WO2019218598A1 - 一种偏甘油酯脂肪酶突变体及其应用 - Google Patents

一种偏甘油酯脂肪酶突变体及其应用 Download PDF

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WO2019218598A1
WO2019218598A1 PCT/CN2018/112112 CN2018112112W WO2019218598A1 WO 2019218598 A1 WO2019218598 A1 WO 2019218598A1 CN 2018112112 W CN2018112112 W CN 2018112112W WO 2019218598 A1 WO2019218598 A1 WO 2019218598A1
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mutant
partial glyceride
glyceride lipase
seq
protein
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王永华
蓝东明
袁红
谭熙钰
杨博
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华南理工大学
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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)
    • C12N9/16—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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

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  • the invention belongs to the field of biotechnology and relates to a preparation method of a partial glyceride lipase mutant with improved thermal stability and hydrogen peroxide tolerance and an application thereof.
  • epoxides are an important class of organic synthesis intermediates, and many high-value compounds can be synthesized by reacting with nucleophiles such as halogens, nitrogen, and sulfur to introduce various types of groups.
  • nucleophiles such as halogens, nitrogen, and sulfur
  • Conventional methods for the preparation of epoxides often use chemical methods, using some toxic and expensive chemical catalysts, such as silica-supported titanium, methyl antimony trioxide, etc., adding hydrogen peroxide or oxygen molecules as oxidants to produce the desired Epoxide.
  • the chemical preparation of epoxides has a certain toxic effect on the environment and limits actual industrial production.
  • the biocatalytic epoxidation reaction is a type of reaction with broad development prospects, and has the advantages of mild reaction conditions and low toxicity.
  • lipase-catalyzed oxidation has received extensive attention. Compared with monooxygenase and haloperoxidase, lipase can effectively catalyze the formation of peroxyacid without adding any cofactors during the preparation process. Economic and environmentally important. It is well known that lipase can catalyze various reactions such as esterification, hydrolysis, transesterification, aminolysis, alcoholysis, acid hydrolysis, etc. and has good ability to withstand high temperatures and organic solvents. Therefore, lipase has a very high industrial application value and has been widely used in medicine, food production, fragrance, chemical, washing and the like.
  • the peroxyacid formed by the organic acid and hydrogen peroxide can directly epoxidize the unsaturated hydrocarbon or cyclic ketone to obtain an epoxide or lactone compound, and does not need With any cofactor.
  • a large amount of side reactions occur with lipase-catalyzed epoxidation of lipid substrates.
  • Lipase catalyzes the hydrolysis of the lipid substrate itself and the lipid epoxidation product to produce fatty acids, which greatly reduces the yield of epoxidized products.
  • diglyceride lipases are a class of substrate-specific lipases that hydrolyze only diglycerides and monoglycerides without hydrolyzing triglycerides. In the process of preparing epoxidized vegetable oil, the occurrence of side reactions can be effectively avoided, and various industrial separation steps such as subsequent deacidification can be reduced.
  • Enzyme engineering is a kind of method that uses biotechnology to modify the enzyme to improve its target characteristics. By analyzing the three-dimensional structure of partial glyceride lipase and establishing the molecular docking model of enzyme-substrate, rational design of mutants is excellent. An enzyme mutant of the target nature.
  • the technical problem to be solved by the present invention is to provide a partial glyceride lipase mutant with improved thermal stability and hydrogen peroxide tolerance, which overcomes the complicated side reactions and low yield of products in industrial applications in the current stage of epoxidation reaction.
  • the enzyme is easily inactivated in a high concentration oxidizing environment.
  • the invention analyzes the three-dimensional structure of the dimeric fatty acid PCL derived from Penicillium (PDB: 5CH8) and establishes a molecular docking model of the enzyme-substrate, rationally designs the mutation site, and prepares the mutant by site-directed mutagenesis.
  • PDB Penicillium
  • tyrosine at position 84 of the protein is mutated to arginine (Y84R);
  • the isoleucine at position 260 of the protein is mutated to glutamic acid or arginine (I260E or I260R).
  • the DNA sequence of the mutant is SEQ NO. 2, SEQ NO. 3, SEQ NO. 4 or SEQ NO. 5.
  • amino acid sequence of the mutant is SEQ NO. 6, SEQ NO. 7, SEQ NO. 8 or SEQ NO.
  • the reaction conditions are: a pH of 5.0 ⁇ 0.5 and a temperature of 40 ⁇ 10 °C.
  • the preparation method of the mutant of the present invention is specifically as follows:
  • Primers were designed using a plasmid containing a partial glyceride lipase gene derived from Penicillium as a template, and site-directed mutagenesis was carried out to obtain a mutant.
  • the obtained mutant plasmid was transformed into E. coli competent cell DH5 ⁇ , and the transformant was applied to an LB plate containing 25 ⁇ g/mL bleomycin (Zeocin), and after culturing overnight at 37 ° C, a single colony grown on the plate was picked.
  • the plasmid was identified and extracted by sequencing.
  • the plasmid obtained above was linearized with DNA restriction endonuclease BlnI, and electrotransformed into Pichia pastoris X-33.
  • the transformant was applied to YPD plates containing 100 ⁇ g/mL bleomycin (Zeocin) and cultured at 37 ° C. At 48 h, the single colony that grew was the expression strain.
  • the PCL wild type retained only 44.0% viability after incubation for 24 hours in 3.3 M H 2 O 2 , and the mutant M209 retained 50.2% viability; Y84R, I260E and I260R retained more than 75% viability, respectively, 81.5%. 77%, 82.5% ( Figure 3).
  • the present invention utilizes an enzymatic engineering method to orient the partial glyceride lipase, the peroxidase activity and the hydrogen peroxide tolerance of the mutant are significantly improved, and the half-life of the enzyme is increased by 3-15 times in terms of thermal stability. Can better meet the needs of industrial production.
  • the partial glyceride lipase mutant of the present invention as a kind of lipase having strict substrate selectivity can effectively prevent the occurrence of side reactions in the lipid epoxidation reaction, and reduce the subsequent multiple
  • Figure 1 shows the structure of partial glyceride lipase PCL.
  • Figure 2 is a graph showing the thermal stability of partial glyceride lipase PCL and its mutants.
  • Figure 3 is a measure of the tolerance of partial glyceride lipase PCL and its mutant hydrogen peroxide.
  • Figure 4 is the effect of pH on the activity of partial glyceride lipase PCL and its mutant peroxidase.
  • Figure 5 is the effect of temperature on the activity of partial glyceride lipase PCL and its mutant peroxidase.
  • Figure 6 is a conversion of partial glyceride lipase PCL and its mutant catalyzed reaction of triolein for 24 h.
  • a mutant primer was designed using the plasmid pGAPZ ⁇ A-PCL derived from Penicillium glycerol lipase as a template, and a plasmid containing the mutant gene was amplified by PCR. After the PCR amplification product was confirmed by 1% agarose gel electrophoresis, a suitable enzyme digestion system was designed, and DpnI was added to remove the original template with methylation. After 4 h of digestion, the digested product was purified by PCR, and the purified product was confirmed by nucleic acid electrophoresis. The obtained purified product was mixed with E.
  • coli competent cell DH5 ⁇ placed on ice for 30 min, heat-shocked at 42 ° C for 90 s, and rapidly placed on ice for 5 min.
  • the competent cells obtained in the previous step were added to 1 ml of LB medium and activated at 37 ° C, 180 rpm for 50 min. After activation, the competent cells were centrifuged at 8000 rpm for 2 min, most of the medium was removed, and the obtained transformant was uniformly coated on LB solid plate containing 25 ⁇ g/mL bleomycin (Zeocin) at 37 ° C.
  • PCR primer sequences are as follows:
  • Y84R-F 5'-CTTGCTTTCAGAGGTTCTCGTTCCGTTAGAAATTGGG-3' (SEQ NO. 10)
  • Y84R-R 5'-CCCAATTTCTAACGGAACGAGAACCTCTGAAAGCAAG-3' (SEQ NO.11)
  • I260R-F 5'-CTGATTTTGAGGCACACAGGTGGTATTTTGTTCAAGTG-3' (SEQ NO. 16)
  • I260R-R 5'-CACTTGAACAAAATACCACCTGTGTGCCTCAAAATCAG-3' (SEQ NO.17)
  • the plasmid of the partial glyceride lipase mutant was linearized by restriction endonuclease BlnI at 37 ° C. After digestion for 5 h, the digested product was confirmed by nucleic acid electrophoresis. After the mutant plasmid was completely cleaved, the resulting digested product was subjected to purification of the PCR product, and finally eluted with sterile water. The purified product was electroporated into Pichia pastoris X-33, and the transformant was applied to a YPD plate containing 100 ⁇ g/mL bleomycin (Zeocin), and cultured at 37 ° C for 48 hours, and the grown single colony was the expression strain.
  • Zeocin bleomycin
  • a single colony in which a partial glyceride lipase mutant was picked was inoculated in 50 ml of YPD medium and cultured at 37 ° C, 200 rpm for 24 hours, and further inoculated into 500 ml of YPD medium for expansion culture. After 72 h, the fermented culture solution was centrifuged at 4 ° C, 12000 rpm for 10 min, and the supernatant was filtered through a 0.45 ⁇ m filter to further remove the bacteria and impurities.
  • the mutant protein is purified by an anion exchange chromatography column, and the specific steps are as follows:
  • Mutant protein concentration assay Mutant protein concentration was determined using the Brandford Protein Assay Kit. A standard curve was prepared by arranging a series of concentrations of bovine serum albumin as a protein concentration standard. Add 10 ⁇ l of different concentrations of standard protein to a 96-well plate, add 200 ⁇ l of Brandford reagent, and react for 5 min at room temperature. The absorbance is measured at 595 nm. The protein concentration standard curve is obtained based on the known protein concentration and the corresponding absorbance. The mutant protein was measured for absorbance at 595 nm under the same reaction conditions, and the test was performed in parallel for three or more times, and the protein concentration of the protein was compared to obtain a mutant protein concentration.
  • PCL purified in Example 2 and its mutants Y84R, M209A, I260E and I260R were incubated at 45 ° C at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, respectively.
  • Samples were taken at 12 h and placed on ice to prevent further loss of enzyme activity and accurate determination of residual enzyme activity.
  • the peroxidase activity of partial glyceride lipase PCL and its mutants was determined by measuring the content of peroxyacid.
  • the reaction system was: 0.1 M valeric acid pH 5.0 buffer (containing 180 ⁇ M MCD, 90 mM NaBr) 80 ⁇ L, 30% H 2 O 2 10 ⁇ L, and then 10 ⁇ L of the enzyme solution was added, and the reaction was carried out at 40 ° C for 5 min. The absorbance was measured at 290 nm and the experiment was repeated three more times. The control group was replaced with a high temperature inactivated enzyme.
  • the purified PCL and its mutants Y84R, M209A, I260E and I260R were incubated in 3.3 M H 2 O 2 , and each enzyme was sampled at 1, 2 , 4, 8, 12 and 24 h for residual viability determination.
  • Example 3 is described. The experimental results are shown in Figure 3.
  • the PCL wild type retained only 44.0% viability after incubation for 24 hours in 3.3 M H 2 O 2
  • the mutant M209 retained 50.2% viability
  • Y84R, I260E and I260R retained more than 75.
  • the vitality of % is 81.5%, 77%, and 82.5%, respectively.
  • valeric acid was selected as organic acid working solution, and purified PCL and its mutants Y84R, M209A, I260E and I260R were measured at pH 4.0, 4.5, 5.0 respectively.
  • the peroxidase activity under the conditions of 5.5, 6.0, and 6.5, and the reaction system are shown in Example 3.
  • the experimental results are shown in Fig. 4.
  • the optimum pH range of PCL wild type and Y84R, M209A, I260E and I260R is 4.5-5.0, and the reaction has the highest activity of peroxidase in this pH range.
  • Example 6 Partially glyceride lipase mutant catalyzed the formation of epoxidized triolein
  • Epoxy value determination method Accurately weigh 0.1g oil sample into 50ml Erlenmeyer flask, add 5ml chloroform to fully dissolve the oil sample, add 5-6 drops of 0.1% crystal violet indicator, mix and calibrate the bromine
  • the hydrogen-glacial acetic acid standard solution was titrated to pale green, and did not fade within 30 s.
  • the consumption of hydrogen bromide-glacial acetic acid solution was recorded, and the three groups were tested in parallel under the same conditions.
  • the control experiment used 0.1 g of triolein instead of 0.1 g of the oil sample for titration.

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Abstract

提供了一种对来源于青霉菌的偏甘油酯脂肪酶进行定点突变获得的偏甘油酯脂肪酶突变体，所述突变体的突变位点为下述任意一种或两种以上：(1)该蛋白84位的酪氨酸突变为精氨酸；(2)该蛋白209位的甲硫氨酸突变为丙氨酸；(3)该蛋白260位的异亮氨酸突变为谷氨酸或精氨酸。该突变体提高了偏甘油酯脂肪酶的热稳定性和过氧化氢耐受性，相较于野生型偏甘油酯脂肪酶，催化酯类进行环氧化反应的能力显著提高，目标产物得率高。

Description

一种偏甘油酯脂肪酶突变体及其应用 技术领域
本发明属于生物技术领域，涉及一种热稳定性和过氧化氢耐受性提高的偏甘油酯脂肪酶突变体的制备方法及其应用。
背景技术
众所周知，环氧化物是一类重要的有机合成中间体，可以通过与含卤素、氮、硫等亲核试剂反应，引入各种类型的基团，合成许多高价值的化合物。制备环氧化物的传统方法常采用化学法，使用一些有毒性且价格昂贵的化学催化剂，如二氧化硅负载钛、甲基三氧化铼等，添加过氧化氢或氧气分子作为氧化剂，生成所需环氧化物。因而，化学法制备环氧化物对环境有一定的毒害作用，并且限制了实际工业生产。生物催化环氧化反应是一类具有广阔发展前景的反应类型，具有反应条件温和、低毒等优势。近年来，脂肪酶催化氧化反应受到了广泛的关注，相较于单加氧酶及卤代过氧化物酶，脂肪酶可以有效催化生成过氧酸，且不需要添加任何辅因子，在制备过程的经济性和环保性上具有重要意义。众所周知，脂肪酶可以催化酯化、水解、酯交换、氨解、醇解、酸解等多种反应且具有较好的耐受高温和有机溶剂的能力。因而，脂肪酶具有极高的工业应用价值，已被广泛应用于医药、食品生产、香料、化工、洗涤等方面。在脂肪酶催化环氧化的过程中，有机酸与过氧化氢生成的过氧酸能直接将不饱和烃类或环酮类环氧化，得到环氧化物或内酯类化合物，且不需要借助任何辅因子。通常情况下，在脂肪酶催化脂类底物发生环氧化反应的过程中，会伴随大量副反应的发生。脂肪酶能催化 脂类底物本身及脂类环氧化产物水解，产生脂肪酸，很大程度上降低环氧化产物得率。由于大多数脂类底物为植物油(大多数为甘油三酯)，甘油二酯脂肪酶作为一类只水解甘油二酯及甘油单酯而不水解甘油三酯的底物专一性脂肪酶，在制备环氧化植物油的过程中能有效避免副反应的发生，减少后续脱酸等多项工业分离步骤。
酶工程是一类利用生物技术对酶进行定向改造从而提高其目标特性的方法，通过分析偏甘油酯脂肪酶的三维结构及建立酶-底物的分子对接模型，理性设计突变体，得到具有优良目标特性的酶突变体。
发明内容
本发明要解决的技术问题是提供一个热稳定性和过氧化氢耐受性提高的偏甘油酯脂肪酶突变体，克服现阶段环氧化反应在工业应用中副反应繁杂、产物得率低、在高浓度氧化环境中酶易失活等问题。
本发明的技术方案如下：
本发明通过分析来源于青霉菌的甘油二酯脂肪酶PCL的三维结构(PDB：5CH8)及建立酶-底物的分子对接模型，理性设计突变位点，采用定点突变的方法制备突变体。
一种偏甘油酯脂肪酶突变体，是对来源于青霉菌的偏甘油酯脂肪酶进行定点突变而得(图1)，其突变位点为下述任意一种或两种以上：
(1)该蛋白84位的酪氨酸突变为精氨酸(Y84R)；
(2)该蛋白209位的甲硫氨酸突变为丙氨酸(M209A)；
(3)该蛋白260位的异亮氨酸突变为谷氨酸或精氨酸(I260E或I260R)。
优选地，所述突变体的DNA序列为SEQ NO.2，SEQ NO.3，SEQ NO.4或 SEQ NO.5。
优选地，所述突变体的氨基酸序列为SEQ NO.6，SEQ NO.7，SEQ NO.8或SEQ NO.9。
所述的偏甘油酯脂肪酶突变体在制备环氧化酯类中的应用。
优选地，在制备环氧化酯类时，反应条件为：pH值为5.0±0.5，温度40±10℃。
本发明所述突变体的制备方法具体如下：
以包含来源于青霉菌的偏甘油酯脂肪酶基因的质粒为模板，设计引物，进行定点突变，得到突变体。
将得到的突变体质粒转化至大肠杆菌感受态细胞DH5α，转化液涂布于含有25μg/mL博来霉素(Zeocin)的LB平板中，37℃培养过夜后，挑平板上长出的单菌落测序鉴定并提取质粒。
将上述所得质粒用DNA限制性内切酶BlnI线性化后电转至毕赤酵母X-33中,转化液涂布于含有100μg/mL博来霉素(Zeocin)的YPD平板中，37℃培养过48h，长出的单菌落为所述表达菌株。
经测定，脂肪酶PCL野生型(其DNA序列为SEQ NO.1)及其突变体的热稳定性、H 2O 2耐受性和催化三油酸甘油酯生成环氧三油酸甘油酯的特点如下：
PCL野生型在45℃下孵育12小时后仅剩余19.7％的活力，M209A和I260E仍剩余30.9％和40.2％的活力；Y84R和I260R在45℃下孵育12小时后依然保有68.2％和82.5％的酶活(图2)。
PCL野生型在3.3M H 2O 2中孵育24小时后只剩下44.0％的活力，突变体M209还残留50.2％的活力；Y84R、I260E和I260R都保留了大于75％的活力，分别为 81.5％、77％、82.5％(图3)。
PCL野生型在反应24h后得到环氧化三油酸甘油酯为31.4％，突变体M209A和Y84R分别得到环氧化三油酸甘油酯54.1％和65.8％；I260E与I260R得到产物环氧化三油酸甘油酯显著增加，分别为80.2％和88.4％。PCL野生型及突变体催化反应所得环氧化三油酸甘油酯。
与现有技术相比，本发明的优点和有益效果在于：
(1)本发明利用酶工程手段定向改造偏甘油酯脂肪酶，所述突变体的过氧化酶活及过氧化氢耐受性得到显著提高，在热稳定性方面酶的半衰期提高3-15倍，能够更好的满足工业生产需求。
(2)本发明所述偏甘油酯脂肪酶突变体作为一类具有严格意义上底物选择性的脂肪酶，在脂类环氧化反应中能非常有效的避免副反应的发生，减少后续多项分离纯化步骤如脱酸，显著提高环氧化产物得率及产物成分的单一性，有效减少实际工业中的生产成本，具有可持续发展的环保意义。
附图说明
图1是偏甘油酯脂肪酶PCL的结构。
图2是偏甘油酯脂肪酶PCL及其突变体的热稳定性测定。
图3是偏甘油酯脂肪酶PCL及其突变体过氧化氢耐受性的测定。
图4是pH对偏甘油酯脂肪酶PCL及其突变体过氧化酶活力的影响。
图5是温度对偏甘油酯脂肪酶PCL及其突变体过氧化酶活力的影响。
图6是偏甘油酯脂肪酶PCL及其突变体催化三油酸甘油酯反应24h的转化率。
具体实施方法
实施例1偏甘油酯脂肪酶突变体的构建
以来源于青霉菌的偏甘油酯脂肪酶的质粒pGAPZαA-PCL为模板，设计突变体引物，利用PCR扩增得到含突变基因的质粒。PCR扩增产物经1％琼脂糖凝胶电泳检验确认后，设计合适的酶切体系，加入DpnI去除带甲基化的原始模板。酶切4h后，酶切产物进行PCR产物纯化，纯化产物经核酸电泳检验确认。将所得纯化产物与大肠杆菌感受态细胞DH5α混合，置于冰上30min，于42℃下热击90s，迅速置于冰上5min。将上步所得感受态细胞加入至1ml LB培养基中，在37℃，180rpm条件下活化50min。活化后感受态细胞于8000rpm条件下离心2min，移除大部分培养基，重悬菌体后所得转化液均匀涂布于含有25μg/mL博来霉素(Zeocin)的LB固体平板上，37℃培养箱中静置培养过夜后，挑取平板上单菌落接种于5ml的LB液体培养基(含25μg/mL Zeocin)中，于37℃，200rpm条件下过夜培养，经基因测序确定扩增结果后提取质粒备用。
PCR引物序列如下：
Y84R-F:5'-CTTGCTTTCAGAGGTTCTCGTTCCGTTAGAAATTGGG-3'(SEQ NO.10)
Y84R-R:5'-CCCAATTTCTAACGGAACGAGAACCTCTGAAAGCAAG-3'(SEQ NO.11)
M209A-F:
5'-GTGAACGTACCCCGCTGACAACAATGGCAGTTTGGGCACT-3'(SEQ NO.12)
M209A-R:
5'-AGTGCCCAAACTGCCATTGTTGTCAGCGGGGTACGTTCAC-3'(SEQ  NO.13)
I260E-F:
5'-GACTGATTTTGAGGCACACGAGTGGTATTTTGTTCAAGTGG-3'(SEQ NO.14)
I260E-R:
5'-CCACTTGAACAAAATACCACTCGTGTGCCTCAAAATCAGTC-3'(SEQ NO.15)
I260R-F:5'-CTGATTTTGAGGCACACAGGTGGTATTTTGTTCAAGTG-3'(SEQ NO.16)
I260R-R:5'-CACTTGAACAAAATACCACCTGTGTGCCTCAAAATCAG-3'(SEQ NO.17)
实施例2偏甘油酯脂肪酶突变体的表达及蛋白纯化
突变体的表达：偏甘油酯脂肪酶突变体的质粒经DNA限制性内切酶BlnI于37℃进行酶切线性化，酶切5h后对酶切产物进行核酸电泳检测确认。突变体质粒被完全切开后，所得酶切产物进行PCR产物纯化，使用无菌水对其进行最终洗脱。将纯化产物电转至毕赤酵母X-33中，转化液涂布于含有100μg/mL博来霉素(Zeocin)的YPD平板中，37℃培养48h，长出的单菌落为所述表达菌株。挑取偏甘油酯脂肪酶突变体的单菌落接种于50ml YPD培养基中于37℃、200rpm条件下培养24h，再进一步接种到500ml YPD培养基中进行扩大培养。72h后，将发酵后的培养液于4℃、12000rpm条件下离心10min，上清液通过0.45μm滤膜抽滤，进一步去除菌体和杂质。
突变体蛋白的纯化：利用切向流超滤膜包对所得发酵液进行回流浓缩，再利 用20mM，PH6.5的磷酸盐缓冲液置换发酵液并将其浓缩至50ml。然后，利用阴离子交换层析柱纯化突变体蛋白，具体步骤如下：
(1)超纯水经0.45μm滤膜抽滤后，冲洗去除用于保护阴离子交换层析柱填料的乙醇，流速为5ml/min。
(2)使用20mM，PH6.5的磷酸盐缓冲液作为平衡缓冲液以平衡阴离子填料，流速为5ml/min。当紫外吸光值及电导率显示平衡后即可。
(3)将浓缩后的突变体蛋白上样至阴离子柱上，流速为3ml/min。待上样完成后，用平衡缓冲液继续洗去未与填料结合的杂蛋白。待基线平衡后，利用含1M NaCl的20mM，PH6.5的磷酸盐缓冲液作为洗脱液进行梯度洗脱(0-1M)，收集洗脱液。
(4)将收集的洗脱液进行SDS-PAGE检测。根据蛋白电泳结果，选择纯度高的突变体蛋白保存备用。
突变体蛋白浓度检测：利用Brandford蛋白质测定试剂盒测定突变体蛋白浓度。配置牛血清蛋白的一系列浓度作为蛋白质浓度标准液，制备标准曲线。加入10μl不同浓度的标准蛋白于96孔板中，再加入200μl Brandford试剂，室温下反应5min后，于595nm处测定吸光值，根据已知蛋白浓度与对应的吸光值，得到蛋白浓度标准曲线。突变体蛋白在相同反应条件下测定595nm下的吸光值，平行试验三次以上，比对蛋白浓度标准曲线后得到突变体蛋白浓度。
实施例3偏甘油酯脂肪酶突变体热稳定性测定
将实施例2纯化后的PCL及其突变体Y84R、M209A、I260E和I260R在45℃下孵育，分别在0.5、1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、12h取样，置于冰上以防进一步损失酶活，精确测定残余酶活。本实验采用测定过氧酸的含 量方法测定偏甘油酯脂肪酶PCL及其突变体的过氧化酶活力。
酶活力的定义：在一定条件下，每分钟氧化1μmol MCD所需要的酶量即为一个酶活单位，用U表示。已知MCD在290nm具有最大吸光值，所检测吸光值比对MCD标准曲线可计算相应酶活力。
反应体系为：0.1M戊酸pH5.0缓冲液(包含180μM MCD，90mM NaBr)80μL、30％H 2O 2 10μL混匀后加入10μL酶液，40℃下反应5min。在290nm测量吸光值，实验重复三次以上。对照组以高温灭活的酶代替。
实验结果如图2所示，PCL野生型在孵育了12小时后只剩下19.7％的活力，M209A和I260E仍剩余30.9％和40.2％的活力；Y84R和I260R在45℃下孵育12小时后依然保有68.2％和82.5％的酶活。
实施例4偏甘油酯脂肪酶突变体过氧化氢耐受性测定
将纯化后的PCL及其突变体Y84R、M209A、I260E和I260R在3.3M H 2O 2中孵育，各酶分别在1、2、4、8、12和24h取样进行残余活力测定，测定方法如实施例3所述。实验结果如图3所示，PCL野生型在3.3M H 2O 2中孵育24小时后只剩下44.0％的活力，突变体M209还残留50.2％的活力；Y84R、I260E和I260R都保留了大于75％的活力，分别为81.5％、77％、82.5％。
实施例5偏甘油酯脂肪酶突变体酶学性质测定
pH对偏甘油酯脂肪酶突变体过氧化酶活力的影响：选取戊酸为有机酸工作液，分别测定纯化后的PCL及其突变体Y84R、M209A、I260E和I260R在pH4.0、4.5、5.0、5.5、6.0、6.5的条件下的过氧化酶活力，反应体系参照实施例3所示。实验结果如图4所示，PCL野生型及Y84R、M209A、I260E、I260R的最适pH范围为4.5～5.0，在该pH范围下反应，过氧化酶活力最高。
温度对偏甘油酯脂肪酶突变体过氧化酶活力的影响：分别测定纯化后的PCL及其突变体Y84R、M209A、I260E和I260R在30、35、40、45、50℃的条件下的过氧化酶活力，反应体系参照实施例3所示。实验结果如图5所示，在不同温度下，PCL与野生型及Y84R、M209A、I260E、I260R的过氧化酶活力变化很小。
实施例6偏甘油酯脂肪酶突变体催化生成环氧化三油酸甘油酯
取1.5ml纯化后的PCL野生型及其突变体酶液(蛋白浓度为1.4mg/ml)、1.5g三油酸甘油酯于10ml锥形瓶中，分次加入1080μl 30％过氧化氢，将锥形瓶置于40℃恒温油浴锅以550rpm搅拌速度进行环氧化反应。反应24h后，移取一定量锥形瓶中上层油样于离心管中，离心后精确称取0.1g上层油样，采用溴化氢-冰醋酸的方法测定环氧值，得到三油酸甘油酯的转化率。
环氧值测定方法：将精确称取的0.1g油样加入至50ml锥形瓶，加入5ml氯仿充分溶解油样，滴加5～6滴0.1％结晶紫指示剂，混匀后用标定后溴化氢-冰醋酸标准溶液滴定至淡绿色，30s内不褪色，记录溴化氢-冰醋酸溶液的消耗量，同样条件下平行实验三组。同时，对照试验使用0.1g三油酸甘油酯替代0.1g油样进行滴定。
实验结果如图6所示，PCL野生型在反应24h后得到环氧化三油酸甘油酯为31.4％，突变体M209A和Y84R分别得到环氧化三油酸甘油酯54.1％和65.8％；I260E与I260R得到产物环氧化三油酸甘油酯显著增加，分别为80.2％和88.4％。PCL野生型及突变体催化反应所得环氧化三油酸甘油酯。

Claims (5)

  1. 一种偏甘油酯脂肪酶突变体，其特征在于，是对来源于青霉菌的偏甘油酯脂肪酶进行定点突变而得，其突变位点为下述任意一种或两种以上：
    (1)该蛋白84位的酪氨酸突变为精氨酸；
    (2)该蛋白209位的甲硫氨酸突变为丙氨酸；
    (3)该蛋白260位的异亮氨酸突变为谷氨酸或精氨酸。
  2. 根据权利要求1所述的突变体，其特征在于，所述突变体的DNA序列为SEQ NO.2，SEQ NO.3，SEQ NO.4或SEQ NO.5。
  3. 根据权利要求1所述的突变体，其特征在于，所述突变体的氨基酸序列为SEQ NO.6，SEQ NO.7，SEQ NO.8或SEQ NO.9。
  4. 权利要求1～3任意一项所述的偏甘油酯脂肪酶突变体在制备环氧化酯类中的应用。
  5. 根据权利要求4所述的应用，其特征在于，在制备环氧化酯类时，反应条件为：pH值为5.0±0.5，温度40±10℃。
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