CN105132487A - Coenzyme regeneration system and establishment method thereof - Google Patents
Coenzyme regeneration system and establishment method thereof Download PDFInfo
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Abstract
The invention provides a coenzyme regeneration system which comprises formate dehydrogenase (FDH), leucine dehydrogenase (LDH) and ammonium formate. The invention further provides an establishment method of the coenzyme regeneration system. The establishment method mainly includes the steps of 1), heterogeneous expression of formate dhydrogenase and leucine dehydrogenase and 2), establishment of the coenzyme regeneration system. The coenzyme regeneration system is high in coenzyme regeneration efficiency and suitable for large-scale industrialized application, the establishment method is simple and convenient, and cost of coenzyme reaction is lowered greatly.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of regenerating coenzyme system and preparation method thereof.
Background technology
Compared to traditional chemical catalysis, biocatalysis because of the chemo-selective of the height of its enzymic catalytic reaction, the advantage such as regioselectivity and stereoselectivity and receive much concern and study.Wherein due to the high efficiency and specificity etc. of oxydo-reductase, they occupy considerable status in biosynthesizing.Oxydo-reductase optionally catalysis can contain carbonyl, aldehydes and ketone compounds.Oxydo-reductase, as catalyst preparing chipal compounds, can consume a certain amount of coenzyme while synthetic product.Such as: S-Leucine prepared by the trimethylammonium pyruvic acid such as Li Shuting, use leucine enzyme and hydrogenlyase coupling system in synthesis, hydrogenlyase can be cozymase regeneration, and the transformation efficiency of product can reach 82%.
Unlike other enzyme, in bioconversion reaction, the relation of auxiliary enzyme-to-substrate is stoichiometric relation.Coenzyme is a kind of consumptive compound, can reuse unlike oxydo-reductase, and in the reaction as the donor of hydrogen, reaction terminates rear coenzyme to be existed with oxidation states.And these coenzyme are often also expensive than the price of prepared product, stability is poor, is difficult to recycling, result in the high of reaction cost in suitability for industrialized production, thus limits the use of oxydo-reductase.In suitability for industrialized production, a large amount of coenzyme can not be added, so the structure of regenerating coenzyme system and the Reusability of coenzyme are the problems that instantly must solve efficiently.
Therefore, those skilled in the art is devoted to a kind of simple, efficient regenerating coenzyme technology of exploitation.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of regenerating coenzyme system is provided.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of regenerating coenzyme system, comprises hydrogenlyase (formatedehydrogenase, FDH), leucine dehydrogenase (leucinedehydrogenase, LDH) and ammonium formiate.
According to one embodiment of present invention, described regenerating coenzyme system also comprises NAD
+and/or NADH.
According to one embodiment of present invention, described regenerating coenzyme system also comprises pyridoxal phosphate.
According to one embodiment of present invention, the hydrogenlyase in described regenerating coenzyme system and leucine dehydrogenase are from engineering bacteria fermentation liquid, and described genetic engineering bacterium expresses hydrogenlyase and leucine dehydrogenase simultaneously.
According to one embodiment of present invention, described genetic engineering bacterium is E.coliBL21 (DE3) bacterial strain of simultaneously expressing hydrogenlyase and leucine dehydrogenase.
Another aspect of the present invention is to provide a kind of preparation method of regenerating coenzyme system, and the method comprises the following steps:
(1) heterogenous expression of hydrogenlyase and leucine dehydrogenase
Build the genetic engineering bacterium of simultaneously expressing FDH and LDH, must containing the enzyme liquid of FDH and LDH through fermentation;
(2) regenerating coenzyme system is prepared
In damping fluid, prepare regenerating coenzyme system, described regenerating coenzyme system comprises: described enzyme liquid, ammonium formiate, NAD containing FDH and LDH
+, and pyridoxal phosphate.
According to one embodiment of present invention, wherein comprise the following steps in step (1):
A () builds FDH carrier
FDH gene is connected into the pET28a carrier using endonuclease Nde I and Xho I double digestion, builds the plasmid pFDH-pET28a obtaining recombinant expressed FDH;
B () builds LDH carrier
LDH gene is connected into the pET21a carrier using endonuclease Xho I and Nde I double digestion, builds and obtain recombinant expression plasmid pLDH-pET21a;
C () builds the genetic engineering bacterium of simultaneously expressing FDH and LDH
Plasmid pFDH-pET28a and plasmid pLDH-pET21a corotation are entered E.coliBL21 (DE3), obtains the bacterial strain E.coliBL21-FDH/LDH of coexpression FDH and LDH.
According to one embodiment of present invention, the method is further comprising the steps of:
D () is fermented described bacterial strain E.coliBL21-FDH/LDH, the described enzyme liquid containing FDH and LDH of preparation
Coexpression bacterial strain E.coliBL21-FDH/LDH is cultivated in containing the LB liquid nutrient medium of Amp and Kan, adding IPTG to final concentration is 0.01 ~ 0.05mmol/L, 16 ~ 27 DEG C of abduction delivering 12 ~ 20h, after fermentation liquor thalline broken wall, namely centrifuging and taking supernatant obtains described FDH/LDH coexpression enzyme liquid.
According to one embodiment of present invention, wherein in step (a) with candiyeast STb gene for template, above downstream primer carries out pcr amplification respectively, thus obtains described FDH gene, wherein:
Upstream primer is 5 '-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3 ', and wherein, line place is NdeI restriction enzyme site; Downstream primer is 5 '-AAACTCGAGTGCGACCTTTTTGTCATTAC-3 ', and wherein, line place is XhoI restriction enzyme site.
According to one embodiment of present invention, wherein adding IPTG to final concentration is 0.02mmol/L, and inducing temperature is 22 DEG C, and the abduction delivering time is 16h, and after fermentation liquor thalline broken wall, namely centrifuging and taking supernatant obtains described FDH/LDH coexpression enzyme liquid.
Advantage of the present invention is: regenerating coenzyme system of the present invention, and regenerating coenzyme efficiency is high, and preparation method is simple and convenient, significantly reduces the cost of coenzyme reaction, is applicable to large-scale industrialization application.
Accompanying drawing explanation
Fig. 1 shows the schematic diagram 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, and swimming lane 1pLDH-pET21a recombinant plasmid digestion verification, swimming lane 2 enzyme cuts the LDH fragment of acquisition.
Embodiment
Following examples are detailed description of the invention.Should be appreciated that the following stated is only the preferred embodiments of the present invention, without departing from the spirit and scope of the present invention, the present invention is modified and is equal to replacement and all should contain within the scope of the present invention.
List the experimental raw that the present invention is used hereinafter.
Table 1 experimental plasmid
Table 2 experimental strain
The PCR primer that table 3 is used in testing
Table 4 experiment reagent
Substratum
LB liquid nutrient medium/gL
-1: peptone 10, yeast extract 5, sodium-chlor 10, pH7.0.
LB solid medium/gL
-1: peptone 10, yeast extract 5, sodium-chlor 10, agar strip 20, pH7.0.
Main solution and damping fluid
(1) plasmid extraction related solution:
Solution I: 0.2973g glucose, 0.091gTris, 0.112gEDTA are settled to 30mL.
Solution II (fresh configuration): 0.2mol/LNaOH, 1%SDS.
Solution III: 44.163g potassium acetate, 17.25mL Glacial acetic acid is settled to 150mL.
(2) 50 × TAE (electrophoretic buffer) (/L)
242gTris57.1mL acetic acid, 100mLEDTA (0.05mol/L).
Embodiment 1:PCR amplifying target genes
(1) pcr amplification primer
According to hydrogenlyase (FDH) threonine deaminase (TD) gene order (Genbanknumber:XM_001525495 and AAB18593) and context design primer thereof.Through screening verification, best hydrogenlyase (FDH) upstream primer is 5 '-AAA
cATATGaAAATCGTTCTCGTTTTGTACTCC-3 ' (line place is NdeI restriction enzyme site); Downstream primer is 5 '-AAA
cTCGAGtGCGACCTTTTTGTCATTAC-3 ' (line place is XhoI restriction enzyme site).Gene order (AAB18593) according to threonine deaminase designs upstream and downstream primer, and through screening verification, best upstream primer is 5 '-AAA
aAGCTTgTTAGCAGCCGGATCTCAG-3 ' (line place is HindIII restriction enzyme site); Downstream primer is 5 '-AAA
cATATGgTATTAAAACAAATTCTTC-3 ' (line place is NdeI restriction enzyme site).
With candiyeast and intestinal bacteria STb gene for template, above downstream primer amplification obtains FDH and TD gene fragment respectively.
(2) PCR reaction system
PrimeSTARHSDNA polysaccharase PCR reaction system is as shown in table 5.
Table 5PCR reaction system
(3) PCR response procedures
PCR response procedures: totally 30 circulations, FDH gene fragment size is about 1300bp, and TD gene fragment size is about 1500bp.
Step1:98 DEG C, 10s sex change
Step2:65 DEG C, 10s anneal
Step3:72 DEG C, 1.5min extend
Embodiment 2: the structure of recombinant plasmid
Plasmid construction flow process (is configured to example with FDH) as shown in Figure 1, with candiyeast STb gene for template, use the gene fragment that specificity restriction endonuclease NdeI and XhoI double digestion increase or the gene fragment be connected with carrier T, double digestion vector plasmid simultaneously, after purifying reclaims, 5h is connected under 16 DEG C of conditions, transform and connect product in competent cell E.coliDH5 α, picking transforms the single bacterium colony on LB solid plate, receive in 5mLLB liquid nutrient medium and (add corresponding microbiotic according to the resistance of plasmid), under 37 DEG C of conditions, in 220r/min shaking table, shaking culture is spent the night.Extracting plasmid, picking plasmid carries out double digestion checking, after checking is correct, the plasmid pFDH-pET28a built is carried out the mensuration of DNA sequence dna.
(1) structure of threonine deaminase expression plasmid
With intestinal bacteria STb gene for template, above downstream primer carries out pcr amplification respectively, and shown in Fig. 2, obtain a specific DNA band, average molecular size is about 1.5kb, conforms to theoretical value.The plasmid pTD-pET28a built is used the checking of HindIII and NdeI double digestion, electrophoresis digestion products, shown in Fig. 2, band conforms to expection, occurs band respectively at 5.4kb and 1.5kb place, and all stripe size meet expection, and expression plasmid builds correct.The plasmid pTD-pET28a built is carried out DNA sequence dna comparison, and after order-checking, comparison is without sudden change.Be transformed into E.coliBL21 (DE3), obtain TD expression strain E.coliBL21-TD, carry out follow-up protein expression.
(2) structure of hydrogenlyase expression plasmid
With candiyeast STb gene for template, above downstream primer carries out pcr amplification respectively, and shown in Fig. 3, obtain a specific DNA band, average molecular size is about 1.3kb, conforms to theoretical value.The plasmid pFDH-pET28a built is used the checking of NdeI and XhoI double digestion, electrophoresis digestion products, shown in Fig. 3, band conforms to expection, occurs band respectively at 5.4kb and 1.3kb place, and expression plasmid builds correct.After order-checking, comparison is without sudden change.The plasmid built can be transformed into E.coliBL21, carries out follow-up protein expression.
(3) structure of leucine dehydrogenase expression plasmid
According to the gene order (Genbanknumber:WP_016086354) of leucine dehydrogenase (LDH), entrust Ying Weijie base biotech firm synthetic gene sequence fragment.Utilize specificity restriction endonuclease NdeI and XhoI double digestion to be connected with the carrier T of LDH fragment, shown in Fig. 4, obtain 3 DNA bands, average molecular size is respectively 2.5kb, 1.1kb, 0.25kb, wherein 2.5kb is the linear strip of carrier T, and 1.1kb is the gene fragment of LDH, and size conforms to theoretical value.The plasmid pLDH-pET21a built is used the checking of NdeI and XhoI double digestion, and shown in Fig. 4, electrophoresis digestion products, band conforms to expection, occurs band respectively at 5.4kb and 1.1kb place, and expression plasmid builds correct.The plasmid pLDH-pET28a built is carried out DNA sequence dna comparison, and after order-checking, comparison is without sudden change.Be transformed into E.coliBL21 (DE3), obtain LDH expression strain E.coliBL21-LDH, carry out follow-up protein expression.
Embodiment 3: the structure of coexpression recombinant bacterial strain
PFDH-pET28a and pLDH-pET21a corotation is dissolved E.coliBL21 (DE3), and coat on LB (each 100 μ g/ml of Amp and Kan) solid plate, single bacterium colony that LB (each 100 μ g/ml of Amp and Kan) solid plate grows, be exactly the bacterial strain simultaneously containing hydrogenlyase and leucine dehydrogenase gene, engineering strain called after E.coliBL21-FDH/LDH will be obtained.This bacterial strain can express hydrogenlyase and leucine dehydrogenase simultaneously.
Embodiment 4: the preparation of crude enzyme liquid
Picking with single colony inoculation of the engineering bacteria of recombinant plasmid in 5mLLB nutrient solution, at 37 DEG C, incubated overnight under 220r/min condition.Respectively with 37 DEG C of cultivations in inoculum size access 100mLLB liquid nutrient medium (adding corresponding microbiotic according to the resistance of plasmid) of 5 ‰.Work as OD
600value reaches 0.6 ~ 0.8, under IPTG optimum induction final concentration and inducing temperature, induce 16h.Fermented liquid after cultivation is abandoning supernatant after 3700r/min frozen centrifugation 15min, by the Na of bacterium mud with appropriate precooling
2hPO
4recentrifuge after (0.1mol/L, pH=8.0) buffer solution.Finally use 20mLNa
2hPO
4the resuspended rear ultrasonication of (0.1mol/L, pH=8.0) damping fluid, ultrasonic power 360W, 10s+20s, 20min, shattering process bacterium liquid remains in ice-water bath.Cytoclasis liquid is through 3700r/min, and 4 DEG C of centrifugal 15min, gained supernatant liquor is crude enzyme liquid, can carry out enzyme activity determination, SDS-PAGE electrophoresis detection and conversion reaction.
Embodiment 5: taking temperature of recombinase protein reaches
The recombinant plasmid of structure is transferred in the competent cell of intestinal bacteria E.coilBL21 by heat shock method, thus obtain can the engineering bacteria of overexpression exogenous enzyme.Picking is inoculated in 3mLLB nutrient solution with the engineering bacteria of recombinant plasmid, incubated overnight under 37 DEG C of conditions.
Getting 1mL bacterium liquid for making negative control, separately getting 1mL and giving over to inoculation use, add IPTG (1mmol/L) induction in remaining 1mL bacterium liquid, express 3h at 37 DEG C.Bacterium liquid after negative control and induction is carried out SDS-PAGE electrophoresis detection.
Select E.coliBL21-FDH, single bacterium colony of E.coliBL21-TD and E.coliBL21-FDH/LDH, incubated overnight under 37 DEG C of conditions, be 1mmol/L at induced concentration, inducing temperature is abduction delivering 3h under the condition of 37 DEG C, and the bacterium liquid after induction is carried out SDS-PAGE electrophoresis detection, result shows, obvious band of expression is had respectively at 47kDa, 500kDa, 42kDa place.Before induction, target protein only has the background of trace to express, and after induction, the expression amount showed increased of soluble proteins, illustrates that target protein can be good at expressing in recombinant bacterium.
Embodiment 6: the optimization of recombinase protein expression condition
The expression of recombinase protein in intestinal bacteria exists in two forms: soluble proteins and inclusion body.And inclusion body protein does not have enzymatic activity, soluble proteins is only had to have enzymatic activity.It is high that carrier pET28a due to selection has expression amount, exists, but also have considerable target protein to exist with soluble proteins even if a large amount of target protein is assembled with inclusion bodies.Affect the solubility expression of albumen because have: induction time, the concentration of IPTG inductor and inducing temperature.For optimizing the condition of protein expression, investigate not isogeneous induction duration respectively, under different IP TG induces final concentration and different inducing temperatures, the solubility expression of E.coliBL21-FDH and E.coliBL21-GDH.
(1) optimization of duration is induced
Generally, induction time is longer, and the expression amount of target protein is higher, until reach maximum sometime, later along with the prolongation of induction time, the expression amount of albumen is tending towards stopping.But induction time is oversize, foreign protein is also expressed more, affects the activity of target protein.Investigate induction duration herein and be respectively 4h, the solubility expression amount of albumen when 8h, 12h, 16h and 20h.
Picking with single colony inoculation of the engineering bacteria of recombinant plasmid in 5mLLB nutrient solution, at 37 DEG C, incubated overnight under 220r/min condition.Respectively with 37 DEG C of cultivations in inoculum size access 30mLLB liquid nutrient medium (containing Kan100 μ g/ml) of 5 ‰.Work as OD
600when value reaches 0.6 ~ 0.8, under optimum IPTG induces final concentration and inducing temperature, abduction delivering (4h, 8h, 12h, 16h and 20h), collected by centrifugation thalline, carries out the solubility expression of SDS-PAGE electrophoresis detection albumen, determines optimum induction duration after process thalline.
Inducible strain E.coliBL21-TD under 16 DEG C of conditions, be respectively 4h to induction duration, when 8h, 12h, 16h and 20h, the solubility expression amount of albumen is investigated, and result shows, before adding inductor, target protein has a small amount of this locality to express.Along with the increase of induction duration, the expression amount of target protein increases thereupon, until 16h expressing quantity is tending towards a maximum value, the expression amount of albumen is tending towards stopping subsequently.Therefore determine that the best induction time length after adding IPTG is 16h.
Inducible strain E.coliBL21-FDH under 22 DEG C of conditions, be respectively 4h to induction duration, when 8h, 12h, 16h and 20h, the solubility expression amount of albumen is investigated, and result shows, before adding inductor, target protein has a small amount of this locality to express.Along with the increase of induction duration, the expression amount of target protein increases thereupon, until 16h expressing quantity is tending towards a maximum value, the expression amount of albumen is tending towards stopping subsequently.Therefore determine that the induction of the optimum after adding IPTG duration is 16h.
Inducible strain E.coliBL21-LDH under 22 DEG C of conditions, be respectively 4h to induction duration, when 8h, 12h, 16h and 20h, the solubility expression amount of albumen is investigated, and result shows, before adding inductor, target protein only has a small amount of local expression.Along with the increase of induction duration, the expression amount of target protein increases thereupon, until 16h expressing quantity is tending towards a maximum value, the expression amount of albumen is tending towards stopping subsequently.Therefore determine that the best induction time after adding IPTG is 16h.
(2) optimization of IPTG induced concentration
In DE3 Host Strains, can change the level of protein expression by changing the concentration of IPTG, expressing can from very low horizontal adjustment to very high level, until the level that pET carrier is induced completely.Therefore investigated the solubility expression amount of albumen when protein induced final concentration is respectively 0.01 ﹑ 0.02 ﹑ 0.05 ﹑ 0.1 ﹑ 0.2mmol/L and 0.5mmol/L herein.
Work as OD
600value reaches 0.6 ~ 0.8, investigate the induction final concentration (0.01 ﹑ 0.02 ﹑ 0.05 ﹑ 0.1 ﹑ 0.2mmol/L and 0.5mmol/L) of 6 kinds of different IP TG, under optimum inducing temperature after abduction delivering 16h, collected by centrifugation thalline, carry out the solubility expression of SDS-PAGE electrophoresis detection albumen after process thalline, determine optimum IPTG induced concentration.
With do not add compared with inductor, after IPTG adds, TD (50kDa) has obvious band of expression, conforms to expection size.But in the scope that induced concentration is 0.02-0.5mmol/L, the expression amount of albumen is roughly the same, therefore the final concentration selecting IPTG is the abduction delivering that 0.02mmol/L carries out albumen.
With do not add compared with inductor, after IPTG adds, FDH (47kDa) has obvious band of expression, conforms to expection size.When IPTG final concentration reaches 0.02mmol/L, during 0.2mmol/L and 0.5mmol/L, target protein all has a large amount of expression.When IPTG final concentration reaches 0.01,0.05mmol/L and 0.1mmol/L, target protein also has the expression of slight amount.Result shows: just can express by inducible protein under IPTG lower concentration, therefore the final concentration selecting IPTG is the abduction delivering that 0.02mmol/L carries out albumen.
With do not add compared with inductor, after IPTG adds, LDH (42kDa) has obvious band of expression, conforms to expection size.When IPTG final concentration reaches 0.01,0.02,0.2mmol/L and 0.5mmol/L, target protein all has a large amount of expression.When IPTG final concentration reaches 0.05mmol/L and 0.1mmol/L, target protein also has the expression of slight amount.Therefore the final concentration selecting IPTG is the abduction delivering that 0.01mmol/L carries out albumen.
(3) optimization of inducing temperature
The expression amount of culture temperature to target protein of bacterium has material impact.The inducing temperature having investigated albumen is herein respectively 16 DEG C, 22 DEG C, 28 DEG C, 32 DEG C and 37 DEG C time albumen solubility expression amount.
Work as OD
600value reaches 0.6 ~ 0.8, final concentration is induced at optimum IPTG, investigate 5 differing tempss (16 DEG C, 22 DEG C, 28 DEG C, 32 DEG C and 37 DEG C) under induce 16h after, collected by centrifugation thalline, carry out the solubility expression of SDS-PAGE electrophoresis detection albumen after process thalline, determine optimum inducing temperature.
In the expression of E.coliBL21-TD, at 16 DEG C, 22 DEG C, 28 DEG C, at 32 DEG C and 37 DEG C of these 5 temperature, the solubility expression of proteolytic enzyme is all higher, there is no the difference of significance, but under the inducing temperature of 22 DEG C, the solubility expression of albumen compares to other temperature and expresses higher, consider economic and high soluble proteins to express, therefore select inducing temperature to be the abduction delivering that 16-28 DEG C (being preferably 22 DEG C) carries out albumen.
In the expression of E.coliBL21-FDH, at 16 DEG C, 22 DEG C, 28 DEG C, at 32 DEG C and 37 DEG C of these 5 temperature, when inducing temperature is 16 DEG C, 22 DEG C, under 28 DEG C of these 3 inducing temperatures, the solubility expression of proteolytic enzyme is all higher, there is no the difference of significance, but under 28 DEG C of conditions, to compare to 16 DEG C and 22 DEG C relative to inclusion body few for soluble proteins amount.When inducing temperature raise be 32 DEG C and above temperature time, solubility expression amount significantly reduces, and most of target protein is all present in precipitation with the form of inclusion body, although this may be because high temperature is conducive to the growth and breeding of bacterium, expressing quantity is also high, but protein expression is too much too fast just defines inclusion body.Consider economic and high soluble proteins to express, therefore select inducing temperature to be 22 DEG C of abduction deliverings carrying out albumen.
In the expression of E.coliBL21-LDH, at 16 DEG C, 22 DEG C, 28 DEG C, at 32 DEG C and 37 DEG C of these 5 temperature, when inducing temperature is 16 DEG C, 22 DEG C, under 28 DEG C of these 3 inducing temperatures, the solubility expression of proteolytic enzyme is all higher, but to compare to 16 DEG C and 22 DEG C relative to inclusion body few for soluble proteins amount under 28 DEG C of conditions.When inducing temperature raise be 32 DEG C and above temperature time, solubility expression amount significantly reduces, and most of target protein is all present in precipitation with the form of inclusion body, although this may be because high temperature is conducive to the growth and breeding of bacterium, expressing quantity is also high, but protein expression is too much too fast just defines inclusion body.Consider economic and high soluble proteins to express, therefore select inducing temperature to be 22 DEG C of abduction deliverings carrying out albumen.
(4) the albumen one piece optimization of coexpression recombinant bacterial strain
The optimum protein expression condition of recombinase E.coliBL21-FDH is: optimum inducing temperature is 22 DEG C, and optimum induced concentration is 0.02mmol/L, and optimum induction duration is 16h.
Associated proteins SDS-PAGE schemes known, because the induction phase of recombinase E.coliBL21-LDH to higher concentration is: 0.01,0.02,0.2mmol/L and 0.5mmol/L, and the induction phase of recombinase E.coliBL21-FDH to higher concentration is: 0.02mmol/L, 0.2mmol/L and 0.5mmol/L, therefore determine that the optimum induced concentration of coexpression bacterial strain E.coliBL21-FDH/LDH is 0.02mmol/L.Therefore finally determine that the optimum protein expression condition of coexpression bacterial strain E.coliBL21-FDH/LDH is: best inducing temperature is 22 DEG C, and best induced concentration is 0.02mmol/L, and best induction duration is 16h.
Induce in optimal conditions, carry out SDS-PAGE electrophoresis, the expression of induction FDH and LDH, has obvious band of expression in 47kDa and 42kDa position respectively, conforms to expection size.Recombinant bacterium E.coil thalline after induction is centrifugal after ultrasonication process, FDH and the LDH major part of E.coil coexpression is present in supernatant liquor, is the albumen of solubility.
Embodiment 7: the mensuration that enzyme is lived
(1) mensuration that hydrogenlyase enzyme is alive
The enzyme using microplate reader to measure hydrogenlyase is herein lived.An enzyme activity unit (U) is defined as under condition determination, and per minute produces the enzyme amount needed for 1 μm of ol product.
The enzyme taking ammonium formiate as substrate survey FDH is alive, and total reaction volume is 1mL, and reaction solution consists of: Na
2hPO
41.67mmol/LNAD is added in (0.1mol/L, pH=8.0) damping fluid
+, 167mmol/LHCOONH
4.Reaction mixture, in 30 DEG C of insulations, starts timing after adding a certain amount of FDH crude enzyme liquid, detects NADH in the increasing amount of 340nm absorbancy every 60s, according to its molar extinction coefficient ε=6220L/ (mol at 340nm
-1cm
-1) calculate hydrogenlyase enzyme live.Oxidizing reaction is as follows:
The method of calculation of enzyme activity are:
In formula, △ A represents the change of 340nm place absorbancy in 1min, Vt represents the volume (ml) of reaction solution, Vs represents the volume (ml) of sample, and d represents colorimetric pool optical path (0.625cm), and 6220 represent molar absorption coefficient (L/mol
-1cm
-1), c represents the concentration (mg/ml) of protein.
With HCOONH
4for substrate, the enzyme that mensuration FDH breaks in bacterium liquid supernatant is lived, and result shows, the activity that FDH breaks bacterium liquid supernatant catalysis generation NADH is about 10U/ml.According to the total protein concentration that Bradford method is measured, the ratio enzyme calculating the crude enzyme liquid of FDH is lived as 13.51U/mg.
(2) mensuration of threonine deaminase and the work of leucine dehydrogenase enzyme
The enzyme taking Threonine as substrate survey threonine deaminase is alive, and total reaction volume is 1mL, adds 0.1g Threonine respectively, 0.03mgPLP, a certain amount of crude enzyme liquid and Na
2hPO
4(0.1mol/LpH=8.0) damping fluid, at 30 DEG C, reacts 2h in 150r/min shaking table, adds isopyknic acetonitrile, then dilute with water 500 times, 12000r/min, with HPLC assay products growing amount after centrifugal 5min, calculates enzyme and lives.
The enzyme surveying leucine dehydrogenase in linked reaction with carbonyl butyric acid for substrate is respectively lived, and total reaction volume is 1mL, adds 400 μm of ol carbonyl butyric acid respectively, 0.2mgNAD
+, 65mgHCOONH
4, a certain amount of crude enzyme liquid and Na
2hPO
4(0.1mol/L, pH=8.0) damping fluid.Reaction mixture, at 30 DEG C, reacts 2h in the shaking table of 150r/min, adds isopyknic acetonitrile, and redilution 5 doubly derives, centrifugal rear HPLC assay products growing amount, calculates enzyme and lives.
An enzyme activity unit (U) is defined as under condition determination, and per minute produces the enzyme amount needed for 1 μm of ol product.
Be that the enzyme that substrate surveys TD is lived with Threonine, result shows, it is 6.67U/ml that TD transforms the ability that Threonine generates carbonyl butyric acid, and the ratio enzyme calculating the crude enzyme liquid of TD is lived as 4.156U/mg.Live with the enzyme that carbonyl butyric acid surveys LDH in E.coliBL21-FDH/LDH linked reaction for substrate, result shows, the ability that LDH transforms carbonyl butyric acid generation L-aminobutyric acid is 7.38U/ml, and the ratio enzyme calculating the crude enzyme liquid of LDH is lived as 6.43U/mg.
Embodiment 8: the mensuration of crude enzyme liquid protein content
Adopt Bradford method to measure the concentration of protein, measure the absorbancy of known bovine serum albumen solution, obtain the typical curve of absorbancy and protein concn.Testing sample is mixed with Bradford solution, at room temperature standing and reacting 5min.Use 96 orifice plates, in the parallel placement of each mixed solution 3 holes, measure the absorbancy of solution under 595nm wavelength subsequently, using the mean value of the absorbancy in these 3 holes as the absorbancy of testing sample.The concentration of protein is calculated according to typical curve.
The absorbancy adopting Bradford method to measure and the typical curve of protein concn.
(1) determination of the protein content of threonine deaminase crude enzyme liquid
The mean value of the absorbancy of the sample recorded is A=0.643.According to typical curve y=4.8618x+0.3310 (R2=0.9906), calculating protein content is 0.0642mg/ml, is 1.605mg/ml according to the protein content that the weaker concn of albumen is calculated.
(2) determination of the protein content of hydrogenlyase crude enzyme liquid
The mean value of the absorbancy of the sample recorded is A=0.475, and calculating protein content is 0.0296mg/ml, is 0.74mg/ml according to the protein content that the weaker concn of albumen is calculated.
(3) mensuration of the protein content of leucine dehydrogenase and hydrogenlyase coexpression
The mean value of the absorbancy of the sample recorded is A=0.554, and calculating protein content is 0.0459mg/ml, is 1.148mg/ml according to the bak protein content that the weaker concn of albumen is calculated.
Embodiment 9: the mensuration of product assay
Use DNF derivatization method to measure L-Gamma-propalanine content, product formation is detected by HPLC.The chromatographic condition generating L-aminobutyric acid is: chromatographic column is C18 post (Φ 4.6mm × 250mm, 5 μm); Column temperature is 30 DEG C; Mobile phase A: Na
2hPO
4(0.02mol/L, pH=7.2) damping fluid, B: acetonitrile, isocratic elution, V (A): V (B)=70: 30; Flow velocity is 1.0ml/min; 360nm ultraviolet detection.Use standard substance, the quantitative assay of production standard curve.The liquid-phase condition generating carbonyl butyric acid is: column temperature is 25 DEG C; Mobile phase A: NaH
2pO
4(0.02mol/L, pH=3.0) damping fluid, B: acetonitrile, isocratic elution, V (A): V (B)=90: 10; Flow velocity is 1.0mL/min; 205nm ultraviolet detection.
Embodiment 10: L-aminobutyric acid is prepared in coexpression system catalysis
Use TD, FDH and LDH coexpression enzyme liquid at same system, catalytic substrate Threonine generates L-aminobutyric acid.Fermentation culture E.coliBL21-TD or E.coliBL21-FDH/LDH in 100mLLB (containing each 100 μ g/ml of Amp and Kan), and prepare crude enzyme liquid, for the synthesis of L-aminobutyric acid.
2.1g Threonine is added successively, 3.0gHCOONH in 250mL triangular flask
4, 19.2mLNa
2hPO
4(0.1mol/L) damping fluid, then add 4.8mLTD crude enzyme liquid, 6mLFDH and LDH coexpression crude enzyme liquid, 10mgNAD
+with 3.215mg pyridoxal phosphate (PLP).Be placed in thermostat container, magnetic agitation, automatical potentiometric titrimeter detects pH value, with the titration of 0.2mol/LNaOH solution.Reaction, to 20h, measures product formation with HPLC.
Prepared in the conversion reaction of L-aminobutyric acid by L-threonine, through great many of experiments, the optimum condition of acquisition is: temperature is 30 DEG C; PH is 8.0; NAD
+starting point concentration be 0.25mg/ml; Adopt the mode of batch charging, maximum substrate charging capacity is 70g/L; Enzyme dosage proportioning is, in the reaction system of 10mL, the add-on of threonine deaminase is 8mL thalline, and the add-on of coexpression hydrogenlyase and leucine dehydrogenase is 10mL thalline; End reaction duration is 16h.Transform preparation L-aminobutyric acid.Charging capacity is when 70g/L, and productive rate can reach 95%.
Sum up
Recombinant protein enzyme E.coliBL21-FDH/LDH is carried out to the optimization of expression condition, obtaining best inducing temperature is 22 DEG C, and best induced concentration is 0.02mmol/L, and best induction duration is 16h.Under optimum condition of the expression, the enzyme recording LDH in this linked reaction is lived as 7.38U/ml.
Prepared in the conversion reaction of L-aminobutyric acid by L-threonine, optimum condition is: temperature is 30 DEG C; PH is 8.0; NAD
+starting point concentration be 0.25mg/ml; Substrate charging capacity is 70g/L; Enzyme dosage proportioning is, in the reaction system of 10mL, the add-on of threonine deaminase is 8mL thalline, and the add-on of coexpression hydrogenlyase and leucine dehydrogenase is 10mL thalline; End reaction duration is 16h, and productive rate can reach 95%.
Regenerating coenzyme system of the present invention, regenerating coenzyme efficiency is high, and preparation method is simple and convenient, significantly reduces the cost of coenzyme reaction, is applicable to large-scale industrialization application.
While specific embodiments of the invention are described in detail, the embodiment that can also find plurality of replaceable design similar in this area related to the present invention and be defined by the claims.
Claims (10)
1. a regenerating coenzyme system, is characterized in that, described regenerating coenzyme system comprises hydrogenlyase (formatedehydrogenase, FDH), leucine dehydrogenase (leucinedehydrogenase, LDH) and ammonium formiate.
2. regenerating coenzyme system according to claim 1, is characterized in that, described regenerating coenzyme system also comprises NAD
+and/or NADH.
3. regenerating coenzyme system according to claim 1, is characterized in that, described regenerating coenzyme system also comprises pyridoxal phosphate.
4. regenerating coenzyme system according to claim 1, it is characterized in that, hydrogenlyase in described regenerating coenzyme system and leucine dehydrogenase are from engineering bacteria fermentation liquid, and described genetic engineering bacterium expresses hydrogenlyase and leucine dehydrogenase simultaneously.
5. regenerating coenzyme system according to claim 1, is characterized in that, described genetic engineering bacterium is E.coliBL21 (DE3) bacterial strain of simultaneously expressing hydrogenlyase and leucine dehydrogenase.
6. a preparation method for the regenerating coenzyme system described in claim 1-5, wherein, said method comprising the steps of:
(1) heterogenous expression of hydrogenlyase and leucine dehydrogenase
Build the genetic engineering bacterium of simultaneously expressing FDH and LDH, must containing the enzyme liquid of FDH and LDH through fermentation;
(2) regenerating coenzyme system is prepared
In damping fluid, prepare regenerating coenzyme system, described regenerating coenzyme system comprises: described enzyme liquid, ammonium formiate, NAD containing FDH and LDH
+, and pyridoxal phosphate.
7. method according to claim 6, is characterized in that, described step comprises the following steps in (1):
A () builds FDH carrier
FDH gene is connected into the pET28a carrier using endonuclease Nde I and Xho I double digestion, builds the plasmid pFDH-pET28a obtaining recombinant expressed FDH;
B () builds LDH carrier
LDH gene is connected into the pET21a carrier using endonuclease Xho I and Nde I double digestion, builds and obtain recombinant expression plasmid pLDH-pET21a;
C () builds the genetic engineering bacterium of simultaneously expressing FDH and LDH
Plasmid pFDH-pET28a and plasmid pLDH-pET21a corotation are entered E.coliBL21 (DE3), obtains the bacterial strain E.coliBL21-FDH/LDH of coexpression FDH and LDH.
8. method according to claim 7, is characterized in that, described method is further comprising the steps of:
D () is fermented described bacterial strain E.coliBL21-FDH/LDH, the described enzyme liquid containing FDH and LDH of preparation
Coexpression bacterial strain E.coliBL21-FDH/LDH is cultivated in containing the LB liquid nutrient medium of Amp and Kan, adding IPTG to final concentration is 0.01 ~ 0.05mmol/L, 16 ~ 27 DEG C of abduction delivering 12 ~ 20h, after fermentation liquor thalline broken wall, namely centrifuging and taking supernatant obtains described FDH/LDH coexpression enzyme liquid.
9. method according to claim 7, is characterized in that, in described step (a) with candiyeast STb gene for template, above downstream primer carries out pcr amplification respectively, thus obtains described FDH gene, wherein:
Upstream primer is 5 '-AAA
cATATGaAAATCGTTCTCGTTTTGTACTCC-3 ', wherein, line place is NdeI restriction enzyme site; Downstream primer is 5 '-AAA
cTCGAGtGCGACCTTTTTGTCATTAC-3 ', wherein, line place is XhoI restriction enzyme site.
10. method according to claim 8, is characterized in that, adding IPTG to final concentration is 0.02mmol/L, 22 DEG C of abduction delivering 16h, and after fermentation liquor thalline broken wall, namely centrifuging and taking supernatant obtains described FDH/LDH coexpression enzyme liquid.
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| CN108103038B (en) * | 2017-12-15 | 2021-03-02 | 江南大学 | Single-cell factory for synthesizing L-phenylglycine and construction and application thereof |
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