CN110643556A

CN110643556A - Recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof

Info

Publication number: CN110643556A
Application number: CN201910784330.7A
Authority: CN
Inventors: 应向贤; 乔艳; 汪钊
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2020-01-03

Abstract

The invention discloses a recombinant gene engineering bacterium for co-expressing olefine aldehyde reductase and glucose dehydrogenase and application thereof in catalyzing isopentenol synthesized by isopentenyl aldehyde. The method has the advantages of high regioselectivity and high activity, 500mM substrate of the isopentenyl aldehyde is completely converted into the product of the isopentenyl alcohol within 3.5h, and no byproduct of saturated alcohol is detected in the reaction process, so that the method is shown to efficiently and specifically catalyze the hydrogenation of C ═ O of the isopentenyl aldehyde to obtain the corresponding isopentenyl alcohol. Meanwhile, the recombinant cell induces and produces D-glucose dehydrogenase by taking glucose as the raw materialThe cosubstrate, glucose dehydrogenase, can continuously convert NADP⁺Converted into NADPH, and no additional coenzyme is needed in the reaction process, so that the production cost is greatly reduced, and the method is more suitable for large-scale industrial production.

Description

Recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof

(I) technical field

The invention relates to a recombinant cell for co-expressing enal reductase and glucose dehydrogenase and application thereof in synthesis of prenol.

(II) background of the invention

Alpha, beta-unsaturated alcohols are very important organic synthesis intermediates, and have wide applications in the production of fragrances, pharmaceuticals and other fine chemicals. Isopentenol is one of the important alpha, beta-unsaturated alcohols, its chemical name is 3-methyl-2-buten-1-ol, its relative molecular weight is 86.13, and its density is 0.848g cm^-3Boiling point 140 deg.C, flash point 43 deg.C, colorless transparent liquid, and water solubility of 170 g.L^-1(20 ℃ C.). Prenols are useful in: (1) synthesizing the intermediate methyl cardiate of the pyrethroid insecticide of the high-efficiency low-toxicity pesticide and downstream products thereof; (2) intermediates in the synthesis of flavors and fragrances (e.g., citral); (3) the thio-geraniol can be widely applied to the flavor blending of various daily necessities.

According to different synthesis processes, the raw materials for synthesizing the isopentenol can respectively comprise isoprene, isobutene, acetone or isopentenal, and the like, wherein the synthesis of the isopentenol by reducing the isopentenal is the most direct synthesis method. However, thermodynamically, C ═ C bonds have lower activation energy than C ═ O bonds, and kinetically, C ═ C bonds are more active than C ═ O bonds, and under the action of general chemical catalysts, the main reduction products of α, β -unsaturated alkenals (ketones) are mostly saturated aldehydes (ketones), and the yield of the more valuable products α, β -unsaturated enols is lower. Unlike chemical catalysts, biocatalysts have excellent regioselectivity and specifically reduce the C ═ O bond of α, β -unsaturated enals (ketones) to give the corresponding α, β -unsaturated enols. In addition, the biological method also has the advantages of mild reaction conditions, environmental friendliness, high reaction efficiency and the like. The key enzyme for preparing the prenol by reducing the prenylaldehyde by a biocatalytic method is an enal reductase (also called enol dehydrogenase). At present, olefine aldehyde reductase and application thereof in alpha, beta-unsaturated enol synthesis are rarely reported. Ying et al used wild bacteria joke's bacteria as biocatalyst to catalytically reduce 50mM of substrates such as crotonaldehyde, 2-hexenal, 2-methyl-2-pentenal, citral and cinnamaldehyde with a conversion rate of 9.45-98.4% (Ying X, WangY, Xiong et al. chromatography of an inductive/benzoyl alcohol dehydrogenase type sp. strain WZY002, an organic porous catalyst for the synthesis of alpha, beta-unsaturated alcohols from alkyl aldehydes and ketones). In addition, patent literature reports that crotonaldehyde and nerol/geraniol are produced by simultaneously catalyzing oxidation of crotyl alcohol (178mM) and reduction of neral (25 mM)/geranial (25mM) using recombinant E.coli expressing an enol dehydrogenase as a biocatalyst; wherein, the yields of nerol and geraniol are respectively 48.5 percent and 48.6 percent, while the yield of crotonaldehyde is only 12.7 percent (Zhang Xiong, Wangzhao, Wang Yifang, etc., an enol dehydrogenase, a coding gene, a vector, an engineering bacterium and application thereof; patent No. ZL 201310578047.1).

At present, no report on the construction of recombinant escherichia coli for co-expressing enal reductase and glucose dehydrogenase is found, and no report on the synthesis of prenol by coupling enal reductase and glucose dehydrogenase to catalyze prenylaldehyde reduction by using the recombinant cell is found.

Disclosure of the invention

The invention aims to provide a recombinant gene engineering bacterium for co-expressing olefine aldehyde reductase and glucose dehydrogenase and a method for catalyzing isoamylene alcohol synthesized from isopentene aldehyde.

The technical scheme adopted by the invention is as follows:

the invention provides a recombinant gene engineering bacterium for co-expressing enal reductase and D-glucose dehydrogenase, which is obtained by introducing enal reductase genes and D-glucose dehydrogenase genes into host bacteria together.

The olefine aldehyde reductase is derived from joker bacteria (Yokenella sp.) WZY002, the amino acid sequence of the olefine aldehyde reductase is shown as SEQ ID No.1, the nucleotide sequence of the coding gene is shown as SEQ ID No.2, and the olefine aldehyde reductase comprises a complementary sequence of the nucleotide sequence shown as SEQ ID No. 2.

The D-glucose dehydrogenase is derived from microbacterium, the amino acid sequence of the D-glucose dehydrogenase is shown as SEQ ID No.3, the nucleotide sequence of the coding gene is shown as SEQ ID No.4, and the D-glucose dehydrogenase comprises a complementary sequence of the nucleotide sequence shown as SEQ ID No. 4.

The recombinant gene engineering bacteria are constructed according to the following method: inserting an enal reductase gene into Nco I and Hind III restriction sites of a first multiple cloning site of a pACYCDuet-1 vector, then inserting a D-glucose dehydrogenase gene into Nde I and Xho I restriction sites of a second multiple cloning site of the pACYCDuet-1 vector to obtain a recombinant vector containing an enal reductase gene and a D-glucose dehydrogenase gene, and introducing the recombinant vector into a host cell to obtain a recombinant gene engineering bacterium. The host cell is preferably e.coli BL21(DE 3).

The invention also provides an application of the recombinant gene engineering bacteria in preparation of prenol by catalyzing prenylaldehyde, and the application method comprises the following steps: the method comprises the steps of taking wet thallus freeze-dried powder obtained by induced culture of recombinant genetic engineering bacteria as a catalyst, taking the isopentenyl aldehyde as a substrate, taking glucose as an auxiliary substrate, taking a buffer solution with pH of 5.5-8.5 (preferably pH of 7.5 and 50mM Tris-HCl buffer solution) as a reaction medium to form a reaction system, carrying out catalytic reaction under the conditions of 20-55 ℃ (preferably 45 ℃) and 0-600rpm (preferably 300rpm), and after the reaction is completed, separating and purifying reaction liquid to obtain the isopentenol.

NADP may be added to the reaction system⁺And NADP in a continuous catalytic reaction System Using the glucose dehydrogenase⁺Conversion to NADPH, NADP⁺The final concentration was added at 0.1-0.8 mM.

In the reaction system, the final concentration of the catalyst is 10-80g/L (preferably 70g/L), the final concentration of the substrate is 50-500mM, and the ratio of the added substances of the substrate and the cosubstrate is 1: 0.5-3 (preferably 1: 2.5).

The catalyst is prepared by the following method: inoculating recombinant genetically engineered bacteria (preferably E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH) into LB liquid medium containing chloramphenicol at a final concentration of 50. mu.g/mL, culturing at 37 ℃ and 200rpm overnight, taking the culture and inoculating the culture at an inoculum size of 2% by volume into 150mL LB liquid medium containing kanamycin at 100. mu.g/mL and chloramphenicol at 50. mu.g/mL, and culturing at 37 ℃ and 200rpm until the OD cell concentration is reached₆₀₀Adding IPTG with final concentration of 0.1-0.5mM (preferably 0.4mM) into the culture, performing induced culture at 16-37 deg.C for 6-14h (preferably 22 deg.C for 12h), centrifuging, collecting wet thallus, and lyophilizing at-80 deg.C for 24h to obtain lyophilized powder。

The reaction liquid separation and purification method comprises the following steps: centrifuging the reaction solution at 12000rpm for 2min, collecting supernatant, adding 4 times of ethyl acetate, extracting at 200rpm and 30 deg.C for 1h, centrifuging at 12000rpm for 1min, and collecting upper organic phase; and adding anhydrous sodium sulfate into the organic phase to remove water, taking 100 mu L of supernatant for gas phase detection, and distilling the rest organic phase to remove ethyl acetate so as to obtain the prenol.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a recombinant genetic engineering bacterium for co-expressing olefine aldehyde reductase and D-glucose dehydrogenase, and an enzyme generated by the recombinant genetic engineering bacterium is used for efficiently catalyzing isopentolefine aldehyde to synthesize isopentenol. The method has the advantages of high regioselectivity and high activity, 500mM substrate of the isopentenyl aldehyde is completely converted into a product of the isopentenyl alcohol within 3.5h, and a byproduct of saturated alcohol is not detected in the reaction process, so that the method is indicated that the C ═ O hydrogenation of the isopentenyl aldehyde is efficiently and specifically catalyzed, and the corresponding isopentenyl alcohol is obtained. Meanwhile, the recombinant cell induces and cultures the D-glucose dehydrogenase, the glucose is taken as a cosubstrate, and the glucose dehydrogenase can continuously carry out NADP⁺Converted into NADPH, and no additional coenzyme is needed to be added in the reaction process, so that the production cost is greatly reduced, and the method is more suitable for large-scale industrial production.

(IV) description of the drawings

FIG. 1 is a schematic diagram of a two-enzyme catalytic process;

FIG. 2 is a schematic diagram of the constructed plasmid pACYCDuet 1-YsADH-EsGDH;

FIG. 3 is a standard curve for protein concentration by BCA;

FIG. 4 is a gel image of SDS-PAGE; from left to right, lane 1 corresponds to Blue plus II protein Maker, lanes 2-5 correspond to induced genetically engineered bacteria, and lane 6 corresponds to pre-induced genetically engineered bacteria;

FIG. 5 shows the effect of induction temperature on the enzyme activity of genetically engineered bacteria;

FIG. 6 shows the effect of the concentration of inducer IPTG on the enzyme activity of the genetically engineered bacteria;

FIG. 7 shows the effect of induction time on enzyme activity of genetically engineered bacteria;

FIG. 8 is a gas chromatograph of a catalytic reduction of 100mM of isopropenal;

FIG. 9 shows the optimum catalytic temperature for catalyzing an enal reduction reaction system;

FIG. 10 is the optimum catalytic pH for a catalytic enal reduction reaction system;

FIG. 11 shows the optimum coenzyme NADP for catalyzing the system of the enal reduction reaction⁺The amount of (c) added;

FIG. 12 shows the optimal concentration ratio of D-glucose as a co-substrate to isopropenylaldehyde as a substrate in a catalytic enal reduction reaction system;

FIG. 13 shows the optimum stirring speed of the catalytic enal reduction reaction system;

FIG. 14 shows the optimum amount of catalyst added for catalyzing the olefine aldehyde reduction reaction system;

FIG. 15 is a gas-mass spectrum (GC-MS) of the reduced product.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to the conventional experimental procedures in the field of molecular biology, such as those described in J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 acquisition of Gene encoding Oakkernella (Yokenella sp.) WZY002 enal reductase

The application of the disclosed enal reductase coding gene (with the GenBank accession number of KF887947) derived from Yokenella sp WZY002 is characterized in that the enal reductase coding gene is artificially synthesized (provided by Jinzhi biotechnology, Suzhou, providing a gene synthesis service), and the amino acid sequence and the nucleotide sequence are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 after codon optimization.

Example 2 acquisition of D-glucose dehydrogenase encoding Gene derived from Microbacterium

The D-glucose dehydrogenase encoding gene (with the GenBank accession number of ACB59697.1) derived from the micro-bacterium (Exiguobacterium sibiricum) is artificially synthesized (provided with gene synthesis service by Jinzhi biotechnology, Suzhou) after codon optimization by using the disclosed D-glucose dehydrogenase encoding gene (with the GenBank accession number of ACB59697.1) derived from the micro-bacterium, and the amino acid sequence and the nucleotide sequence are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

Example 3 construction of recombinant Gene engineering bacteria Co-expressing Enaldehyde reductase and D-glucose dehydrogenase

The enol reductase encoding gene (SEQ ID NO.2) and the D-glucose dehydrogenase encoding gene (SEQ ID NO.4) are inserted between two pairs of enzyme cutting sites of Nco I, Hind III, Nde I and Xho I on the pACYCDuet1 vector through one-step cloning to obtain the recombinant plasmid pACYCDuet-1-YsADH-EsGDH (figure 2). The recombinant plasmid pACYCDuet-1-YsADH-EsGDH is transferred into E.coli BL21(DE3) to obtain the genetically engineered bacterium E.coli BL21(DE 3)/pACYCDuet-1-YsADH-EsGDH.

The recombinant genetically engineered bacterium E.coliBL21(DE3)/pACYCDuet-1-YsADH-EsGDH is inoculated on LB solid culture medium containing 50 mu g/mL chloramphenicol for streak separation, a single colony is selected to be inoculated in 50mL LB liquid culture medium, the final concentration of 50 mu g/mL chloramphenicol is added, shaking culture is carried out at the constant temperature of 37 ℃ and 200rpm for 10h, and wet thalli are collected. The sequencing of plasmid extracted from the genetically engineered bacterium E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH shows that the coding gene of the double enzymes is inserted without errors. Composition of LB liquid medium: 10g/L of peptone, 5g/L of yeast extract, 5g/L of NaCl, 5g/L of NaCl and deionized water as a solvent, wherein the pH value is 7.0-7.5. LB solid medium composition: 15g of agar powder per liter was added to the LB liquid medium.

Preparing a crude enzyme solution: adding 50mM Tris-HCl buffer solution (pH 8.0) with the mass of 20 times that of the fresh wet thalli into the fresh wet thalli collected by centrifugation, stirring the mixture into bacterial suspension by using a glass rod, carrying out ultrasonic crushing for 10min under the condition of ice bath (0 ℃), carrying out ultrasonic working for 1s and intermittent working for 2s and carrying out ultrasonic power of 250W, and centrifuging the bacterial suspension subjected to ultrasonic crushing for 10min at 12000rpm and 4 ℃, wherein the obtained supernatant is the crude enzyme solution.

Example 4 determination of Enal reductase and glucose dehydrogenase Activity

The enzyme activity of the enal reductase is calculated by measuring the change of the absorbance value of NADPH at 340nm by a single-factor kinetic method of a spectrophotometer. The enzyme activity detection system is as follows: 50mM of isopropenal, 0.4mM of NADPH, 50. mu.L of the crude enzyme solution, and 50mM of Tris-HCl (pH 6.5) were added to make up to 1 mL. Definition of enzyme activity unit (U): the amount of enzyme required to oxidize 1. mu. mol NADPH per minute at 30 ℃. Three parallel experiments were performed each time, and the mean and standard error were calculated. The volume enzyme activity and specific activity calculation formula of the enal reductase (YsADH) is shown as formula 1 and formula 2:

(i. delta. A is the change in absorbance within 1min

V1 and V2 are respectively the total volume of the reaction solution and the volume of the added enzyme solution, mL;

③ 6220 is the molar extinction coefficient of NAD (P) H at 340nm,

l is the optical path distance and is 1 cm; t is reaction time, 1 min;

fifthly, mg is the protein mass unit in the reaction system.

Method for measuring NADP by adopting single-factor kinetic method of spectrophotometer for glucose dehydrogenase⁺The change in absorbance at 340nm was used to calculate the enzyme activity. The enzyme activity detection system is as follows: 50mM D-glucose, 0.4mM NADP ⁺50. mu.L of the crude enzyme solution was supplemented with Tris-HCl buffer (pH 6.5) to make up 1 mL. Definition of enzyme activity unit (U): the amount of enzyme required to produce 1. mu. mol NADPH per minute at 30 ℃. Three parallel experiments were performed each time, and the mean and standard error were calculated. The volume enzyme activity and specific activity of the glucose dehydrogenase are calculated by the formula 1 and the formula 2.

In addition, protein concentration was determined by BCA method. The preparation of the BCA reagent includes: reagent a, 1L: 10g of BCA (disodium 2, 2-biquinoline-4, 4-dicarboxylate, 1%), and 20g of Na were weighed out separately₂CO₃·H₂O(2％)，1.6g Na₂C₄H₄O₆·2H₂O(0.16％)，4g NaOH(0.4％)，9.5g NaHCO₃(0.95%) water was added to 1L and NaOH or solid NaHCO was used₃The pH was adjusted to 11.25. Reagent B, 50 mL: taking 2g of CuSO₄·5H₂O (4%), distilled water was added to 50 mL. ③ BCA reagent: 50 parts of reagent A and 1 part of reagent B are uniformly mixed. The reagent was stable for one week. Standard protein solution preparation: weighing 0.5g of bovine serum albumin, dissolving the bovine serum albumin in distilled water, and fixing the volume to 100mL to prepare a solution of 5 mg/mL; it is diluted ten times. During measurement, the BCA reagent is uniformly mixed with a proper amount of protein solution, then is subjected to heat preservation at 37 ℃ for 30min, and then is subjected to colorimetric determination at 562 nm.

Drawing a protein concentration standard curve according to the BCA method protein concentration determination kit, drawing the standard curve by taking the protein content as an abscissa and the absorbance as an ordinate, and as shown in FIG. 3, measuring a linear relation formula of Y ═ 0.0029X +0.1124, wherein Y: is the absorbance value at 562nm, X: in BSA solution concentration (. mu.g/mL), standard deviation R²0.9979. And when measuring the activities of the enal reductase and the glucose dehydrogenase, determining and calculating the protein concentration of the crude enzyme solution according to a protein concentration standard curve, and further calculating the specific enzyme activity. Each time two sets of replicates were performed, the mean and standard error were calculated.

Example 5 Induction expression of recombinant Gene engineering bacteria Co-expressing Enaldehyde reductase and glucose dehydrogenase

1. Inducing expression: coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH was inoculated into LB liquid medium containing 50. mu.g/mL chloramphenicol at the final concentration, cultured overnight at 37 ℃ and 200rpm, the culture was inoculated into 150mL LB liquid medium containing 100. mu.g/mL kanamycin and 50. mu.g/mL chloramphenicol at an inoculum size of 2% by volume, and cultured at 37 ℃ and 200rpm until the OD cell concentration was reached₆₀₀To 0.623, IPTG was added to the culture at a final concentration of 0.3mM and induction-cultured at 22 ℃ for 12 hours to obtain an induction culture solution. Under the same conditions, the culture medium without IPTG addition was used as an uninduced control culture medium.

2. Preparation of SDS-PAGE assay samples: the non-induced control culture solution and the induced culture solution were each taken 1mL, centrifuged at 12000rpm for 1min, and the supernatant was discarded to leave the cells. The cells were then resuspended in a suspension by adding 100. mu.L of ultrapure water to each cell. Then, 18. mu.L of each suspension was added to 6. mu.L of 4X Protein Loading Buffer, mixed well, and boiled for 10 min. After boiling, the mixture was centrifuged at 12000rpm for 1min, and 15. mu.L of each supernatant was used for SDS-PAGE detection, and the Protein Marker was Blueplus Protein Marker (14-120 kDa). As shown in FIG. 4 (lanes 2-5 correspond to the induced genetically engineered bacteria, and lane 6 corresponds to the non-induced genetically engineered bacteria), SDS-PAGE detection shows that both the enal reductase and the glucose dehydrogenase are successfully expressed in E.coli.

3. And optimizing the induction expression conditions of the recombinant genetic engineering bacteria E.coli BL21(DE3)/pACYCDUet-1-YsADH-EsGDH by investigating the activities of the enal reductase and the glucose dehydrogenase after the recombinant genetic engineering bacteria are induced and expressed.

(1) Optimization of induction temperature: the steps of induction expression were carried out as described in this example except that the induction temperatures in step 1 were set to 16 ℃, 20 ℃, 23 ℃, 25 ℃, 28 ℃ and 37 ℃, and the enzyme activities of the collected cells were measured for their enal reductase (ADH) and Glucose Dehydrogenase (GDH) activities by the method described in example 4. As shown in FIG. 5, when the temperature is 20 ℃, the maximum specific enzyme activity of the corresponding enal reductase YsADH is 1955U/g, and the maximum specific enzyme activity of the glucose dehydrogenase EsADH is 1231U/g.

(2) Optimization of Inducer (IPTG) concentration: the steps for induction expression were carried out in the same manner as in the example except that the concentration of the Inducer (IPTG) in step 1 was changed to 0.1mM, 0.2mM, 0.3mM, 0.4mM and 0.5mM, and the enzyme activities of the collected cells were measured for their enal reductase (ADH) and Glucose Dehydrogenase (GDH) activities by the method described in example 4. As shown in FIG. 6, when the concentration of the inducer is 0.4mM, the maximum specific enzyme activity of the corresponding enal reductase YsADH is 1684U/g, and the maximum specific enzyme activity of the glucose dehydrogenase EsADH is 2235U/g.

(3) Optimization of induction time: the induction expression step was carried out in the same manner as described in this example except that the induction time in step 1 was set to 6 hours, 8 hours, 10 hours, 12 hours and 14 hours, and the enzyme activities of the collected cells were measured for their enal reductase (ADH) and Glucose Dehydrogenase (GDH) activities by the method described in example 4. As shown in FIG. 7, when the induction time is 12h, the maximum specific enzyme activity of the corresponding enal reductase YsADH is 1415U/g, and the maximum specific enzyme activity of the glucose dehydrogenase EsADH is 1485U/g.

Therefore, under the optimal induction condition (the induction temperature is 20 ℃, the induction time is 12h, and the concentration of an inducer is 0.4mMIPTG), the maximum specific enzyme activity of the corresponding enal reductase YsADH is 1415U/g, and the maximum specific enzyme activity of the glucose dehydrogenase EsADH is 1485U/g.

Example 6 biocatalyst

The recombinant genetically engineered bacterium E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH was inoculated into LB liquid medium containing 50. mu.g/mL chloramphenicol at the final concentration, cultured overnight at 37 ℃ and 200rpm, the culture was inoculated into 150mL LB liquid medium containing 100. mu.g/mL kanamycin and 50. mu.g/mL chloramphenicol at an inoculum size of 2% by volume, and cultured at 37 ℃ and 200rpm until the OD cell concentration was reached₆₀₀And when the concentration is 0.623, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.4mM into the culture, carrying out induction culture at 20 ℃ for 12h, then centrifugally collecting 20g of wet thalli, carrying out freeze-drying at-80 ℃ for 24h to obtain 2.213g of freeze-dried powder, and calculating the water removal rate of the freeze-dried powder to be 88.93% according to a formula 3.

And then carrying out induction expression under the optimal condition, centrifugally collecting thalli, and preparing freeze-dried powder for optimizing an isopropenylaldehyde reduction system, wherein the freeze-dried powder comprises the pH value of a catalytic reaction system, the reaction temperature, the addition amount of a biocatalyst, the addition amount of coenzyme, the addition amount of an auxiliary substrate D-glucose, the optimal substrate concentration and the like. The wet cell of 20g recombinant cell E.coli BL21(DE3)/pACYCDuet 1-YsADH-EsGDH was lyophilized, the mass was called 2.213g, and was substituted into formula 3, and the water removal rate after 24h of lyophilization at-80 ℃ was calculated to be 88.93%.

Example 7 optimum temperature for synthesizing isopentenol from isopentenal catalyzed by freeze-dried powder

The chromatographic conditions for detecting the isopentenal and the isopentenol by using the gas chromatography are as follows:

gas phase column, Agilent 19091J-413-HP-5(30 m.times.0.32 mm.times.0.25 μm); detector FID, 250 ℃; carrier gas, N₂(ii) a Flow of carrier gas2.27 mL/min; the split ratio is 1: 100; the sample amount is 0.2 mu L; the injection port temperature was 250 ℃. Temperature rising procedure: the temperature is maintained at 40 ℃ for 10min, and then the temperature is raised to 140 ℃ at 20 ℃/min for 15 min. As shown in FIG. 8, the retention times of the isoprenal and the prenol were 3.619min and 3.671min, respectively.

Taking the freeze-dried powder prepared in the example 6 as a catalyst, taking the isopentenal as a substrate, taking the D-glucose as an auxiliary substrate, and adding NADP⁺A5 mL reaction system was constructed using 50mM Tris-HCl buffer solution at pH 6.5 as the reaction medium, with a final substrate concentration of 100mM, a final co-substrate concentration of 200mM, and NADP⁺The final concentration is 0.4mM, and the final concentration of the freeze-dried powder is 40 g/L.

Reacting at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C and 55 deg.C respectively at 600rpm for 30min, centrifuging 300 μ L of reaction solution at 12000rpm for 3min, and extracting 200 μ L of supernatant with 800 μ L of ethyl acetate for 30 min. And after extraction is finished, centrifuging at 12000rpm for 1min, taking 600 mu L of the upper organic phase, adding 0.8g of anhydrous sodium sulfate, then centrifuging at 12000rpm for 1min, taking 100 mu L of supernatant, adding ethyl acetate to a final volume of 0.5mL, and detecting the content of the isopentenol and the isopropenal in the sample by using a gas chromatography. Two replicates were run each time and the mean and standard error were calculated and the results are shown in figure 9.

As shown in fig. 9, the conversion of the product was 13.73% catalyzed at 20 ℃. As the temperature increases, the conversion of the product also increases; when the catalytic temperature was 45 ℃, the conversion of the product had reached 66.17%. When the temperature was increased to 50 ℃ and 55 ℃, the conversion of the product was also reduced to 60.48% and 60.45%. From this, it was found that the optimum temperature of the catalyst system was 45 ℃.

EXAMPLE 8 optimum pH of the catalytic System

The same procedure as in example 7 was repeated, except that the reaction system of example 7 was changed to pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 and the reaction temperature was 45 ℃. Two parallel experiments were performed each time, and the mean and standard error were calculated. The results are shown in FIG. 10, where the pH is 5.5, the conversion of the product is 45.51%. The conversion of the product increased with increasing pH, and reached 69.49% within 30min at pH 7.5. However, as the pH of the reaction solution continued to increase, there was a downward trend in the conversion of the product, which was 65.41% and 16.94% at pH 8.0 and 8.5, respectively. It can be seen that the optimum catalytic pH is 7.5.

EXAMPLE 9 optimum coenzyme addition amount of catalytic System

The pH of the reaction system of example 7 was set to 7.5, and NADP was selected as a coenzyme⁺The reaction was carried out at a concentration of 0mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM and 0.8mM at 45 ℃ and 600rpm for 30 minutes, and the same procedure as in example 7 was otherwise carried out. Two parallel experiments were performed each time, and the mean and standard error were calculated.

As shown in FIG. 11, NADP was added when no coenzyme was added⁺When the conversion of the product was 59.92%, along with NADP⁺When the concentration of (2) was increased to 0.8mM, the conversion of the product was increased from 59.92% to 70.81%, and NADP was added at a high concentration⁺The product conversion rate is only improved by about 1.1 times compared with that without the coenzyme, and the improvement range is not large. Taking the cost factor of coenzyme addition into consideration, the catalytic system does not add coenzyme NADP⁺Is preferred.

Example 10 molar ratio of co-substrate to substrate for the catalytic System

The pH of the reaction system of example 7 was set to 7.5, and NADP as a coenzyme was removed⁺The final concentration of isopentenal was 100mM, the molar concentration ratios of D-glucose as a co-substrate and isoamylenal as a substrate were selected to be 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1 and 3:1, and the reaction was carried out at 45 ℃ and 600rpm for 30 minutes, respectively, in the same manner as in example 7. Two parallel experiments were performed each time, and the mean and standard error were calculated.

Whether the coenzyme cycle can be efficiently operated or not is related to the concentration ratio between the cosubstrate and the substrate. D-glucose is selected as a cosubstrate. As shown in fig. 12, when the co-substrate D-glucose: when the substrate, i.e., the pentenal, was 0.5:1, the conversion of the product was 29.17%. The conversion of the product increased from 29.17% to 69.89% with the ratio from 0.5:1 to 1:1, 1.5:1, 2:1, 2.5:1, and then decreased from 69.89% to 51.57% with the ratio adjusted to 3: 1. Thus, the optimum co-substrate to substrate concentration ratio for the catalytic system is 2.5: 1.

EXAMPLE 11 optimum stirring speed of the catalytic System

Example 7 reaction System, substrate Final concentration 100mM, Co-substrate Final concentration 250mM, and No NADP addition⁺The final concentration of the freeze-dried powder is 40g/L, and the reaction is carried out for 30min at the temperature of 45 ℃. The rotation speed was selected to take 0rpm, 100rpm, 200rpm, 300rpm, 400rpm, 500rpm and 600rpm, and other operations were the same as example 7, and two sets of parallel experiments were performed each time to calculate the average value and the standard error.

As shown in FIG. 13, when the reaction system was in a state of rest (0rpm), the conversion of the product at 30min was 52.16%. When the rotation speed is increased to 300rpm, the conversion rate of the product is increased to 73.29%, and then when the rotation speed is gradually increased from 300rpm to 600rpm, the conversion rate of the product is reduced to 66.47%. Therefore, the optimum speed of the catalyst system may be 300 rpm.

EXAMPLE 12 optimum biocatalyst addition for the catalytic System

Example 7 reaction System, substrate Final concentration 100mM, Co-substrate Final concentration 250mM, and No NADP addition⁺The addition amounts of the lyophilized powders were 0.05g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, and 0.4g, and the reactions were performed at 45 ℃ and 300rpm for 30min, and the other operations were the same as example 7, and two parallel experiments were performed each time to calculate the average value and the standard error.

As shown in FIG. 14, the yield of the product is increased with the increase of the addition amount of the biocatalyst, and when the addition amount of the dry bacterial powder is 10g/L, the yield of the product reaches 11.39%. When the adding amount of the catalyst is increased to 60g/L, the product conversion rate is up to 87.61%, and when the adding amount of the biocatalyst is subsequently increased to 70g/L, the product conversion rate is 100%, so when the whole cell catalytic system catalyzes 100mM substrate, the adding amount of the biocatalyst should be 70 g/L.

Example 13 Effect of substrate concentration on the catalytic reduction of Isopentenal to Isopentenol by E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH

The lyophilized powder prepared in example 6 was used as a catalyst, the isopropenylaldehyde was used as a substrate, D-glucose was used as a co-substrate, and 50mM Tris-HCl buffer solution at pH 7.5 was used as a reaction medium to constitute a 5mL reaction system, whichThe final concentration of the medium substrate is 50mM, 100mM, 250mM or 500mM, the quantity ratio of the auxiliary substrate to the substrate substance is 2.5:1, the final concentration of the freeze-dried powder is 40g/L, the mixture is magnetically stirred and reacted for 12 hours at the temperature of 45 ℃ and the rpm of 300, and 4M Na is used in the catalysis process₂CO₃The pH was maintained constant. After the completion of the reaction, 300. mu.L of the reaction mixture was taken and subjected to gas chromatography (same as in example 7).

The gas phase detection result shows that when 50mM, 100mM, 250mM or 500mM of the isopropenal is used as a reaction substrate, the substrate can be completely converted into a product after a certain time of catalysis, and the conversion rate reaches 100%. However, during the course of the reaction, it was found that the catalytic time increases with increasing substrate concentration. When the substrate concentration is 50mM, the conversion rate reaches 100% after the reaction time is 0.5 h; when the substrate concentration is 100mM, the conversion rate reaches 100% after the reaction time is 1.5 h; when the substrate concentration is 250mM and the reaction time is 3h, the conversion rate reaches 100%; when the substrate concentration is 500mM, the conversion reaches 100% after a reaction time of 6 h. No by-product saturated alcohol was detected in all reactions. Low concentrations of substrate will be completely converted to product in a short time, while high concentrations of substrate require longer reaction times, indicating that an increase in substrate concentration inhibits catalytic activity to some extent.

Example 14 Effect of substrate addition on the catalytic efficiency of E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH

The reaction system was 5mL, containing 500mM of isopropenal, 1.25M D-glucose, 0.2g of lyophilized powder prepared in example 6, and 50mM of Tris-HCl (pH 7.5), respectively. The substrate addition mode is as follows: the initial concentration of the reaction substrate was 250mM, and after 2 hours of reaction, substrate was supplemented to a substrate concentration of 500 mM. Adding the reaction solution into a three-neck flask, maintaining the reaction pH at 7.5 under the magnetic stirring of 300rpm at 45 ℃, and using 4M Na for the catalytic process₂CO₃The pH was maintained constant. After 12h of reaction, samples were taken for gas chromatography and gas-mass spectrometry. The chromatographic conditions of example 7 were used for the hue chromatography analysis, and the mass spectrometry parameters in the gas-mass spectrometry were set as follows: auxiliary heating temperature, 250 ℃; quadrupole temperature, 150 ℃; ion source temperature, 230 ℃; scanning mass range, 30-500 amu; the current is emitted and the current is measured,200 muA; electron energy, 70 eV. As shown in FIG. 15, the molecular weight of the peak of the product was 86 by GC-MS analysis, which is consistent with that of prenol. The gas chromatography result shows that the conversion rate of the product reaches 80.8% after the reaction is carried out for 2.5h, and the conversion rate of the product reaches 100% after the sampling detection is carried out for 3.5 h. Compared with example 13, the time for 500mM substrate to be completely converted is shortened from 6h to 3.5h, and no by-product is generated in the reaction. Therefore, the mode of adding the substrate in batches is beneficial to relieving the inhibition of high-concentration substrate on the catalytic activity, and the catalytic efficiency is obviously improved.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Zhejiang industrial university

<120> recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 339

<212> PRT

<213> Unknown (Unknown)

<400> 1

Met Ser Ile Ile Lys Ser Tyr Ala Ala Lys Glu Ala Gly Ser Glu Leu

1 5 10 15

Glu Leu Tyr Glu Tyr Asp Ala Gly Glu Leu Arg Pro Glu Asp Val Glu

20 25 30

Val Gln Val Asp Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile

35 40 45

Asp Asn Glu Trp Gly Phe Ser Gln Tyr Pro Leu Val Ala Gly His Glu

50 55 60

Val Ile Gly Arg Val Ala Ala Leu Gly Ser Ala Ala Gln Glu Lys Gly

65 70 75 80

Val Lys Val Gly Gln Arg Val Gly Val Gly Trp Thr Ala Arg Ser Cys

85 90 95

Gly His Cys Asp Ala Cys Ile Ser Gly Asn Gln Ile Asn Cys Leu Glu

100 105 110

Gly Ala Val Ala Thr Ile Leu Asn Arg Gly Gly Phe Ala Glu Lys Leu

115 120 125

Arg Ala Asp Trp Gln Trp Val Ile Pro Leu Pro Glu Ser Ile Asp Ile

130 135 140

Glu Ser Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Phe Lys Pro

145 150 155 160

Leu Leu Met His His Ile Thr Ala Thr Ser Arg Val Gly Val Ile Gly

165 170 175

Ile Gly Gly Leu Gly His Ile Ala Ile Lys Leu Leu His Ala Met Gly

180 185 190

Cys Glu Val Thr Ala Phe Ser Ser Asn Pro Ser Lys Glu Gln Glu Val

195 200 205

Leu Ala Met Gly Ala Asp Lys Val Val Asn Ser Arg Asp Pro Asp Ala

210 215 220

Leu Asn Ala Leu Ala Gly Gln Phe Asp Leu Ile Ile Asn Thr Val Asn

225 230 235 240

Val Asp Leu Asp Trp Gln Pro Tyr Phe Glu Ala Leu Ala Tyr Gly Gly

245 250 255

His Phe His Thr Val Gly Ala Val Met Lys Pro Leu Pro Val Pro Ala

260 265 270

Phe Thr Leu Ile Ala Gly Asp Arg Ser Ile Ser Gly Ser Ala Thr Gly

275 280 285

Thr Pro Tyr Glu Leu Arg Lys Leu Met Lys Phe Ala Gly Arg Ser Lys

290 295 300

Val Ser Pro Thr Thr Glu Leu Phe Pro Met Ser Gln Ile Asn Glu Ala

305 310 315 320

Ile Gln His Val Arg Asp Gly Lys Ala Arg Tyr Arg Val Val Leu Gln

325 330 335

Ala Asp Phe

<210> 2

<211> 1020

<212> DNA

<213> Unknown (Unknown)

<400> 2

atgtctatta taaaaagcta tgccgcaaaa gaggcgggca gcgaactcga actttacgaa 60

tatgatgccg gtgaactcag gccggaagat gtcgaggtgc aggtcgacta ctgcggtatc 120

tgccattccg atctttccat gatcgacaac gaatggggat tctctcagta tccgctggtt 180

gccgggcatg aagtgattgg ccgcgtggcg gcgctcggca gtgcggcgca ggaaaaaggg 240

gtgaaagttg gtcagcgcgt gggcgtaggc tggacggcgc gcagctgtgg gcattgcgat 300

gcatgtatca gcggtaatca gattaactgc ctggaaggcg ccgtagccac cattctcaac 360

cgtggcggtt ttgccgagaa actgcgggca gactggcagt gggtgatccc gcttccggag 420

agcatcgata ttgagtcggc aggtcctctg ttatgcggcg gtattacggt ttttaaacct 480

ctgctgatgc accacatcac cgcgaccagt cgcgtggggg tgatcggcat cggcggtctt 540

gggcacattg ccattaaact gttgcacgca atgggctgtg aagtgaccgc attcagctcg 600

aatccgtcga aagaacagga agtgctggca atgggggcgg ataaagtcgt gaacagtcgc 660

gatccagacg cgttaaatgc gctggcaggc cagtttgatc tcattatcaa caccgttaat 720

gtcgacctcg actggcagcc ctactttgaa gcgctggcct atggcggcca tttccacacc 780

gtcggcgcag tgatgaagcc gctgccggtt ccggcgttta cattgattgc tggcgatcgc 840

agcatctccg gctcagcaac cggtacgccc tatgagctgc gcaaattgat gaagtttgcc 900

gggcgcagca aggtctcgcc gacgacagag ctgttcccaa tgtcgcaaat caacgaagcc 960

atccagcacg ttcgcgacgg caaagcgcgt taccgcgtgg tactgcaagc cgacttttga 1020

<210> 3

<211> 262

<212> PRT

<213> Unknown (Unknown)

<400> 3

Met Gly Tyr Asn Ser Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly

1 5 10 15

Ser Met Gly Ile Gly Glu Ala Ile Ile Arg Arg Tyr Ala Glu Glu Gly

20 25 30

Met Arg Val Val Ile Asn Tyr Arg Ser His Pro Glu Glu Ala Lys Lys

35 40 45

Ile Ala Glu Asp Ile Lys Gln Ala Gly Gly Glu Ala Leu Thr Val Gln

50 55 60

Gly Asp Val Ser Lys Glu Glu Asp Met Ile Asn Leu Val Lys Gln Thr

65 70 75 80

Val Asp His Phe Gly Gln Leu Asp Val Phe Val Asn Asn Ala Gly Val

85 90 95

Glu Met Pro Ser Pro Ser His Glu Met Ser Leu Glu Asp Trp Gln Lys

100 105 110

Val Ile Asp Val Asn Leu Thr Gly Ala Phe Leu Gly Ala Arg Glu Ala

115 120 125

Leu Lys Tyr Phe Val Glu His Asn Val Lys Gly Asn Ile Ile Asn Met

130 135 140

Ser Ser Val His Glu Ile Ile Pro Trp Pro Thr Phe Val His Tyr Ala

145 150 155 160

Ala Ser Lys Gly Gly Val Lys Leu Met Thr Gln Thr Leu Ala Met Glu

165 170 175

Tyr Ala Pro Lys Gly Ile Arg Ile Asn Ala Ile Gly Pro Gly Ala Ile

180 185 190

Asn Thr Pro Ile Asn Ala Glu Lys Phe Glu Asp Pro Lys Gln Arg Ala

195 200 205

Asp Val Glu Ser Met Ile Pro Met Gly Asn Ile Gly Lys Pro Glu Glu

210 215 220

Ile Ser Ala Val Ala Ala Trp Leu Ala Ser Asp Glu Ala Ser Tyr Val

225 230 235 240

Thr Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser

245 250 255

Phe Gln Ala Gly Arg Gly

260

<210> 4

<211> 789

<212> DNA

<213> Unknown (Unknown)

<400> 4

atgggttata attctctgaa aggcaaagtc gcgattgtta ctggtggtag catgggcatt 60

ggcgaagcga tcatccgtcg ctatgcagaa gaaggcatgc gcgttgttat caactatcgt 120

agccatccgg aggaagccaa aaagatcgcc gaagatatta aacaggcagg tggtgaagcc 180

ctgaccgtcc agggtgacgt ttctaaagag gaagacatga tcaacctggt gaaacagact 240

gttgatcact tcggtcagct ggacgtcttt gtgaacaacg ctggcgttga gatgccttct 300

ccgtcccacg aaatgtccct ggaagactgg cagaaagtga tcgatgttaa tctgacgggt 360

gcgttcctgg gcgctcgtga agctctgaaa tacttcgttg aacataacgt gaaaggcaac 420

attatcaata tgtctagcgt ccacgaaatc atcccgtggc ctactttcgt acattacgct 480

gcttctaagg gtggcgttaa actgatgacc cagactctgg ctatggaata tgcaccgaaa 540

ggtatccgca ttaacgctat cggtccaggc gcgatcaaca ctccaattaa tgcagaaaaa 600

ttcgaggatc cgaaacagcg tgcagacgtg gaaagcatga tcccgatggg caacatcggc 660

aagccagagg agatttccgc tgtcgcggca tggctggctt ctgacgaagc gtcttacgtt 720

accggcatca ccctgttcgc agatggtggc atgaccctgt acccgagctt tcaggctggc 780

cgtggttga 789

Claims

1. A recombinant gene engineering bacterium for co-expressing enal reductase and glucose dehydrogenase is characterized in that the engineering bacterium is obtained by introducing enal reductase genes and D-glucose dehydrogenase genes into host bacteria together.

2. The recombinant genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the enal reductase gene is represented by SEQ ID No. 2.

3. The recombinant genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the D-glucose dehydrogenase gene is represented by SEQ ID No. 4.

4. The recombinant genetically engineered bacterium of claim 1, wherein the recombinant genetically engineered bacterium is constructed by the following method: inserting the enal reductase gene into Nco I and Hind III restriction sites of a first multiple cloning site of a pACYCDuet-1 vector, inserting the polynucleotide of the D-glucose dehydrogenase gene into Nde I and Xho I restriction sites of a second multiple cloning site of the pACYCDuet-1 vector to obtain a recombinant vector containing the enal reductase gene and the D-glucose dehydrogenase gene, and introducing the recombinant vector into a host cell to obtain the recombinant gene engineering bacterium.

5. The recombinant genetically engineered bacterium of claim 4, wherein the host cell is E.coli BL21(DE 3).

6. An application of the recombinant genetically engineered bacterium of claim 1 in catalyzing isopentenyl aldehyde to prepare isopentenyl alcohol.

7. The use according to claim 6, characterized in that the method of application is: the method comprises the steps of forming a reaction system by using wet thallus freeze-dried powder obtained by induced culture of recombinant genetic engineering bacteria as a catalyst, using isopropenal as a substrate, using glucose as an auxiliary substrate and using a buffer solution with pH of 5.5-8.5 as a reaction medium, carrying out catalytic reaction at 20-55 ℃ and 0-600rpm, and after the reaction is completed, separating and purifying reaction liquid to obtain the isopentenol.

8. The use according to claim 7, wherein the final concentration of the catalyst in the reaction system is 10-80g/L, the final concentration of the substrate is 50-500mM, and the ratio of the added substances of the substrate and the co-substrate is 1: 0.5-3.

9. The use according to claim 7, wherein the reaction temperature is 45 ℃ and the rotation speed is 300 rpm.

10. The use according to claim 7, wherein the catalyst is prepared by the following process: inoculating the recombinant genetically engineered bacteria into LB liquid culture medium containing 50. mu.g/mL chloramphenicol at final concentration, culturing overnight at 37 ℃ and 200rpm, taking the culture and inoculating the culture with an inoculum size of 2% of the volume concentration into LB liquid culture medium containing 100. mu.g/mL kanamycin and 50. mu.g/mL chloramphenicol, and culturing at 37 ℃ and 200rpm until the thallus concentration OD₆₀₀And (3) adding IPTG with the final concentration of 0.1-0.5mM into the culture, carrying out induction culture at 16-37 ℃ for 6-14h, then centrifugally collecting wet bacteria, and freeze-drying at-80 ℃ for 24h to obtain freeze-dried powder.