CN107653259B - A kind of method of external enzyme reaction production D- (-) -3-hydroxy-2-butanone - Google Patents

A kind of method of external enzyme reaction production D- (-) -3-hydroxy-2-butanone Download PDF

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CN107653259B
CN107653259B CN201710972637.0A CN201710972637A CN107653259B CN 107653259 B CN107653259 B CN 107653259B CN 201710972637 A CN201710972637 A CN 201710972637A CN 107653259 B CN107653259 B CN 107653259B
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butanone
acetolactate
pet28a
alpha
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陈涛
崔真真
赵玉姣
毛雨丰
王智文
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Tianjin University
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Abstract

The invention discloses a kind of method that external enzyme reaction produces D () 3-hydroxy-2-butanone, step is：(1) carrier pET28a is connect to obtain pET28a alsS with α acetolactate synthestase encoding genes alsS and imports Escherichia coli, fermented, purified, concentration obtains α acetolactate synthestase concentrates；Carrier pET28a is connect to obtain pET28a alsD with coding for alpha acetolactate decarboxylase gene alsD and imports Escherichia coli, is fermented, is purified, concentration obtains α acetolactate decarboxylase concentrates；(2) by above two concentrate, Sodium Pyruvate, Mg2+It is uniformly mixed with b1thiaminpyrophosphate, reaction obtains D () 3-hydroxy-2-butanone.The present invention realizes that the external efficient production of high added value chiral D () 3-hydroxy-2-butanone, product chiral purity are high using cheap Sodium Pyruvate as substrate.

Description

A kind of method of external enzyme reaction production D- (-) -3-hydroxy-2-butanone
Technical field
The invention belongs to biotechnologies and application field, more particularly to a kind of external enzyme reaction production D- (-)-second The method of acyloin.
Background technology
3-hydroxy-2-butanone, also known as be 3- hydroxy-2-butanones, it is a kind of colourless or weak yellow liquid, monomer is colourless or faint yellow Liquid is in milk fragrance, is a kind of common food grade spice, is added in food, promotes the milk fragrance in food.Meanwhile second Acyloin is one of the platform chemicals of U.S. Department of Energy preferential development, especially chiral 3-hydroxy-2-butanone (D- (-) -3-hydroxy-2-butanone and L- (+) -3-hydroxy-2-butanone) it is widely used in functional material, the fields such as chiral drug and chemical intermediate synthesis.
Currently, the main method of 3-hydroxy-2-butanone industrialized production is chemical synthesis, include oxidation, the butanone of 2,3-butanediol Chlorinolysis and diacetyl the methods of partial hydrogenation.The operation of these production technologies is easier, but energy consumption is big, and yield is low And have certain pollution to environment, and 3-hydroxy-2-butanone obtained is mostly the mixture of two kinds of chiral isomers.In recent years, many is ground Sight is turned to both economically and environmentally beneficial Production by Microorganism Fermentation chirality 3-hydroxy-2-butanone by the person of studying carefully.The bacterial strain of production 3-hydroxy-2-butanone has at present Escherichia coli, saccharomyces cerevisiae, bacillus subtilis, serratia marcescens, Corynebacterium glutamicum etc..Second is synthesized using microbial method There are many report of acyloin, and yield is higher, but the 3-hydroxy-2-butanone chiral purity produced is not high, and microbial metabolism is complicated, synthesize in vivo Not easy-regulating.Bae etc.[1]α-acetolactic acid of Bacillus subtilis is introduced in Saccharomyces cerevisiae The hydration nadh oxidase of synzyme, alpha -acetolactate decarboxylase and Lactococcus lactis, and by the synthesis of by-product Approach blocks so that the ultimate output of 3-hydroxy-2-butanone reaches 100.1g/L, but author does not detect the chiral purity of 3-hydroxy-2-butanone.Guo Deng[2]People co-expresses meso-2,3- butanediol dehydrogenases, nadh oxidase using whole-cell catalytic technology in Escherichia coli With the transparent bacillus hemoglobin that quivers.Finally, the meso-2 of 93.73g/L, 3- butanediol produce 86.74g/LD- (-) -3-hydroxy-2-butanone, Chiral purity is up to 97.89%.But to substrate meso-2, the purity requirement of 3- butanediols is higher, expensive, is unfavorable for industry Production.
In contrast, the synthesising biological system based on purifying enzyme has apparent advantage：Efficiency of pcr product is high, and process expends Low, reaction rate is fast, easy to control, and product is easily separated.This environmentally friendly production platform has been successfully applied to many lifes The production of the object energy and biochemicals, such as hydrogen, n- butanol, 1,3-PD etc..Jiao etc.[3]People is synthesized with acetolactic acid Enzyme and acetolactate decarboxylase are attempted to utilize Pyruvate production 3-hydroxy-2-butanone for the first time, as a result obtain 3.36mM (0.296g/ in vitro L 3-hydroxy-2-butanone).Although author has screened acetolactate synthestase and acetolactate decarboxylase with good thermal stability, Acetolactate synthestase from Caldicellulosiruptor owensensis OL and come from Bacillus It is full not make us the compatibility of substrate, substrate specificity and enzyme activity for the acetolactate decarboxylase of subtilis IPE5-4 Meaning.Therefore, the yield of 3-hydroxy-2-butanone, conversion ratio and yield are relatively low.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of external enzyme reaction production D- (-) -3-hydroxy-2-butanone Method.
Technical scheme of the present invention is summarized as follows：
A kind of method of external enzyme reaction production D- (-) -3-hydroxy-2-butanone, includes the following steps：
(1) protein expression vector pET28a is connect to obtain plasmid with α-acetolactate synthestase encoding gene alsS The plasmid pET28a-alsS is imported in Escherichia coli Escherichia coli BL21 (DE3) and is obtained by pET28a-alsS Bacterial strain BL21-1 purifies bacterial strain BL21-1 cultivation and fermentations express alpha-acetolactate synthestase, and concentration obtains α-acetolactic acid Synzyme concentrate；Protein expression vector pET28a is connect to obtain plasmid with alpha -acetolactate decarboxylase encoding gene alsD The plasmid pET28a-alsD is imported in Escherichia coli Escherichia coli BL21 (DE3) and is obtained by pET28a-alsD Bacterial strain BL21-2, expresses alpha -acetolactate decarboxylase to bacterial strain BL21-2 cultivation and fermentations, purifies, and concentration obtains α-acetolactic acid Decarboxylase concentrate；(2) by α-acetolactate synthestase concentrate, alpha -acetolactate decarboxylase concentrate, Sodium Pyruvate, Mg2+ It is uniformly mixed with TPP (b1thiaminpyrophosphate, Thiamine pyrophosphate), reaction obtains D- (-) -3-hydroxy-2-butanone.
D- (-)-second method of the invention, it is possible to using relatively inexpensive Sodium Pyruvate as substrate, realize high added value is even The high yield pulp1 produced in vitro of relation by marriage, chiral purity 99.9%, and yield is up to 129.5g/L, yield is up to 32.4g/L/h.Reaction The core component acetolactate synthestase and alpha -acetolactate decarboxylase of system pass through reasonably combined, the realization D- of two kinds of enzymes The production of (-) -3-hydroxy-2-butanone.
Description of the drawings
Fig. 1 is the collection of illustrative plates of the pET28a-alsS plasmid vectors of structure.
Fig. 2 is the collection of illustrative plates of the pET28a-alsD plasmid vectors of structure.
Fig. 3 is the SDS-PAGE running gel figures of α-acetolactate synthestase and alpha -acetolactate decarboxylase.
Fig. 4 is 3-hydroxy-2-butanone formation curve.
Fig. 5 is the gas chromatographic detection of chiral 3-hydroxy-2-butanone, wherein A is the standard specimen of two kinds of chiral 3-hydroxy-2-butanones in gas-chromatography In peak figure；B reacts peak figure of the sample in gas-chromatography.
Specific implementation mode
With reference to embodiment, the present invention will be further described, and following embodiments are to make those skilled in the art Member can be better understood from the present invention, but be not intended to be limited in any to the present invention.
α-acetolactate synthestase encoding gene alsS, Genbank number of logging in selected by the present invention is 936852.By it α-acetolactate synthestase of coding can be catalyzed Sodium Pyruvate and generate α-acetolactic acid while discharge CO2, process needs Mg2+With The participation of TPP, reaction equation are：
Alpha -acetolactate decarboxylase encoding gene alsD, the Genbank number of logging in 936857 selected by the present invention.By its volume The alpha -acetolactate decarboxylase of code can be catalyzed α-acetolactic acid and generate D- (-) -3-hydroxy-2-butanone, while have CO2Release, reaction Formula is：
The sources original plasmid pET28a are biovector (http://www.biovector.net/)；
168 sources original strain B.subtilis be BGSC (Bacillus Genetic Stock Center, http://www.bgsc.org/)；
E.coli BL21 (DE3) competence source be NEB (http://www.neb-china.com/)；
3-hydroxy-2-butanone standard items are from sigma companies (http://www.sigmaaldrich.com/sigma-aldrich) purchase It buys.
Restriction enzyme used, dephosphorylation enzyme, DNA ligase etc., molecular biology reagents are purchased from thermo companies Buy (http://www.thermoscientificbio.com/fermentas).
Sodium Pyruvate used other biochemical reagents (such as tryptone, yeast extract, NaCl, TRIS, imidazoles, TPP, MgCl2Deng) from Sangon Biotech (Shanghai) Co., Ltd. buy (http://www.sangon.com/).
Embodiment 1 is overexpressed α-acetolactate synthestase (alsS) using commercial protein expression vector pET28a
Using 168 genomes of bacillus subtilis B.subtilis as template, with primer p-alsS1 and p-alsS2 for expanding Increase gene alsS segments (about 1.7kp).AlsS segments and pET28a plasmids are used into Thermo Fast digest XhoI/ BamHI double digestions obtain the expression vector pET28a-alsS (see Fig. 1) of alsS genes, sequencing detection nothing after connecting, converting Accidentally.The correct plasmid of sequencing result is transferred to commercialization competent E.coli E.coli BL21 by traditional chlorinated calcium method (DE3), the BL21-1 of α-acetolactate synthestase (alsS) overexpression is obtained.
Embodiment 2 is overexpressed alpha -acetolactate decarboxylase (alsD) using commercial protein expression vector pET28a
Using 168 genomes of bacillus subtilis B.subtilis as template, with primer p-alsD1 and p-alsD2 for expanding Increase gene alsD segments (768bp).Then alsD segments and pET28a plasmids are used into Thermo Fast digest XhoI/ EcoRI double digestions obtain the expression vector pET28a-alsD (see Fig. 2) of alsD genes, sequencing detection nothing after connecting, converting Accidentally.The correct plasmid of sequencing result is transferred to commercialization competent E.coli E.coli BL21 by traditional chlorinated calcium method (DE3) in, the BL21-2 of α-acetolactate decarboxylase (alsD) overexpression is obtained.
1 strain construction the primer sequence of table
The purifying of 3 α of embodiment-acetolactate synthestase and alpha -acetolactate decarboxylase concentrates
1, α-acetolactate synthestase purifying concentration the specific steps are：
1) e. coli bl21-1 is inoculated into 400mL LB culture mediums, 37 DEG C of shaking table, 220rpm is cultivated to OD600 It is 0.6, derivant IPTG (isopropylthiogalactoside) is added to final concentration of 0.5mM, 16 DEG C of culture 12h, 4 DEG C, 4200rpm centrifuges 20min and collects thalline, and 20mL buffer A is used in combination to suspend.
2) collection step 1) the obtained suspension of BL21-1, smudge cells under the action of high-pressure cell crusher, 4 DEG C, 1200bar, oil pressure 18Kg/cm3Under the conditions of handle 3 times, be crushed after 4 DEG C, 8000rpm centrifuge 30min, collect supernatant obtain Crude enzyme liquid.
3) crude enzyme liquid that will be obtained in step 2) utilizes gravity ni-sepharose purification method, purifying protein.It, will under the conditions of 4 DEG C Crude enzyme liquid all flows through the pillar equipped with nickel filler, then the different imidazole concentrations prepared with buffer A and buffer B The elution of (20Mm, 50mM, 100mM, 150mM, 200mM, 250Mm, 500mM) collects the outflow of 50-250mM concentration Liquid obtains α-acetolactate synthestase solution of high-purity.
4) the destination protein solution for obtaining step 3) utilizes the super filter tube protein concentrate of aperture 10KD.By the stream of collection Go out liquid, in 4800rpm, 4 DEG C of centrifugations.It is finally washed 2 times with 5mL PBS (pH=7.0) buffer solution, continuing centrifugation to surplus is 2mL, packing, obtains α-acetolactate synthestase solution.Recycle Bradford methods[4]It is dense to measure α-acetolactate synthestase Degree, it is final concentration of：23mg/mL.
5) α for obtaining step 4)-acetolactate synthestase solution carries out SDS-PAGE electrophoresis, confirms that α-acetolactic acid closes It is correct (see Fig. 3, M at enzyme size:Marker；Swimming lane 2:α-acetolactate synthestase (64kDa))
2, alpha -acetolactate decarboxylase purifying concentration the specific steps are：
1) e. coli bl21-2 is inoculated into 400mL LB culture mediums, 37 DEG C of shaking table, 220rpm is cultivated to OD600 It is 0.6, derivant IPTG to final concentration of 0.5mM is added, 16 DEG C of culture 12h, 4 DEG C, 4200rpm centrifuges 20min and collects bacterium Body is used in combination 20mL buffer A to suspend.
2) collection step 1) obtained BL21-2 suspension, smudge cells under the action of high-pressure cell crusher, 4 DEG C, 1200bar, oil pressure 18Kg/cm3Under the conditions of handle 3 times, be crushed after 4 DEG C, 8000rpm centrifuge 30min, collect supernatant obtain Crude enzyme liquid.
3) crude enzyme liquid that will be obtained in step 2) utilizes gravity ni-sepharose purification method, purifying protein.It, will under the conditions of 4 DEG C Crude enzyme liquid all flows through the pillar equipped with nickel filler, then the different imidazole concentrations with buffer A and buffer B configurations The elution of (20mM, 50mM, 100mM, 150mM, 200mM, 250mM, 500mM) collects the outflow of 50-250mM concentration Liquid obtains the alpha -acetolactate decarboxylase solution of high-purity.
4) the destination protein solution for obtaining step 3) utilizes aperture 10kD super filter tube protein concentrates.By the outflow of collection Liquid, in 4800rpm, 4 DEG C of centrifugations.It is finally washed 2 times with 5mL PBS (pH=7.0) buffer solution, continuing centrifugation to surplus is 2mL, packing, obtains alpha -acetolactate decarboxylase solution.Bradford methods are recycled to measure alpha -acetolactate decarboxylase concentration, It is final concentration of：58mg/mL.
5) α for obtaining step 4) -- acetolactate decarboxylase solution carries out SDS-PAGE electrophoresis, confirms α-acetolactic acid Decarboxylase size it is correct (see Fig. 3, M:Marker；Swimming lane 1:α -- acetolactate decarboxylase (32.6kDa))
LB culture medium prescriptions are：10g/L tryptones, 5g/L yeast extracts, 10g/L NaCl adjust pH to 7.0. Sterilize 20min under 0.1Mpa pressure.
PBS (pH=7.0) buffer formulation is：Solution I：100mM NaH2PO3, 150Mm NaCl；Solution II：100mM Na2HPO3, 150Mm NaCl, solution I and solution II by volume 19:31 mixing.
Buffer A are formulated：25mM PBS, 150mM NaCl, 20mM imidazoles adjusts PH to 7.0.
Buffer B are formulated：25mM PBS, 150mM NaCl, 500mM imidazoles adjusts PH to 7.0.
The measurement of 4 α of embodiment-acetolactate synthestase and alpha -acetolactate decarboxylase specific enzyme activity
1, the specific enzyme activity of α-acetolactate synthestase measures, the specific steps are：
1) substrate buffer solution is configured：The Sodium Pyruvate of each component concentration 20mM, 10mM MgCl2, 0.2mM TPP
2) enzyme solution after 100 μ L dilute, 37 DEG C of water-bath 30min are added in the substrate buffer solution of step 1)；
3) 0.1mL 50%H are added in the reaction solution of step 2)2SO4Reaction is terminated, is incubated 25 minutes at 37 DEG C, 13000rpm centrifuges 5min；
4) it takes the reaction mixture 0.55mL that step 3) obtains to be added in the reaction tube containing 0.45mL 2.5M NaOH, mixes It is even.The solution after 0.25ml mixings is taken to add to 1mL mixed liquor As (5% alpha-Naphthol and 0.5% sarcosine, V again：V=1：1), room temperature Lower shake 30min.
5) solution for obtaining step 4) measures the absorption value under 530nm wavelength with spectrophotometric.1U acetolactic acids synthesize Enzyme is defined as the enzyme amount needed for 1 μm of ol acetolactic acids/3-hydroxy-2-butanone of generation per minute.
As a result the specific enzyme activity for measuring α-acetolactate synthestase is 51U/mg.
2, the specific enzyme activity of alpha -acetolactate decarboxylase measures, the specific steps are：
1) substrate buffer solution is prepared：100 μ L 2- acetylmethyl ethyl acetoacetates are added in 6mL 0.5M NaOH, Then sustained oscillation 20min is settled to 50mL with 0.5M HCl tune pH to 7.0 with PBS buffer solution, which need to make With preceding configuration.
2) it takes the substrate buffer solution that step 1) obtains to be mixed with the enzyme solution after 100 μ L dilutions, is reacted in 37 DEG C of metal baths 20min。
3) taking the 100 μ L of mixed liquor that step 2) obtains to add to 1mL color developing agents, (0.1% creatine, 1% alpha-Naphthol are dissolved in In 1mol/LNaOH), 30min develops the color in 30 DEG C of metal baths.
4) solution for obtaining step 3) measures the absorption value under 530nm wavelength with spectrophotometric.1U acetolactic acid decarboxylations Enzyme is defined as:The enzyme amount per minute for making 1 μm of ol α-acetolactic acid decarboxylation form 1 μm of ol 3-hydroxy-2-butanone.
As a result the specific enzyme activity for measuring Α-acetolactate decarboxylase is 52U/mg.
5 external dual-enzyme system of embodiment produces D- (-) -3-hydroxy-2-butanone
With the coupling for two reactions that α-acetolactate synthestase and alpha -acetolactate decarboxylase are catalyzed, design external Enzyme reaction system produces D- (-) -3-hydroxy-2-butanone, and design parameter is as shown in table 2,35 DEG C, is reacted under static conditions, and 4h terminates reaction. 3-hydroxy-2-butanone formation curve is as shown in Figure 4.
2. reaction system of table
The chiral purity that 3-hydroxy-2-butanone is detected using GC-FID, uses chiral column HP-chiral 20b column (Agilent Technologies).Fig. 5 is shown in the chromatography detection of chiral D-(-) -3-hydroxy-2-butanone, wherein it is that two kinds of chiral second are even that A, which schemes (standard items), The peak figure (being followed successively by D- (-) -3-hydroxy-2-butanone, L (+) -3-hydroxy-2-butanone) of the mixture of relation by marriage；B figures are the detection peak of the present embodiment reaction sample Figure.
From fermentation results as can be seen that the total output of 3-hydroxy-2-butanone is 1.47M (129.5g/L), reach theoretical yield 98%, and D- (-) -3-hydroxy-2-butanone chiral purity is up to 99.9%, yield is up to 32.4g/L/h.It is external double constructed by the present invention Enzyme reaction system can realize the production of external chiral 3-hydroxy-2-butanone, and production process is environmentally protective, D- (-) -3-hydroxy-2-butanone yield and Rate reaches high level and chiral purity is also higher, has very good application prospect.
Bibliography：
[1]Bae SJ,Kim S,Hahn JS.Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase[J].Scientific Reports,2016,6:27667.
[2]Guo Z,Zhao X,He Y,et al.Efficient(3R)-acetoin production from meso-2,3-butanediol using a new whole-cell biocatalyst with co-expression of meso-2,3-butanediol dehydrogenase,NADH oxidase and Vitreoscilla hemoglobin [J].Journal of microbiology and biotechnology,2016.
[3]Jia X,Liu Y,Han Y.A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate[J].Scientific Reports,2017,7(1): 4333.
[4]Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1-2):248-254。
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Claims (1)

1. a kind of method of external enzyme reaction production D- (-) -3-hydroxy-2-butanone, it is characterized in that including the following steps：
(1) protein expression vector pET28a is connect to obtain plasmid with α-acetolactate synthestase encoding gene alsS The plasmid pET28a-alsS is imported in Escherichia coli Escherichia coli BL21 (DE3) and is obtained by pET28a-alsS Bacterial strain BL21-1 purifies bacterial strain BL21-1 cultivation and fermentations express alpha-acetolactate synthestase, and concentration obtains α-acetolactic acid Synthesize enzyme solutions；Protein expression vector pET28a is connect to obtain plasmid with alpha -acetolactate decarboxylase encoding gene alsD The plasmid pET28a-alsD is imported in Escherichia coli Escherichia coli BL21 (DE3) and is obtained by pET28a-alsD Bacterial strain BL21-2, expresses alpha -acetolactate decarboxylase to bacterial strain BL21-2 cultivation and fermentations, purifies, and concentration obtains α-acetolactic acid Decarboxylation enzyme solutions；
(2) by α-acetolactate synthestase, alpha -acetolactate decarboxylase, Sodium Pyruvate, Mg2+It is equal with b1thiaminpyrophosphate mixing Even, reaction obtains D- (-) -3-hydroxy-2-butanone；
The α-acetolactate synthestase encoding gene alsS, Genbank number of logging in is 936852；
Alpha -acetolactate decarboxylase encoding gene alsD, the Genbank number of logging in 936857.
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CN116024201B (en) * 2022-12-23 2024-03-19 天津大学 Alpha-acetolactate decarboxylase mutant and application thereof in acetoin production
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