WO2022100011A1 - 基于分子动力学计算修饰的2709碱性蛋白酶突变体及其应用 - Google Patents

基于分子动力学计算修饰的2709碱性蛋白酶突变体及其应用 Download PDF

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WO2022100011A1
WO2022100011A1 PCT/CN2021/089618 CN2021089618W WO2022100011A1 WO 2022100011 A1 WO2022100011 A1 WO 2022100011A1 CN 2021089618 W CN2021089618 W CN 2021089618W WO 2022100011 A1 WO2022100011 A1 WO 2022100011A1
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alkaline protease
protease mutant
mutant
amino acid
molecular dynamics
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张剑
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山西大学
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    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • 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
    • C11D3/38618—Protease or amylase in liquid compositions only
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

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  • the invention belongs to the technical field of protein engineering, in particular to a 2709 alkaline protease mutant modified by molecular dynamics calculation and its application in liquid detergents.
  • Enzyme preparations have been used in the detergent industry for more than 40 years as an important adjuvant for detergent preparations.
  • Proteases are one of the most important enzymes in these formulations, although other enzymes including lipases, amylases, cellulases or enzyme mixtures are also used in detergents.
  • proteases with altered properties such as higher low temperature activity, higher thermostability, higher specific pH at a given pH
  • Activity altered Ca 2+ dependence, unaffected in the presence of other detergent ingredients and can maintain higher stability, etc.
  • proteases with altered properties includes the discovery of naturally occurring proteases (the so-called wild-type proteases) and the modification of well-known proteases (eg, by genetic manipulation of the nucleic acid sequences encoding the proteases), which have been the search for efficient and applied modification of enzymes.
  • proteases naturally occurring proteases
  • modification of well-known proteases eg, by genetic manipulation of the nucleic acid sequences encoding the proteases
  • protease A variety of enzyme preparations often used in detergents is protease. At present, the research on alkaline protease in China is still in its infancy. There are few types of alkaline protease enzymes that have been industrialized, and they are similar to foreign ones in terms of product performance and production cost. The product gap is very large, far from meeting the growing domestic market demand.
  • the present invention provides an alkaline protease mutant.
  • the present invention uses molecular dynamics methods to study and evaluate the dynamic structure of alkaline protease 2709 through computer simulation of the structure of alkaline protease 2709 derived from Bacillus licheniformis. This is the only effective method to study the relationship between the structure, molecular dynamics and enzyme function of 2709 alkaline protease at the atomic level. After molecular dynamics calculations, appropriate sites were selected for protein engineering, and mutant proteases suitable for detergent industrial applications were obtained.
  • the protease mutant of the invention has significantly improved enzyme activity and better stability under alkaline conditions, not only has good decontamination effect, but also has good compatibility with various components in detergents, and can be widely used in washing industrial field.
  • alkaline protease 2709 For alkaline protease 2709, it is necessary to obtain detailed information on its molecular motion characteristics and kinetics, whether before or after binding to the substrate, to fully understand its function. In view of the limitation of experimental methods, it is not possible to directly observe the dynamic characteristics of proteins. Therefore, the dynamic structural characteristics of alkaline protease 2709 were studied by molecular dynamics method through computer simulation, which is currently the only protease structure that can be studied from the atomic level. - An efficient approach to kinetic-function relationships. We performed long-term molecular dynamics simulations of protease 2709 without substrate binding and protease 2709 with substrate binding to observe the changes in molecular motion induced before and after substrate binding to explore its dynamic catalytic mechanism, and then optimize on this basis.
  • Mutating the gene structure of protease 2709 and performing calculations to select the most suitable mutation point for industrial washing can change the pH and stability of 2709 alkaline protease, and significantly improve the effect of decomposing washing stains.
  • the agent compatibility is good to facilitate the present invention.
  • the present invention provides a 2709 alkaline protease mutant modified based on molecular dynamics calculation, the parent protein of which is the 2709 alkaline protease of Bacillus licheniformis.
  • the alkaline protease mutant contains a deletion of amino acids in the region 11-33 or at least one of amino acid modification substitutions at the following positions: 9, 12, 15, 16, 17, 18, 19, 33, 38, 39, 40, 41, 42, 46, 47, 48, 49, 50, 58, 59, 60, 67, 79, 96, 97, 98, 99, 107, 108, 109, 110, 111, 116, 131, 132, 133, 134, 139, 140, 152, 153, 163, 164, 165, 166, 173, 188, 189, 190, 191, 193, 195, 198, 201, 203, 211, 221, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 255, 256, 257, 259, 260, 262, 268, 273, wherein the positions correspond to the positions of the amino acid sequence SEQ ID NO: 1.
  • the alkaline protease mutant comprises at least one amino acid modification substitution in G79D, N116D, I173D, P9R, Q273R, wherein the position corresponds to the position of the amino acid sequence SEQ ID NO: 1.
  • the alkaline protease mutant comprises at least one amino acid modification substitution in G46P, N96P, I110P, D139P, N140P, P166S, A198P, A201P, V203P, T211S, L239P, G256P, G262P/S and V268P, wherein said position corresponds to the position of the amino acid sequence SEQ ID NO: 1.
  • the alkaline protease mutant contains amino acid deletions at positions 17-33 or 15-33, and at least one amino acid modification substitution in K12N/Q, Q19S/R, T33H/Y/K, wherein all Said position corresponds to the position of the amino acid sequence SEQ ID NO: 1.
  • the present invention provides a plasmid carrying the gene encoding the above-mentioned 2709 alkaline protease mutant.
  • the present invention provides a recombinant Bacillus licheniformis, which is constructed by transferring the above-mentioned plasmid carrying the gene encoding the 2709 alkaline protease mutant into Bacillus licheniformis.
  • the present invention provides a detergent composition comprising the above-mentioned 2709 alkaline protease mutant.
  • the present invention provides the application of a modified 2709 alkaline protease mutant based on molecular dynamics calculation, which is applied in the field of cleaning or washing.
  • the present invention has the following advantages:
  • the present invention studies the dynamic structural characteristics of 2709 alkaline protease by using molecular dynamics method through computer simulation, selects suitable sites for protein engineering, and obtains a 2709 alkaline protease mutant suitable for detergent industrial applications.
  • the 2709 alkaline protease mutant of the present invention has better thermal stability. Without adding any protective agent and stabilizer, 2709 alkaline protease and its mutants were incubated at 50°C for half an hour. It can be clearly seen that the residual activity of the 2709 alkaline protease mutant was significantly higher than that of the 2709 alkaline protease, even if the incubation was longer. For a long time, the residual viability of mutants after mutation improvement is always high.
  • the 2709 alkaline protease mutant of the present invention has good stability under alkaline pH conditions and has a wider application range. Without adding any protective agent and stabilizer, the alkaline protease and its mutants were incubated at different pH for 1 hour. It can be clearly seen that the residual activity of the mutant was significantly higher than that of the original 2709 alkaline protease after the pH was greater than 9.
  • the 2709 alkaline protease mutant enzyme of the present invention has better stability in commercial concentrated powder and standard liquid washing system. Without adding any protective agent and stabilizer, adding the same amount of 2709 alkaline protease and its mutants in the commercially available concentrated powder and standard liquid washing system, it can be clearly seen that the 2709 alkaline protease mutant is compared with 2709 Alkaline protease activity is more stable than both in the above two detergent systems. It is helpful to expand the application range of alkaline protease and lay a foundation for its wider application in the washing industry.
  • the 2709 alkaline protease mutant of the present invention has better retention activity under extreme conditions. Without adding any protective agent and stabilizer, the hygrothermal stability of 2709 alkaline protease and its mutants was determined under the harsh environment of 40°C surface fully exposed and 75% humidity, and it can be seen that the 2709 alkaline protease mutant The residual activity of 2709 alkaline protease was always higher than that of the original 2709 alkaline protease, and the residual enzymatic activity of protease mutants after two weeks was all above 75%, while the residual enzymatic activity of 2709 alkaline protease was only about 35%.
  • the 2709 alkaline protease mutant of the present invention has a significantly improved decontamination effect on protein stains, and has achieved unexpected technical effects.
  • the 2709 alkaline protease mutant of the present invention is in In the presence of other detergents, it can still maintain a high detergency without being affected.
  • Figure 1 SDS-PAGE electropherogram of 2709 alkaline protease protein purification; where Mr is the protein Marker; lane 1 is the expression of 2709 alkaline protease, and lane 2 is the 2709 alkaline protease mutant (corresponding amino acid sequence is the expression of SEQ ID NO: 2).
  • Figure 2 is a graph showing the enzyme activity stability curves of 2709 alkaline protease mutant and 2709 alkaline protease at 50°C.
  • Figure 3 is a graph showing the effect of pH on the enzymatic activity of 2709 alkaline protease mutant and 2709 alkaline protease.
  • Figure 4 is a graph showing the stability of 2709 alkaline protease mutant and 2709 alkaline protease in different detergents.
  • Figure 5 is a graph of the damp heat stability before and after mutation of 2709 alkaline protease.
  • the amino acid sequence corresponding to 2709 alkaline protease mutant 1 in the figure is the sequence shown in SEQ ID NO: 2
  • the amino acid sequence corresponding to 2709 alkaline protease mutant 2 is the sequence shown in SEQ ID NO: 3
  • the 2709 alkaline protease mutant The corresponding amino acid sequence of 3 is the sequence shown in SEQ ID NO: 4.
  • the amino acid sequence of the original 2709 alkaline protease is the sequence shown in SEQ ID NO: 1
  • the amino acid sequence of the 2709 alkaline protease mutant 1 is the sequence shown in SEQ ID NO: 2
  • the amino acid sequence of the 2709 alkaline protease mutant 2 It is the sequence shown in SEQ ID NO: 3
  • the amino acid sequence of 2709 alkaline protease mutant 3 is the sequence shown in SEQ ID NO: 4.
  • Alkaline protease enzyme activity determination method refer to GB/T 23527-2009 Appendix B Fulin method, the specific reaction process is as follows: first take out a series of empty test tubes, mark one test tube as the control group in each group, and mark the remaining three test tubes. for the experimental group.
  • test tubes In all test tubes, add 1 mL of 1% casein solution prepared with buffer, and incubate the test tubes at 40 °C for 2 min; add 1 mL of crude enzyme solution to the test tubes except for the blank to make the enzyme solution and substrate React for 10min; add 2mL 0.4mol/L trichloroacetic acid to stop the reaction; add 1mL enzyme solution to the control group; centrifuge after standing for 10min, take 1mL supernatant each in a new test tube; add 5mL sodium carbonate, 1mL Folin reagent; Incubate at 40°C for 20min. Absorbance was measured at 680 nm.
  • Molecular dynamics simulations were completed by Amber software and carried out on the GPU computing cluster of Shanxi University Computing Center, using 1 GPU graphics card, and the 24-hour simulation time unit scale was 100ns.
  • the specific parameters of the molecular dynamics simulation are set as: the time step is 2 femtoseconds (fs); the electrostatic interaction is described by the PME algorithm, and the truncation radius of the electrostatic interaction is set as
  • the cutoff radius for van der Waals interactions is set to Solutes (ie, protein molecules) and solvents (ie, water molecules and sodium ions) were separately subjected to thermal bath treatment, and the temperature was set to 300K; the pressure was set to one standard atmosphere.
  • the initial velocities of atoms were randomly generated according to the Maxwell distribution at 300k temperature.
  • the duration of the production molecular dynamics simulation is 300 ns and contains a total of 30,000 frames. Because the research on alkaline protease is mostly based on the alignment of primary amino acid sequences, the three-dimensional structure is less considered. After calculating the three-dimensional structure of the enzyme preparation with significant washing application effect by means of the three-dimensional structure simulation of computational chemical molecular dynamics, site-directed mutagenesis was carried out and the energy and stability of the alkaline protease mutant after the site-directed mutation were calculated in the decomposition substrate and water environment. sex. The alkaline protease mutant structure calculated by Amber software according to molecular dynamics simulation is more effective.
  • alkaline protease mutant of the present invention can be constructed and expressed by methods well known to those skilled in the art.
  • the invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT ROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art.
  • the present invention is not limited to the specific methods, experimental protocols and reagents described in the examples, but also includes those skilled in the art.
  • the upstream and downstream primers were designed according to the Fast Mutagenesis System of Beijing Quanshi Gold Biotechnology Co., Ltd., the recombinant plasmid pBE 2R-AP of the constructed alkaline protease 2709 mutant was used as the template, and the corresponding mutant primers were used for PCR amplification; The amplified PCR product was subjected to agarose electrophoresis and purified to recover the PCR product.
  • the PCR amplification reaction system is: 2 ⁇ PCR SuperMix 25 ⁇ L, primer Up (10 ⁇ mol/L) 1 ⁇ L, primer Dn (10 ⁇ mol/L) 1 ⁇ L, template (1/30) 1 ⁇ L, add water to make up to 50 ⁇ L.
  • the amplification reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 4 min, 25 cycles; extension at 72°C for 10 min. Store at 4°C.
  • Transfer the correctly sequenced recombinant plasmids into competent cells WB600, and the specific transformation process is as follows: Pick up single cells grown on LB (peptone 1%, NaCl 1%, yeast powder 0.5%, agar powder 1.5%) plate with a pipette tip. Colonies were cultured in 2 mL GMI for 12 h;
  • GMI preparation method 10mL solution A, 1.5mL solution B, 25mL solution C, 100uL solution D, 25mL solution G, add sterile water to 100mL.
  • solution A preparation method 0.4g yeast extract, 0.08g casein hydrolyzate, dissolved in 40mL water
  • solution B preparation method 5g glucose, dissolved in 10mL water
  • solution C preparation method 4.8g KH2PO4, 11.2g K2HPO4 , 0.16g MgSO4 7H2O, 0.8g trisodium citrate, 1.6g (NH4)2SO4, dissolved in 200mL water
  • solution D 0.9g MnCl2 4H2O, 1.415g boric acid, 0.68g FeSO4 7H2O, 13.45mg CuCl2 2H2O , 23.5mg ZnSO4 7H2O, 20.2mg CoCl2 6H2O, 12.6mg sodium monomolybdate, 0.855g sodium tartrate, dissolved in 500
  • the bacterial liquid cultured overnight was added to 98 mL of GMI, and incubated at 37°C at 200 rpm for about 4 h; 10 mL of bacterial liquid was added to 90 mL of GMII (GMII preparation method: 98 mL of GMI, 1 mL of solution E, 1 mL of solution F, and mixed.
  • solution E prepared Method 2.16g MgCl2 ⁇ 6H2O, dissolved in 20mL water; solution F preparation method: 147mg CaCl2 dissolved in 20mL water), cultured at 37°C, 200rpm for about 90min; cell ice water bath for 30min, 4000rpm, 4°C centrifugation for 30min, go on Add 10 mL of GMIII (GMIII preparation method: 9 mL of GMII, 1 mL of glycerol), and mix well to obtain competent cells.
  • GMIII GMIII preparation method: 9 mL of GMII, 1 mL of glycerol
  • Alkaline protease mutant Bacillus licheniformis recombinant engineering bacteria were inoculated in 5mL LB liquid medium (peptone 1%, NaCl 1%, yeast powder 0.5%), 37 °C, 200rpm shaking culture for 12h, the bacterial liquid was inoculated according to 2%. Bacteria were transferred to fermentation enzyme production medium (dextrin 1%, soluble starch 2%, yeast powder 1%, NaCl 0.5%, pH 7.0), 37 °C, 200rpm shaking culture for 84h.
  • the fermentation broth was centrifuged at 13000 r/min for 15 min, and then the supernatant was filtered on a positive pressure filter with a 0.22 ⁇ m membrane to remove residual Bacillus licheniformis.
  • the supernatant was slowly added with ammonium sulfate powder to the crude enzyme solution of the mutant alkaline protease, so that the concentration of ammonium sulfate reached 70%. After the ammonium sulfate powder was completely dissolved, let it stand overnight in a chromatographic cabinet at 4°C, and then at 13,000 rpm.
  • the protein concentration was 0.2 mg/ml, and the protein buffer was 50 mM Tris-HCl, 100 mM NaCl, pH 8.0.
  • the enzyme activity determination method was carried out with reference to the Fulin method in Appendix B of GB/T 23527-2009.
  • the daily washing temperature is generally 30 °C or 40 °C.
  • the activity and stability of 2709 alkaline protease and its mutants were tested at 50 °C to confirm its Provides stable stain removal at normal washing temperatures.
  • the samples were incubated at 50 °C for 3 h, and samples were taken every 0.5 h to measure the enzyme activity.
  • the enzyme activity determination method was carried out with reference to the Fulin method in Appendix B of GB/T 23527-2009.
  • the 2709 alkaline protease of the present invention has good heat-resistant performance, its enzyme activity is obviously improved under alkaline conditions, and its damp-heat stability is good.
  • the 2709 alkaline protease mutant has good detergency when used as a detergent builder, and retains activity better than the parent protease under extreme conditions, and can be used at higher temperatures and in stronger alkaline environments . It shows that the 2709 alkaline protease mutant of the present invention can be widely used in the field of washing industry.
  • the 2709 alkaline protease mutant 1 of the present invention enzyme activity 5000U/ml;
  • the 2709 alkaline protease mutant 2 of the present invention the enzyme activity is 5000 U/ml;
  • the 2709 alkaline protease mutant 3 of the present invention the enzyme activity is 5000 U/ml;
  • the original 2709 alkaline protease of the present invention the enzyme activity is 5000U/ml.
  • Washing equipment vertical decontamination machine (RHLQ II), China Daily Chemical Industry Research Institute
  • Detergent concentration standard laundry liquid 2g/L; commercial detergent concentrate 1g/L
  • Reflection value measuring instrument whiteness meter (WSD-3C)
  • Test soiling cloth JB02 national standard protein soiling cloth
  • the 2709 alkaline protease mutant of the present invention has a significantly improved decontamination effect on protein stains compared with the original 2709 alkaline protease, and an unexpected technical effect has been achieved.
  • the 2709 alkaline protease mutant of the present invention has In the presence of other detergents, the body can still maintain high detergency without being affected. It shows that the 2709 alkaline protease mutant can be used in the field of cleaning or washing.

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Abstract

提供一种基于分子动力学计算修饰的2709碱性蛋白酶突变体及其在液体洗涤剂中的应用。对来源于地衣芽孢杆菌的2709碱性蛋白酶通过计算机模拟，用分子动力学方法研究2709碱性蛋白酶的动态结构特征，通过分子动力学计算后，选择合适位点进行蛋白质工程改造，获得了适合洗涤剂工业应用的2709碱性蛋白酶突变体。

Description

基于分子动力学计算修饰的2709碱性蛋白酶突变体及其应用 技术领域
本发明属于蛋白质工程改造技术领域，具体涉及一种基于分子动力学计算修饰的2709碱性蛋白酶突变体及其在液体洗涤剂中的应用。
背景技术
酶制剂在洗涤剂工业中作为洗涤制剂的重要助剂已经使用了40多年。蛋白酶是这些制剂中最重要酶之一，当然其他酶包括脂肪酶、淀粉酶、纤维素酶或酶混合物也用于洗涤剂中。
为改进蛋白酶的成本和/或性能，寻找具有改变特性的蛋白酶一直是近年来的研究热点，这些特性如：更高的低温活性、更高的热稳定性、在给定pH下更高的比活性、改变的Ca 2+依赖性、在其他洗涤剂成份存在下不受影响且能保持更高的稳定性等。
寻找具有改变特性的蛋白酶包括发现天然存在的蛋白酶(即所谓野生型蛋白酶)和改造已熟知的蛋白酶(如通过对编码所述蛋白酶的核酸序列的基因操作)一直是寻求酶的高效和应用改性的两个重要方向。近年来计算化学的快速进展对蛋白质三维结构和功能之间关系的认识提高了评估改变蛋白质的哪一区域来影响蛋白质特定性状的能力。通过计算化学来评估并改造高效能的酶变的越来越有可能性。
经常在洗涤剂中使用的一个酶制剂品种是蛋白酶，目前国内对于碱性蛋白酶的研究还处于起步阶段，已产业化的碱性蛋白酶酶种类较少，且在产品性能和生产成本方面与国外同类产品的差距很大，远远不能满足国内日益增长的市 场需要。
发明内容
针对上述问题本发明提供了一种碱性蛋白酶突变体，本发明通过对来源于地衣芽孢杆菌的2709碱性蛋白酶结构进行计算机模拟，用分子动力学方法研究和评估改变碱性蛋白酶2709的动态结构特征的哪一区域来影响其特定性状及酶效能的能力，这也是目前唯一能够从原子水平上来研究2709碱性蛋白酶结构与分子动力学与酶功能关系的有效方法。通过分子动力学计算后，选择合适位点进行蛋白质工程改造，获得了适合洗涤剂工业应用的突变体蛋白酶。本发明的蛋白酶突变体在碱性条件下的酶活力得到显著提高，且稳定性更好，不仅具有良好的去污作用，还具有和洗涤剂中各组分兼容性好，可广泛应用于洗涤工业领域。
为了达到上述目的，本发明采用了下列技术方案：
尽管目前的蛋白酶晶体学研究为研究蛋白酶的催化机制提供了宝贵的结构信息，但是酶的晶体结构仅提供了底物结合前和结合后的静态构象，是基于一级氨基酸序列的比对做出的，较少考虑三维结构。同时酶与底物的结合本身是一个动态过程，会涉及一系列的构象变化和相互作用。例如，底物是如何与酶结合并定位到正确的位置。催化完成后产物如何释放？以及这些动力学过程如何调节的？
对于碱性蛋白酶2709而言，无论是与底物结合前还是结合后的结构，获取他们的分子运动特征和动力学细节信息对于全面理解其功能都是非常有必要的。鉴于实验手段的限制，直接观察蛋白质的动力学特征还无法实现，因此，通过计算机模拟，用分子动力学方法研究碱性蛋白酶2709的动态结构特征，这也是目前唯一能够从原子水平上来研究蛋白酶结构-动力学-功能关系的有效方 法。我们分别对没有底物结合的蛋白酶2709和有底物结合的蛋白酶2709进行长时间分子动力学模拟，以观察底物结合前后所诱导的分子运动变化探究其动态催化机理，然后在此基础上优化突变蛋白酶2709的基因结构并进行计算选择出最适洗涤工业应用的突变点，可使2709碱性蛋白酶的作用pH和稳定性发生改变，并显著提升其分解洗涤污渍的效果，且与洗涤剂助剂相容性良好从而促成本发明。
本发明提供一种基于分子动力学计算修饰的2709碱性蛋白酶突变体，其亲本蛋白为地衣芽孢杆菌的2709碱性蛋白酶。
所述碱性蛋白酶突变体含有11-33区域内氨基酸的缺失或至少包含有以下位置的氨基酸修饰置换中的一个：9、12、15、16、17、18、19、33、38、39、40、41、42、46、47、48、49、50、58、59、60、67、79、96、97、98、99、107、108、109、110、111、116、131、132、133、134、139、140、152、153、163、164、165、166、173、188、189、190、191、193、195、198、201、203、211、221、234、235、236、237、238、239、240、241、242、243、244、245、255、256、257、259、260、262、268、273，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
进一步，所述碱性蛋白酶突变体至少包含G79D、N116D、I173D、P9R、Q273R中的一个位置上的氨基酸修饰置换，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
进一步，所述碱性蛋白酶突变体至少包含G46P、N96P、I110P、D139P、N140P、P166S、A198P、A201P、V203P、T211S、L239P、G256P、G262P/S和V268P中的一个位置上的氨基酸修饰置换，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
进一步，所述碱性蛋白酶突变体含有位置17-33或15-33的氨基酸缺失，同时至少含有K12N/Q、Q19S/R、T33H/Y/K中的一个位置上的氨基酸修饰置换，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
本发明提供一种质粒，该质粒携带有编码上述2709碱性蛋白酶突变体的基因。
本发明提供一种重组地衣芽孢杆菌，该地衣芽孢杆菌是将上述携带有编码2709碱性蛋白酶突变体的基因的质粒转入地衣芽孢杆菌中构建的。
本发明提供一种洗涤剂组合物，该洗涤剂组合物含有上述2709碱性蛋白酶突变体。
本发明提供一种基于分子动力学计算修饰的2709碱性蛋白酶突变体的应用，应用于清洁或洗涤领域。
与现有技术相比本发明具有以下优点：
1.本发明通过计算机模拟，用分子动力学方法研究2709碱性蛋白酶的动态结构特征，选择合适位点进行蛋白质工程改造，获得了适合洗涤剂工业应用的2709碱性蛋白酶突变体。
2.本发明2709碱性蛋白酶突变体具有较好的热稳定性。在不加任何保护剂和稳定剂的前提下，2709碱性蛋白酶及其突变体50℃保温半小时，可以明显看出2709碱性蛋白酶突变体剩余活力明显高于2709碱性蛋白酶，即使保温更长时间，始终是突变改进后的突变体的残余活力高。
3.本发明2709碱性蛋白酶突变体在碱性pH条件下稳定性好，适用范围更加宽泛。在不加任何保护剂和稳定剂的前提下，碱性蛋白酶及其突变体在不同pH下保温1小时，可以明显看出突变体剩余活力在pH大于9后明显高于原2709碱性蛋白酶。
4.本发明2709碱性蛋白酶突变体酶相比2709碱性蛋白酶在市售浓缩粉和标准液体洗涤体系中有更好的稳定性。在不加任何保护剂和稳定剂的前提下，添加相同量的2709碱性蛋白酶及其突变体在市售浓缩粉和标准液体洗涤体系中，可以明显看出2709碱性蛋白酶突变体相比2709碱性蛋白酶活在上述两种洗涤剂体系中酶活均较均更稳定。有助于拓展碱性蛋白酶的使用范围，为其更广泛的应用于洗涤工业领域奠定了基础。
5.本发明2709碱性蛋白酶突变体在极端条件下有更好的保留活性。在不加任何保护剂和稳定剂的前提下，测定2709碱性蛋白酶及其突变体在40℃表面充分暴露、75％湿度的苛刻环境下的湿热稳定性，可以看出2709碱性蛋白酶突变体的剩余活力始终高于原始2709碱性蛋白酶，且蛋白酶突变体两周后的残余酶活力都在75％以上，而2709碱性蛋白酶残余酶活仅有35％左右。
6.本发明的2709碱性蛋白酶突变体较原始型2709碱性蛋白酶对蛋白污渍的去污效果得到显著提升，取得了意料不到的技术效果，同时本发明所述2709碱性蛋白酶突变体在其它洗涤剂存在的情况下不受影响仍能保持较高的去污能力。
附图说明
图1 2709碱性蛋白酶蛋白纯化SDS-PAGE电泳图；其中Mr所示为蛋白Marker；泳道1所示为2709碱性蛋白酶表达情况，泳道2所示为2709碱性蛋白酶突变体(对应的氨基酸序列为SEQ ID NO：2)的表达情况。
图2为50℃条件下，2709碱性蛋白酶突变体与2709碱性蛋白酶酶活力稳定性曲线图。
图3为pH对2709碱性蛋白酶突变体与2709碱性蛋白酶的酶活力影响曲线图。
图4为2709碱性蛋白酶突变体与2709碱性蛋白酶在不同洗涤剂中稳定性曲线图。
图5为2709碱性蛋白酶突变前后湿热稳定性图。
图中2709碱性蛋白酶突变体1对应的氨基酸序列为SEQ ID NO：2所示序列，2709碱性蛋白酶突变体2对应的氨基酸序列为SEQ ID NO：3所示序列，2709碱性蛋白酶突变体3对应的氨基酸序列为SEQ ID NO：4所示序列。
具体实施方式
下面结合具体实施例对本发明做进一步的阐述，下述实例中所用方法如无特别说明均为分子克隆、蛋白质纯化以及酶分析的常规方法。其中，原始2709碱性蛋白酶的氨基酸序列为SEQ ID NO：1所示序列，2709碱性蛋白酶突变体1的氨基酸序列为SEQ ID NO：2所示序列，2709碱性蛋白酶突变体2的氨基酸序列为SEQ ID NO：3所示序列，2709碱性蛋白酶突变体3的氨基酸序列为SEQ ID NO：4所示序列。
实施例1
碱性蛋白酶酶活力的测定：
碱性蛋白酶酶活测定方法：参照GB/T 23527-2009附录B福林法进行，具体反应过程如下：首先取出一系列空试管，每组将一个试管标记为对照组，将其余三个试管标记为实验组。在所有试管中，加入1mL用缓冲液配制的1％的酪素溶液，并将试管在40℃下保温2min；向除了空白之外的试管中加入1mL的粗酶液，使酶液和底物反应10min；加入2mL 0.4mol/L三氯乙酸以终止反应；对照组加入1mL酶液；静置10min后离心，各取1mL上清液于新的试管中；加入5mL碳酸钠，1mL Folin试剂；置于40℃显色20min。在680nm处测定吸光值。 酶活计算公式如下：X＝A×K×4/10×n，其中X代表蛋白酶酶活力(U/mL)；A表示样品平行试验的平均吸光度；K代表吸光常数(实验室测定值K＝97)；4表示反应试剂的总体积(mL)；10表示反应时间10min；n为稀释倍数。
实施例2
2709碱性蛋白酶突变体基因的筛选及合成
分子动力学模拟通过Amber软件完成，在山西大学计算中心GPU计算集群上进行，使用1个GPU显卡，24个小时的模拟时间单位尺度为100ns。分子动力学模拟的具体参数设置为：时间步长为2飞秒(fs)；静电相互作用由PME算法描述，静电相互作用的截断半径设置为
Figure PCTCN2021089618-appb-000001
范德华相互作用的截断半径设置为
Figure PCTCN2021089618-appb-000002
溶质(即蛋白质分子)和溶剂(即水分子和钠离子)被分别进行热浴处理，温度设置为300K；压力设置为一个标准大气压。原子的初始速率根据300k温度下的麦克斯维尔(Maxwell)分布随机生成。生产分子动力学模拟的时长为300ns，共含有30000帧。由于碱性蛋白酶的研究多是基于一级氨基酸序列的比对作出的，较少考虑三维结构。借助于运用计算化学分子动力学的三维结构模拟计算出洗涤应用效果显著的酶制剂三维结构后，进行定点突变并计算定点突变后碱性蛋白酶突变体在分解底物及水环境下的能量及稳定性。根据分子动力学模拟通过Amber软件计算完成的碱性蛋白酶突变体结构，更具有效性。
实施例3
碱性蛋白酶突变体的构建和表达：
本发明的碱性蛋白酶突变体的构建可通过本领域技术人员熟知的方法构建和表达。发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING：A LABORATORY MANUAL，3nd Ed.(Sambrook，2001) 和CURRENT ROTOCOLS IN MOLECULAR BIOLOGY(Ausubel，2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。而且本发明不限定于实施例中记载的具体方法、实验方案和试剂，还包括本领域的技术人员。
按照北京全氏金生物技术有限公司的Fast Mutagenesis System设计上下游引物，以构建好的碱性蛋白酶2709突变体的重组质粒pBE 2R-AP为模板，以相应的突变体引物进行PCR扩增；将扩增好的PCR产物进行琼脂糖电泳并纯化回收PCR产物。
PCR扩增反应体系为：2×PCR SuperMix 25μL，引物Up(10μmol/L)1μL，引物Dn(10μmol/L)1μL，模板(1/30)1μL，加水补足至50μL。
扩增反应条件为：94℃预变性3min；94℃变性20s，55℃退火20s，72℃延伸4min，25个循环；72℃延伸10min。4℃保存。
加1μL DMT酶于PCR产物中，混匀，37℃孵育1h。加2-5μL的DMT酶消化产物，用热激法转入地衣芽孢杆菌，提质粒进行测序确定。
将测序正确的重组质粒转入感受态细胞WB600中，具体转化过程如下：用枪头挑取在LB(蛋白胨1％，NaCl 1％，酵母粉0.5％，琼脂粉1.5％)平板上生长的单菌落于2mLGMI中，培养12h；
GMI配制方法：10mL溶液A，1.5mL溶液B，25mL溶液C，100uL溶液D，25mL溶液G，加灭菌水至100mL。其中，溶液A配制方法：0.4g酵母提取物，0.08g酪蛋白水解物，溶于40mL水中；溶液B配制方法：5g葡萄糖，溶于10mL水中；溶液C配制方法：4.8g KH2PO4，11.2g K2HPO4，0.16g MgSO4·7H2O，0.8g柠檬酸三钠，1.6g(NH4)2SO4，溶于200mL水中；溶液 D：0.9g MnCl2·4H2O，1.415g硼酸，0.68g FeSO4·7H2O，13.45mg CuCl2·2H2O，23.5mg ZnSO4·7H2O，20.2mg CoCl2·6H2O，12.6mg高钼酸钠，0.855g酒石酸钠，溶于500mL水中；溶液G配制方法：36.5g山梨醇，溶于100mL水中。
将过夜培养的菌液加到98mLGMI中，37℃，200rpm培养约4h；取10mL菌液加入90mL GMII(GMII配制方法：98mL GMI，1mL溶液E，1mL溶液F，混匀。其中，溶液E配制方法：2.16g MgCl2·6H2O，溶于20mL水中；溶液F配制方法：147mg CaCl2溶于20mL水中)中，37℃，200rpm培养约90min；菌体冰水浴30min，4000rpm，4℃离心30min，去上清；加10mL GMIII(GMIII配制方法：9mL GMII，1mL甘油)，混匀，即为感受态细胞。
在500μL感受态细胞中加入5μL质粒，直接将感受态细胞置于37℃，200rpm摇床培养1.5h，低速离心3min，弃部分上清，均匀涂布于含40μg/mL卡那霉素的脱脂奶粉培养基平板上，37℃恒温培养箱培养12h。次日平板上的单菌落即为含碱性蛋白酶突变体的重组菌株。
碱性蛋白酶突变体地衣芽孢杆菌重组工程菌接种于5mL LB液体培养基(蛋白胨1％，NaCl 1％，酵母粉0.5％)中，37℃，200rpm振荡培养12h，将菌液按2％的接菌量分别转接于发酵产酶培养基(糊精1％，可溶性淀粉2％，酵母粉1％，NaCl 0.5％，pH值为7.0)中，37℃，200rpm振荡培养84h。
发酵完成后，发酵液13000r/min离心15min，然后上清液用0.22μm膜在正压滤器上过滤去除残留地衣芽孢杆菌。上清液分别缓慢加硫酸铵粉末到碱性蛋白酶突变体的粗酶液中，使硫酸铵浓度至70％，待硫酸铵粉末完全溶解，在4℃层析柜中过夜静置，再以13000rpm离心30min，收集沉淀，将沉淀于50mM Tris-HCl，100mM NaCl，pH＝8的缓冲液透析并用超滤杯超滤浓缩。上样至用 相同缓冲液(50mM Tris-HCl，100mM NaCl，pH 8)平衡好的Superdex75凝胶层析柱(购自GE公司)，收集2709碱性蛋白酶突变体。在SDS-PAGE电泳，图1结果显示为单一条带的蛋白样品。
实施例4
碱性蛋白酶突变体的稳定性实验：
蛋白浓度为0.2mg/ml，蛋白缓冲液为50mM Tris-HCl，100mM NaCl，pH8.0。酶活测定方法参照GB/T 23527-2009附录B福林法进行。
1.日常洗涤温度一般为30℃或40℃，本实验在不加任何保护剂和稳定剂的前提下，测试2709碱性蛋白酶及其突变体在50℃条件下的活力稳定性情况，确认其可在正常洗涤温度中稳定发挥去污作用。将试样在50℃条件下保温3h，每隔0.5h取样测定酶活性。酶活测定方法参照GB/T 23527-2009附录B福林法进行。从图2可以看出在不加任何保护剂和稳定剂的前提下，2709碱性蛋白酶及其突变体在50℃条件下保温半小时，2709碱性蛋白突变体的剩余活力明显高于原始2709碱性蛋白酶，并且即使保温更长时间，突变体的残余活力始终高于原始2709碱性蛋白酶。
2.在不加任何保护剂和稳定剂的前提下，测试2709碱性蛋白酶及其突变体1和2在pH为5-13范围的酶活力，酶活测定方法参照GB/T 23527-2009附录B福林法进行。从图3可以明显看出突变体的剩余活力在pH大于9后明显高于原始2709碱性蛋白酶。
3.在不加任何保护剂和稳定剂的前提下，分别添加相同量的2709碱性蛋白酶和其突变体1和2于市售浓缩粉和标准液体洗涤体系中，测其酶活力，酶活测定方法参照GB/T 23527-2009附录B福林法进行。从图4可以明显看出2709碱性蛋白酶突变体相比2709碱性蛋白酶在上述两种洗涤剂体系中有更好的稳定 性。
4、在不加任何保护剂和稳定剂的前提下，测定2709碱性蛋白酶及其突变体1、2和3在40℃表面充分暴露、75％湿度的苛刻环境下的湿热稳定性，图5可以看出2709碱性蛋白酶突变体的剩余活力始终高于原始2709碱性蛋白酶，且蛋白酶突变体两周后的残余酶活力都在75％以上，而2709碱性蛋白酶残余酶活仅有35％左右。
综合表明本发明所述的2709碱性蛋白酶具有较好的耐热性能，其酶活力在碱性条件下有明显提高，且湿热稳定性较好。2709碱性蛋白酶突变体作为洗涤助剂使用时，具有良好的去污作用，比亲本蛋白酶在极端条件下能更好的保留活性，可以在更高的温度下以及更强的碱性环境下使用。表明本发明所述2709碱性蛋白酶突变体可广泛应用于洗涤工业领域。
实施例5
2709碱性蛋白酶突变体应用于洗涤剂中的蛋白污渍去污效果验证实验
1、实验材料：
1)碱性蛋白酶样品：
市售国外碱性蛋白酶产品：酶活5000U/ml；
本发明所述2709碱性蛋白酶突变体1：酶活5000U/ml；
本发明所述2709碱性蛋白酶突变体2：酶活5000U/ml；
本发明所述2709碱性蛋白酶突变体3：酶活5000U/ml；
本发明所述原始2709碱性蛋白酶：酶活5000U/ml。
2)市售洗衣浓缩粉和标准液体洗涤剂
3)JB02国标蛋白污布GB/T 7568.2-2008
2、去污力洗涤实验条件：
将上述碱性蛋白酶样品分别按比例(表1方式)添加到市售洗涤浓缩粉及标准液体洗涤剂中，通过循环洗涤的方法(GB/T 13174-2008)确定2709碱性蛋白酶突变体对JB02国标蛋白污布的去污效果。
洗涤设备：立式去污机(RHLQ II)，中国日用化学工业研究院
洗涤温度：30℃
洗涤时间：20min
搅拌速度：120r/min
水质硬度：250mg/kg(nCa2+/nMg2+＝3/2)
洗涤剂浓度：标准洗衣液2g/L；市售洗涤浓缩粉1g/L
反射值测量仪：白度仪(WSD-3C)
测试污布：JB02国标蛋白污布
R457白度：蓝光白度Wr
3、实验结果如下：
表1不同碱性蛋白酶对JB02国标蛋白污布洗前洗后白度差值
Figure PCTCN2021089618-appb-000003
Figure PCTCN2021089618-appb-000004
表2不同碱性蛋白酶对JB02国标蛋白污布的去污比值
Figure PCTCN2021089618-appb-000005
备注：洗后与洗前的白度差值越大，去污效果越明显；去污比值越大，去污效果越好。
从表1和表2的数据可以看出，添加2709碱性蛋白酶突变体样品的实验组2-5对JB02国标蛋白污布洗后与洗前白度差值较大，说明2709碱性蛋白酶突变体对蛋白污渍确实具有一定的去污效果；其中，添加2709碱性蛋白酶突变体的实验组3-5和8-10对蛋白污渍的去污力明显高于原始型2709碱性蛋白酶实验组2和7以及对照组1和2，与国外市售蛋白酶产品对蛋白污渍的去污能力相当。从而说明，本发明所述2709碱性蛋白酶突变体较原始型2709碱性蛋白酶对蛋 白污渍的去污效果得到显著提升，取得了意料不到的技术效果，同时本发明所述2709碱性蛋白酶突变体在其它洗涤剂存在的情况下不受影响仍能保持较高去污能力。表明该2709碱性蛋白酶突变体可用于清洁或洗涤领域。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干变型和改进，都包含在本发明的保护范围之内。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
Figure PCTCN2021089618-appb-000006
Figure PCTCN2021089618-appb-000007
Figure PCTCN2021089618-appb-000008

Claims (8)

  1. 一种基于分子动力学计算修饰的2709碱性蛋白酶突变体，其特征在于：所述碱性蛋白酶突变体的亲本蛋白为地衣芽孢杆菌的2709碱性蛋白酶，所述碱性蛋白酶突变体含有11-33区域内氨基酸的缺失或至少包含有以下位置的氨基酸修饰置换中的一个：9、12、15、16、17、18、19、33、38、39、40、41、42、46、47、48、49、50、58、59、60、67、79、96、97、98、99、107、108、109、110、111、116、131、132、133、134、139、140、152、153、163、164、165、166、173、188、189、190、191、193、195、198、201、203、211、221、234、235、236、237、238、239、240、241、242、243、244、245、255、256、257、259、260、262、268、273，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
  2. 根据权利要求1所述的一种基于分子动力学计算修饰的2709碱性蛋白酶突变体，其特征在于：所述碱性蛋白酶突变体至少包含G79D、N116D、I173D、P9R、Q273R中的一个位置上的氨基酸修饰置换，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
  3. 根据权利要求1所述的一种基于分子动力学计算修饰的2709碱性蛋白酶突变体，其特征在于：所述碱性蛋白酶突变体至少包含G46P、N96P、I110P、D139P、N140P、P166S、A198P、A201P、V203P、T211S、L239P、G256P、G262P/S和V268P中的一个位置上的氨基酸修饰置换，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
  4. 根据权利要求1所述的一种基于分子动力学计算修饰的2709碱性蛋白酶突变体，其特征在于：所述碱性蛋白酶突变体含有位置17-33或15-33的氨基酸缺失，同时至少含有K12N/Q、Q19S/R、T33H/Y/K中的一个位置上的氨基酸修饰置换，其中所述位置对应于氨基酸序列SEQ ID NO：1的位置。
  5. 一种质粒，其特征在于：所述的质粒携带有编码权利要求1-4任一项所述的2709碱性蛋白酶突变体的基因。
  6. 一种重组地衣芽孢杆菌，其特征在于：所述的地衣芽孢杆菌是将权利要求5所述的质粒转入地衣芽孢杆菌中构建的。
  7. 一种洗涤剂组合物，其特征在于：含有权利要求1-4中任一项所述的2709碱性蛋白酶突变体。
  8. 一种基于分子动力学计算修饰的2709碱性蛋白酶突变体的应用，其特征在于：用于清洁或洗涤领域。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116240153A (zh) * 2022-10-09 2023-06-09 天津科技大学 一种缺失羊毛硫肽基因簇的地衣芽孢杆菌及其应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112301023B (zh) * 2020-11-14 2022-09-23 山西大学 基于分子动力学计算修饰的2709碱性蛋白酶突变体及其应用
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections
CN114908074B (zh) * 2022-06-21 2023-11-14 山西大学 水溶液稳定性碱性蛋白酶2709突变体及其制备方法和应用

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060121455A1 (en) * 2003-04-14 2006-06-08 California Institute Of Technology COP protein design tool
CN1946841A (zh) * 2004-04-23 2007-04-11 汉高两合股份公司 新型碱性蛋白酶以及含有该新型碱性蛋白酶的洗涤剂和清洁剂
CN103087145A (zh) * 2013-02-20 2013-05-08 福州大学 一种基于理性设计的蛋白质分子热稳定性改造方法
CN107384892A (zh) * 2017-07-14 2017-11-24 江南大学 一种南极假丝酵母脂肪酶b突变体、改造方法及其应用
CN108623652A (zh) * 2018-03-13 2018-10-09 广西科学院 一种蛋白质热稳定性改造的方法及其在普鲁兰酶中的应用
CN109666666A (zh) * 2019-01-21 2019-04-23 天津科技大学 一种基于分子动力学的酶柔性分析提高肝素酶i热稳定性的突变体及其制备方法
CN110016390A (zh) * 2019-05-28 2019-07-16 河南歌瑞斯企业管理咨询有限公司 一种去除甲醛的洗衣液及其制备方法
CN110205314A (zh) * 2019-06-04 2019-09-06 山西大学 含2709碱性蛋白酶的稳定酶制剂及其制备工艺和应用
CN110777136A (zh) * 2019-11-26 2020-02-11 山西大学 一种洗涤用的碱性蛋白酶突变体及其在液体洗涤剂中的应用
CN110923221A (zh) * 2019-12-13 2020-03-27 中国科学院天津工业生物技术研究所 一种新型的来源于地衣芽孢杆菌的碱性蛋白酶高温突变体
CN111909979A (zh) * 2019-05-09 2020-11-10 武汉英凯生物技术研究院有限公司 L-草铵膦的制备方法
CN112301023A (zh) * 2020-11-14 2021-02-02 山西大学 基于分子动力学计算修饰的2709碱性蛋白酶突变体及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4143274B2 (ja) * 2001-04-12 2008-09-03 花王株式会社 アルカリプロテアーゼ
EP3464599A1 (en) * 2016-05-31 2019-04-10 Danisco US Inc. Protease variants and uses thereof

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060121455A1 (en) * 2003-04-14 2006-06-08 California Institute Of Technology COP protein design tool
CN1946841A (zh) * 2004-04-23 2007-04-11 汉高两合股份公司 新型碱性蛋白酶以及含有该新型碱性蛋白酶的洗涤剂和清洁剂
CN103087145A (zh) * 2013-02-20 2013-05-08 福州大学 一种基于理性设计的蛋白质分子热稳定性改造方法
CN107384892A (zh) * 2017-07-14 2017-11-24 江南大学 一种南极假丝酵母脂肪酶b突变体、改造方法及其应用
CN108623652A (zh) * 2018-03-13 2018-10-09 广西科学院 一种蛋白质热稳定性改造的方法及其在普鲁兰酶中的应用
CN109666666A (zh) * 2019-01-21 2019-04-23 天津科技大学 一种基于分子动力学的酶柔性分析提高肝素酶i热稳定性的突变体及其制备方法
CN111909979A (zh) * 2019-05-09 2020-11-10 武汉英凯生物技术研究院有限公司 L-草铵膦的制备方法
CN110016390A (zh) * 2019-05-28 2019-07-16 河南歌瑞斯企业管理咨询有限公司 一种去除甲醛的洗衣液及其制备方法
CN110205314A (zh) * 2019-06-04 2019-09-06 山西大学 含2709碱性蛋白酶的稳定酶制剂及其制备工艺和应用
CN110777136A (zh) * 2019-11-26 2020-02-11 山西大学 一种洗涤用的碱性蛋白酶突变体及其在液体洗涤剂中的应用
CN110923221A (zh) * 2019-12-13 2020-03-27 中国科学院天津工业生物技术研究所 一种新型的来源于地衣芽孢杆菌的碱性蛋白酶高温突变体
CN112301023A (zh) * 2020-11-14 2021-02-02 山西大学 基于分子动力学计算修饰的2709碱性蛋白酶突变体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAWEJA MEHAK, SINGH PUNEET KUMAR, SADAF AYESHA, TIWARI RAMESHWAR, NAIN LATA, KHARE SUNIL KUMAR, SHUKLA PRATYOOSH: "Cost effective characterization process and molecular dynamic simulation of detergent compatible alkaline protease from Bacillus pumilus strain MP27", PROCESS BIOCHEMISTRY, ELSEVIER LTD, GB, vol. 58, 1 July 2017 (2017-07-01), GB , pages 199 - 203, XP055930504, ISSN: 1359-5113, DOI: 10.1016/j.procbio.2017.04.024 *
CHAO ZHANG, LV 1S, XU-DONG, MA 1S, 1S YU-FEI, FEI YAN, WANG 1S, CUI-HONG, ZHANG 2S, 1S MEI-LING: "The Ｒegional Selectivity of Lauric Acid Catalyzed by Cytochrome P 450 4Z1 Based on Quantitative Calculations and Molecular Dynamics Simulations", ZHONGGUO SHENGWU HUAXUE YU FENZI SHENGWU XUEBAO - CHINESE JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, BEIJING DAXUE YIXUEBU, CN, vol. 36, no. 6, 1 June 2020 (2020-06-01), CN , pages 689 - 698, XP055930496, ISSN: 1007-7626 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116240153A (zh) * 2022-10-09 2023-06-09 天津科技大学 一种缺失羊毛硫肽基因簇的地衣芽孢杆菌及其应用

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