CN105132487B

CN105132487B - A kind of regenerating coenzyme system and preparation method thereof

Info

Publication number: CN105132487B
Application number: CN201510442408.9A
Authority: CN
Inventors: 李航; 徐军; 阙利民; 江岳恒; 蔡彤�
Original assignee: ABA Chemicals Corp
Current assignee: ABA Chemicals Corp
Priority date: 2015-07-24
Filing date: 2015-07-24
Publication date: 2019-04-09
Anticipated expiration: 2035-07-24
Also published as: CN105132487A; WO2017016442A1

Abstract

The present invention provides a kind of regenerating coenzyme system comprising: hydrogenlyase (formate dehydrogenase, FDH), leucine dehydrogenase (leucine dehydrogenase, LDH) and ammonium formate.The present invention additionally provides the preparation method of the regenerating coenzyme system simultaneously, mainly includes the heterogenous expression of step (1) hydrogenlyase and leucine dehydrogenase；2) regenerating coenzyme system is prepared.Regenerating coenzyme system of the invention, regenerating coenzyme is high-efficient, and preparation method is simple and convenient, significantly reduces the cost of coenzyme reaction, is suitable for large-scale industrialization application.

Description

A kind of regenerating coenzyme system and preparation method thereof

Technical field

The present invention relates to field of biotechnology more particularly to a kind of regenerating coenzyme system and preparation method thereof.

Background technique

Compared to traditional chemical catalysis, chemo-selective of the biocatalysis because of the height of its enzymic catalytic reaction, region choosing The advantages such as selecting property and stereoselectivity and be concerned and study.Wherein due to the high efficiency of oxidoreducing enzyme and specificity etc., They occupy considerable status in biosynthesis.The catalysis of oxidoreducing enzyme energy selectivity contains carbonyl, aldehydes and ketone Compound.For oxidoreducing enzyme as catalyst preparation chipal compounds, synthetic product while, can consume a certain amount of coenzyme. Such as: Li Shuting etc. prepares S-Leucine with trimethyl pyruvic acid, is coupled in synthesis using leucine enzyme and hydrogenlyase System, hydrogenlyase can be cozymase regeneration, and the conversion ratio of product can achieve 82%.

Unlike other enzymes, in bioconversion reaction, the relationship of auxiliary enzyme-to-substrate is the relationship of stoichiometry.It is auxiliary Enzyme is a kind of consumptive compound, can be reused unlike oxidoreducing enzyme, in the reaction as the donor of hydrogen, instead Coenzyme exists after answering with oxidation states.And these coenzyme are often also more expensive than the price of prepared product, stablize Property is poor, it is difficult to reuse, the high of reaction cost in industrialized production be resulted in, to limit oxidoreducing enzyme It uses.In industrialized production, it is impossible to a large amount of coenzyme is added, so building and the coenzyme of efficient regenerating coenzyme system Reusability is must to solve the problems, such as instantly.

Therefore, those skilled in the art is dedicated to developing a kind of simple, efficient regenerating coenzyme technology.

Summary of the invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of regenerating coenzyme systems.

Above-mentioned purpose of the invention is achieved through the following technical solutions:

A kind of regenerating coenzyme system, including hydrogenlyase (formate dehydrogenase, FDH), leucine dehydrogenation Enzyme (leucine dehydrogenase, LDH) and ammonium formate.

According to one embodiment of present invention, the regenerating coenzyme system further includes NAD⁺And/or NADH.

According to one embodiment of present invention, the regenerating coenzyme system further includes phosphopyridoxal pyridoxal phosphate.

According to one embodiment of present invention, the hydrogenlyase and leucine dehydrogenase in the regenerating coenzyme system From engineering bacteria fermentation liquid, and the genetic engineering bacterium expresses hydrogenlyase and leucine dehydrogenase simultaneously.

According to one embodiment of present invention, the genetic engineering bacterium is while expressing hydrogenlyase and leucine dehydrogenation E.coli BL21 (DE3) bacterial strain of enzyme.

Another aspect of the present invention is to provide a kind of preparation method of regenerating coenzyme system, method includes the following steps:

(1) heterogenous expression of hydrogenlyase and leucine dehydrogenase

Building while the genetic engineering bacterium for expressing FDH and LDH, it is fermented that the enzyme solution containing FDH and LDH is made；

(2) regenerating coenzyme system is prepared

Prepare regenerating coenzyme system in buffer, the regenerating coenzyme system include: the enzyme solution containing FDH and LDH, Ammonium formate, NAD⁺And phosphopyridoxal pyridoxal phosphate.

According to one embodiment of present invention, wherein in step (1) the following steps are included:

(a) FDH carrier is constructed

FDH gene is connected into the pET28a carrier using I double digestion of endonuclease Nde I and Xho, building is recombinated Express the plasmid pFDH-pET28a of FDH；

(b) LDH carrier is constructed

LDH gene is connected into the pET21a carrier using I double digestion of endonuclease Xho I and Nde, building is recombinated Expression plasmid pLDH-pET21a；

(c) construct while expressing the genetic engineering bacterium of FDH and LDH

Plasmid pFDH-pET28a and plasmid pLDH-pET21a corotation are entered into E.coli BL21 (DE3), co-expressed The bacterial strain E.coli BL21-FDH/LDH of FDH and LDH.

According to one embodiment of present invention, this method is further comprising the steps of:

(d) ferment the bacterial strain E.coli BL21-FDH/LDH, prepares the enzyme solution containing FDH and LDH

Coexpression bacterial strain E.coli BL21-FDH/LDH is cultivated in the LB liquid medium containing Amp and Kan, is added IPTG to final concentration of 0.01~0.05mmol/L, 16~27 DEG C of 12~20h of inducing expression, fermentation liquid are centrifuged after thallus broken wall Supernatant is taken to co-express enzyme solution up to the FDH/LDH.

According to one embodiment of present invention, wherein in step (a) using Candida total DNA as template, respectively with upper and lower It swims primer and carries out PCR amplification, to obtain the FDH gene, in which:

Upstream primer is 5 '-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3 ', wherein is NdeI enzyme at scribing line Enzyme site；Downstream primer is 5 '-AAACTCGAGTGCGACCTTTTTGTCATTAC-3 ', wherein is XhoI digestion position at scribing line Point.

According to one embodiment of present invention, wherein IPTG to final concentration of 0.02mmol/L, inducing temperature 22 is added DEG C, the inducing expression time be 16h, fermentation liquid after thallus broken wall centrifuging and taking supernatant up to the FDH/LDH co-express enzyme solution.

The invention has the advantages that regenerating coenzyme system of the invention, regenerating coenzyme is high-efficient, and preparation method is simple and convenient, The cost of coenzyme reaction is significantly reduced, large-scale industrialization application is suitable for.

Detailed description of the invention

Fig. 1 shows the schematic diagram of building plasmid pFDH-pET28a；

Fig. 2 shows the digestion verification figure of pTD-pET28a, the pcr amplification product of swimming lane 1TD, swimming lane 2pTD-pET28a Recombinant plasmid digestion verification；

Fig. 3 shows the digestion verification figure of pFDH-pET28a, the pcr amplification product of swimming lane 1FDH, swimming lane 2pFDH- PET28a recombinant plasmid digestion verification；

Fig. 4 shows the digestion verification figure of pLDH-pET21a, swimming lane 1pLDH-pET21a recombinant plasmid digestion verification, swimming The LDH segment that 2 digestion of road obtains.

Specific embodiment

Following embodiment is detailed description of the invention.It should be appreciated that as described below is only preferred implementation of the invention Example modifies to the present invention and equivalent replacement should all cover at this without departing from the spirit and scope of the present invention Within the scope of invention.

Experimental raw used in the present invention is listed below.

1 experimental plasmid of table

2 experimental strain of table

Used PCR primer in the experiment of table 3

4 experiment reagent of table

Culture medium

LB liquid medium/gL^-1: peptone 10, yeast extract 5, sodium chloride 10, pH 7.0.

LB solid medium/gL^-1: peptone 10, yeast extract 5, sodium chloride 10, agar strip 20, pH 7.0.

Main solution and buffer

(1) plasmid extraction related solution:

I: 0.2973g glucose of Solution, 0.091g Tris, 0.112g EDTA are settled to 30mL.

Solution II (fresh configuration): 0.2mol/L NaOH, 1%SDS.

III: 44.163g potassium acetate of Solution, 17.25mL glacial acetic acid are settled to 150mL.

(2) 50 × TAE (electrophoretic buffer) (/L)

242g Tris 57.1mL acetic acid, 100mL EDTA (0.05mol/L).

Embodiment 1:PCR amplifying target genes

(1) PCR amplification primer

According to hydrogenlyase (FDH) threonine deaminase (TD) gene order (Genbank number:XM_ 001525495 and AAB18593) and its context design primer.By screening verification, on optimal hydrogenlyase (FDH) Trip primer is 5 '-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3 ' (being NdeI restriction enzyme site at scribing line)；Draw in downstream Object is 5 '-AAACTCGAGTGCGACCTTTTTGTCATTAC-3 ' (being XhoI restriction enzyme site at scribing line).According to threonine deaminase The gene order (AAB18593) of enzyme designs upstream and downstream primer, and by screening verification, optimal upstream primer is 5 '- AAAAAGCTTGTTAGCAGCCGGATCTCAG-3 ' (being HindIII restriction enzyme site at scribing line)；Downstream primer is 5 '- AAACATATGGTATTAAAACAAATTCTTC-3 ' (being NdeI restriction enzyme site at scribing line).

Using Candida and Escherichia coli total DNA as template, expand to obtain FDH and TD gene piece respectively with upstream and downstream primer Section.

(2) PCR reaction system

PrimeSTAR HS archaeal dna polymerase PCR reaction system is as shown in table 5.

5 PCR reaction system of table

(3) PCR response procedures

PCR response procedures: totally 30 circulations, FDH genetic fragment size is about 1300bp, and TD genetic fragment size is about 1500bp。

Step1:98 DEG C, 10s denaturation

Step2:65 DEG C, 10s annealing

Step3:72 DEG C, 1.5min extends

Embodiment 2: the building of recombinant plasmid

Plasmid construction process (by taking FDH is constructed as an example) as shown in Figure 1 uses specificity using Candida total DNA as template The genetic fragment that restriction endonuclease NdeI and XhoI double digestion expands or the genetic fragment being connected with carrier T, while double digestion carrier Plasmid after purification and recovery, connects 5h under the conditions of 16 DEG C, converts connection product in competent cell E.coli DH5 α, picking It converts to be connected in the single colonie on LB solid plate in 5mL LB liquid medium and (resist according to the addition of the resistance of plasmid is corresponding Raw element), under the conditions of 37 DEG C, shaken cultivation is stayed overnight in 220r/min shaking table.Plasmid is extracted, picking plasmid carries out double digestion and tests The plasmid pFDH-pET28a built after verifying is correct, is carried out the measurement of DNA sequence dna by card.

(1) building of threonine deaminase expression plasmid

Using Escherichia coli total DNA as template, PCR amplification is carried out with upstream and downstream primer respectively, shown in Fig. 2, obtains Yi Tiaote Anisotropic DNA band, average molecular size is about 1.5kb, is consistent with theoretical value.The plasmid pTD-pET28a built is used HindIII and NdeI double digestion verifying, electrophoresis digestion products, shown in Fig. 2, band with expection be consistent, respectively 5.4kb with Occurs band at 1.5kb, all stripe sizes meet expection, and expression plasmid building is correct.The plasmid pTD- that will be built PET28a carries out DNA sequence dna comparison, compares after being sequenced without mutation.It is transformed into E.coli BL21 (DE3), obtains TD expression bacterium Strain E.coli BL21-TD, carries out subsequent protein expression.

(2) building of hydrogenlyase expression plasmid

Using Candida total DNA as template, PCR amplification is carried out with upstream and downstream primer respectively, shown in Fig. 3, obtains Yi Tiaote Anisotropic DNA band, average molecular size is about 1.3kb, is consistent with theoretical value.The plasmid pFDH-pET28a built is made It is verified with NdeI and XhoI double digestion, electrophoresis digestion products, shown in Fig. 3, band is consistent with expection, respectively in 5.4kb and 1.3kb There is band in place, and expression plasmid building is correct.It compares after being sequenced without mutation.The plasmid built can be transformed into E.coli BL21 carries out subsequent protein expression.

(3) building of leucine dehydrogenase expression plasmid

According to the gene order (Genbank number:WP_016086354) of leucine dehydrogenase (LDH), the English Weihe River is entrusted Jie Ji biotech firm synthetic gene sequence segment.The T of LDH segment is connected with using specific restriction endonuclease NdeI and XhoI double digestion Carrier shown in Fig. 4, obtains 3 DNA bands, and average molecular size is respectively 2.5kb, 1.1kb, 0.25kb, and wherein 2.5kb is T The linear strip of carrier, 1.1kb are the genetic fragment of LDH, and size is consistent with theoretical value.The plasmid pLDH- that will be built PET21a is verified using NdeI and XhoI double digestion, and shown in Fig. 4, electrophoresis digestion products, band is consistent with expection, is existed respectively Occurs band at 5.4kb and 1.1kb, expression plasmid building is correct.The plasmid pLDH-pET28a built is subjected to DNA sequence dna It compares, compares after being sequenced without mutation.It is transformed into E.coli BL21 (DE3), obtains LDH expression bacterial strain E.coli BL21- LDH carries out subsequent protein expression.

Embodiment 3: the building of recombinant bacterial strain is co-expressed

PFDH-pET28a and pLDH-pET21a corotation is dissolved into E.coli BL21 (DE3), and be coated on LB (Amp and Each 100 μ g/ml of Kan) on solid plate, the single colonie grown on LB (Amp and each 100 μ g/ml of Kan) solid plate is exactly Bacterial strain containing hydrogenlyase and leucine dehydrogenase gene simultaneously is named as E.coli for engineering strain is obtained BL21-FDH/LDH.The bacterial strain can express hydrogenlyase and leucine dehydrogenase simultaneously.

Embodiment 4: the preparation of crude enzyme liquid

Picking is inoculated into 5mL LB culture solution with the single colonie of engineering bacteria of recombinant plasmid, at 37 DEG C, 220r/min Under the conditions of be incubated overnight.It (is added respectively according to the resistance of plasmid corresponding with 5 ‰ inoculum concentration access 100mL LB liquid medium Antibiotic) in 37 DEG C culture.Work as OD₆₀₀Value reaches 0.6~0.8, induces under the optimal induction final concentration of IPTG and inducing temperature 16h.Fermentation liquid after culture discards supernatant liquid after 3700r/min refrigerated centrifuge 15min, by the suitable pre-cooling of bacterium mud Na₂HPO₄It is centrifuged again after the washing of (0.1mol/L, pH=8.0) buffer.Finally use 20mL Na₂HPO₄(0.1mol/L, pH= 8.0) ultrasonication after buffer is resuspended, ultrasonic power 360W, 10s+20s, 20min, shattering process bacterium solution remain at ice In water-bath.Clasmatosis liquid is through 3700r/min, and 4 DEG C of centrifugation 15min, gained supernatant is crude enzyme liquid, can carry out enzyme activity survey It is fixed, SDS-PAGE electrophoresis detection and conversion reaction.

Embodiment 5: it recombinates taking temperature for zymoprotein and reaches

The recombinant plasmid of building is transferred in the competent cell of Escherichia coli E.coilBL21 by heat shock method, thus Obtaining can be with the engineering bacteria of overexpression exogenous enzymes.Picking is inoculated into 3mL LB culture solution with the engineering bacteria of recombinant plasmid, It is incubated overnight under the conditions of 37 DEG C.

It takes 1mL bacterium solution is standby to make negative control, separately takes 1mL to give over to inoculation and use, IPTG is added in remaining 1mL bacterium solution (1mmol/L) induction, in 37 DEG C of expression 3h.Bacterium solution after negative control and induction is subjected to SDS-PAGE electrophoresis detection.

Select E.coli BL21-FDH, the single colonie of E.coli BL21-TD and E.coli BL21-FDH/LDH, 37 It is incubated overnight under the conditions of DEG C, is 1mmol/L in induced concentration, inducing temperature is inducing expression 3h under conditions of 37 DEG C, will be induced Bacterium solution afterwards carries out SDS-PAGE electrophoresis detection, the results show that there is obvious expression item at 47kDa, 500kDa, 42kDa respectively Band.Before induction, destination protein only has micro background expression, and after induction, the expression quantity of soluble protein be increased significantly, and illustrate mesh Mark albumen can be good at expressing in recombinant bacterium.

Embodiment 6: the optimization of recombination enzyme protein expression condition

It recombinates expression of the zymoprotein in Escherichia coli to exist in two forms: soluble protein and inclusion body.And forgive Body protein does not have enzymatic activity, and only soluble protein has enzymatic activity.Since the carrier pET28a of selection has The high feature of expression quantity even if a large amount of destination protein aggregations exist with inclusion bodies, but also has considerable destination protein Exist with soluble protein.Influence the solubility expression of albumen because being known as: induction time and lures at the concentration of IPTG inducer Lead temperature.For the condition for optimizing protein expression, not isogeneous induction duration has been investigated respectively, different IPTG induction final concentrations and different Under inducing temperature, the solubility expression of E.coli BL21-FDH and E.coli BL21-GDH.

(1) optimization of duration is induced

Under normal circumstances, induction time is longer, and the expression quantity of destination protein is higher, reaches maximum until sometime putting, Later with the extension of induction time, the expression quantity of albumen tends to stop.But if induction time is too long, foreign protein is also expressed It is more, influence the activity of destination protein.That has investigated albumen when induction duration is respectively 4h, 8h, 12h, 16h and 20h herein can Dissolubility expression quantity.

Picking is inoculated into 5mL LB culture solution with the single colonie of engineering bacteria of recombinant plasmid, at 37 DEG C, 220r/min Under the conditions of be incubated overnight.Respectively with 37 DEG C of trainings in 5 ‰ inoculum concentration access 30mL LB liquid medium (the 100 μ g/ml containing Kan) It supports.Work as OD₆₀₀When value reaches 0.6~0.8, in the case where optimal IPTG induces final concentration and inducing temperature, inducing expression (4h, 8h, 12h, 16h and 20h), thalline were collected by centrifugation, handles the solubility expression of progress SDS-PAGE electrophoresis detection albumen after thallus, really Fixed optimal induction duration.

Inducible strain E.coli BL21-TD under the conditions of 16 DEG C is respectively 4h, 8h, 12h, 16h and 20h to induction duration When albumen amount of soluble expression investigated, the results showed that, before inducer is added, destination protein has a small amount of local expression. With the increase of induction duration, the expression quantity of destination protein increases therewith, until 16h expressing quantity tends to a maximum value, The expression quantity of subsequent albumen tends to stop.It is thus determined that a length of 16h of best induction time after IPTG is added.

Inducible strain E.coliB L21-FDH under the conditions of 22 DEG C is respectively 4h, 8h, 12h to induction duration, 16h and The amount of soluble expression of albumen is investigated when 20h, the results showed that, before inducer is added, destination protein has this small amount of earth's surface It reaches.With the increase of induction duration, the expression quantity of destination protein increases therewith, until 16h expressing quantity tends to a maximum Value, the expression quantity of subsequent albumen tend to stop.It is thus determined that be added after IPTG optimal induction when a length of 16h.

Inducible strain E.coli BL21-LDH under the conditions of 22 DEG C is respectively 4h, 8h, 12h to induction duration, 16h and The amount of soluble expression of albumen is investigated when 20h, the results showed that, before inducer is added, this only a small amount of earth's surface of destination protein It reaches.With the increase of induction duration, the expression quantity of destination protein increases therewith, until 16h expressing quantity tends to a maximum Value, the expression quantity of subsequent albumen tend to stop.It is thus determined that the best induction time being added after IPTG is 16h.

(2) optimization of IPTG induced concentration

In DE3 host strain, the concentration by changing IPTG can change the level of protein expression, and expression can be from very low Level tune to very high level, until the level that pET carrier induces completely.Therefore protein induced final concentration point has been investigated herein Not Wei 0.01 ﹑, 0.02 ﹑, 0.05 ﹑, 0.1 ﹑ 0.2mmol/L and 0.5mmol/L when albumen amount of soluble expression.

Work as OD₆₀₀Value reaches 0.6~0.8, investigates induction final concentration (0.01 ﹑, 0.02 ﹑, 0.05 ﹑, 0.1 ﹑ of 6 kinds of difference IPTG 0.2mmol/L and 0.5mmol/L), under optimal inducing temperature after inducing expression 16h, thalline were collected by centrifugation, and processing thallus is laggard The solubility expression of row SDS-PAGE electrophoresis detection albumen, determines optimal IPTG induced concentration.

With not plus compared with inducer, after IPTG is added, TD (50kDa) has obvious band of expression, is consistent with expected size.But It is in the range of induced concentration is 0.02-0.5mmol/L, the expression quantity of albumen is roughly the same, therefore selects the final concentration of IPTG The inducing expression of albumen is carried out for 0.02mmol/L.

With not plus compared with inducer, after IPTG is added, FDH (47kDa) has obvious band of expression, is consistent with expected size. When IPTG final concentration reaches 0.02mmol/L, 0.2mmol/L and 0.5mmol/L, destination protein has a large amount of expression.When When IPTG final concentration reaches 0.01,0.05mmol/L and 0.1mmol/L, destination protein also has the expression of slight amount.The result shows that: It just can induce protein expression under IPTG low concentration, therefore select the induction table of the final concentration of 0.02mmol/L progress albumen of IPTG It reaches.

With not plus compared with inducer, after IPTG is added, LDH (42kDa) has obvious band of expression, is consistent with expected size. When IPTG final concentration reaches 0.01,0.02,0.2mmol/L and 0.5mmol/L, destination protein has a large amount of expression.When When IPTG final concentration reaches 0.05mmol/L and 0.1mmol/L, destination protein also has the expression of slight amount.Therefore select the end of IPTG Concentration is the inducing expression that 0.01mmol/L carries out albumen.

(3) optimization of inducing temperature

The cultivation temperature of bacterium has a major impact the expression quantity of destination protein.The inducing temperature point of albumen has been investigated herein Wei not be 16 DEG C, the amount of soluble expression of albumen at 22 DEG C, 28 DEG C, 32 DEG C and 37 DEG C.

Work as OD₆₀₀Value reaches 0.6~0.8, optimal IPTG induce final concentration, investigate 5 different temperatures (16 DEG C, 22 DEG C, 28 DEG C, 32 DEG C and 37 DEG C) under induce 16h after, thalline were collected by centrifugation, handle thallus after carry out SDS-PAGE electrophoresis detection egg White solubility expression determines optimal inducing temperature.

In the expression of E.coli BL21-TD, at 16 DEG C, 22 DEG C, 28 DEG C, 32 DEG C and 37 DEG C this 5 temperature, albumen The solubility expression of enzyme is higher, not the difference of conspicuousness, but under 22 DEG C of inducing temperature, the solubility expression of albumen It is higher compared to the expression of other temperature, comprehensively consider economic and high soluble protein and express, therefore selects inducing temperature for 16-28 DEG C (preferably 22 DEG C) carry out the inducing expression of albumen.

In the expression of E.coli BL21-FDH, at 16 DEG C, 22 DEG C, 28 DEG C, 32 DEG C and 37 DEG C this 5 temperature, when luring Leading temperature is 16 DEG C, and 22 DEG C, under 28 DEG C of this 3 inducing temperatures, the solubility expression of protease is higher, not the difference of conspicuousness Different, still, soluble protein amount is few compared to 16 DEG C and 22 DEG C relative to inclusion body under the conditions of 28 DEG C.When inducing temperature raising is 32 DEG C and when temperatures above, amount of soluble expression significantly reduces, and most of destination protein is all present in the form of inclusion body In precipitating, although this may be since high temperature is conducive to the growth and breeding of bacterium, expressing quantity is also high, and protein expression is excessive It is too fast to be formed inclusion body.Comprehensively consider economic and high soluble protein to express, therefore inducing temperature is selected to carry out for 22 DEG C The inducing expression of albumen.

In the expression of E.coli BL21-LDH, at 16 DEG C, 22 DEG C, 28 DEG C, 32 DEG C and 37 DEG C this 5 temperature, when luring Leading temperature is 16 DEG C, and 22 DEG C, under 28 DEG C of this 3 inducing temperatures, the solubility expression of protease is higher, still, 28 DEG C of conditions Lower soluble protein amount is few compared to 16 DEG C and 22 DEG C relative to inclusion body.It is 32 DEG C and temperatures above when inducing temperature increases When, amount of soluble expression significantly reduces, and most of destination protein is all present in precipitating in the form of inclusion body, this may be Although expressing quantity is also high since high temperature is conducive to the growth and breeding of bacterium, protein expression is excessively too fast to be formed packet Contain body.Comprehensively consider economic and high soluble protein to express, therefore selects inducing temperature for the inducing expression of 22 DEG C of progress albumen.

(4) the albumen condition optimizing of recombinant bacterial strain is co-expressed

The optimal protein expression condition of recombinase E.coli BL21-FDH are as follows: optimal inducing temperature is 22 DEG C, optimal induction Concentration is 0.02mmol/L, when optimal induction a length of 16h.

Binding protein SDS-PAGE figure it is found that due to recombinase E.coli BL21-LDH the relatively high concentration of induction are as follows: 0.01,0.02,0.2mmol/L and 0.5mmol/L, and the relatively high concentration of induction of recombinase E.coli BL21-FDH are as follows: 0.02mmol/L, 0.2mmol/L and 0.5mmol/L, therefore determine the optimal induction of coexpression bacterial strain E.coli BL21-FDH/LDH Concentration is 0.02mmol/L.Therefore the final optimum protein expression condition for determining coexpression bacterial strain E.coli BL21-FDH/LDH are as follows: Best inducing temperature is 22 DEG C, best induced concentration a length of 16h when being 0.02mmol/L, most preferably induction.

Induce in optimal conditions, carry out SDS-PAGE electrophoresis, induce the expression of FDH and LDH, respectively in 47kDa and There is apparent band of expression in the position 42kDa, is consistent with expected size.Recombinant bacterium E.coil thallus after induction passes through ultrasonication It is centrifuged after processing, the FDH and LDH of E.coil coexpression are mostly present in supernatant, for soluble albumen.

Embodiment 7: the measurement of enzyme activity

(1) measurement of hydrogenlyase enzyme activity

Microplate reader used herein measures the enzyme activity of hydrogenlyase.One enzyme activity unit (U) is defined as measuring Under the conditions of, enzyme amount needed for generating 1 μm of ol product per minute.

The enzyme activity of FDH, total reaction volume 1mL, reaction solution composition are as follows: Na are surveyed using ammonium formate as substrate₂HPO₄(0.1mol/ L, pH=8.0) 1.67mmol/L NAD is added in buffer⁺, 167mmol/L HCOONH₄.Reaction mixture is kept the temperature in 30 DEG C, Start timing after a certain amount of FDH crude enzyme liquid is added, every 60s detection NADH 340nm absorbance incrementss, according to it Molar extinction coefficient ε=6220L/ (mol of 340nm^-1·cm^-1) calculate the enzyme activity of hydrogenlyase.Oxidation reaction is as follows:

The calculation method of enzyme activity are as follows:

△ A indicates the variation of absorbance at 340nm in 1min in formula, and Vt indicates the volume (ml) of reaction solution, and Vs indicates sample The volume (ml) of product, d indicate colorimetric pool optical path (0.625cm), and 6220 indicate molar absorption coefficient (L/mol^-1·cm^-1), c table Show the concentration (mg/ml) of protein.

With HCOONH₄For substrate, the enzyme activity in the broken supernatant of bacteria solution of FDH is measured, the results showed that, FDH breaks supernatant of bacteria solution catalysis The activity for generating NADH is about 10U/ml.According to the total protein concentration that Bradford method is measured, the ratio of the crude enzyme liquid of FDH is calculated Enzyme activity is 13.51U/mg.

(2) measurement of threonine deaminase and leucine dehydrogenase enzyme activity

The enzyme activity of threonine deaminase is surveyed using threonine as substrate, total reaction volume 1mL is separately added into 0.1g Soviet Union ammonia Acid, 0.03mg PLP, a certain amount of crude enzyme liquid and Na₂HPO₄(0.1mol/L pH=8.0) buffer, at 30 DEG C, 150r/min 2h is reacted in shaking table, isometric acetonitrile is added, then is diluted with water 500 times, 12000r/min, is analyzed after being centrifuged 5min with HPLC Product formation calculates enzyme activity.

The enzyme activity of leucine dehydrogenase in coupling reaction is surveyed using carbonyl butyric acid as substrate respectively, total reaction volume 1mL divides Not Jia Ru 400 μm of ol carbonyl butyric acid, 0.2mg NAD⁺, 65mg HCOONH₄, a certain amount of crude enzyme liquid and Na₂HPO₄(0.1mol/ L, pH=8.0) buffer.Reaction mixture reacts 2h in 30 DEG C, the shaking table of 150r/min, and isometric acetonitrile is added, then 5 times of dilution is derived, and analyzes product formation with HPLC after centrifugation, calculates enzyme activity.

One enzyme activity unit (U) is defined as under determination condition, enzyme amount needed for generating 1 μm of ol product per minute.

The enzyme activity of TD is surveyed using threonine as substrate, the results showed that, the ability that TD conversion threonine generates carbonyl butyric acid is 6.67U/ml, the specific enzyme activity for calculating the crude enzyme liquid of TD is 4.156U/mg.E.coli BL21- is surveyed by substrate of carbonyl butyric acid The enzyme activity of LDH in FDH/LDH coupling reaction, the results showed that, the ability that LDH conversion carbonyl butyric acid generates L- aminobutyric acid is 7.38U/ml, the specific enzyme activity for calculating the crude enzyme liquid of LDH is 6.43U/mg.

Embodiment 8: the measurement of crude enzyme liquid protein content

Using the concentration of Bradford method measurement protein, the absorbance of known bovine serum albumen solution is measured, is obtained The standard curve of absorbance and protein concentration.Sample to be tested is uniformly mixed with Bradford solution, is being stored at room temperature reaction 5min.Using 96 orifice plates, each mixed liquor is placed in parallel in 3 holes, then measures the absorbance of solution under 595nm wavelength, with Absorbance of the average value of the absorbance in this 3 holes as sample to be tested.The concentration of protein is calculated according to standard curve.

Using the absorbance of Bradford method measurement and the standard curve of protein concentration.

(1) determination of the protein content of threonine deaminase crude enzyme liquid

The average value of the absorbance of the sample measured is A=0.643.According to standard curve y=4.8618x+0.3310 (R2 =0.9906) it is 0.0642mg/ml that protein content, which, is calculated, according to the counted protein content of the diluted concentration of albumen For 1.605mg/ml.

(2) determination of the protein content of hydrogenlyase crude enzyme liquid

The average value of the absorbance of the sample measured is A=0.475, and it is 0.0296mg/ml that protein content, which is calculated, It is 0.74mg/ml according to the counted protein content of the diluted concentration of albumen.

(3) measurement of leucine dehydrogenase and the protein content of hydrogenlyase coexpression

The average value of the absorbance of the sample measured is A=0.554, and it is 0.0459mg/ml that protein content, which is calculated, It is 1.148mg/ml according to the counted bak protein content of the diluted concentration of albumen.

Embodiment 9: the measurement of product assay

L- Gamma-propalanine content is measured using 2,4-dinitrofluorobenzene derivatization method, product formation is detected by HPLC. Generate the chromatographic condition of L- aminobutyric acid are as follows: chromatographic column is C18 column (Φ 4.6mm × 250mm, 5 μm)；Column temperature is 30 DEG C；Flowing Phase A:Na₂HPO₄(0.02mol/L, pH=7.2) buffer, B: acetonitrile, isocratic elution, V (A): V (B)=70: 30；Flow velocity is 1.0ml/min；360nm ultraviolet detection.Using standard items, standard curve quantitative determination is made.Generate the liquid phase item of carbonyl butyric acid Part are as follows: column temperature is 25 DEG C；Mobile phase A: NaH₂PO₄(0.02mol/L, pH=3.0) buffer, B: acetonitrile, isocratic elution, V (A): V (B)=90: 10；Flow velocity is 1.0mL/min；205nm ultraviolet detection.

Embodiment 10: coexpression system catalysis preparation L- aminobutyric acid

Using TD, FDH and LDH coexpression enzyme solution in same system, catalysis substrate threonine generates L- aminobutyric acid.? Fermented and cultured E.coli BL21-TD or E.coli BL21-FDH/LDH in 100mL LB (each 100 μ g/ml containing Amp and Kan), And crude enzyme liquid is prepared, the synthesis for L- aminobutyric acid.

2.1g threonine, 3.0g HCOONH are sequentially added in 250mL triangular flask₄, 19.2mL Na₂HPO₄(0.1mol/ L) buffer, adds 4.8mL TD crude enzyme liquid, and 6mL FDH and LDH co-express crude enzyme liquid, 10mg NAD⁺With 3.215mg phosphoric acid Pyridoxal (PLP).It is placed in insulating box, magnetic agitation, automatical potentiometric titrimeter detects pH value, with 0.2mol/L NaOH solution Titration.Reaction measures product formation with HPLC to 20h.

In the conversion reaction for preparing L- aminobutyric acid by L-threonine, by many experiments, the optimum condition of acquisition are as follows: temperature Degree is 30 DEG C；PH is 8.0；NAD⁺Initial concentration be 0.25mg/ml；By the way of batch charging, maximum substrate inventory For 70g/L；Enzyme dosage proportion is, in the reaction system of 10mL, the additional amount of threonine deaminase is 8mL thallus, coexpression The additional amount of hydrogenlyase and leucine dehydrogenase is 10mL thallus；A length of 16h when end reaction.Conversion preparation L- amino fourth Acid.For inventory in the case where 70g/L, yield is up to 95%.

It summarizes

The optimization of expression condition is carried out to recombinant protease E.coli BL21-FDH/LDH, obtaining best inducing temperature is 22 DEG C, best induced concentration a length of 16h when being 0.02mmol/L, most preferably induction.Under optimum condition of the expression, it is anti-to measure this coupling The enzyme activity for answering middle LDH is 7.38U/ml.

In the conversion reaction for preparing L- aminobutyric acid by L-threonine, optimum condition are as follows: temperature is 30 DEG C；PH is 8.0； NAD⁺Initial concentration be 0.25mg/ml；Substrate inventory is 70g/L；Enzyme dosage proportion is, in the reaction system of 10mL, The additional amount of threonine deaminase is 8mL thallus, and the additional amount for co-expressing hydrogenlyase and leucine dehydrogenase is 10mL bacterium Body；A length of 16h when end reaction, yield is up to 95%.

Regenerating coenzyme system of the invention, regenerating coenzyme is high-efficient, and preparation method is simple and convenient, significantly reduces coenzyme The cost of reaction is suitable for large-scale industrialization application.

While specific embodiments of the present invention are described in detail, it has also been found that related to the present invention Similar embodiment plurality of replaceable design and be defined by the claims in the art.

Claims

1. a kind of regenerating coenzyme system, which is characterized in that the regenerating coenzyme system includes hydrogenlyase (formate Dehydrogenase, FDH), leucine dehydrogenase (leucine dehydrogenase, LDH) and ammonium formate and phosphoric acid pyrrole It trembles aldehyde, the hydrogenlyase and leucine dehydrogenase in the regenerating coenzyme system come from engineering bacteria fermentation liquid, the base Because engineering bacteria expresses hydrogenlyase and leucine dehydrogenase simultaneously, there is plasmid pFDH-pET28a in the genetic engineering bacterium With plasmid pLDH-pET21a, the regenerating coenzyme system is prepared by the method included the following steps:

(1) heterogenous expression of hydrogenlyase and leucine dehydrogenase

Building while the genetic engineering bacterium for expressing FDH and LDH, it is fermented that the enzyme solution containing FDH and LDH is made, including the following steps:

(a) FDH carrier is constructed

FDH gene is connected into the pET28a carrier using I double digestion of endonuclease Nde I and Xho, building is recombinantly expressed The plasmid pFDH-pET28a of FDH；

(b) LDH carrier is constructed

LDH gene is connected into the pET21a carrier using I double digestion of endonuclease Xho I and Nde, building is recombinantly expressed Plasmid pLDH-pET21a；

(c) construct while expressing the genetic engineering bacterium of FDH and LDH

Plasmid pFDH-pET28a and plasmid pLDH-pET21a corotation are entered into E.coli BL21 (DE3), obtain coexpression FDH with The bacterial strain E.coli BL21-FDH/LDH of LDH；

Coexpression bacterial strain E.coli BL21-FDH/LDH is cultivated in the LB liquid medium containing Amp and Kan, IPTG is added To final concentration of 0.01~0.05mmol/L, 16~27 DEG C of 12~20h of inducing expression, fermentation liquid is after thallus broken wall in centrifuging and taking It is clear to co-express enzyme solution up to the FDH/LDH；

(2) regenerating coenzyme system is prepared

Regenerating coenzyme system is prepared in buffer, the regenerating coenzyme system includes: enzyme solution, the formic acid containing FDH and LDH Ammonium, NAD⁺And phosphopyridoxal pyridoxal phosphate.

2. regenerating coenzyme system according to claim 1, which is characterized in that the regenerating coenzyme system further includes NADH。

3. regenerating coenzyme system according to claim 1, which is characterized in that with Candida total DNA in the step (a) For template, PCR amplification is carried out with upstream and downstream primer respectively, to obtain the FDH gene, in which:

Upstream primer is 5 '-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3 ', wherein be NdeI digestion position at scribing line Point；Downstream primer is 5 '-AAACTCGAGTGCGACCTTTTTGTCATTAC-3 ', wherein be XhoI restriction enzyme site at scribing line.

4. regenerating coenzyme system according to claim 1, which is characterized in that IPTG to final concentration of 0.02mmol/ is added L, 22 DEG C of inducing expression 16h, fermentation liquid after thallus broken wall centrifuging and taking supernatant up to the FDH/LDH co-express enzyme solution.