CN105821090B - Application of symbiotic bacillus thermophilus meso-diaminopimelate dehydrogenase mutant - Google Patents

Abstract

The invention discloses a bacillus subtilis (B) derived from thermophilic symbiosis bacillusSymbiobacterium thermophilum）mesoThe method for synthesizing the D-alanine by using the diaminopimelate dehydrogenase mutant as the biocatalyst, NAD (H) as a coenzyme and alpha-keto acid as a substrate successfully utilizes the cheaper NAD (H) as a cofactor for the first time compared with the prior art, and the biocatalyst can be prepared from crude enzyme by a one-step heating method, so the method has the advantages of simple operation, mild reaction conditions, environmental friendliness, high product yield and high optical purity.

Description

Thermophilic symbiotic bacillusmesoApplication of diaminopimelate dehydrogenase mutant

Technical Field

The invention belongs to the field of biocatalysis, and relates to a method for utilizing thermophilic symbiotic bacillus (Bacillus (B) (R))Symbiobacterium thermophilum）mesoMethod for synthesizing D-alanine using diaminopimelate dehydrogenase mutant as biocatalyst, NAD (H) as coenzyme, alpha-keto acid as substrate, and optical purity of product D-alanine>98% and a method for preparing the biocatalyst by one-step heat treatment of the crude enzyme.

Background

D-amino acid accounts for about 10% of more than 400 amino acids and analogues thereof found in nature (Jianlingao, LiuGuilan. asymmetric synthesis of D-phenylalanine, journal of amino acid, 1988, 2: 1-3). Among the various D-amino acids, D-alanine is more widely used. In the field of food additives, D-alanine and L-aspartic acid synthesize tianalane dipeptide (trade name: alitame), the sweetness of which is 2000 times of that of cane sugar, the sweetness is stable to heat and pH value, the tianan dipeptide can be widely used as a sweetening agent in food industry and daily chemical industry, and the tianan dipeptide has excellent market prospect (Ellis, J.W. [ J.W. ]] Journal of Chemical Education1995,72 (8): 671-675). D-alanine can be used as one of synthetic raw materials of bactericide metalaxyl-M (Zhao Ke Jian, Chinese chemical medicine)]And second edition. Beijing: new era press, 1999). Alanine is also used in the medical field in a very wide range of applications.

In the biosynthesis of D-alanine, the enzymes currently used are primarily transaminases (Koma D., Sawai T., Hara, R., Harayama S., Kino, K.,Appl. Microbiol. Biot. 2008, 79775-784), D-aminoacylase and D-hydantoinase/N-carbamoyl-D-aminoamidohydrolase combination enzyme systems (Turner R. J., AikensJ., Royer S., DeFilippi L., Yap A., Holzle D., Somers N., Fotheringham I. G.,Eng. Life Sci. 2004, 4, 517-520），mesoDiaminopimelate dehydrogenase (DAPDH) (Gao x., Chen x., Liu w., Feng j., Wu q., Hua l., Zhu d.,Appl. Environ. Microbiol. 2012, 788595-8600, patent application no: 201210334554.6), etc., and these enzymatic synthesis methods are practically applicable. DAPDH catalyzes the reversible oxidative deamination/reductive amination of amino acids in the presence of the coenzyme NADP (H) (Ohshima T., SodaK., in)Bioprocesses and Applied Enzymology, Vol. 42Springer Berlin/Heidelberg, 1990, pp. 187-209). However, the coenzyme preference of all currently known DAPDH enzymes and mutants is NADP (H) -dependent. The coenzyme NAD (H) has better stability, lower price and wider coenzyme cycle method compared with NADP (H), and NAD (H) -dependent amino acid dehydrogenase can use Formate Dehydrogenase (FDH) and ammonia formate as coenzyme cycle system. They can be started from prochiral keto acids, using free NH₄ ⁺As an amino donor, chiral amino acids are synthesized in the presence of a coenzyme circulation system. Thus, NAD (H) -dependent ADPDH is more atom-economical than NADP (H) -dependent DAPDH, a green-economical method of amino acid synthesis (Zhu d., Hua l.,Biotech. J. 2009, 4, 1420-1431）。

Disclosure of Invention

The invention utilizes an StDapdh mutant with coenzyme preference modified into NAD (H) dependence as a biocatalyst, directly prepares a catalyst for catalytic reaction by a method of crushing supernatant through one-step heat treatment, and applies the catalyst to the synthesis of D-alanine, and the ee value of the product is more than 98%.

Stdapdh mutant reaction scheme

The biocatalyst used in the present invention is obtained by the following steps:

1. Introducing R35E single-site mutation by using a Quick Change Mutagenesis Kit with pET32-StDapdh plasmid as a template;

2. Introducing R35E/R36V two-site combined mutation by using a Quick Change Mutagenesis Kit with pET32-StDapdh plasmid as a template;

3. Introducing R35D/R36V two-site combined mutation by using a Quick Change Mutagenesis Kit with pET32-StDapdh plasmid as a template;

4. Introducing R35D/R36Q two-site combined mutation by using a Quick Change Mutagenesis Kit with pET32-StDapdh plasmid as a template;

5. introducing R35D/R36Q/Y76V three-site combined mutation by using a Quick Change Mutagenesis Kit (Quick Change Mutagenesis Kit) by taking pET32-StDapdh plasmid as a template;

6. culturing the constructed mutant engineering bacteria, and performing induced expression on the target protein;

7. Respectively collecting thalli from the single-site, double-site and three-site combined mutants in the step 6, then carrying out heavy suspension by using a buffer solution, carrying out high-pressure crushing, centrifuging, taking a crushed supernatant, and purifying by using a Ni-NTA chromatographic column;

8. And (4) collecting thalli from the three-site combined mutant in the step 6, then carrying out heavy suspension by using a buffer solution, carrying out high-pressure crushing, and centrifuging to obtain a crushed supernatant. And (4) performing heat treatment on the crushed supernatant in a water bath at 70 ℃ for 30min, and centrifuging again to obtain the heat-treated supernatant.

the method for performing D-amino acid synthesis reaction on the StDapdh mutant purified by the Ni column or the StDapdh mutant subjected to heat treatment comprises the following steps:

the method comprises the steps of forming a reaction system by using a substrate alpha C ~ keto acid, an amino compound and a solvent, adding an NAD (H) cofactor and a biocatalyst StDapdh mutant, and reacting for 4C ~ 72 hours at 20C ~ 60 ℃ and at the pH value of 6C ~ 11 to obtain the optically pure D C ~ alanine.

the dosage of the biocatalyst in each liter of reaction system is 0.1 g ~ 100 g, and the solvent is a single solvent or a plurality of solvents.

And (3) determining and detecting the configuration of the reaction product:

Adding perchloric acid denatured protein into the reaction product, derivatizing the amino acid of the reaction product, and determining the product configuration and ee value by using a derivatized L, D-alanine standard sample as a control.

The StDapdh mutant enzyme in the method has coenzyme preference of NAD (H) -dependent type, and can be used for catalyzing and generating D-alanine by one-step simple heat treatment, and the optical purity is more than 98 percent

Description of the drawings:

FIG. 1: SDS-PAGE electrophoretogram of heat treatment process of StDapdh mutant protein. In the figure, lane M: protein molecular weight Marker, lane 1: expressed mutant primary enzyme; lane 2: the mutant supernatant was subjected to primary enzymatic heat treatment for 10min, lane 3: the supernatant of the mutant is subjected to primary enzyme heat treatment for 20min, the supernatant of the mutant is subjected to primary enzyme heat treatment for 30min, and the supernatant of the mutant is subjected to primary enzyme heat treatment for 40 min.

FIG. 2 is a drawing: FDAA derived D-alanine standards.

FIG. 3: FDAA derived L-alanine standards.

FIG. 4 is a drawing: FDAA derived control reaction (primary enzyme without heat treatment) assay results.

FIG. 5: carrying out catalytic reaction detection on the crude FDAA derived mutant enzyme after heat treatment at 70 ℃ for 10 min.

FIG. 6: carrying out catalytic reaction detection on the crude FDAA derived mutant enzyme after heat treatment at 70 ℃ for 20 min.

FIG. 7: carrying out catalytic reaction detection on the crude FDAA derived mutant enzyme after heat treatment at 70 ℃ for 30 min.

FIG. 8: carrying out catalytic reaction detection on the crude FDAA derived mutant enzyme after heat treatment at 70 ℃ for 40 min.

FIG. 9: the crude enzyme heat treatment time and the ee value of the catalytic product of the StDapdh R35E/R36V/Y76V mutant correspond to each other.

Detailed Description

the invention is further illustrated by the following specific examples, but these examples are not intended to limit the scope of the inventionThe examples are not to be construed as limiting the invention. The materials used in the following examples were purchased from Sigma, unless otherwise specified. DNA and protein Marker were both from Fermentas and protein purified Ni-NTA filler from GE. The Quick Change mutagenesis Kit was purchased from Agilent, and the mutant primers were designed according to the Kit requirements. The primer synthesis and DNA sequencing were carried out by Huada Gene Co., Ltd. (Beijing).pET₃₂Plasmid vector, TOP10, BL21(DE3) high efficiency competent (Novagen). The liquid chromatography was carried out using an Agilent-1200 chromatograph, and the column was an Eclipse XDB-C18 column (4.6X 150 mm).

example 1: obtaining of mutants

use ofStDapdhthe gene is Genbank with the number of AP006840.1, which is synthesized and connected topET₃₂Obtaining a plasmid on the vector:pET₃₂StDapdh, and soluble expression of the wild-type gene in E.coli BL21(DE3), the expressed protein being 6 × his tag at the N-terminus for subsequent Ni-NTA purification. PCR mutation primers used in Table 1 were synthesized according to the site to be mutated with reference to the QuickChange Mutagenesis Kit, and PCR product amplification, Dpn1 cleavage and subsequent nucleic acid recovery were performed according to the Kit instructions.

Table 1: mutant PCR primers

to be provided withpET32-StDapdh plasmid is used as a template, R35E/R36V double mutation is introduced into the plasmid by using primers 1 and 2, the plasmid is transformed into escherichia coli TOP10 competence, the plasmid is extracted and sequenced to confirm to obtain double mutation plasmidpET₃₂StDapdhR 35E/R36V. And taking the obtained double-mutation plasmid as a template, and continuously introducing a Y76V mutation into the mutant by using primers 3 and 4 to obtain a triple-mutation plasmid:pET₃₂StDapdhR35E/R36V/Y76V, transformed into E.coli BL21(DE3) high-efficiency competence, and confirmed by plasmid sequencing.

Example 2: expression of mutant enzyme, preparation by Heat shock treatment

coli BL21(DE3) containing the triple mutant plasmid was cultured in 2L of LB liquid medium and cultured at 37 ℃ to OD₆₀₀After about 0.8, isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added thereto at a final concentration of 0.5 mM for induction expression at an induction temperature of 30 ℃ for 20 hours. After the induction expression was completed, the cells were centrifuged at 5000 rpm for 5 minutes to collect the cells. A part of the cell sample was taken for purification and activity determination, the cells were resuspended and washed with buffer A (20 mM Tris-Cl pH 8.0, 500 mM sodium chloride), disrupted by high pressure homogenization, and disrupted by centrifugation at 14000 rpm for 30 minutes to remove disrupted precipitates. Of the product by other hetero-enzymes in the crude enzyme (L-amino acid dehydrogenases, carbonyl reductases, etc.)eeBoth the value and the product purity have an influence. The StDAPDH mutant has better thermal stability, and hybrid enzyme which influences the reaction is removed under the condition of maintaining StDAPDH activity by carrying out heat treatment on the crude enzyme. The supernatant was heat-treated in a 70 ℃ water bath, and 100. mu.L of the supernatant was sampled every 10min and continuously sampled for 40 min. After each sampling, the supernatant was centrifuged and a portion of the supernatant was subjected to SDS-PAGE (see FIG. 1).

Example 3: establishment of catalytic reaction system and determination of ee value of product

To 1 mL of sodium carbonate/sodium bicarbonate buffer (100 mM, pH 9.0), 50 mM of the substrate pyruvate, 20 mg of ammonium formate, 1 mg of glucose dehydrogenase GDH, 1 mM of coenzyme NAD were added⁺And a proper amount of mutant enzyme treated supernatant, adjusting the pH value of the system to 9.0, and reacting for 24 hours at the rotating speed of 200 rpm and 30 ℃. The reaction is ended by adding 10 mul perchloric acid into a catalytic reaction system or the reaction is ended by heating, denatured protein is removed by centrifugation, 200 mul of reaction solution is taken, 400 mul of FDAA derivative reagent is added, and 80 mul of 1M NaHCO is added₃After 1 h reaction at 40 ℃ 40. mu.L of 2M HCl was added and 0.45 μ M membrane filtration HPLC analysis was performed.

Alanine enantiomer retention times were: t is t_R(D-alanine) = 14.9 min, t_R(L-alanine) = 7.7 min. The ee values of the supernatant reaction after different heat treatment times are shown in figures 2, 3, 4, 5, 6, 7 and 8, and the crude enzyme heat treatment time and catalytic product of the mutantThe correspondence of the ee value of the substance is shown in FIG. 9. We can see from fig. 9: when the heat treatment is not carried out, a large amount of L-type products exist in the crushed supernatant catalytic reaction products, the amount of the L-type products is gradually reduced along with the prolonging of the treatment time, when the treatment is carried out at 70 ℃ for 20min, the L-type content in the treated supernatant catalytic reaction products is very little, and the ee value of the D-type products is already>95 percent; when the reaction product is treated at 70 ℃ for 30min or more, the ee values of the treated supernatant catalytic reaction products are all>98%。

SEQUENCE LISTING

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> application of symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutant

<130> 2015

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 33

<212> DNA

<213> Symbiobacterium thermophilum

<400> 1

tggttggtgt tgttgaagtg aaagttctgg cgg 33

<210> 2

<211> 34

<212> DNA

<213> Symbiobacterium thermophilum

<400> 2

gccgccagaa ctttcacttc aacaacacca acca 34

<210> 3

<211> 29

<212> DNA

<213> Symbiobacterium thermophilum

<400> 3

gttctgttcc ggaagtcgcg gaagcgatg 29

<210> 4

<211> 29

<212> DNA

<213> Symbiobacterium thermophilum

<400> 4

catcgcttcc gcgacttccg gaacagaac 29

Claims (1)

1. A method for synthesizing D ~ alanine with optical purity of more than 98% by using a meso ~ diaminopimelate dehydrogenase mutant derived from thermophilic symbiotic bacillus (Symbiobacterium thermophilum) as a biocatalyst comprises a reaction system formed by alpha ~ keto acid, alpha ~ ketonate and an amino compound serving as substrates and a solvent, adding the biocatalyst and a coenzyme NAD + circulating system to perform catalytic reduction amination reaction, wherein the reaction temperature is 20 ~ 60 ℃, the reaction pH value is 6 ~ 11, and the reaction time is 4 ~ 72 hours, so that D ~ amino acid with optical purity of more than 98% is prepared, wherein the meso ~ diaminopimelate dehydrogenase mutant is obtained by replacing arginine (R) at the 35 th position of an amino acid sequence corresponding to Genbank number AP006840.1 with glutamic acid (E), replacing arginine (R) at the 36 th position with valine (V), and replacing tyrosine (Y) at the 76 th position with valine (V), namely R35E/R36V/Y76V.