R、S343Y、G275A、Q332F、D310V、L180H、F316C\G340C、S178C\Q238C、F316C\G340C\S178C\Q238C、S235A\S343Y\Q332F\D310V、S151N\K171R\G275A\L180H、 S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H、S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H\F316C\G340C\S178C\Q238C、D159R或T319V，分别命名为L1、L2、L3、L4、L5、L6、L7、L8、L9、L10、L11、L12、L13、L14、L15、L16以及L17。
实施例3：不同脂肪酶的酶学性质研究
具体步骤如下：
1、最适温度(T opt)
分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃的条件下测定实施例1获得的野生型和实施例2获得的L1、L2、L3、L4、L5、L6、L7、L8、L9、L10、L11、L12、L13、L14、L15、L16以及L17的脂肪酶酶活，以酶活最高的为100％，其余酶活与之相比计算相对酶活，以考察酶的最适作用温度(检测结果见图3-4)。
由图3-4可知，野生型的最适温度均为40℃；L1、L2、L3、L4、L5、L6、L8、L9、L10、L16和L17的最适温度也均为40℃，与野生型相比没有提升；而L7的最适温度提升至45℃；L13的最适温度提升至43℃；L11和L12的最适温度提升至45℃；L14的最适温度提升至48℃；L15的最适温度提升最多，为50℃，较野生型提升了10℃。
2、变性温度(T m)
将实施例1获得的野生型和实施例2获得的L1、L2、L3、L4、L5、L6、L7、L8、L9、L10、L11、L12、L13、L14、L15、L16以及L17先分别于40℃～70℃的恒温水浴中处理30min，然后分别冰浴20min，最后于40℃下测定脂肪酶酶活，当酶活为初始酶活的50％时，该热处理温度即为酶的变性温度(检测结果见表1)。
由表2可知，与野生型相比，L1～L15的变性温度均有不同程度的提升，其中，提高程度较大的为L11～L15，变性温度分别为49.2℃、50.3℃、53.8℃、60.8℃和68.8℃，分别比野生型提高了4.2℃、5.3℃、8.8℃、15.8℃和23.8℃；而L16的变性温度为40.1℃，L17的变性温度为38.5℃，分别较野生型降低了4.9℃和6.5℃。
表1不同脂肪酶的变性温度(T m)
编号 变性温度(T m)
野生型 45.0℃
L1 47.8℃
L2 46.9℃
L3 45.7℃
L4 48.1℃
L5 47.2℃
L6 46.3℃
L7 46.9℃
L8 47.3℃
L9 48.1℃
L10 46.2℃
L11 49.2℃
L12 50.3℃
L13 53.8℃
L14 60.8℃
L15 68.8℃
L16 40.1℃
L17 38.5℃
3、半衰期(t 1/2)
将实施例1获得的野生型和实施例2获得的L1、L2、L3、L4、L5、L6、L7、L8、L9、L10、L11、L12、L13、L14、L15、L16以及L17分别置于65℃恒温水浴中，每隔一段时间取样一次，测其残留酶活，比较其热稳定性(检测结果见表2)。
由表3可知，与野生型相比，L1～L15在65℃下半衰期均有不同程度的延长，其中，延长程度较大的为L11～L15，半衰期分别为3.9min、4.9min、7.6min、19.8min和56.2min，分别比野生型延长了4.59倍、5.76倍、8.94倍、23.29倍和66.11倍；而L16的半衰期为0.75min，L17的半衰期为0.5min，分别较野生型降低了11.76％和41.18％。
表2不同脂肪酶在65℃下处理后的半衰期
编号 半衰期
野生型 0.85min
L1 2.8min
L2 2.1min
L3 1.2min
L4 3.2min
L5 2.5min
L6 1.9min
L7 2.0min
L8 2.6min
L9 3.3min
L10 1.9min
L11 3.9min
L12 4.9min
L13 7.6min
L14 19.8min
L15 56.2min
L16 0.75min
L17 0.5min
4、最适pH
分别配置浓度为0.05mol/L的柠檬酸缓冲液(pH 6)、浓度为0.05mol/L的磷酸盐缓冲液(pH 6～8)、浓度为0.05mol/L的Tris-HCl缓冲液(pH 8～9)以及浓度为0.05mol/L的碳酸盐缓冲(pH 9～10)代替脂肪酶活力测定方法中的缓冲液，在40℃下测定实施例1获得的野生型和实施例2获得的L1、L2、L3、L4、L5、L6、L7、L8、L9、L10、L11、L12、L13、L14、L15、L16以及L17的脂肪酶酶活，以酶活最高的为100％，其余酶活与之相比计算相对酶活，以考察酶的最适作用pH(检测结果见图5-6)。
由图5-6可知，野生型的最适pH为8.0；L1、L2、L3、L4、L5、L6、L7、L8、L10、L12、L13、L14、L16和L17的最适pH也为8.0，与野生型相比没有变化；L9的最适pH均上升为8.5，较野生型提升了0.5；L11和L15的最适pH均上升为9.0，较野生型提升了1，更加偏好碱性条件。
5、耐碱能力
根据4中的结果，计算实施例1获得的野生型和实施例2获得的L1、L2、L3、L4、L5、L6、L7、L8、L9、L10、L11、L12、L13、L14、L15、L16以及L17在pH为10的条件下的酶活占最适pH条件下酶活的比例，计算结果见表3。
由表3可知，L9、L11和L15在pH为10的条件下的酶活占最适pH条件下酶活的90.6％、80.5％和75.2％，分别较野生型分别提高了12.1倍、10.7倍和10.0倍；L16和L17在pH为10的条件下的酶活占最适pH条件下酶活的7.2％和6.9％，与野生型相比略有降低；其余突变体在pH为10的条件下的酶活占最适pH条件下酶活的比例与野生型相差不大或略有上升。
表3不同脂肪酶在pH为10的条件下的酶活占最适pH条件下酶活的比例
编号 比例
野生型 7.5％
L1 8.5％
L2 16.5％
L3 17.6％
L4 21.0％
L5 24.5％
L6 9.9％
L7 17.8％
L8 15.6％
L9 90.6％
L10 13.4％
L11 80.5％
L12 5.7％
L13 30.3％
L14 25.6％
L15 75.2％
L16 7.2％
L17 6.9％
6、pH稳定性
配置浓度为0.05mol/L、pH为10的碳酸盐缓冲代替脂肪酶活力测定方法中的缓冲液，将实施例1获得的野生型和实施例2获得的L1、L2、L3、L4、L5、L6、L7、L8、L9、L10、L11、L12、L13、L14、L15、L16以及L17分别在上述缓冲体系下于25℃保存30min后，在40℃下测定脂肪酶活力，以初始酶活为100％，保存后酶活与之相比计算残留酶活，以考察其pH稳定性(检测结果见表4)。
由表4可知，L5在pH为10的条件下处理30min后的残余酶活为65.0％，较野生型提高了57.3％；L7在pH为10的条件下处理30min后的残余酶活为55.3％，较野生型提高了34.2％；L9在pH为10的条件下处理30min后的残余酶活为87.4％，较野生型提高了112％；L11在pH为10的条件下处理30min后的残余酶活为98.8％，较野生型提高了139.8％；L12在pH为10的条件下处理30min后的残余酶活为55.8％，较野生型提高了35.4％；L13和L14在pH为10的条件下处理30min后的残余酶活为65.2％和65.8％，分别较野生型提高了58.3％和59.7％；L15在pH为10的条件下处理30min后的酶活无损失，仍保持100％，较野生型提高了142.7％；而L16和L17在pH为10的条件下处理30min后的残余酶活为39.4％和38.5％，比野生型略有降低；其余突变体在pH为10的条件下处理30min后的残余酶活与野生型相差 不大或略有上升。
表4不同脂肪酶在pH为10的条件下处理30min后的残余酶活
编号 残余酶活
野生型 41.2％
L1 40.6％
L2 48.3％
L3 44.4％
L4 47.2％
L5 65.0％
L6 37.8％
L7 55.3％
L8 42.1％
L9 87.4％
L10 40.5％
L11 90.8％
L12 55.8％
L13 65.2％
L14 65.8％
L15 100％
L16 39.4％
L17 38.5％
实施例3：不同脂肪酶的洗涤性能评估
具体步骤如下：
1、油污布的制作
将棉布裁剪成6×6cm大小的方形棉布块，在沸腾的三氯甲烷中煮沸脱脂4h，得到脱脂棉布；将橄榄油加入到甲苯中，得到浓度为200mg/mL的油污液；将油污液以每次0.1mL的量分两次滴加到脱脂棉布的两侧，得到滴加了油污液的脱脂棉布；将滴加了油污液的脱脂棉布先在通风橱里晾干，然后放置在60℃的烘箱中烘干1h，得到油污布。
2、洗涤剂的制作
配置浓度为0.1mol/L的磷酸缓冲液(pH 9.0)，浓度为0.1mol/L的Tris缓冲液(pH 10.0、10.5、11.0)以及浓度为0.5％(m/m)的SDS溶液与实施例1获得的野生型和实施例2获得的L9、L11、L15、L16以及L17配制成洗涤液，洗衣液最终体积均为100mL，配方如下：
洗涤剂1：缓冲液50mL，去离子水50mL；
洗涤剂2～7：缓冲液50mL，实施例1获得的野生型和实施例2获得的L9、L11、L15、L16或L17100U，添加去离子水至总体积100mL；
洗涤剂8：缓冲液50mL，SDS溶液25mL，添加去离子水至总体积100mL；
洗涤剂9～14：缓冲液50mL，实施例1获得的野生型和实施例2获得的L9、L11、L15、L16或L17100U，SDS溶液25mL，添加去离子水至总体积100mL。
3、油污布的洗涤
经油污布分别放入洗衣液1～14中，在50℃、200rpm的条件下洗涤30min，洗涤完成后，将油污布取出，分别用100mL的纯净水在40℃、200rpm的条件下洗涤3次，每次2min，晾干后，将油污布放置60℃烘箱中继续烘干1h。
4、洗涤性能的评估
用精密天平称取洗前和洗后油污布的质量，去油率以洗前和洗后的油重量为衡量标准：
去油率(％)＝[(洗前油重-洗后油重)/洗前油重]×100；
每个条件下做15块重复油污布，不同脂肪酶对油污布的洗涤效果如表5。
由表5可知，在pH为9.0和10.0、10.5和11.0的四个条件下加入野生型后，去油率与未添加相比没有提升，还略有降低，说明野生型并无洗涤性能；
在pH为9.0和10.0、10.5和11.0的四个条件下加入L9后，去油率比仅用缓冲液洗涤时提升近一倍，当洗涤剂中含有SDS时，再加入L9，去油率可以由79.9％、78.6％、75.8％和76.1％分别上升至88.1％、90.5％、92.8％和85.5％，提升幅度为10.3％、15.3％、22.4％和12.4％；在pH为9.0和10.0、10.5和11.0的四个条件下加入L11后，去油率比仅用缓冲液洗涤时提升一倍，当洗涤剂中含有SDS时，再加入L11，去油率可以由79.9％、78.6％、75.8％和76.1％分别上升至87.5％、90.4％、93.5％和86.3％，提升幅度为9.9％、15.0％、23.4％和13.4％；在pH为9.0和10.0、10.5和11.0的四个条件下加入L15后，去油率比仅用缓冲液洗涤时提升一倍，当洗涤剂中含有SDS时，再加入L15，去油率可以由79.9％、78.6％、75.8％和76.1％分别上升至87.1％、89.1％、93.6％和86.5％，提升幅度为9.0％、13.4％、23.4％和13.7％，说明L9、L11、L15具有非常良好的洗涤效果，应用前景广泛；
在pH为9.0和10.0、10.5和11.0的四个条件下加入L16或L17后，去油率与未添加相比没有提升，说明L16和L17也并无洗涤性能。
表5含有不同脂肪酶的洗涤剂在不同pH下的洗涤性能
组别 洗净率(％)
  pH 9 pH 10 pH 10.5 pH 11
洗涤剂1 16.4 19.3 17.4 16.8
洗涤剂2 14.8 15.9 22.2 23.4
洗涤剂3 37.1 38.3 33.8 33.0
洗涤剂4 38.1 38.9 33.1 32.9
洗涤剂5 37.7 39.4 32.2 31.5
洗涤剂6 15.1 15.5 16.6 16.4
洗涤剂7 12.9 14.7 16.5 15.6
洗涤剂8 79.9 78.6 75.8 76.1
洗涤剂9 76.9 75.5 75.5 76.3
洗涤剂10 88.1 90.5 92.8 85.5
洗涤剂11 87.5 90.4 93.5 86.3
洗涤剂12 87.1 89.1 93.6 86.5
洗涤剂13 75.7 76.3 74.2 73.4
洗涤剂14 73.3 75.7 74.4 73.8
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. 一种脂肪酶突变体，其特征在于，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位、第235位、第171位、第343位、第275位、第332位、第310位或第180位氨基酸进行突变得到的；
    或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位以及第340位氨基酸同时进行突变得到的；
    或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第178位以及第238位氨基酸同时进行突变得到的；
    或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位、第340位、第178位以及第238位氨基酸同时进行突变得到的；
    或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位、第343位、第332位以及第310位氨基酸同时进行突变得到的；
    或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位、第171位、第275位以及第180位氨基酸同时进行突变得到的；
    或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位、第343位、第332位、第310位、第151位、第171位、第275位以及第180位氨基酸同时进行突变得到的；
    或者，所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位、第343位、第332位、第310位、第151位、第171位、第275位、第180位、第316位、第340位、第178位以及第238位氨基酸同时进行突变得到的。
  2. 如权利要求1所述的一种脂肪酶突变体，其特征在于，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位丝氨酸突变为天冬酰胺得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第171位赖氨酸突变为精氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第343位丝氨酸突变为酪氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第275位甘氨酸突变为丙氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第332位谷氨酰胺突变为苯丙氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第310位天冬氨酸突变为缬氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第180位亮氨酸突变为组氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位苯丙氨酸突变为半胱氨酸以及第340位甘氨酸突变为半胱氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第178位丝氨酸突变为半胱氨酸以及第238位谷氨酰胺突变为半胱氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第316位苯丙氨酸突变为半胱氨酸、第340位甘氨酸突变为半胱氨酸、第178位丝氨酸突变为半胱氨酸以及第238位谷氨酰胺突变为半胱氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸、第343位丝氨酸突变为酪氨酸、第332位谷氨酰胺突变为苯丙氨酸以及第310位天冬氨酸突变为缬氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第151位丝氨酸突变为天冬酰胺、第171位赖氨酸突变为精氨酸、第275位甘氨酸突变为丙氨酸以及第180位亮氨酸突变为组氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸、第343位丝氨酸突变为酪氨酸、第332位谷氨酰胺突变为苯丙氨酸、第310位天冬氨酸突变为缬氨酸、第151位丝氨酸突变为天冬酰胺、第171位赖氨酸突变为精氨酸、第275位甘氨酸突变为丙氨酸以及第180位亮氨酸突变为组氨酸得到的；
    或者，所述脂肪酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的脂肪酶的第235位丝氨酸突变为丙氨酸、第343位丝氨酸突变为酪氨酸、第332位谷氨酰胺突变为苯丙氨酸、第310位天冬氨酸突变为缬氨酸、第151位丝氨酸突变为天冬酰胺、第171位赖氨酸突变为精氨酸、第275位甘氨酸突变为丙氨酸、第180位亮氨酸突变为组氨酸、第316位苯丙氨酸突变为半胱氨酸、第340位甘氨酸突变为半胱氨酸、第178位丝氨酸突变为半胱氨酸以及第238位谷氨酰胺突变为半胱氨酸得到的。
  3. 一种基因，其特征在于，所述基因编码权利要求1或2所述的脂肪酶突变体。
  4. 一种重组质粒，其特征在于，所述重组质粒携带权利要求3所述的基因。
  5. 如权利要求4所述的一种重组质粒，其特征在于，所述重组质粒的表达载体为pPIC9K载体、pPIC3.5K载体、pPICZα载体或pPICZ载体。
  6. 一种宿主细胞，其特征在于，所述宿主细胞携带权利要求3所述的基因或权利要求4或5所述的重组质粒。
  7. 如权利要求6所述的一种宿主细胞，其特征在于，所述宿主细胞为真菌或细菌。
  8. 如权利要求6或7所述的一种宿主细胞，其特征在于，所述宿主细胞为毕赤酵母。
  9. 一种洗涤剂，其特征在于，所述洗涤剂含有权利要求1或2所述的脂肪酶突变体。
  10. 如权利要求9所述的一种洗涤剂，其特征在于，所述洗涤剂含有权利要求1或2所述的脂肪酶突变体、表面活性剂以及缓冲液。
  11. 如权利要求10所述的一种洗涤剂，其特征在于，所述表面活性剂可为十二烷基硫酸钠(SDS)、乙氧基化烷基硫酸钠(AES)、脂肪醇聚氧乙烯醚(AEO-9)和/或月桂基磺化琥珀酸单酯二钠(DLS)。
  12. 如权利要求10或11所述的一种洗涤剂，其特征在于，所述缓冲液可为磷酸缓冲液、Tris缓冲液、柠檬酸盐缓冲液或碳酸钠盐缓冲液。
  13. 如权利要求9-12任一所述的一种洗涤剂，其特征在于，所述洗涤剂中权利要求1或2所述脂肪酶突变体的浓度为10～400U/L。
  14. 一种去污方法，其特征在于，所述方法为在需去污物品中添加权利要求1或2所述的脂肪酶突变体；或者，所述方法为在需去污物品中添加权利要求9-13任一所述的洗涤剂。
  15. 权利要求1或2所述的脂肪酶突变体或权利要求3所述的基因或权利要求4或5所述的重组质粒或权利要求6-8任一所述的宿主细胞在油脂加工、乳品加工、面食加工、肉类加工、药物合成、柴油合成、高分子合成、手性化合物合成、皮革生产、制备洗涤剂或造纸方面的应用。
  16. 权利要求1或2所述的脂肪酶突变体或权利要求9-13任一所述的洗涤剂或权利要求14所述的去污方法在去污方面的应用。
PCT/CN2020/079438 2019-05-31 2020-03-16 一种脂肪酶突变体及其在去污方面的应用 Ceased WO2020238339A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910468809.XA CN110172455B (zh) 2019-05-31 2019-05-31 一种脂肪酶突变体及其在去污方面的应用
CN201910468809.X 2019-05-31

Publications (1)

Publication Number Publication Date
WO2020238339A1 true WO2020238339A1 (zh) 2020-12-03

Family

ID=67696853

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/079438 Ceased WO2020238339A1 (zh) 2019-05-31 2020-03-16 一种脂肪酶突变体及其在去污方面的应用

Country Status (2)

Country Link
CN (5) CN110172455B (zh)
WO (1) WO2020238339A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110172455B (zh) * 2019-05-31 2020-03-17 江南大学 一种脂肪酶突变体及其在去污方面的应用
CN112574975B (zh) * 2020-09-30 2022-04-01 华南理工大学 甘油酯脂肪酶突变体g28c-p206c及其编码基因与应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974499A (zh) * 2010-08-13 2011-02-16 江南大学 热稳定性提高的脂肪酶突变体
CN102839164A (zh) * 2012-09-06 2012-12-26 江南大学 基于二硫键强化折叠的热稳定性脂肪酶突变体及其构建方法
CN102994471A (zh) * 2012-12-10 2013-03-27 江南大学 一种最适温度提高的脂肪酶突变体及其应用
CN107034205A (zh) * 2017-06-08 2017-08-11 江南大学 一种利用表面活性剂制备高酯化活性脂肪酶的方法
CN110172455A (zh) * 2019-05-31 2019-08-27 江南大学 一种脂肪酶突变体及其在去污方面的应用

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6410994A (en) * 1987-07-03 1989-01-13 Kao Corp Production of lower alcohol fatty acid ester
JPH07143883A (ja) * 1993-11-24 1995-06-06 Showa Denko Kk リパーゼ遺伝子及び変異体リパーゼ
JPH0984584A (ja) * 1995-09-21 1997-03-31 Fuji Oil Co Ltd 耐熱性リパーゼおよびその製造方法
WO2002031124A2 (en) * 2000-10-13 2002-04-18 Novozymes A/S Alpha-amylase variant with altered properties
CN1552866A (zh) * 2003-05-27 2004-12-08 中国科学院微生物研究所 热稳定脂肪酶及其编码基因与应用和专用工程菌
CN101960008B (zh) * 2008-02-29 2016-04-13 诺维信公司 具有脂肪酶活性的多肽和编码该多肽的多核苷酸
CN102102093B (zh) * 2009-12-16 2013-02-13 福建福大百特科技发展有限公司 一种高温脂肪酶突变体的制备及其用途
CN102653743B (zh) * 2010-08-13 2013-09-18 江南大学 通过定向进化构建的热稳定性提高的脂肪酶突变体
CN102653742B (zh) * 2010-08-13 2014-03-26 江南大学 一种耐高温的华根霉脂肪酶突变体
KR20120077750A (ko) * 2010-12-31 2012-07-10 지에스칼텍스 주식회사 ＣａｌＢ 변이체 및 이를 이용한 글리세롤 카보네이트의 제조방법
US20130345392A1 (en) * 2011-03-04 2013-12-26 Pfizer Inc Edn3-like peptides and uses thereof
JP5987824B2 (ja) * 2011-05-02 2016-09-07 宇部興産株式会社 改変型リパーゼ及びその製造方法、並びに当該酵素を用いた反応
US20130123162A1 (en) * 2011-11-10 2013-05-16 The Procter & Gamble Company Consumer products
CN102660517B (zh) * 2011-12-08 2013-04-03 上海交通大学 热稳定性提高的脂肪酶突变体及其构建方法
US20150299622A1 (en) * 2012-11-05 2015-10-22 Novozymes A/S Enzyme Compositions Enabling Re-use of Water in Laundry
US9890367B2 (en) * 2014-02-26 2018-02-13 Jiangnan University Bifunctional lipase mutant and methods of using same
CN103966186B (zh) * 2014-04-08 2016-04-13 南京工业大学 一种提高枯草芽孢杆菌脂肪酶a的热稳定性的方法
CN104498450B (zh) * 2014-12-18 2017-02-22 上海交通大学 褶皱假丝酵母脂肪酶1突变体及其基因
CN105950585B (zh) * 2016-04-29 2019-09-10 华南农业大学 一种热稳定的脂肪酶及制备方法与应用
CN106047838B (zh) * 2016-06-07 2019-09-10 华南农业大学 一种高催化活性的耐热突变脂肪酶及其制备方法与应用
TWI870338B (zh) * 2017-06-05 2025-01-21 美商艾澤西公司 粘附分子-4結合蛋白及其使用方法
CN107858337B (zh) * 2017-11-29 2020-05-15 华南农业大学 一种耐热突变脂肪酶及制备方法与应用
CN108220270B (zh) * 2018-01-31 2020-07-07 江南大学 一种催化活性提高的酸性脂肪酶突变体
CN108359655B (zh) * 2018-02-08 2020-04-24 刘丹妮 一种热稳定性高的脂肪酶突变体TDL-mut及其编码基因
CN108841806B (zh) * 2018-06-29 2021-07-06 深圳市夏盟生物科技有限公司 一种热稳定性提高的脂肪酶突变体
CN108841807B (zh) * 2018-06-29 2021-09-17 广东丰绿源生物医药科技有限公司 一种热稳定性提高的脂肪酶突变体
CN109929822B (zh) * 2019-04-10 2021-04-06 浙江工业大学 一种米曲霉脂肪酶突变体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974499A (zh) * 2010-08-13 2011-02-16 江南大学 热稳定性提高的脂肪酶突变体
CN102839164A (zh) * 2012-09-06 2012-12-26 江南大学 基于二硫键强化折叠的热稳定性脂肪酶突变体及其构建方法
CN102994471A (zh) * 2012-12-10 2013-03-27 江南大学 一种最适温度提高的脂肪酶突变体及其应用
CN107034205A (zh) * 2017-06-08 2017-08-11 江南大学 一种利用表面活性剂制备高酯化活性脂肪酶的方法
CN110172455A (zh) * 2019-05-31 2019-08-27 江南大学 一种脂肪酶突变体及其在去污方面的应用

Also Published As

Publication number Publication date
CN111117981B (zh) 2021-07-13
CN110172455A (zh) 2019-08-27
CN110951712A (zh) 2020-04-03
CN110904074A (zh) 2020-03-24
CN111117981A (zh) 2020-05-08
CN110172455B (zh) 2020-03-17
CN110904073A (zh) 2020-03-24
CN110904073B (zh) 2020-12-01
CN110951712B (zh) 2021-07-13
CN110904074B (zh) 2020-12-01

Similar Documents

Publication Publication Date Title
Choi et al. Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96
CN103789227B (zh) 一株高产碱性蛋白酶的枯草芽孢杆菌菌株
CN101168735A (zh) 一种耐热角质酶及其编码基因和表达
CN110904073B (zh) 一种脂肪酶突变体及其在去污方面的应用
Chen et al. Overexpression of Bacillus circulans alkaline protease in Bacillus subtilis and its potential application for recovery of protein from soybean dregs
CN117925579B (zh) 一种α-半乳糖苷酶突变体及其应用
Tong et al. Characterization of a new sn‐1, 3‐regioselective triacylglycerol lipase from Malbranchea cinnamomea
CN103555596B (zh) 一种表达脂肪酶的黑曲霉菌株
CN110511897B (zh) 一种沙雷氏菌及其在蛋白酶生产中的应用
CN102653743B (zh) 通过定向进化构建的热稳定性提高的脂肪酶突变体
JPH04228070A (ja) 新規エステラーゼ及びその製法
CN102653742A (zh) 一种耐高温的华根霉脂肪酶突变体
JP2025100716A (ja) トランス脂肪酸含有エステル体を分解する新規エステラーゼ
Ozcan et al. Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates
CN102994471A (zh) 一种最适温度提高的脂肪酶突变体及其应用
CN107058135A (zh) 一种产木聚糖酶的菌株及其应用
CN102676395A (zh) 一株宇佐美曲霉突变菌株及其在制备酸性蛋白酶中的应用
Abidi et al. Purification and characterization of an alkaline protease Prot 1 from Botrytis cinerea: biodetergent catalyst assay
CN107916257A (zh) T1脂肪酶突变体和应用
CN116790561B (zh) 一种几丁质酶突变体ChiPcM及其应用
CN110938554B (zh) 一种稳定高产脂肪酶的黑曲霉突变菌株
CN111378583A (zh) 一种里氏木霉及其应用
CN110846341B (zh) 一种脂肪酶高产菌株及其应用
CN102409033B (zh) 一种l-ncc酰胺水解酶和编码基因及其重组表达蛋白质的应用
Srinivasan et al. Characterization of an efficient waste fat hydrolysing and detergent compactible lipase from newly isolated Pseudomonas mosselii

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20814697

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20814697

Country of ref document: EP

Kind code of ref document: A1