WO2023142178A1 - 一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用 - Google Patents

一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用 Download PDF

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WO2023142178A1
WO2023142178A1 PCT/CN2022/076360 CN2022076360W WO2023142178A1 WO 2023142178 A1 WO2023142178 A1 WO 2023142178A1 CN 2022076360 W CN2022076360 W CN 2022076360W WO 2023142178 A1 WO2023142178 A1 WO 2023142178A1
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glutamic acid
seq
mutated
mutant
tolerance
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饶志明
韩瑾
杨套伟
徐美娟
张显
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Jiangnan University
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    • C12Y401/01015—Glutamate decarboxylase (4.1.1.15)

Definitions

  • the invention belongs to the technical field of bioengineering, in particular to a glutamic acid decarboxylase mutant with improved pH tolerance and its application in the synthesis of gamma-aminobutyric acid.
  • GABA ⁇ -aminobutyric acid
  • GABA ⁇ -aminobutyric acid
  • the preparation methods of GABA mainly include chemical synthesis and biosynthesis. According to the different types of biocatalysts, the latter can be further subdivided into plant transformation methods, microbial transformation methods and enzymatic methods.
  • One is to use potassium phthalimide and ⁇ -chlorobutane as substrates. After reacting at 180°C, the product is hydrolyzed with concentrated sulfuric acid under reflux, and GABA is obtained by crystallization and purification.
  • Another method is to use pyrrolidone as a raw material, react at 121°C-135°C, and prepare GABA through hydrolysis and ring opening of Ca(OH)2 and NH4HCO3.
  • GABA Glutamic acid decarboxylase
  • GAD widely exists in various microorganisms such as bacteria, yeast and filamentous fungi, and mediates the synthesis of intracellular GABA.
  • the use of microorganisms containing GAD to transform and prepare GABA is not limited by resources, environment and space, and has become one of the most effective means of industrial production of GABA, which has attracted widespread attention.
  • the optimum pH of GAD in nature is generally between 4.0-5.0, and the enzyme activity drops sharply at neutral pH.
  • the catalytic activity of the bacteria tends to decrease with the fermentation liquid.
  • the accumulation of GABA leads to the rise and decrease of the pH value, resulting in a low conversion rate of the substrate. Therefore, broadening the pH range of glutamic acid decarboxylase can help to improve the substrate conversion rate.
  • the present invention aims to provide a glutamic acid decarboxylase mutant with improved pH tolerance and its application in the synthesis of ⁇ -aminobutyric acid. Conversion efficiency of butyric acid.
  • the glutamic acid decarboxylase mutant with improved pH tolerance is obtained by mutating the glutamic acid decarboxylase shown in SEQ ID NO.3 in any of the following ways:
  • Serine at position 24 is mutated to arginine, and the amino acid sequence is shown in SEQ ID NO.7;
  • Glutamic acid decarboxylase shown in SEQ ID NO.3 is compared with GB 01-21 strain glutamic acid decarboxylase (amino acid sequence is shown in SEQ ID NO.5, nucleotide sequence is shown in SEQ ID NO.6)
  • amino acid residues There are four mutations in amino acid residues, namely the mutation of proline at position 72 to threonine, the mutation of alanine at position 154 to threonine, the mutation of methionine at position 185 to valine and the mutation of position 412 Leucine is mutated to phenylalanine.
  • SEQ ID NO.3 The nucleotide sequence of the gene encoding glutamic acid decarboxylase shown in SEQ ID NO.3 is shown in SEQ ID NO.4.
  • the second aspect of the present invention provides a gene encoding the above-mentioned glutamic acid decarboxylase mutant with improved pH tolerance.
  • nucleotide sequence of the gene is shown in SEQ ID NO.2, SEQ ID NO.8, SEQ ID NO.10 or SEQ ID NO.12.
  • the coding amino acid sequence is as shown in SEQ ID NO.1 glutamic acid decarboxylase mutant gene, its nucleotide sequence is as shown in SEQ ID NO.2; the coding amino acid sequence is as shown in SEQ ID NO.7.
  • the gene of amino acid decarboxylase mutant, its nucleotide sequence is as shown in SEQ ID NO.8;
  • the gene of coding aminoacid sequence as shown in SEQ ID NO.9 glutamic acid decarboxylase mutant, its nucleotide sequence is as shown in Shown in SEQ ID NO.10;
  • coding amino acid sequence is shown in the gene of glutamic acid decarboxylase mutant as shown in SEQ ID NO.11, and its nucleotide sequence is shown in SEQ ID NO.12.
  • the third aspect of the present invention provides a recombinant expression vector carrying the above-mentioned genes.
  • pET-28a, PMA5 or PXMJ-19 was used as the original expression vector.
  • the fourth aspect of the present invention provides a recombinant bacterium containing the above-mentioned recombinant expression vector.
  • the recombinant bacteria use Escherichia coli, Bacillus subtilis or Corynebacterium glutamicum as host bacteria.
  • Bacillus subtilis includes Bacillus subtilis 168 (Bacillus subtilis 168, BS168).
  • the fifth aspect of the present invention provides the application of the above-mentioned glutamic acid decarboxylase mutant, gene, recombinant expression vector or recombinant bacteria with improved pH tolerance in the synthesis of ⁇ -aminobutyric acid.
  • L-glutamic acid or L-sodium glutamate is used as a substrate, and the above-mentioned glutamate decarboxylase mutant or recombinant bacteria with improved pH tolerance is used as a catalyst to generate ⁇ -aminobutyric acid.
  • the temperature is 25-60°C.
  • the invention mutates glutamic acid decarboxylase, not only broadens the enzyme activity of GAD at the optimum pH, but also broadens its enzyme activity at partial neutral pH, and enhances the ability of the enzyme to synthesize ⁇ -aminobutyric acid , making it more suitable for industrial production;
  • the glutamic acid decarboxylase mutant of the present invention has an increased enzymatic activity than the parental glutamic acid decarboxylase, especially the mutation mode is that the 24th serine is mutated into arginine, and the 24th serine is mutated into arginine,
  • the enzyme activity at pH 6.5 is increased to that of the original (shown in SEQ ID NO.3) 178%, fed 1000g substrate in batches for 12h in a 5L tank, the final yield was as high as 688.13g/L, about 52% higher than the original glutamic acid decarboxylase, and the final molar conversion rate could reach 98.2%.
  • Figure 1 is the SDS-PAGE diagram of the mutant strain.
  • Figure 2 is the relative enzyme activity of different mutants.
  • Figure 3 is the relative enzyme activity of the three combination mutants at different pH.
  • Figure 4 is the relative enzyme activity of the three combined mutant strains at different temperatures.
  • the gene fragments upstream and downstream of the mutation site were amplified respectively with primers containing the mutation site, and the fusion PCR method was used to construct the mutant gene.
  • the combined mutants use the single mutant plasmid as a template to amplify the upstream and downstream gene fragments of the mutation site with primers containing the mutation site, and then use the fusion PCR method to finally obtain a series of genes. details as follows:
  • the expression plasmid pET-28a was digested with restriction endonuclease EcoR I/Hind III to obtain a linearized vector, the target gene was connected with the linearized vector to obtain a recombinant plasmid, and the recombinant plasmid was transformed into Escherichia coli by chemical transformation In JM109, spread on LB plates containing kanamycin resistance, culture overnight at 37°C, randomly pick clones for colony PCR identification and sequencing verification, the results show that there is a recombination of the gene encoding glutamic acid decarboxylase mutant
  • the expression vector was successfully transformed into the cloning vector Escherichia coli JM109, and the recombinant plasmid was extracted and stored in a -20°C refrigerator after sequencing verified that the mutation was successful. The sequencing work was completed by Suzhou Jinweizhi.
  • the pET-28a recombinant plasmid containing the mutant gene was transformed into Escherichia coli BL21 by chemical transformation method. Recombinant Escherichia coli was induced at 16°C for 12 hours and then centrifuged to collect broken cells. The nickel column was used for protein purification, and the obtained purified enzyme was stored at 4°C with 10% glycerol added.
  • the purified enzyme was analyzed by SDS-PAGE, and the results of E.coli BL21/pET-28a-lpgad S24R/D88R/Y309K are shown in Figure 1, where M, protein marker; lane 1, pET28a; lane 2, lpgad S24R/D88R /Y309K mutant supernatant; lane 3, purified lpgad S24R/D88R/Y309K mutant. The results showed that the electrophoretic pure recombinant glutamic acid decarboxylase mutant was obtained.
  • Example 3 Enzyme activity assay of glutamate decarboxylase and HPLC detection of gamma-aminobutyric acid
  • the enzyme activity unit is defined as: 1 enzyme activity unit (U) is equal to the amount of enzyme required to catalyze L-glutamic acid to generate 1 nmol ⁇ -aminobutyric acid within 1 min at 30°C.
  • the specific enzyme activity is the enzyme activity of 1 mg of wet cells, and the unit is U/mg.
  • the concentrations of the substances used in the 1mL reaction system in which the enzyme and the substrate undergo decarboxylation reactions are: 0.01mM PLP, 100mM L-sodium glutamate. Before the reaction, the buffer solution and the enzyme solution were preheated at 30°C for 5 minutes, then mixed, reacted at 30°C for 30 minutes, and finally boiled quickly to terminate the reaction and centrifuged. Dilute it 5 times with 5% trichloroacetic acid (TCA), precipitate the protein in a 4°C refrigerator for about 3 hours, and then detect it by HPLC.
  • TCA trichloroacetic acid
  • HPLC The reaction solution was diluted 5 times with 5% trichloroacetic acid (TCA), centrifuged at 4° C. to precipitate protein for about 3 hours, and the supernatant was filtered through a 0.22 ⁇ membrane. Then inject.
  • Chromatographic column dimosoil C18 (5 ⁇ L, 250mm ⁇ 4.6mm), mobile phase: A: 0.1% formic acid aqueous solution, B: 100% acetonitrile, detector: UV Detector, detection wavelength: 360nm, column temperature: 25°C, injection volume : 10 ⁇ L, flow rate: 1.0 mL/min.
  • the target gene was amplified with primers P1/P2, and the expression plasmid PMA5 was digested with restriction endonuclease BamHI/Mlu I
  • To obtain a linearized vector connect the target gene to the linearized vector to obtain the recombinant plasmid PMA5-lpgad S24R/D88R/Y309K , transform the recombinant plasmid into E.
  • the successfully constructed PMA5-lpgad S24R/D88R/Y309K recombinant plasmid was transformed into Bacillus subtilis BS168 by the chemical transformation method to construct the BS168/PMA5-lpgad S24R/D88R/Y309K recombinant strain, which was coated on kanamycin-resistant LB plates were cultured overnight at 37°C, and clones were randomly selected for colony PCR identification. The successful transformation was verified by adding glycerol to the bacterial solution and storing it in a -80°C refrigerator.
  • the BS168/PMA5-lpgad S24R/D88R/Y309K mutant engineered bacteria in Example 4 were transformed into L-glutamic acid substrate.
  • a 5L tank use 1L 0.9% NaCl solution as the buffer system, control the OD 600nm of the bacteria to 16, add 100g/l L-glutamic acid in batches, and react at 30°C for 12h, lpgad S24R/D88R/Y309K engineering bacteria
  • the final yield of 1000g substrate in batch feed is as high as 688.13g/L, which is about 52% higher than that of the original enzyme, and the molar conversion rate can reach up to 98.2%.
  • the results show that the catalytic efficiency of the mutant enzyme is significantly improved, and it has broad industrial application prospects.

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Abstract

提供一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用，该突变体通过将氨基酸序列如SEQ ID NO.3所示的谷氨酸脱羧酶突变得到；该突变体在pH 6.5时的酶活提高至原始(SEQ ID NO.3所示)的178％，在5L罐中12h分批补料1000g底物最终产量高达688.13g/L，比原始谷氨酸脱羧酶生产性能提高约52％，最终摩尔转化率可达98.2％。

Description

一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用 技术领域
本发明属于生物工程技术领域，具体涉及一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用。
背景技术
GABA(γ-氨基丁酸)是一种天然存在的非蛋白质氨基酸，作为哺乳动物中枢神经系统中主要的抑制性神经递质，具有抗焦虑、降血压、镇定安神、增强记忆、预防肥胖及糖尿病等多种重要的生理功能，因而被广泛应用于医药及食品领域。
目前，有关GABA的制备方法主要有化学合成法和生物合成法两大类。根据生物催化剂种类的不同，后者又可进一步细分为植物转化法、微生物转化法和酶法。常见的GABA化学合成法主要有两种。一种是以邻苯二甲酰亚胺钾和γ－氯丁氰为底物，在180℃条件下反应后，产物再与浓硫酸回流水解，经结晶提纯得到GABA。另一种方法是以吡咯烷酮作为原料，在121℃-135℃下反应，经Ca(OH)2和NH4HCO3水解开环而制得GABA。由于工业生产反应条件苛刻，且反应过程容易产生有毒物质，因此GABA的生产主要为微生物酶法合成，利用谷氨酸脱羧酶(GAD)能催化L-谷氨酸脱羧生成γ-氨基丁酸，反应过程中消耗一个质子。
GAD广泛存在于细菌、酵母和丝状真菌等各种微生物中，介导了胞内GABA的合成。利用含有GAD的微生物转化制备GABA，不受资源、环境和空间等限制，成为工业化生产GABA最为有效的手段之一，受到人们普遍关注。目前，自然界中存在的GAD最适pH普遍在4.0-5.0之间，在中性pH下酶活力急剧下降，而在微生物自然发酵或转化过程中，由于菌体催化活性往往会随着酵液中 GABA积累所导致pH值的上升而降低，从而致使底物转化率偏低。因此拓宽谷氨酸脱羧酶的pH作用范围可有助于底物转化率的提高。
发明内容
本发明旨在提供一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用，在偏中性条件下的酶活提高，能够提高制备γ-氨基丁酸的转化效率。
按照本发明的技术方案，所述pH耐受性提高的谷氨酸脱羧酶突变体，通过将氨基酸序列如SEQ ID NO.3所示的谷氨酸脱羧酶经以下任一方式突变得到：
(1)第24位丝氨酸突变为精氨酸，氨基酸序列如SEQ ID NO.7所示；
(2)第24位丝氨酸突变为精氨酸，第88位的天冬氨酸突变为精氨酸，氨基酸序列如SEQ ID NO.9所示；
(3)第24位丝氨酸突变为精氨酸，第309位的酪氨酸突变为赖氨酸，氨基酸序列如SEQ ID NO.11所示；
(4)第24位丝氨酸突变为精氨酸，第88位的天冬氨酸突变为精氨酸以及第309位的酪氨酸突变为赖氨酸，氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.3所示谷氨酸脱羧酶与GB 01-21菌株谷氨酸脱羧酶(氨基酸序列如SEQ ID NO.5所示，核苷酸序列如SEQ ID NO.6所示)相比具有四个氨基酸残基突变，分别是第72位脯氨酸突变为苏氨酸、第154位丙氨酸突变为苏氨酸、第185位甲硫氨酸突变为缬氨酸以及第412位亮氨酸突变为苯丙氨酸。
编码SEQ ID NO.3所示谷氨酸脱羧酶的基因的核苷酸序列如SEQ ID NO.4所示。
本发明的第二方面提供了编码上述pH耐受性提高的谷氨酸脱羧酶突变体的基因。
进一步的，所述基因的核苷酸序列如SEQ ID NO.2、SEQ ID NO.8、SEQ ID NO.10或SEQ ID NO.12所示。
具体的，编码氨基酸序列如SEQ ID NO.1所示谷氨酸脱羧酶突变体的基因， 其核苷酸序列如SEQ ID NO.2所示；编码氨基酸序列如SEQ ID NO.7所示谷氨酸脱羧酶突变体的基因，其核苷酸序列如SEQ ID NO.8所示；编码氨基酸序列如SEQ ID NO.9所示谷氨酸脱羧酶突变体的基因，其核苷酸序列如SEQ ID NO.10所示；编码氨基酸序列如SEQ ID NO.11所示谷氨酸脱羧酶突变体的基因，其核苷酸序列如SEQ ID NO.12所示。
本发明的第三方面提供了携带上述基因的重组表达载体。
进一步的，以pET-28a、PMA5或PXMJ-19作为原始表达载体。
本发明的第四方面提供了含有上述重组表达载体的重组菌。
进一步的，所述重组菌以大肠杆菌、枯草芽孢杆菌或谷氨酸棒杆菌作为宿主菌。
进一步的，所述枯草芽孢杆菌包括枯草芽孢杆菌168(Bacillus subtilis 168，BS168)。
本发明的第五方面提供了上述pH耐受性提高的谷氨酸脱羧酶突变体、基因、重组表达载体或重组菌在合成γ-氨基丁酸中的应用。
进一步的，以L-谷氨酸或L谷氨酸钠为底物，上述pH耐受性提高的谷氨酸脱羧酶突变体或重组菌为催化剂，生成γ-氨基丁酸。
进一步的，以0.9％NaCl溶液为转化体系，不需要调节pH，转化以后产物提取简单。
进一步的，温度为25-60℃。
本发明的技术方案相比现有技术具有以下优点：
本发明通过将谷氨酸脱羧酶进行突变，不仅拓宽了GAD在最适pH下的酶活，还拓宽其在偏中性pH下的酶活，增强了该酶合成γ-氨基丁酸的能力，使其更适应于工业生产；
试验结果表明，在不同pH条件下本发明的谷氨酸脱羧酶突变体比亲本谷氨酸脱羧酶的酶活均有所上升，特别是突变方式为第24位丝氨酸突变为精氨酸，第88位天冬氨酸突变为精氨酸以及第309位酪氨酸残基突变成赖氨酸残基时， 在pH 6.5时的酶活提高至原始(SEQ ID NO.3所示)的178％，在5L罐中12h分批补料1000g底物最终产量高达688.13g/L，比原始谷氨酸脱羧酶提高约52％，最终摩尔转化率可达98.2％。
附图说明
图1是突变株的SDS-PAGE图。
图2为不同突变体的相对酶活。
图3是为三组合突变株在不同pH下的相对酶活。
图4是为三组合突变株在不同温度下的相对酶活。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1 含编码谷氨酸脱羧酶突变体基因的重组表达载体的构建
以含有SEQ ID NO：4所示核苷酸序列的pET-28a重组质粒为模板，分别以含有突变位点的引物扩增出突变位点上下游的基因片段，采用融合PCR法构建突变基因，其中组合突变体通过以单突变质粒为模板以含有突变位点的引物扩增出突变位点上下游基因片段，再采用融合PCR法，最终可得到一系列基因。具体如下：
引物 基因
P1/P2、P3/P20 lpgad S24R
P1/P4、P5/P20 lpgad D88R
P1/P6、P7/P20 Lpgad L135K
P1/P8、P9/P20 Lpgad E170R
P1/P10、P11/P20 Lpgad H196R
P1/P12、P13/P20 Lpgad A225K
P1/P14、P15/P20 Lpgad Y309K
P1/P16、P17/P20 Lpgad A359R
P1/P18、P19/P20 Lpgad E417K
P1/P2、P3/P4、P5/P20 lpgad S24R/D88R
P1/P2、P3/P6、P7/P20 lpgad S24R/L135K
P1/P2、P3/P8、P9/P20 lpgad S24R/E170R
P1/P2、P3/P10、P11/P20 lpgad S24R/H196R
P1/P2、P3/P12、P13/P20 lpgad S24R/A225K
P1/P2、P3/P14、P15/P20 lpgad S24R/Y309K
P1/P2、P3/P16、P17/P20 lpgad S24R/A359R
P1/P2、P3/P18、P19/P20 lpgad S24R/E417K
P1/P2、P3/P4、P5/P14、P15/P20 lpgad S24R/D88R/Y309K
P1：ATGGCAATGTTATACGGTAAACACAAT
P2：CTTAGGAAGATCATGTTGTTCGCGAGG
P3：GGTGCGCCTCGCGAACAACATGATCTT
P4：TCAGATTTCCGGATGGCATTCTTTCGC
P5：TGCCATCCGGAAATCTGAGTACCCCCG
P6：CATTGCTTTACCGCCTAACATACAAGC
P7：AGGCGGTAAAGCAATGAAATTCGCCTG
P8：AAACTTACGCCAGCAAACTTGATAGCC
P9：TTGCTGGCGTAAGTTTTGTGTCTACTG
P10：TAAGACACGGTTAACGTCAAGGACCAT
P11：GTTAACCGTGTCTTAGACTACGTGGAC
P12：ATCGAGTTTGGCTAGGTCGTCATATTG
P13：CTAGCCAAACTCGATAAGGTCGTTACT
P14：CCCACCTAATTTACTAACTTTGAAGAC
P15：AAAGTTAGTAAATTAGGTGGGGAGTTG
P16：CAGAGCGCGTGCCAGGTAGCGGGCAAC
P17：CTGGCACGCGCTCTGGATAAAGTTGGT
P18：TTGTTGTTTCAGATTAGCAGGGAAAGG
P19：AATCTGAAACAACAAGTCATCCAACGA
P20：TCAGTGTGTGAATAGGTATTTCTTAGG
利用限制性内切酶EcoR I/Hind III对表达质粒pET-28a进行酶切处理以获得线性化载体，目的基因与线性化载体连接后获得重组质粒，将重组质粒用化学转化法转化至大肠杆菌JM109中，涂布于含卡那霉素抗性的LB平板，37℃下培养过夜，随机挑取克隆进行菌落PCR鉴定和测序验证，结果表明含有编码谷氨酸脱羧酶突变体的基因的重组表达载体成功转化至克隆载体大肠杆菌JM109中，经测序验证突变成功的菌液提取重组质粒保存于-20℃冰箱。测序工作由苏州金唯智完成。
实施例2 在大肠BL21中进行GAD突变体的表达及纯化
将含有突变基因的pET-28a重组质粒采用化学转化法转化至大肠杆菌BL21中。重组大肠杆菌经过16℃诱导12h后离心收集菌体破碎细胞，采用镍柱进行蛋白纯化，所得纯化酶添加10％的甘油于4℃贮存备用。纯化后的酶用SDS-PAGE进行分析，其中E.coli BL21/pET-28a-lpgad S24R/D88R/Y309K结果参见图1，其中M，蛋白质Marker；泳道1，pET28a；泳道2，lpgad S24R/D88R/Y309K突变体上清；泳道3，纯化lpgad S24R/D88R/Y309K突变体。结果表明得到电泳纯的重组谷氨酸脱羧酶突变体。
实施例3 谷氨酸脱羧酶的酶活测定及γ-氨基丁酸的HPLC检测
测定了实施例2中所得一系列纯化酶(谷氨酸脱羧酶突变体)在最适pH 4.5以及pH 6.0时与L-谷氨酸反应时的相对酶活(以SEQ ID NO.3所示酶活为100％)，如图2所示，结果显示：lpgad S24R、lpgad S24R/D88R、lpgad S24R/Y309K和lpgad S24R/D88R/Y309K的相对酶活较高，且pH 6.0条件下相对酶活高于pH 4.5。
同时测定谷氨酸脱羧酶lpgad S24R/D88R/Y309K突变体在pH为4.0、4.5、5.0、5.5、6.0、6.5、7.0(如图3所示，可以看出其在pH6.5时相对酶活最高，pH5.0-7.0时相对酶活均处于较高水平)以及在25℃、30℃、35℃、40℃、50℃、55℃、60℃时的相对酶活(如图4所示)。反应30min后取样用HPLC法测产物γ-氨基丁酸的含量。
定义酶活单位为：1个酶活单位(U)等于在30℃下，在1min内催化L-谷氨酸生成1nmolγ-氨基丁酸所需的酶量。比酶活为1mg湿细胞的酶活，单位为U/mg。酶与底物发生脱羧反应的1mL反应体系采用的各物质浓度为：0.01mM PLP、100mM L-谷氨酸钠。在反应之前分别把缓冲液和酶液在30℃下预热5min，然后混匀，在30℃下反应30min，最后迅速煮沸终止反应，离心。用5％三氯乙酸(TCA)稀释5倍，在4℃冰箱沉淀蛋白3h左右，然后用HPLC检测。
HPLC：取反应液用5％三氯乙酸(TCA)稀释5倍，在4℃冰箱沉淀蛋白3h左右离心，取上清液用0.22μ膜进行过滤处理。然后进样。色谱柱：dimosoil C18(5μL,250mm×4.6mm)，流动相：A：0.1％甲酸水溶液，B：100％乙腈，检测器：UV Detector，检测波长：360nm，柱温：25℃，进样量：10μL，流速：1.0mL/min。过程：0～22min:15％B→50％B；22～22.1min:50％B→15％B；22.1～26min:15％B。
实施例4 lpgad S24R/D88R/Y309K在枯草芽孢杆菌中的构建
以实施例1构建的pET-28a-lpgad S24R/D88R/Y309K质粒为模板，以引物P1/P2扩增获得目的基因，并利用限制性内切酶BamHI/Mlu I对表达质粒PMA5进行酶切处理以获得线性化载体，目的基因与线性化载体连接后获得重组质粒PMA5-lpgad S24R/D88R/Y309K，将重组质粒用化学转化法转化至大肠杆菌JM109中，涂布于含氨苄霉素抗性的LB平板，37℃下培养过夜，随机挑取克隆进行菌落PCR鉴定，结果表明含有编码谷氨酸脱羧酶突变体基因的重组质粒成功转化至克隆载体大肠杆菌JM109中，挑取经验证为阳性菌落接种于5mL LB液体培养 基中于37℃下培养过夜，菌液提取重组质粒保存于-20℃冰箱。
P1：ATGGCAATGTTATACGGTAAACACAAT
P2：TCAGTGTGTGAATAGGTATTTCTTAGG
采用化学转化法将构建成功的PMA5-lpgad S24R/D88R/Y309K重组质粒转化至枯草芽孢杆菌BS168中构建BS168/PMA5-lpgad S24R/D88R/Y309K重组菌株，涂布于含卡那霉素抗性的LB平板，37℃下培养过夜，随机挑取克隆进行菌落PCR鉴定，验证转化成功的菌液加入甘油并于-80℃冰箱保藏。
实施例5 BS168/PMA5-lpgad S24R/D88R/Y309K工程菌制备γ-氨基丁酸
将实施例4中的BS168/PMA5-lpgad S24R/D88R/Y309K突变型工程菌进行L-谷氨酸底物的转化。在5L罐中以1L 0.9％NaCl溶液为缓冲体系，菌量OD 600nm控制为16，分批添加100g/l L-谷氨酸，在30℃下反应12h，lpgad S24R/D88R/Y309K工程菌分批补料1000g底物最终产量高达688.13g/L，比原始酶提高约52％，摩尔转化率最高可达98.2％。结果表明该突变酶的催化效率明显提升，具有广阔的工业化应用前景。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种pH耐受性提高的谷氨酸脱羧酶突变体，其特征在于，通过将氨基酸序列如SEQ ID NO.3所示的谷氨酸脱羧酶经以下任一方式突变得到：
    (1)第24位丝氨酸突变为精氨酸；
    (2)第24位丝氨酸突变为精氨酸，第88位的天冬氨酸突变为精氨酸；
    (3)第24位丝氨酸突变为精氨酸，第309位的酪氨酸突变为赖氨酸；
    (4)第24位丝氨酸突变为精氨酸，第88位的天冬氨酸突变为精氨酸以及第309位的酪氨酸突变为赖氨酸。
  2. 编码权利要求1所述的pH耐受性提高的谷氨酸脱羧酶突变体的基因。
  3. 如权利要求2所述的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.2、SEQ ID NO.8、SEQ ID NO.10或SEQ ID NO.12所示。
  4. 携带权利要求2或3所述基因的重组表达载体。
  5. 如权利要求4所述的重组表达载体，其特征在于，以pET-28a、PMA5或PXMJ-19作为原始表达载体。
  6. 含有权利要求4或5所述重组表达载体的重组菌。
  7. 如权利要求6所述的重组菌，其特征在于，所述重组菌以大肠杆菌、枯草芽孢杆菌或谷氨酸棒杆菌作为宿主菌。
  8. 如权利要求7所述的重组菌，其特征在于，所述枯草芽孢杆菌包括枯草芽孢杆菌168。
  9. 如权利要求1所述的pH耐受性提高的谷氨酸脱羧酶突变体、权利要求2或3所述的基因、权利要求4或5所述的重组表达载体或权利要求6、7或8所述的重组菌在合成γ-氨基丁酸中的应用。
  10. 如权利要求9所述的应用，其特征在于，以L-谷氨酸或L谷氨酸钠为底物，权利要求1所述的pH耐受性提高的谷氨酸脱羧酶突变体或权利要求8所述的重组菌为催化剂，生成γ-氨基丁酸。
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