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CN105062997A - L-asparaginase mutant with improved enzyme activity and construction method thereof - Google Patents

L-asparaginase mutant with improved enzyme activity and construction method thereof Download PDF

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CN105062997A
CN105062997A CN201510526470.6A CN201510526470A CN105062997A CN 105062997 A CN105062997 A CN 105062997A CN 201510526470 A CN201510526470 A CN 201510526470A CN 105062997 A CN105062997 A CN 105062997A
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asparaginase
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饶志明
龙水清
张显
杨套伟
徐美娟
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Jiangnan University
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Rugao Jiangda Food Biotechnology Research Institute Co Ltd
Jiangnan University
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Abstract

The invention discloses an L-asparaginase mutant with the improved enzyme activity and a construction method thereof, and belongs to the field of gene engineering. According to the L-asparaginase mutant, on the basis of amino acid shown in the SEQ ID NO.2, the 107th glycine is mutated into aspartic acid. The obtained mutant is expressed in bacillus subtilis, fermentation is performed in a shake flask for 24 h and then the enzyme activity is 961 U/mL; the enzyme activity of the mutant is improved by 80%, the appetency of a substrate is decreased by 50% compared with protoenzyme, the catalytic efficiency is improved by 84%, and meanwhile the specific enzyme activity is improved by 83%. According to the L-asparaginase mutant, it is shown that the 107th amino acid residue has a great influence on the enzyme catalytic action, a certain foundation is provided for research on the enzyme catalytic mechanism, and the enzyme industrial application potential is improved.

Description

一种酶活提高的L-天冬酰胺酶突变体及其构建方法A kind of L-asparaginase mutant with improved enzyme activity and its construction method

技术领域technical field

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。The invention relates to an L-asparaginase mutant with improved enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering.

背景技术Background technique

L-天冬酰胺酶(L-asparaginaseamidohydrolase,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。L-asparaginase (L-asparaginaseamidohydrolase, E.C.3.5.1.1) can hydrolyze and deaminate 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 Hodgkinson's disease. In recent years, studies have found that L-asparaginase can also reduce the formation of acrylamide in fried foods. The size, structure and properties of L-asparaginase vary from source to source. L-asparaginase has a wide range of sources, and L-asparaginase is found in guinea pig serum, plants and microorganisms.

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。The prominent problems of heterologous expression of L-asparaginase are low protein expression and low activity of L-asparaginase. Therefore, site-directed mutagenesis to transform L-asparaginase and improve extracellular enzyme activity is of great significance for improving the industrial application prospect of L-asparaginase.

发明内容Contents of the invention

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SEQIDNO.1所示的序列。The present invention firstly provides an L-asparaginase mutant with improved enzyme activity, the amino acid sequence of which is the sequence shown in SEQ ID NO.1.

编码所述突变体的核苷酸序列是SEQIDNO.3所示的序列。The nucleotide sequence encoding the mutant is the sequence shown in SEQ ID NO.3.

所述突变体是在氨基酸如序列SEQIDNO.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。The mutant is based on the amino acid shown in the sequence of SEQ ID NO.2, and the 107th amino acid is mutated from glycine to aspartic acid.

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。The invention also provides a genetically engineered bacterium expressing the L-asparaginase mutant.

所述基因工程菌的制备方法,是在SEQIDNO.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。The preparation method of the genetically engineered bacteria is to mutate the codon encoding the 107th glycine into the codon encoding aspartic acid on the basis of the nucleotide sequence shown in SEQ ID NO.4 to obtain the recombinant gene The recombinant gene is connected to the expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a Bacillus subtilis host bacterium to obtain a Bacillus subtilis genetically engineered bacterium.

在本发明的一种实施方式中,所述表达载体是pMA5。In one embodiment of the present invention, the expression vector is pMA5.

在本发明的一种实施方式中,所述的制备方法,具体是:In one embodiment of the present invention, the preparation method is specifically:

(1)以SEQIDNO.4所示核苷酸序列为模板,Flprimer(序列如SEQIDNO.5所示),Rlprimer(序列如SEQIDNO.6所示)为引物,进行PCR即得到SEQIDNO.3所示的重组基因G107D。(1) With the nucleotide sequence shown in SEQIDNO.4 as a template, Flprimer (sequence shown in SEQIDNO.5), and Rlprimer (sequence shown in SEQIDNO.6) as primers, PCR is performed to obtain the nucleotide sequence shown in SEQIDNO.3 Recombinant gene 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.subtilis168,获得重组枯草芽孢杆菌工程菌株,命名为pMA5-G107D/B.subtilis168。(2) Connect the recombinant gene sequence obtained in the previous step into the pMA5 expression vector to obtain the recombinant plasmid pMA5-G107D, transform the recombinant plasmid into B. subtilis168, and obtain the recombinant Bacillus subtilis engineering strain, named pMA5-G107D/B. subtilis168.

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。On the basis of the natural L-asparaginase, the invention transforms the molecular structure of the L-asparaginase through site-directed mutation biotechnology, and the specific enzyme activity of the pure enzyme liquid of the mutant enzyme is increased by 83% compared with that before the mutation. The substrate affinity K m of the mutant enzyme G107D decreased by 50% compared with that before the mutation, while the catalytic efficiency (ratio of k cat to K m ) increased by 84%. The invention shows that the 107th amino acid residue has great influence on the catalysis of the enzyme, provides a certain basis for the research on the catalysis mechanism of the enzyme, and improves the industrial application potential of the enzyme. The obtained product of the invention can be used to prepare medicines for treating acute lymphocytic leukemia and Hodgkinson's disease, and can also be used to reduce the generation of acrylamide in fried food.

具体实施方式Detailed ways

实施例1含L-天冬酰胺酶突变体的重组载体的构建Embodiment 1 Contains the construction of the recombinant vector of L-asparaginase mutant

(1)G107D突变体的获得:以SEQIDNO.4所示核苷酸序列为模板,Fprimer(序列如SEQIDNO.5所示)、Rprimer(序列如SEQIDNO.6所示)为引物,进行PCR即得到SEQIDNO.3所示的重组基因。(1) Obtaining the G107D mutant: using the nucleotide sequence shown in SEQIDNO.4 as a template, Fprimer (sequence shown in SEQIDNO.5) and Rprimer (sequence shown in SEQIDNO.6) as primers, PCR is performed to obtain The recombinant gene shown in SEQ ID NO.3.

(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。(2) Digest the recombinant gene and pMA5 with BamHI and MluI, respectively, and ligate with T4 DNA ligase overnight at 16°C after purification. The ligation product was chemically transformed into JM109 competent cells. The transformation solution was applied to an LB plate containing kanamycin (50 mg/L), the plasmid was extracted, and the recombinant plasmid constructed was verified by double enzyme digestion, which was named pMA5-G107D. The sequencing work was completed by Shanghai Sangong.

实施例2产L-天冬酰胺酶枯草芽孢杆菌工程菌构建Example 2 Production of L-asparaginase Bacillus subtilis Engineering Bacteria Construction

将实施例1得到的重组质粒pMA5-G107D化学法转化入B.subtilis168感受态细胞,具体方法如下:The recombinant plasmid pMA5-G107D obtained in Example 1 was chemically transformed into B. subtilis168 competent cells, and the specific method was as follows:

(1)转化实验所需溶液如下(g/L):(1) The solution required for the conversion experiment is as follows (g/L):

Sp-A:(NH4)2SO44,K2HPO428,柠檬酸钠12Sp-B:MgSO4·7H2O0.4Sp-A: (NH 4 ) 2 SO 4 4, K 2 HPO 4 28, sodium citrate 12Sp-B: MgSO 4 7H 2 O0.4

100×CAYE:Casaminoacid20,酵母粉100SpI培养基:Sp-A49%,Sp-B49%,50%葡萄糖2%,100×CAYE2%SpII培养基:SpI培养基98%,50mmol/LCaCl21%,250mmol/LMgCl21%。115℃湿热灭菌。100×CAYE: Casaaminoacid20, yeast powder 100SpI medium: Sp-A49%, Sp-B49%, 50% glucose 2%, 100×CAYE2% SpII medium: SpI medium 98%, 50mmol/LCaCl 2 1%, 250mmol /LMgCl2 1 %. 115°C damp heat sterilization.

(2)将B.Subtilis168的单菌落接种至2mLSpI培养基中(50mL离心管),37℃、200r/min培养过夜;(2) Inoculate a single colony of B.Subtilis168 into 2mL SpI medium (50mL centrifuge tube), and culture overnight at 37°C and 200r/min;

(3)取100μL培养液至5mLSpI培养基中,37℃、200r/min培养至对数期(OD600值为1左右),约4~5h;(3) Take 100 μL of the culture solution into 5 mL of SpI medium, and culture at 37°C and 200 r/min until the logarithmic phase (OD600 value is about 1), about 4 to 5 hours;

(4)取200μL培养液至2mLSpII培养基中,37℃、200r/min培养90min,取出后加入20μL10mmol/LEGTA,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.subtilis168。(4) Take 200 μL of culture solution into 2 mL of SpII medium, incubate at 37°C and 200r/min for 90min, add 20μL of 10mmol/LEGTA after taking it out, continue to cultivate at 37°C and 200r/min for 10min, then divide into 500μL tubes, add 5μL The recombinant plasmid pMA5-G107D was mixed, cultured at 37°C and 200r/min for 90min, and the bacterial solution was taken to coat the resistant plate. Incubate at 37°C for 12 hours, and pick positive transformants for verification. The recombinant strain pMA5-G107D/B.subtilis168 was obtained.

实施例3重组菌pMA5-G107D/B.subtilis168L-天冬酞胺酶高效表达及酶活测定。Example 3 High expression and enzyme activity determination of recombinant bacteria pMA5-G107D/B.subtilis168L-asparaginase.

(1)将实施例2构建的重组菌pMA5-G107D/B.subtilis168与表达未突变的酶的对照菌株pMA5-ansz/B.subtilis168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。(1) The recombinant bacteria pMA5-G107D/B.subtilis168 constructed in Example 2 and the control strain pMA5-ansz/B.subtilis168 expressing unmutated enzymes were inoculated in 10 mL of LB medium containing kanamycin respectively, 37 Shake culture overnight at ℃, transfer to Bacillus subtilis fermentation medium at 4% inoculum the next day, culture at 37℃ for 24h, take the fermentation broth and centrifuge at 10000r/min for 10min at 4℃, the supernatant is crude extracellular enzyme liquid, and the supernatant of broken cells was the intracellular crude enzyme solution, which was used for the determination of enzyme activity.

(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,NaCl5g/L。调节pH6.8-7.0。(2) Bacillus subtilis fermentation medium: soybean peptone 10g/L, K 2 HPO 4 2.3g/L, KH 2 PO 4 1.7g/L, corn steep liquor 15g/L, urea 3g/L, glucose 40g/L , MgSO 4 0.75g/L, NaCl 5g/L. Adjust pH6.8-7.0.

(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1μmolNH3所需要的酶量为一个酶活单位。(3) Definition of enzyme activity: under the reaction condition of 40°C, the amount of enzyme required to catalyze the conversion of L-asparagine into 1 μmol NH per minute is an enzyme activity unit.

(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:400μL25mML-天冬酰胺(溶解于50mMpH7.5Tris-HCl);400μL50mMpH7.5Tris-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100μL15%(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。(4) L-asparaginase enzyme activity assay method: with L-asparagine as substrate, the enzyme activity is determined by measuring the amount of NH3 released in the catalytic reaction. The reaction mixture (1 mL) consisted of: 400 μL 25 mM L-asparagine (dissolved in 50 mM pH 7.5 Tris-HCl); 400 μL 50 mM pH 7.5 Tris-HCl; 100 μL enzyme solution of appropriate concentration. The reaction mixture was reacted at 40° C. and pH 7.5 for 15 min, and then 100 μL of 15% (W/V%) trichloroacetic acid solution was added to terminate the reaction. The reaction solution in which trichloroacetic acid was added to terminate the reaction before the enzyme reaction was used as a blank control. The reaction mixture was centrifuged at 20000g for 10min, and 200uL supernatant was added to 4.8mL deionized water. Add 200 μL of Nessler's reagent to the above system, measure the absorbance at a wavelength of 450 nm, and measure the amount of NH3 released by the enzyme reaction through a color reaction.

(3)结果表明重组菌pMA5-G107D/B.subtilis168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/.subtilis168(534.2U/mL)L-天冬酰胺酶酶活提高80%。(3) The results showed that the total enzyme activity (sum of intracellular and extracellular enzyme activity) of L-asparaginase expressed by the recombinant strain pMA5-G107D/B.subtilis168 was 961U/mL, which was higher than that of the control strain pMA5-ansz/. subtilis168 (534.2U/mL) L-asparaginase activity increased by 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。(4) The extracellular crude enzyme liquid obtained in step (1) is purified to obtain L-asparaginase G107D ansz , and the enzymatic properties of the purified recombinant L-asparaginase G107D ansz are analyzed, as shown in Table 1, substrate The affinity K m is reduced by 50% compared with that before the mutation, the catalytic efficiency k cat /K m is increased by 84%, and the specific enzyme activity is increased by 83%. The specific enzymatic activity of G107D ansz was increased due to the enhanced catalytic efficiency.

表1G107Dansz反应动力学参数Table 1G107D ansz reaction kinetic parameters

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。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

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SEQIDNO.1所示。1. An L-asparaginase mutant, characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO.1. 2.编码权利要求1所述突变体的基因。2. A gene encoding the mutant of claim 1. 3.一种含有权利要求2所述基因的重组表达载体。3. A recombinant expression vector containing the gene of claim 2. 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。4. A genetic engineering bacterium expressing the L-asparaginase mutant described in claim 1. 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SEQIDNO.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。5. A method for preparing the genetically engineered bacterium according to claim 4, characterized in that, on the basis of the sequence shown in SEQ ID NO.4, the 107th glycine is mutated into aspartic acid to obtain the recombinant gene, and the recombinant gene Connecting to the expression vector to obtain a recombinant plasmid, and transforming the recombinant plasmid into a Bacillus subtilis host bacterium to obtain a Bacillus subtilis genetically engineered bacterium. 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SEQIDNO.4所示核酸序列为模板,以序列如SEQIDNO.5、SEQIDNO.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。6. the preparation method according to claim 5, is characterized in that, described method is specifically: (1) take the nucleic acid sequence shown in SEQIDNO.4 as template, with the primer shown in SEQIDNO.5, SEQIDNO.6 with sequence , carry out PCR, promptly obtain the G107D mutant gene sequence that the 107th amino acid of coding is mutated into aspartic acid by glycine; (2) the recombinant gene sequence obtained in the previous step is connected to the pMA5 expression vector to obtain the recombinant plasmid pMA5-G107D , transforming B. Subtilis with recombinant plasmids to obtain recombinant Bacillus subtilis genetically engineered bacteria. 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。7. The application of the L-asparaginase mutant described in claim 1 in the preparation of medicines for the treatment of acute lymphoblastic leukemia and Hodgkinson's disease. 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。8. The application of the L-asparaginase mutant described in claim 1 in reducing the generation of acrylamide in fried food.
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WO2019113965A1 (en) * 2017-12-15 2019-06-20 江南大学 Thermophilic l-asparaginase mutant and screening and fermentation method therefor
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CN112941059A (en) * 2021-02-23 2021-06-11 江南大学 L-asparaginase mutant and expression thereof in bacillus subtilis

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