WO2017031839A1 - 一种酶活提高的l-天冬酰胺酶突变体及其构建方法 - Google Patents

一种酶活提高的l-天冬酰胺酶突变体及其构建方法 Download PDF

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WO2017031839A1
WO2017031839A1 PCT/CN2015/094554 CN2015094554W WO2017031839A1 WO 2017031839 A1 WO2017031839 A1 WO 2017031839A1 CN 2015094554 W CN2015094554 W CN 2015094554W WO 2017031839 A1 WO2017031839 A1 WO 2017031839A1
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recombinant
asparaginase
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gene
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饶志明
龙水清
张显
杨套伟
徐美娟
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江南大学
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  • the invention relates to an L-asparaginase mutant with improved enzyme activity and a construction method thereof, and belongs to the technical field of genetic engineering.
  • L-asparaginase amidohydrolase (E.C. 3.5.1.1) is capable of hydrolyzing deamination of L-asparagine to form L-aspartic acid and ammonia.
  • L-asparaginase has anti-tumor activity and has been used in the treatment of acute lymphoblastic leukemia and Hodgkin's disease. In recent years, it has been found that L-asparaginase can also reduce the formation of acrylamide in fried foods.
  • the size, structure and nature of L-asparaginase vary from source to source. L-asparaginase is widely available, and L-asparaginase is found in guinea pig serum, plants and microorganisms.
  • L-asparaginase The problem of heterologous expression of L-asparaginase is that the protein expression is low and the L-asparaginase enzyme activity is low. Therefore, site-directed mutagenesis of L-asparaginase and enhancement of extracellular enzyme activity are of great significance for improving the industrial application prospect of L-asparaginase.
  • the present invention first provides an L-asparaginase mutant having improved enzymatic activity, the amino acid sequence of which is the sequence shown in SEQ ID NO.
  • the nucleotide sequence encoding the mutant is the sequence shown in SEQ ID NO.
  • the mutant is based on an amino acid such as the amino acid shown in SEQ ID NO. 2, and amino acid 107 is mutated from glycine to aspartic acid.
  • the present invention also provides a genetically engineered bacteria expressing the L-asparaginase mutant.
  • the method for preparing the genetically engineered bacteria is to mutate the codon encoding the 107th glycine into a codon encoding aspartic acid based on the nucleotide sequence shown in SEQ ID NO.
  • the recombinant gene is ligated to the expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a Bacillus subtilis host strain to obtain a Bacillus subtilis genetically engineered strain.
  • the expression vector is pMA5.
  • the preparation method is specifically:
  • the present invention modifies the molecular structure of L-asparaginase by site-directed mutagenesis, and the pure enzyme solution of the mutant enzyme is 83% higher than that before the mutation.
  • the substrate affinity K m of the mutant enzyme G107D was reduced by 50% compared to before the mutation, and the catalytic efficiency was increased (the ratio of k cat to K m ) was 84%.
  • the invention shows that the 107 amino acid residue has a great influence on the catalytic action of the enzyme, provides a certain basis for the research on the catalytic mechanism of the enzyme, and improves the industrial application potential of the enzyme.
  • the invention can be used for preparing medicines for treating acute lymphocytic leukemia and Hawkinson's disease, and can also be used for reducing the formation of acrylamide in fried foods.
  • the recombinant gene and pMA5 were digested with BamHI and MluI, respectively, and purified, and then ligated with T4 DNA ligase at 16 ° C overnight.
  • the ligation product chemically transforms JM109 competent cells.
  • the transformant was coated with kanamycin (50 mg/L) LB plate, the plasmid was extracted, and the constructed recombinant plasmid was verified by double digestion, and named pMA5-G107D.
  • the sequencing work was completed by Shanghai Biotech.
  • the recombinant plasmid pMA5-G107D obtained in Example 1 was chemically transformed into B. subtilis 168 competent cells by the following methods:
  • Sp-A (NH 4 ) 2 SO 4 4, K 2 HPO 4 28, sodium citrate 12Sp-B: MgSO 4 ⁇ 7H 2 O 0.4
  • yeast powder 100Sp I medium: Sp-A 49%, Sp-B 49%, 50% glucose 2%, 100 ⁇ CAYE 2%
  • Sp II medium Sp I medium 98%, 50 mmol/LCaCl 2 1%, 250 mmol/L MgCl 2 1%. Damp heat sterilization at 115 °C.
  • Example 3 High-efficiency expression and enzyme activity assay of recombinant strain pMA5-G107D/B.subtilis 168L-aspartate.
  • the recombinant strain pMA5-G107D/B.subtilis 168 constructed in Example 2 and the control strain pMA5-ansz/B.subtilis 168 expressing the unmutated enzyme were inoculated into 10 mL of LB medium containing kanamycin, respectively.
  • the culture was shaken overnight at 37 ° C, and transferred to the fermentation medium of Bacillus subtilis by 4% inoculation on the next day, and cultured at 37 ° C for 24 h.
  • the fermentation broth was centrifuged at 4 ° C, 10000 r / min for 10 min, and the supernatant was extracellular.
  • the crude enzyme solution and the cell disrupted supernatant are intracellular crude enzyme solutions for the determination of enzyme activity.
  • Fermentation medium of Bacillus subtilis soybean protein ⁇ 10g / L, K 2 HPO 4 2.3g / L, KH 2 PO 4 1.7g / L, corn syrup 15g / L, urea 3g / L, glucose 40g / L , MgSO 4 0.75 g / L, NaCl 5 g / L. Adjust pH 6.8-7.0.
  • Enzyme activity definition The amount of enzyme required to catalyze the conversion of L-aspartamide to 1 ⁇ mol of NH 3 per minute under a reaction condition of 40 ° C is an enzyme unit.
  • L-asparaginase enzyme activity assay method The enzyme activity was determined by measuring the amount of NH 3 released in the catalytic reaction using L-asparagine as a substrate.
  • the reaction mixture (1 mL) was composed of: 400 ⁇ L of 25 mM L-asparagine (dissolved in 50 mM pH 7.5 Tris-HCl); 400 ⁇ L of 50 mM pH 7.5 Tris-HCl; 100 ⁇ L of an appropriate concentration of the enzyme solution.
  • the reaction mixture was reacted at 40 ° C, pH 7.5 for 15 min, and then the reaction was terminated by adding 100 ⁇ L of a 15% (W/V%) trichloroacetic acid solution.
  • the reaction solution in which the reaction was terminated by adding trichloroacetic acid before the enzyme reaction was used as a blank control.
  • the reaction mixture was centrifuged at 20,000 g for 10 min, and 200 uL of the supernatant was added to 4.8 mL of deionized water.
  • 200 ⁇ L of the Nessler reagent was added to the above system, and the absorbance was measured at a wavelength of 450 nm, and the amount of NH 3 released by the enzyme reaction was measured by a color reaction.
  • the extracellular crude enzyme solution obtained in the step (1) is purified to obtain L-asparaginase G107D ansz , and the purified L-asparaginase G107D ansz enzymatic properties are analyzed, as shown in Table 1, substrate
  • the affinity K m was reduced by 50% before the mutation, the catalytic efficiency k cat /K m was increased by 84%, and the enzyme activity was increased by 83%. Due to the increase in catalytic efficiency, the specific enzyme activity of G107D ansz is increased.

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Abstract

提供了一种酶活提高的L-天冬酰胺酶突变体及其构建方法，所述突变体是在SEQ ID NO.2所示的氨基酸序列的基础上，将107位氨基酸由甘氨酸突变成天冬氨酸而构建的。还提供了一种表达所述L-天冬酰胺酶突变体的基因工程菌。

Description

[根据细则26改正22.03.2016]　一种酶活提高的L-天冬酰胺酶突变体及其构建方法 技术领域
本发明涉及一种酶活提高的L-天冬酰胺酶突变体及其构建方法，属于基因工程技术领域。
背景技术
L-天冬酰胺酶(L-asparaginase amidohydrolase，E.C.3.5.1.1)能够将L-天冬酰胺水解脱氨基形成L-天冬氨酸和氨。L-天冬酰胺酶具有抗肿瘤活性，目前已应用于治疗急性淋巴细胞白血病及霍金森病等，近年来研究发现L-天冬酰胺酶还可以减少油炸食品中丙烯酰胺的生成。L-天冬酰胺酶大小、结构及性质因来源不同而有所不同。L-天冬酰胺酶来源比较广泛，豚鼠血清、植物以及微生物中都发现含有L-天冬酰胺酶。
异源表达L-天冬酰胺酶突出的问题是，蛋白表达量低、L-天冬酰胺酶酶活低。因此，定点突变改造L-天冬酰胺酶，提高胞外酶活，对于提高L-天冬酰胺酶工业化应用前景具有重要意义。
发明内容
本发明首先提供了一种酶活提高的L-天冬酰胺酶突变体，其氨基酸序列是SEQ ID NO.1所示的序列。
编码所述突变体的核苷酸序列是SEQ ID NO.3所示的序列。
所述突变体是在氨基酸如序列SEQ ID NO.2所示的氨基酸的基础上，将107位氨基酸由甘氨酸突变成天冬氨酸。
本发明还提供了一种表达所述L-天冬酰胺酶突变体的基因工程菌。
所述基因工程菌的制备方法，是在SEQ ID NO.4所示核苷酸序列的基础上，将编码第107位的甘氨酸的密码子突变成了编码天冬氨酸的密码子，得到重组基因，将重组基因连接到表达载体得到重组质粒，重组质粒转化到枯草芽孢杆菌宿主菌中即得到枯草芽孢杆菌基因工程菌。
在本发明的一种实施方式中，所述表达载体是pMA5。
在本发明的一种实施方式中，所述的制备方法，具体是：
(1)以SEQ ID NO.4所示核苷酸序列为模板，Flprimer(序列如SEQ ID NO.5所示)，Rlprimer(序列如SEQ ID NO.6所示)为引物，进行PCR即得到SEQ ID NO.3所示的重组基因 G107D。
(2)将上一步得到的重组基因序列，连接到pMA5表达载体中，得到重组质粒pMA5-G107D，重组质粒化转化B.subtilis 168，获得重组枯草芽孢杆菌工程菌株，命名为pMA5-G107D/B.subtilis 168。
本发明在天然L-天冬酰胺酶的基础上，通过定点突变生物技术改造L-天冬酰胺酶分子结构，突变体酶的纯酶液比酶活较突变前提高83％。突变体酶G107D的底物亲和力Km较突变前降低50％，而催化效率提高(kcat与Km的比值)84％。本发明表明107位氨基酸残基对酶的催化作用有较大影响，对该酶的催化机理的研究提供了一定的基础，并提高了该酶的工业应用潜力。本发明所得可用于制备治疗急性淋巴细胞白血病及霍金森病的药物，也可用于减少油炸食品中丙烯酰胺的生成。
具体实施方式
实施例1含L-天冬酰胺酶突变体的重组载体的构建
(1)G107D突变体的获得：以SEQ ID NO.4所示核苷酸序列为模板，Fprimer(序列如SEQ ID NO.5所示)、Rprimer(序列如SEQ ID NO.6所示)为引物，进行PCR即得到SEQ ID NO.3所示的重组基因。
(2)将重组基因与pMA5分别用BamHI、MluI双酶切，纯化后用T4DNA连接酶16℃过夜连接。连接产物化学法转化JM109感受态细胞。转化液涂布含卡那霉素(50mg/L)LB平板，提取质粒，双酶切验证构建的重组质粒，命名为pMA5-G107D。测序工作由上海生工完成。
实施例2产L-天冬酰胺酶枯草芽孢杆菌工程菌构建
将实施例1得到的重组质粒pMA5-G107D化学法转化入B.subtilis 168感受态细胞，具体方法如下：
(1)转化实验所需溶液如下(g/L)：
Sp-A：(NH4)2SO44，K2HPO428，柠檬酸钠12Sp-B：MgSO4·7H2O 0.4
100×CAYE：Casamino acid 20，酵母粉100Sp I培养基：Sp-A 49％，Sp-B 49％，50％葡萄糖2％，100×CAYE 2％Sp II培养基：Sp I培养基98％，50mmol/LCaCl21％，250mmol/L MgCl21％。115℃湿热灭菌。
(2)将B.Subtilis 168的单菌落接种至2mL Sp I培养基中(50mL离心管)，37℃、200r/min培养过夜；
(3)取100μL培养液至5mL Sp I培养基中，37℃、200r/min培养至对数期(OD600值 为1左右)，约4～5h；
(4)取200μL培养液至2mL Sp II培养基中，37℃、200r/min培养90min，取出后加入20μL 10mmol/L EGTA，于37℃、200r/min继续培养10min，然后分装成500μL每管，加入5μL重组质粒pMA5-G107D，混匀，37℃、200r/min培养90min，取菌液涂布抗性平板。37℃培养12h，挑取阳性转化子验证。得到重组菌pMA5-G107D/B.subtilis 168。
实施例3重组菌pMA5-G107D/B.subtilis 168L-天冬酞胺酶高效表达及酶活测定。
(1)将实施例2构建的重组菌pMA5-G107D/B.subtilis 168与表达未突变的酶的对照菌株pMA5-ansz/B.subtilis 168分别接种于l0mL含卡那霉素的LB培养基中，37℃振荡培养过夜，次日按4％的接种量转接于枯草芽抱杆菌发酵培养基中，37℃培养24h，取发酵液于4℃、10000r/min离心l0min，上清为胞外粗酶液，细胞破碎上清液为胞内粗酶液，用于酶活力的测定。
(2)枯草芽抱杆菌发酵培养基：大豆蛋白胨10g/L，K2HPO42.3g/L，KH2PO41.7g/L，玉米浆15g/L，尿素3g/L，葡萄糖40g/L，MgSO40.75g/L，NaCl 5g/L。调节pH 6.8-7.0。
(3)酶活定义:在40℃反应条件下，每分钟内能催化L-天冬酞胺转化为1μmol NH3所需要的酶量为一个酶活单位。
(4)L-天冬酰胺酶酶活测定方法：以L-天冬酰胺为底物，通过测定在催化反应中释放的NH3的量来测定酶活。反应混合物(1mL)组成为：400μL 25mM L-天冬酰胺(溶解于50mM pH 7.5Tris-HCl)；400μL50mM pH 7.5Tris-HCl；100μL适当浓度的酶溶液。反应混合物在40℃，pH7.5条件下，反应15min后，加入100μL 15％(W/V％)三氯乙酸溶液终止反应。以酶反应前加入三氯乙酸终止反应的反应液作为空白对照。反应混合物在20000g条件下离心10min,取200uL上清液加入到4.8mL的去离子水中。向上述体系中加入200μL的奈斯勒试剂，测定在450nm波长下测吸光度，通过显色反应测酶反应所释放的NH3的量。
(3)结果表明重组菌pMA5-G107D/B.subtilis 168表达的L-天冬酰胺酶的总酶活(胞内与胞外酶活的总和)为961U/mL，比对照菌株pMA5-ansz/.subtilis 168(534.2U/mL)L-天冬酰胺酶酶活提高80％。
(4)步骤(1)得到的胞外粗酶液经纯化后得到L-天冬酰胺酶G107Dansz，分析纯化后的重组L-天冬酰胺酶G107Dansz酶学性质，如表1，底物亲和力Km较突变前降低50％，催化效率kcat/Km提高84％，同时比酶活提高83％。由于催化效率的提高，增加了G107Dansz的比酶活。
表1 G107Dansz反应动力学参数
Figure PCTCN2015094554-appb-000001
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。
Figure PCTCN2015094554-appb-000002
Figure PCTCN2015094554-appb-000003
Figure PCTCN2015094554-appb-000004
Figure PCTCN2015094554-appb-000005
Figure PCTCN2015094554-appb-000006
Figure PCTCN2015094554-appb-000007
Figure PCTCN2015094554-appb-000008

Claims (8)

  1. 一种L-天冬酰胺酶突变体，其特征在于，所述突变体的氨基酸序列如SEQ ID NO.1所示。
  2. 编码权利要求1所述突变体的基因。
  3. 一种含有权利要求2所述基因的重组表达载体。
  4. 一种表达权利要求1所述L-天冬酰胺酶突变体的基因工程菌。
  5. 一种制备权利要求4所述基因工程菌的方法，其特征在于，是在SEQ ID NO.4所示序列的基础上，将第107位甘氨酸突变成天冬氨酸，得到重组基因，将重组基因连到表达载体得到重组质粒，重组质粒转化到枯草芽孢杆菌宿主菌中即得到枯草芽孢杆菌基因工程菌。
  6. 根据权利要求5所述的制备方法，其特征在于，所述方法具体是:(1)以SEQ ID NO.4所示核酸序列为模板，以序列如SEQ ID NO.5、SEQ ID NO.6所示的引物，进行PCR，即得到编码的107位氨基酸由甘氨酸突变成天冬氨酸的G107D突变体基因序列；(2)将上一步得到的重组基因序列，连接到pMA5表达载体中，得到重组质粒pMA5-G107D，重组质粒化转化B.Subtilis，获得重组枯草芽孢杆菌基因工程菌。
  7. 权利要求1所述L-天冬酰胺酶突变体在制备治疗急性淋巴细胞白血病及霍金森病的药物中的应用。
  8. 权利要求1所述L-天冬酰胺酶突变体在减少油炸食品中丙烯酰胺生成中的应用。
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CN107828768B (zh) * 2017-12-13 2020-10-09 江南大学 一种l-天冬酰胺酶突变体及其构建方法
CN107988194B (zh) * 2017-12-15 2020-08-04 江南大学 一种酶活提高的L-天冬氨酸α-脱羧酶变体及其构建方法
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