WO2021184809A1 - 一种l-氨基酸脱氨酶突变体的制备及其应用 - Google Patents

一种l-氨基酸脱氨酶突变体的制备及其应用 Download PDF

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WO2021184809A1
WO2021184809A1 PCT/CN2020/130330 CN2020130330W WO2021184809A1 WO 2021184809 A1 WO2021184809 A1 WO 2021184809A1 CN 2020130330 W CN2020130330 W CN 2020130330W WO 2021184809 A1 WO2021184809 A1 WO 2021184809A1
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amino acid
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deaminase
ketoisovaleric
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吴静
裴杉杉
刘佳
宋伟
陈修来
罗秋玲
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Jiangnan University
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    • C12Y104/03002—L-Amino-acid oxidase (1.4.3.2)

Definitions

  • the invention relates to the preparation and application of an L-amino acid deaminase mutant, and belongs to the technical field of genetic engineering.
  • L-amino acid deaminase is a flavin oxidase that can catalyze the formation of ⁇ -keto acids from L-amino acids.
  • LAAD L-amino acid deaminase
  • researchers have done a lot of research on the spatial structure of L-amino acid deaminase, its substrate specificity, and its catalytic ability on non-natural substrates. The application gradually attracted the attention of researchers.
  • Alpha-keto acid is an important intermediate, which is mainly used in food, medicine, cosmetics and other fields, which makes enzymatic conversion of alpha-keto acid widely used in industrial production.
  • L-amino acid deaminase is a flavin protease.
  • the oxidative deamination reaction of L-amino acid can be divided into two steps: firstly , the hydrogen on amino acid C ⁇ is transferred to FAD. Alpha-keto acid and water. Then, after FADH 2 is oxidized by oxygen molecules, it becomes reduced FAD.
  • L-amino acid deaminase has a wide substrate spectrum and high catalytic efficiency, which provides the possibility for heterologous expression of amino acid deaminase to convert L-valine to produce ⁇ -ketoisovalerate.
  • the present invention provides a LAAD mutant that can efficiently prepare LAAD mutant and its transformation method, and use the mutant protein to catalyze L-valine to produce ⁇ -ketoisovaleric acid.
  • the strain constructed by the present invention is It has high catalytic activity during preparation, which greatly improves the production efficiency of industrial production.
  • the present invention provides a mutant of L-amino acid deaminase.
  • the mutant uses L-amino acid deaminase (pmirLAAD) derived from Proteus mirabilis as a parent, and the parent L-amino acid deaminase
  • the amino acid sequence is shown in SEQ ID NO.1.
  • nucleotide sequence encoding the L-amino acid deaminase parent is shown in SEQ ID NO.2.
  • the mutant is mutated at the 98th amino acid, serine is changed to alanine, and the mutant S98A is obtained.
  • the mutant has a mutation at the 105th amino acid of the pmirLAAD parent, and threonine is changed to alanine to obtain mutant T105A.
  • the mutant is mutated at its 106th amino acid and serine is mutated to alanine to obtain mutant S106A.
  • the mutant is mutated at the 341th amino acid, leucine is mutated to alanine, and the mutant L341A is obtained.
  • the mutant changes the 98th serine to alanine and the 105th threonine to alanine relative to the pmirLAAD parent to obtain the mutant S98A/T105A.
  • the mutant changes the 98th serine to alanine, the 105th threonine to alanine, and the 106th serine to Alanine, the mutant S98A/T105A/S106A was obtained.
  • the mutant changes the 98th serine to alanine, the 105th threonine to alanine, and the 106th serine to Alanine and threonine at position 341 were changed to alanine to obtain mutant S98A/T105A/S106A/L341A.
  • amino acid sequences of the mutants S98A, T105A, S106A, L341A, S98A/T105A, S98A/T105A/S106A, S98A/T105A/S106A/L341A are as shown in SEQ ID NO. 3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15 are shown.
  • the present invention provides a gene encoding the mutant, nucleotides encoding the gene of the mutant S98A, T105A, S106A, L341A, S98A/T105A, S98A/T105A/S106A, S98A/T105A/S106A/L341A
  • the sequences are shown in SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16.
  • the present invention provides a recombinant plasmid containing the gene of the mutant, and the recombinant plasmid uses a pET series vector, a pRSFDuet series or a pGEX series vector as an expression vector.
  • the present invention provides a method for obtaining the pmirLAAD mutant, the method comprising:
  • the host cell is E. coli BL21 (DE3).
  • the present invention provides a host bacteria expressing the mutant, or the gene, or containing the recombinant vector.
  • the present invention provides a method for producing ⁇ -ketoisovaleric acid yield.
  • the method uses L-valine as a reaction substrate and adds the host bacteria to the reaction solution at pH 7.5-9.5, 20-40 The reaction is carried out for 22 to 26 hours under the conditions of °C and aeration of 1 to 5 vvm.
  • the reaction is conducted for 22-25 hours under the conditions of pH 8.5-9.5, 25-35° C., and aeration rate of 1-2 vvm.
  • the final concentration of the host bacteria in the system is 10-30 g/L.
  • the final concentration of the host bacteria in the system is 10-15 g/L.
  • the present invention provides a method for producing the yield of ⁇ -ketoisovaleric acid.
  • the method uses L-valine as the reaction substrate, and adds the mutant to the reaction solution at pH 7.5-9.5, 20-40 The reaction is carried out for 22 to 26 hours under the conditions of °C and aeration of 1 to 5 vvm.
  • the reaction is conducted for 22-25 hours under the conditions of pH 8.5-9.5, 25-35° C., and aeration rate of 1-2 vvm.
  • the present invention provides the mutant, or the gene encoding the mutant, or the application of the recombinant plasmid in the preparation of ⁇ -ketoisovaleric acid or the improvement of the production of ⁇ -ketoisovaleric acid in the fields of food, medicine, and chemical industry .
  • the present invention provides the application of the host bacteria or the method for preparing ⁇ -ketoisovaleric acid in the preparation of ⁇ -ketoisovaleric acid in the fields of food, medicine, and chemical industry.
  • the present invention constructs an L-amino acid deaminase mutant and its preparation method, which are used to catalyze the production of ⁇ -ketoisovaleric acid.
  • the catalytic efficiency (1.61 mM -1 ⁇ min -1 ) and product inhibition constant (5.4 mM) of the present invention are respectively increased by 5.2 times and 5.7 times compared with the control.
  • the production capacity of the unit catalyst is improved, and the production cost is effectively reduced.
  • the reaction solution uses only water as the catalytic medium. It has the advantages of mild reaction conditions, simple operation, easy separation, and environmental friendliness, and the process is simple, easy to control, and easy to promote application.
  • the mutant obtained by the present invention uses L-valine as the substrate in a 3L fermentor, the yield of ⁇ -ketoisovaleric acid can reach 96.5g/L, and the conversion rate is >97%, which is the highest yield currently. , Hastened the industrialization process of producing ⁇ -ketoisovaleric acid by enzyme conversion method.
  • Figure 1 is a graph showing the relationship between the concentration of whole-cell catalyst and the production of ⁇ -ketoisovaleric acid.
  • Gene source the biological enzyme pmirLAAD gene involved in this patent is derived from Proteus mirabilis, pET28a(+) plasmid was purchased from Novagen (Madison, WI, USA), restriction endonuclease, T4DNA ligase, primeSTAR, etc. were purchased from TaKaRa( Dalian, China). Standard samples were purchased from SIGMA.
  • the pmirLAAD mutants are all obtained from molecular modification, and the rest of the reagents are all purchased from the market.
  • LB medium 10g peptone, 5g yeast powder, 10g sodium chloride, distilled water to a volume of 1L.
  • Fermentation medium (TB medium): peptone 12g, yeast extract 24g, glycerol 4mL, potassium dihydrogen phosphate 2.31g, dipotassium hydrogen phosphate 16.42g, distilled water to make the volume to 1L.
  • Sample preparation for the determination of ⁇ -ketoisovaleric acid content by HPLC method Take 1 mL of the converted conversion solution, centrifuge at 12000 rpm for 5 min, take the supernatant and dilute it, filter it through a 0.45 ⁇ m membrane filter, and use the filtrate for liquid chromatographic analysis.
  • HPLC method to determine the content of ⁇ -ketoisovaleric acid waters high performance liquid chromatograph (with UV-visible detector), using Bó Lé Aminex HPX-87H (300 ⁇ 7.8mm, 9 ⁇ m) chromatographic column, the mobile phase is 2.5mmol/ L dilute sulfuric acid, the mobile phase was filtered through a 0.22 ⁇ m filter membrane, ultrasonically degassed, the flow rate was 0.6mL/min, the column temperature was 35°C, and the UV detection wavelength was 210nm.
  • Mutant construction Design primers for the 98th, 105th, 106th, and 341th mutation sites, as shown in Table 1, and construct them by full plasmid PCR.
  • reaction PCR amplification system PrimSTAR enzyme 0.5 ⁇ L, 5 ⁇ PrimeSTAR Buffer 10 ⁇ L, dNTP 4 ⁇ L each of the two primers at each mutation site 1 ⁇ L, template (the nucleotide sequence of pmirLAAD) 4 ⁇ L, water 32.5 ⁇ L; reaction conditions are as follows: 194°C3min;298°C10s;355°C30s;472°C3min;5cycle the three steps 2 ⁇ 4 29 times; 672°C5min; 712°C heat preservation.
  • the conversion conditions are: the conversion temperature is 25°C, the reaction pH is 8.0, and the conversion time is 24h.
  • step (1) in Example 1 refers to step (1) in Example 1, and 6 double mutants PmirLAAD S98A/T105A were prepared .
  • PmirLAAD S98A/S106A PmirLAAD S98A/L341A
  • PmirLAAD T105A/S106A PmirLAAD T105A/L341A
  • PmirLAAD S106A/L341A PmirLAAD S106A/L341A .
  • step (1) in Example 3 refers to step (1) in Example 3 to prepare four triple mutants PmirLAAD S98A/T105A/S106A , PmirLAAD S98A/T105A/L341A , PmirLAAD S98A/ S106A/L341A , and PmirLAAD T105A/S106A/L341A .
  • Mutation of L341A on the basis of mutant PmirLAAD S98A/T105A/S106A Using mutant PmirLAAD S98A/T105A/S106A as a template, using mutant primers L341A-S and L341A-A (table 3) Perform full-plasmid PCR, and digest the PCR product.
  • the PCR system is the same as the digestion system as in Example 3.
  • Example 3 Determination of the kinetic parameters and product inhibition constants of the parent enzyme and the mutant
  • the present invention determined the kinetic parameters of the mutant parent PmirLAAD WT and mutants PmirLAAD M1 , PmirLAAD M2 , PmirLAAD M3 , and PmirLAAD M4 at 25°C.
  • k cat /K m is calculated by measuring the initial rate of ⁇ -ketoisovaleric acid produced by hydrolyzing L-valine substrates at different concentrations at 25°C. Determine the product inhibition of the parent enzyme and the mutant through the product inhibition constant determination experiment during the transformation process.
  • the PmirLAAD WT parent enzyme strain and the mutant strain were each added to the reaction solution at a final concentration of 10g/L wet cells, and the concentration was 60mM.
  • V 0 initial reaction rate
  • V max maximum reaction rate
  • K m Michaelis constant
  • [S] substrate concentration
  • [P] product concentration
  • K PI product inhibition constant.
  • the K cat /K m value of all mutants was increased compared with the parent enzyme at 25°C.
  • the k cat /K m value of mutant PmirLAAD M4 was increased by 5.2 times, resulting in the catalytic activity of PmirLAAD. The efficiency becomes greater.
  • the product inhibition constant of each mutant was improved compared with the parent enzyme, and the product inhibition constant of the mutant PmirLAAD M4 (hereinafter referred to as the M4 strain) was increased by 5.7 times.
  • Example 4 1L system level optimization L-valine to produce ⁇ -ketoisovaleric acid
  • the conversion reaction was carried out under the conditions of 25° C., 600 rpm, and an aeration rate of 1 vvm, and the total volume after the conversion reaction was 1 L.
  • a whole-cell catalyst concentration of 10g/L also has a higher ⁇ -ketoisovaleric acid concentration and a high product/catalyst ratio (that is, the production of ⁇ -ketoisovaleric acid per cell) Valeric acid capacity) 7.6g/g, which was used in the transformation experiment.
  • the concentration of the whole cell agent is controlled to 10g/L
  • the concentration of L-valine is controlled to 100g/L
  • the aeration rate is 1vvm
  • the pH during the conversion reaction is controlled to 7.5, 8.0, 8.5, 9.0, 9.5.
  • Table 7 It is more suitable for the synthesis of ⁇ -ketoisovaleric acid under weak acid-base conditions. Therefore, the pH of the selective conversion is controlled at 8.5 ⁇ 9.0. At this time, the concentration of ⁇ -ketoisovaleric acid is 78.9g/L .
  • the specific steps are the same as (1), the transformation pH is controlled to 8.5, the concentration of the whole cell agent is controlled to 10g/L, the aeration rate is 1vvm, and the temperature during the transformation reaction is controlled to 20°C, 25°C, 30°C, 35 °C, 40°C.
  • the results are shown in Table 8.
  • the pH of the conversion temperature is selected to be around 30°C.
  • the concentration of ⁇ -ketoisovaleric acid is 78.9 g/L.
  • the specific steps are the same as (1), the transformation pH is controlled to 8.5, the concentration of the whole cell agent is controlled to 10g/L, the temperature is controlled to 30°C, and the ventilation rate is controlled to 1vvm, 1.5vvm, 2vvm, 2.5vvm, 3vvm.
  • the results are shown in Table 9.
  • the ventilation volume is 1.5 vvm, it is more suitable for the synthesis of ⁇ -ketoisovaleric acid. Therefore, the ventilation volume is selected to control about 1.5 vvm, and the concentration of ⁇ -ketoisovaleric acid is 96.5 g/L at this time.
  • Example 4 Specific embodiments refer to Example 4, the difference is that the mutant M4 strain is replaced with a wild-type WT strain to carry out fermentation and transformation experiments. After the conversion, a portion of the conversion solution was centrifuged at 12,000 ⁇ g for 15 minutes, and the supernatant was filtered with a 0.22 ⁇ m microfiltration membrane and then analyzed by HPLC. The HPLC chromatogram result showed that the yield of ⁇ -ketoisovaleric acid was 40 g/L, and the conversion rate was 40.3%.

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Abstract

提供一种L-氨基酸脱氨酶突变体的制备及其应用，通过分析pmirLAAD产物结合位点周围柔性Loop环结构，对蛋白进行改造，并通过实验验证，最终得到最佳突变体pmirLAADM4，催化效率(1.61mM -1·min -1)和产物抑制常数(5.4mM)较对照分别提高了5.2倍和5.7倍；α-酮异戊酸的产量可达到96.5g/L，转化率>97％，加快了酶转化法生产α-酮异戊酸的工业化进程。

Description

一种L-氨基酸脱氨酶突变体的制备及其应用 技术领域
本发明涉及一种L-氨基酸脱氨酶突变体的制备及其应用，属于基因工程技术领域。
背景技术
L-氨基酸脱氨酶(LAAD)是一种能够催化L-氨基酸生成α-酮酸的黄素氧化酶。研究人员对L-氨基酸脱氨酶的空间结构、底物特异性、以及其对非天然底物的催化能力方面进行过大量的研究，近年来，该酶在生物转化生产α-酮酸方面的应用逐渐引起研究人员的关注。
α-酮酸是重要的中间体，它主要应用于食品、医药、化妆品等领域，这使得酶法转化α-酮酸在工业生产上有着广泛的应用。L-氨基酸脱氨酶是黄素蛋白酶，L-氨基酸的氧化脱氨反应可以分为两个步骤：首先将氨基酸C α上的氢转移给FAD，氨基酸变成亚氨基酸，亚氨基酸不稳定分解成α-酮酸和水。然后FADH 2被氧分子氧化后，变成了还原型FAD。L-氨基酸脱氨酶底物谱广泛，催化效率高，这为异源表达氨基酸脱氨酶转化L-缬氨酸生产α-酮异戊酸提供了可能。
然而，目前利用L-氨基酸改造的方法一定程度上提高了α-酮异戊酸的产量和转化率，但是产量依然较低，因此，为提高α-酮异戊酸的产量和转化率，增强其工业应用价值，急需提高L-氨基酸脱氨酶对L-缬氨酸的催化效率。
发明内容
本发明提供了一种可以高效制备LAAD突变体及其改造方法，并利用该突变体蛋白催化L-缬氨酸制备α-酮异戊酸，本发明所构建的菌株在α-酮异戊酸制备中具有高催化活性，极大提高了工业化生产的生产效率。
本发明提供了一种L-氨基酸脱氨酶的突变体，所述突变体以来源于奇异变形杆菌(Proteus mirabilis)的L-氨基酸脱氨酶(pmirLAAD)为亲本，亲本L-氨基酸脱氨酶氨基酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，编码所述L-氨基酸脱氨酶亲本的核苷酸序列如SEQ ID NO.2所示。
在本发明的一种实施方式中，所述突变体相对于pmirLAAD亲本，将其第98位氨基酸发生突变，将丝氨酸变为丙氨酸，获得突变体S98A。
在本发明的一种实施方式中，所述突变体相对于pmirLAAD亲本，将其第105位氨基酸发生突变，将苏氨酸变为丙氨酸，获得突变体T105A。
在本发明的一种实施方式中，所述突变体相对于pmirLAAD亲本，将其第106位氨基酸发生突变，将丝氨酸突变为丙氨酸，获得突变体S106A。
在本发明的一种实施方式中，所述突变体相对于pmirLAAD亲本，将其第341位氨基酸发生突变，将亮氨酸突变为丙氨酸，获得突变体L341A。
在本发明的一种实施方式中，所述突变体相对于pmirLAAD亲本，将第98位丝氨酸变为丙氨酸并将第105苏氨酸变为丙氨酸，获得突变体S98A/T105A。
在本发明的一种实施方式中，所述突变体相对于pmirLAAD亲本，将第98位丝氨酸变为丙氨酸、将第105位苏氨酸变为丙氨酸并将第106位丝氨酸突变为丙氨酸，获得突变体S98A/T105A/S106A。
在本发明的一种实施方式中，所述突变体相对于pmirLAAD亲本，将第98位丝氨酸变为丙氨酸、将第105位苏氨酸变为丙氨酸、将第106位丝氨酸突变为丙氨酸并将第341位苏氨酸变为丙氨酸，获得突变体S98A/T105A/S106A/L341A。
在本发明的一种实施方式中，所述突变体S98A、T105A、S106A、L341A、S98A/T105A、S98A/T105A/S106A、S98A/T105A/S106A/L341A的氨基酸序列分别如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11，SEQ ID NO.13，SEQ ID NO.15所示。
本发明提供了一种编码所述突变体的基因，编码所述突变体S98A、T105A、S106A、L341A、S98A/T105A、S98A/T105A/S106A、S98A/T105A/S106A/L341A的基因的核苷酸序列分别如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12，SEQ ID NO.14，SEQ ID NO.16所示。
本发明提供了含有所述突变体的基因的重组质粒，所述重组质粒以pET系列载体、pRSFDuet系列或pGEX系列载体为表达载体。
本发明提供了一种获得所述pmirLAAD突变体的方法，所述方法包括：
(1)在奇异变形杆菌L-氨基酸脱氨酶pmirLAAD氨基酸序列的基础上确定突变位点；设计定点饱和突变和定点突变的引物，以携带L-氨基酸脱氨酶pmirLAAD基因的载体为模板进行定点突变；构建含所述突变体的重组质粒；
(2)将突变体质粒转化进宿主细胞；
(3)挑选阳性克隆进行发酵培养、诱导，进而获得pmirLAAD突变体的蛋白，用于后续转化实验。
在本发明的一种实施方式中，所述宿主细胞为E.coli BL21(DE3)。
本发明提供了一种表达所述突变体、或所述基因、或含有所述重组载体的宿主菌。
本发明提供了一种生产α-酮异戊酸产量的方法，所述方法以L-缬氨酸为反应底物，将所述宿主菌加入反应液，在pH7.5～9.5、20～40℃、通气量为1～5vvm条件下反应22～26h。
在本发明的一种实施方式中，在pH8.5～9.5、25～35℃、通气量为1～2vvm条件下反应22～25h。
在本发明的一种实施方式中，所述宿主菌在体系中的终浓度为10～30g/L。
在本发明的一种实施方式中，所述宿主菌在体系中的终浓度为10～15g/L。
本发明提供了一种生产α-酮异戊酸产量的方法，所述方法以L-缬氨酸为反应底物，将所述突变体加入反应液，在pH7.5～9.5、20～40℃、通气量为1～5vvm条件下反应22～26h。
在本发明的一种实施方式中，在pH8.5～9.5、25～35℃、通气量为1～2vvm条件下反应22～25h。
本发明提供了所述突变体，或编码所述突变体的基因，或所述重组质粒在食品、医药、化工领域中制备α-酮异戊酸或提高α-酮异戊酸产量中的应用。
本发明提供了所述宿主菌，或所述制备α-酮异戊酸的方法在食品、医药、化工领域中制备α-酮异戊酸中的应用。
本发明的有益效果
本发明构建了一种L-氨基酸脱氨酶突变体及其制备办法，用于催化生产α-酮异戊酸。本发明的催化效率(1.61mM -1·min -1)和产物抑制常数(5.4mM)较对照分别提高了5.2倍和5.7倍。提高了单位催化剂的生产能力，有效降低了生产成本，并且反应溶液仅以水为催化介质，具有反应条件温和，操作简便，易于分离，对环境友好等优势，且工艺简单，便于控制，易于推广应用。本发明得到的突变体在3L发酵罐中以L-缬氨酸为底物时，α-酮异戊酸的产量可达到96.5g/L，转化率>97％，该产量是目前最高的产量，加快了酶转化法生产α-酮异戊酸的工业化进程。
附图说明
图1为全细胞催化剂浓度和α-酮异戊酸生产量的关系图。
具体实施方式
基因来源：本专利所涉及到的生物酶pmirLAAD基因来源于Proteus mirabilis，pET28a(+)质粒购自Novagen(Madison,WI,U.S.A.)，限制性内切酶、T4DNA连接酶、primeSTAR等购自TaKaRa(Dalian,China)。标样购自SIGMA。pmirLAAD突变体均为分子改造所得，其余试剂均为市场购买所得。
LB培养基：蛋白胨10g，酵母粉5g，氯化钠10g，蒸馏水定容到1L。
发酵培养基：(TB培养基)：蛋白胨12g，酵母提取物24g，甘油4mL，磷酸二氢钾2.31g，磷酸氢二钾16.42g，蒸馏水定容到1L。
HPLC法测定α-酮异戊酸含量的样品制备：取1mL转化后的转化溶液，在12000rpm下离心5min，取上清液稀释后，经过0.45μm滤膜过滤，过滤液供液相色谱分析。HPLC法测定α-酮异戊酸的含量：waters高效液相色谱仪(配紫外可见检测器)，采用伯乐Aminex HPX-87H(300×7.8mm，9μm)色谱柱，流动相为浓度2.5mmol/L的稀硫酸，流动相经0.22μm滤膜过滤，超声脱气，流速为0.6mL/min，柱温为35℃，在紫外检测波长210nm下检测。
实施例1：单突变体的构建和筛选
(1)突变体构建：设计第98、第105位、106位、341位突变位点的引物，如表1所示，通过全质粒PCR进行构建。
表1突变引物序列
Figure PCTCN2020130330-appb-000001
构建反应PCR扩增体系：PrimSTAR酶0.5μL、5×PrimeSTAR Buffer 10μL、dNTP 4μL每个突变位点的两条引物各1μL、模板(pmirLAAD的核苷酸序列)4μL、水32.5μL；反应条件为：①94℃3min；②98℃10s；③55℃30s；④72℃3min；⑤将②～④三个步骤循环29次；⑥72℃5min；⑦12℃保温。
将上述反应体系在37℃孵育3h以消化质粒模板(消化体系为：DpnI 0.5μL、上述反应PCR产物45μL、10×T Buffer 5μL)，消化结束后得到的消化产物通过化学转化方法导入大肠杆菌BL21感受态细胞，化学转化法具体步骤：
1)将10μl同源重组产物导入100μl BL21感受态细胞；
2)冰浴15-30min；
3)42℃水浴热激90s，取出后迅速放入冰中静置冰浴3-5min；
4)加入800μl无抗性LB培养基混匀，于37℃，200rpm培养1h；
5)5000rpm离心2min收菌；
6)移去上清，剩余100-200μl吹吸混匀涂布至含0.05mg/mL卡那霉素抗性平板上，37℃恒温培养12h左右。
7)挑取单克隆于含0.05mg/mL卡那霉素抗性LB中，200rpm、37℃恒温培养12h后，送去公司测序，测序正确的即为阳性转化子(突变体菌株)，突变的位点分别为将第98位的S突变为A，将第105位的T突变为A，将第106位的S突变为A，将第341位的L突变为A。
(2)单突突变体的摇瓶筛选：将得到的4个突变体菌株和含有野生型L-氨基酸脱氨酶的菌株分别接种至LB种子培养基，200rpm、37℃培养培养10h左右，分别以培养基体积的5％的接种量接种至摇瓶发酵培养基，在200rpm、37℃培养至OD 600＝0.8左右，加入终浓度为0.04mmol/L的IPTG诱导，诱导条件为200rpm、25℃诱导14h，6000rpm离心8min收集菌体，离心收集的菌体用于后期转化实验。
转化条件为：转化温度25℃，反应pH为8.0，转化时间为24h。
将转化结束后的转化液用HPLC方法进行测定α-酮异戊酸的产量，结果如表2所示，最终S98A生产得到α-酮异戊酸产量最高。
表2单突变体摇瓶筛选结果
Figure PCTCN2020130330-appb-000002
实施例2：双突、三突及四突突变体的构建和筛选
(1)双突突变体构建：在突变体PmirLAAD S98A的基础上，利用突变引物T105A-S和T105A-A、S106A-S和S106A-A、L341A-S和L341A-A，分别构建双突突变体；在突变体PmirLAAD T105A的基础上，利用突变引物S106A*-S和S106A*-A、L341A-S和L341A-A，分别构建双突突变体；在突变体PmirLAAD S106A的基础上，利用突变引物L341A-S和L341A-A(表3)；通过全质粒PCR进行双突突变体构建，具体实施方式参见实施例1中步 骤(1)，制备得到6个双突突变体PmirLAAD S98A/T105A、PmirLAAD S98A/S106A、PmirLAAD S98A/L341A、PmirLAAD T105A/S106A、PmirLAAD T105A/L341A、PmirLAAD S106A/L341A。
表3双突变体突变引物序列
Figure PCTCN2020130330-appb-000003
(2)双突突变体的筛选：具体实施方式参见实施例3中步骤(3)，结果如表4所示，最终S98A/T105A生产得到α-酮异戊酸产量最高。
表4双突变体摇瓶筛选结果
Figure PCTCN2020130330-appb-000004
(3)三突突变体的构建：在突变体PmirLAAD S98A/T105A的基础上，利用突变引物S106A*-S和S106A*-A、L341A-S和L341A-A，分别构建三突突变体；在突变体PmirLAAD S98A/S106A和PmirLAAD T105A/S106A的基础上，利用突变引物L341A-S、L341A-A，分别构建三突突变体(表4)，通过全质粒PCR进行三突变体的构建，具体实施方式参见实施例3中步骤(1)，制备得到四种三突突变体PmirLAAD S98A/T105A/S106A、PmirLAAD S98A/T105A/L341A、PmirLAAD S98A/ S106A/L341A、PmirLAAD T105A/S106A/L341A。
(4)三突变体的筛选：具体实施方式参见实施例1中步骤(2)，结果如表5所示，最终S98A/T105A/S106A生产得到α-酮异戊酸产量最高。
表5三突变体摇瓶筛选结果
Figure PCTCN2020130330-appb-000005
Figure PCTCN2020130330-appb-000006
(5)四突突变体构建：在突变体PmirLAAD S98A/T105A/S106A基础上对L341A进行突变：以在突变体PmirLAAD S98A/T105A/S106A为模板，利用突变引物L341A-S和L341A-A(表3)进行全质粒PCR，PCR产物进行消化，PCR体系与消化体系和实施例3相同。
实施例3：亲本酶及突变体的动力学参数、产物抑制常数测定
为了对突变体进行评价，本发明测定了突变亲本PmirLAAD WT及突变体PmirLAAD M1、PmirLAAD M2、PmirLAAD M3、PmirLAAD M4在25℃下的动力学参数。
k cat/K m通过在25℃条件下测定不同浓度的水解L-缬氨酸底物所产生α-酮异戊酸的初始利率计算。通过转化过程中的产物抑制常数测定实验来确定亲本酶和突变体的产物抑制情况，分别将PmirLAAD WT亲本酶菌株和突变体菌株各以10g/L终浓度的湿细胞加入反应液中，以60mM的L-缬氨酸为底物，在转化体系中加入10-100mM的α-酮异戊酸，反应30min左右测定初始反应率V 0，反应2h左右测定最大反应率V max，并根据如下公式计算产物抑制常数K PI：
Figure PCTCN2020130330-appb-000007
V 0：初始反应速率，V max：最大反应速率，K m：米氏常数，[S]：底物浓度，[P]：产物浓度，K PI：产物抑制常数。
如表6所示，在25℃下，所有的突变体与亲本酶相比，K cat/K m值均提高，其中突变体PmirLAAD M4的k cat/K m值提高5.2倍，导致PmirLAAD的催化效率变大。与之一致的是各突变体的产物抑制常数与亲本酶相比均得到提高，其中突变PmirLAAD M4(以下简称M4菌株)的产物抑制常数增加了5.7倍。
表6 PmirLAAD亲本酶及其突变体的动力学参数
Figure PCTCN2020130330-appb-000008
Figure PCTCN2020130330-appb-000009
实施例4：1L体系水平优化L-缬氨酸生产α-酮异戊酸
将平板上测序正确的突变体M4菌株接种至含0.05mg/mL卡那霉素抗性LB中，200rpm、37℃培养10-12h，以体积比5％的接种量接种至TB培养基中，200rpm、37℃培养至OD 600为3时加入0.04mmol/L终浓度IPTG诱导，诱导温度为25℃，诱导时间14h后以6,000×g离心8min收集细胞，放置-40℃冰箱用于转化。
(1)不同全细胞催化剂浓度对α-酮异戊酸浓度的影响
在发酵罐中配制转化反应体系：将L-缬氨酸溶解于一定量Tris-HCl缓冲液中，将L-缬氨酸溶液倒入发酵罐中(使得L-缬氨酸在反应体系中的终浓度为160g/L)，调节温度至25℃，转速为300rpm，10g、15g、20g、25g、30g突变体湿菌体细胞(即为全细胞催化剂)同样使用缓冲液溶解均匀，待转化液温度升至25℃，将溶解的菌液倒入发酵罐(全细胞催化剂的浓度分别为10g/L、15g/L、20g/L、25g/L、30g/L)。
在25℃、600rpm、通气量为1vvm条件下进行转化反应，转化反应后总体积为1L。
转化反应结束后取部分转化液12,000×g离心15min，上清液用0.22μm的微滤膜过滤后进行HPLC分析。结果如图1所示，当催化剂的浓度从10g/L提高至30g/L，α-酮异戊酸的浓度从76.7g/L提高至154.3g/L。但是产物/催化剂的比值从7.6g/g降低至5.1g/g。考虑到工业对高产量以及低催化剂(菌体)用量，10g/L的全细胞催化剂浓度同时具备较高α-酮异戊酸浓度及高产物/催化剂比例(即单位菌体生产α-酮异戊酸能力)7.6g/g，被用于转化实验。
(2)不同转化反应pH对α-酮异戊酸浓度的影响
具体步骤同(1)，将全细胞化剂浓度控制为10g/L，将L-缬氨酸浓度控制为100g/L，通气量为1vvm，且将转化反应过程中的pH分别控制为7.5、8.0、8.5、9.0、9.5。结果如表7所示，在弱酸碱性的条件下更适合α-酮异戊酸的合成，因此选择转化pH控制在8.5～9.0，此时α-酮异戊酸浓度为78.9g/L。
表7转化pH优化结果
Figure PCTCN2020130330-appb-000010
(3)不同转化反应温度对α-酮异戊酸浓度的影响
具体步骤同(1)，将转化pH控制为8.5，全细胞化剂浓度控制为10g/L，通气量为1vvm，将转化反应过程中的温度分别控制为20℃、25℃、30℃、35℃、40℃。结果如表8所示，在温度为30℃时更适合α-酮异戊酸的合成，因此选择转化温度pH控制在30℃左右，此时α-酮异戊 酸浓度为78.9g/L。
表8转化温度优化结果
Figure PCTCN2020130330-appb-000011
(4)不同转化反应通气量对α-酮异戊酸浓度的影响
具体步骤同(1)，将转化pH控制为8.5，全细胞化剂浓度控制为10g/L，温度控制为30℃、将通气量控制为1vvm、1.5vvm、2vvm、2.5vvm、3vvm。结果如表9所示，在通气量为1.5vvm时更适合α-酮异戊酸的合成，因此选择通气量控制在1.5vvm左右，此时α-酮异戊酸浓度为96.5g/L。
表9转化通气量优化结果
Figure PCTCN2020130330-appb-000012
对比例1
具体实施方式参见实施例4，不同之处在于，将突变体M4菌株替换为野生型WT菌株去进行发酵与转化实验。转化结束后，取部分转化液12,000×g离心15min，上清液用0.22μm的微滤膜过滤后进行HPLC分析。HPLC色谱图结果显示α-酮异戊酸产量为40g/L，转化率为40.3％。
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。

Claims (15)

  1. 一种L-氨基酸脱氨酶的突变体，其特征在于，所述突变体是将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第98位、第105位、第106位和第341位进行突变，所述突变体的氨基酸序列如SEQ ID NO.15。
  2. 一种L-氨基酸脱氨酶的突变体，其特征在于，所述突变体是在氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第98位、第105、第106位、第341位的氨基酸中的一个或多个位点突变。
  3. 根据权利要求2所述的突变体，其特征在于，所述突变体为以下(a)～(g)任一：
    (a)将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第98位突变为丙氨酸；
    (b)将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第105位突变为丙氨酸；
    (c)将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第106位突变为丙氨酸；
    (d)将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第341位突变为丙氨酸；
    (e)将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第98位和105位均突变为丙氨酸；
    (f)将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第98位、第105位和第106位进行突变，获得的突变体氨基酸序列如SEQ ID NO.13；
    (g)将氨基酸序列如SEQ ID NO.1所示的L-氨基酸脱氨酶的第98位、第105位、第106位和第341位进行突变，获得的突变体氨基酸序列如SEQ ID NO.15。
  4. 编码权利要求1-3任一所述突变体的基因。
  5. 含有权利要求4所述基因的重组质粒。
  6. 表达权利要求1-3任一所述突变体，或含有权利要求4所述载体的宿主菌。
  7. 一种生产α-酮异戊酸的方法，其特征在于，利用权利要求1-3任一所述突变体催化L-缬氨酸转化生成α-酮异戊酸。
  8. 根据权利要求7所述的方法，其特征在于，将所述突变体加入含有100-200g/L L-缬氨酸的转化体系中，在通气量为1-5vvm的条件下反应22-26h。
  9. 根据权利要求8所述的方法，其特征在于，所述转化体系的反应条件为20-40℃、pH7.5～9.5。
  10. 一种生产α-酮异戊酸的方法，其特征在于，以权利要求6所述的宿主菌作为催化剂，催化L-缬氨酸得到α-酮异戊酸。
  11. 根据权利要求10所述的方法，其特征在于，将所述宿主菌添加至含有100-200g/L L-缬氨酸的转化体系中，在通气量为1-5vvm的条件下反应22-26h。
  12. 根据权利要求11所述的方法，其特征在于，所述宿主菌在转化体系中的终浓度为 10～30g/L。
  13. 根据权利要求10所述的方法，其特征在于，所述转化体系的反应条件为20-40℃、pH7.5～9.5
  14. 权利要求1-3任一所述突变体，或权利要求4所述基因，或权利要求5所述重组质粒在食品、医药、化工领域制备α-酮异戊酸中的应用。
  15. 权利要求6所述宿主菌，或权利要求7-13任一所述方法在食品、医药、化工领域制备α-酮异戊酸中的应用。
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