CN118291408A - Enzyme combination, recombinant engineering bacterium, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an enzyme combination, recombinant engineering bacteria, a method for preparing the same and application of the recombinant engineering bacteria. The enzyme combination includes an L-pantolactone dehydrogenase, a ketopantolactone reductase, a glucose dehydrogenase, and a ketopantolactone reductase. The recombinant engineering bacteria comprise the enzyme combination or a recombinant vector containing a nucleotide sequence for encoding the enzyme combination. The recombinant engineering bacteria of the invention can be used for preparing D-pantolactone, which can obviously improve the selectivity and the reaction efficiency of the L-pantolactone dehydrogenase under the condition of high concentration substrate, obviously reduce the accumulation of intermediate product ketopantoic acid and improve the conversion rate of products.

Description

Enzyme combination, recombinant engineering bacterium, and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an enzyme combination and recombination engineering bacterium, and a preparation method and application thereof.

Background

D-pantolactone is a pharmaceutical intermediate for synthesizing vitamin D-panthenol and neurotrophic D-calcium pantothenate, and is used as a feed additive and a synthesis precursor of daily chemical products, and the annual yield exceeds tens of thousands tons.

The current methods for synthesizing D-pantolactone include chemical synthesis, chemical enzyme method and recombinant cell utilization. The industrialized synthesis of D-pantolactone adopts a technical route combining a chemical method and a hydrolytic enzyme splitting method, takes cheap isobutyraldehyde and formaldehyde as starting materials, and forms hydroxypivalaldehyde by aldol condensation of the isobutyraldehyde and the formaldehyde under alkaline and high temperature conditions; then hydrocyanic acid is added for cyanidation, and alcohol cyanidation reaction is carried out under the acidic condition to form cyanohydrin; and (3) hydrolyzing and cyclizing cyanohydrin under an acidic condition to obtain DL-pantolactone, and then resolving the prepared DL-pantolactone by utilizing a microbial enzyme method to obtain D-pantolactone. The microbial enzyme method for resolving DL-pantolactone to obtain D-pantolactone comprises the following steps: 1) Microbial enzyme selectively hydrolyzes D-pantolactone in DL-pantolactone to obtain L-pantolactone and D-pantoic acid; 2) The mixed liquid is extracted by using an organic reagent to realize the separation of L-pantolactone and D-pantoic acid; 3) D-pantoic acid is subjected to lactonization treatment to obtain D-pantoic acid lactone, and L-pantoic acid lactone is subjected to chemical racemization to be changed into DL-pantoic acid lactone, and then is split. The method has the following defects: firstly, the separation steps are more, and the separation efficiency is low; secondly, a large amount of pigment and other impurities are generated in the chemical racemization process, the D-pantolactone prepared by resolution is darker in color and poorer in appearance quality; thirdly, chemical racemization can generate a large amount of sulfate to form solid waste, thereby causing environmental pollution.

Based on the defects of the industrialized synthesis method, researchers develop a method for catalyzing and synthesizing D-pantolactone by using recombinant cell biological enzyme. CN110423717B discloses a recombinant cell of multiple enzymes and a method for synthesizing D-pantolactone by multi-enzyme cascade catalysis, which converts DL-pantolactone to ketopantolactone by expressing L-pantolactone dehydrogenase, ketopantolactone reductase and glucose dehydrogenase by the recombinant cell, and further generates D-pantolactone. The method can convert DL-pantolactone into D-pantolactone by a one-step method, avoids complicated resolution procedures, and still has the following defects: when the concentration of the substrate is high, the product yield is obviously reduced, and when the concentration of the substrate is up to 0.75M, the product yield can still be kept at 99%; when the substrate concentration reaches 1M, namely the substrate concentration reaches 130g/L, the product yield is more than 90 percent. With increasing substrate concentration to 1.25M, a relatively significant drop in product yield occurred. The reason for this is analyzed, on the one hand, it is possible that the L-pantolactone dehydrogenase has poor selectivity for L-pantolactone and its catalytic reaction, which catalyzes both the L-pantolactone to produce ketopantolactone and the D-pantolactone to produce ketopantolactone, and the L-pantolactone and D-pantolactone compete simultaneously for binding to the active site of the L-pantolactone dehydrogenase, and the D-pantolactone falls into the cyclic reaction, thereby resulting in a decrease in enzyme activity; on the other hand, as the concentration of the substrate increases, D-pantolactone in the substrate can inhibit the activity of ketopantolactone reductase, so that ketopantolactone cannot be rapidly reduced, spontaneous hydrolysis of ketopantolactone can occur as the accumulation concentration increases, and ketopantoic acid cannot be further converted by ketopantolactone dehydrogenase, so that a considerable part of ketopantoic acid intermediate products accumulate in the reaction product, and the product quality is difficult to ensure.

Therefore, how to develop high-selectivity L-pantolactone dehydrogenase and reduce accumulation of ketopantoic acid in the reaction process, thereby being beneficial to improving concentration of reaction substrates, purity and quality of D-pantolactone, reaction efficiency, resource utilization rate and other recombinant vectors and engineering bacteria thereof are urgent problems to be solved in the field.

Disclosure of Invention

The invention aims to solve the technical problem that the yield of a product is obviously reduced when the concentration of a substrate is high in the prior art, and provides an enzyme combination, recombinant engineering bacteria containing the enzyme combination, a method for preparing the same and application of the recombinant engineering bacteria.

The invention solves the technical problems through the following technical proposal.

In a first aspect the invention provides an enzyme combination comprising an L-pantolactone dehydrogenase, a ketopantolactone reductase, a glucose dehydrogenase and a ketopantolactone reductase.

In some embodiments of the invention, the L-pantolactone dehydrogenase is derived from any one of actinomycetes, nocardia, rhodococcus, mycobacteria, streptomyces; the ketopantolactone reductase is derived from any one of Saccharomyces cerevisiae, candida, micromonospora and streptomyces; the glucose dehydrogenase is derived from any one of bacillus subtilis, bacillus pumilus, burkholderia, escherichia coli and aeromonas.

In some embodiments of the invention, the L-pantolactone dehydrogenase is selected from Rhodococcus collecticus (Rhodococcus fascians), the ketopantolactone reductase is selected from Rhodococcus longus (Lodderomyces elongisporus), the glucose dehydrogenase is selected from Microbacterium (Exiguobacterium sibiricum), and the ketopantolactone reductase is selected from E.coli.

In some embodiments of the invention, the amino acid sequence of the L-pantolactone dehydrogenase is shown as SEQ ID NO. 5, the amino acid sequence of the ketopantolactone reductase is shown as SEQ ID NO. 6, the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO. 7, and the amino acid sequence of the ketopantolactone reductase is shown as SEQ ID NO. 8.

In a second aspect the invention provides a recombinant vector comprising nucleotide sequences encoding the enzyme combinations according to the first aspect of the invention.

In some embodiments of the invention, the nucleotide sequence encoding the L-pantolactone dehydrogenase is shown as SEQ ID NO. 1, the nucleotide sequence encoding the ketopantolactone reductase is shown as SEQ ID NO. 2, the nucleotide sequence encoding the glucose dehydrogenase is shown as SEQ ID NO. 3, and the nucleotide sequence encoding the ketopantolactone reductase is shown as SEQ ID NO. 4.

In some embodiments of the invention, the nucleotide sequences encoding the enzyme combinations are on the same vector or separate vectors.

In some preferred embodiments of the invention, the separate vectors are a first vector comprising a nucleotide sequence encoding an L-pantolactone dehydrogenase, a second vector comprising a nucleotide sequence encoding a ketopantolactone reductase and a glucose dehydrogenase, and a third vector comprising a nucleotide sequence encoding a glucose dehydrogenase and a ketopantolactone reductase.

In some embodiments of the invention, the backbone plasmid of the first vector is pET21a, the backbone plasmid of the second vector is pRSFDuet-1, and the backbone plasmid of the third vector is pACYCDuet-1.

In a third aspect, the invention provides a recombinant engineering bacterium comprising an enzyme combination according to the first aspect of the invention or a recombinant vector according to the second aspect of the invention.

In some embodiments of the invention, the recombinant engineering bacteria comprise a first vector, a second vector and a third vector, wherein the first vector comprises a nucleotide sequence encoding an L-pantolactone dehydrogenase, the second vector comprises a nucleotide sequence encoding a ketopantolactone reductase and a glucose dehydrogenase, and the third vector comprises a nucleotide sequence encoding a glucose dehydrogenase and a ketopantolactone reductase.

The fourth aspect of the invention provides a recombinant engineering bacterium combination, which comprises a first recombinant engineering bacterium and a second recombinant engineering bacterium, wherein the first recombinant engineering bacterium comprises a first vector and a second vector, and the second recombinant engineering bacterium comprises a third vector; wherein the first vector comprises a nucleotide sequence encoding an L-pantolactone dehydrogenase, the second vector comprises a nucleotide sequence encoding a ketopantolactone reductase and a glucose dehydrogenase, and the third vector comprises a nucleotide sequence encoding a glucose dehydrogenase and a ketopantolactone reductase.

In a fifth aspect, the invention provides a method for constructing a recombinant engineering bacterium, comprising introducing the recombinant vector according to the second aspect of the invention into a host cell.

In some embodiments of the invention, the host cell of the recombinant engineering bacterium is selected from any one of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutinosa, escherichia coli, pantoea ananatis.

In some embodiments of the invention, the host cell is selected from E.coli.

In some embodiments of the invention, the host cell is Escherichia coli BL21 (DE 3).

The sixth aspect of the invention provides a method for culturing recombinant engineering bacteria or recombinant engineering bacteria combinations, which comprises fermenting and culturing the recombinant engineering bacteria according to the third aspect of the invention or the recombinant engineering bacteria according to the fourth aspect of the invention in a culture medium.

In some embodiments of the invention, seed culture is further included before fermentation culture to obtain seed liquid, and IPTG is added for induction after fermentation culture.

In some embodiments of the invention, the culture medium for fermentation culture is TB and the culture medium for seed culture is LB.

In some embodiments of the invention, the recombinant engineering bacteria or the recombinant engineering bacteria combination are inoculated into LB culture medium according to 0.1-5% of inoculum size, and the seed solution is inoculated into TB culture medium according to 1-10% of inoculum size.

In a seventh aspect, the present invention provides a method of preparing an enzyme combination according to the first aspect of the invention, comprising the steps of:

(1) Culturing recombinant engineering bacteria or a combination of recombinant engineering bacteria using a method according to the sixth aspect of the invention;

(2) And collecting thalli to obtain bacterial suspension.

In an eighth aspect the invention provides an enzyme reaction system comprising an enzyme combination according to the first aspect of the invention.

In some embodiments of the invention, one or more of DL-pantolactone and glucose are also included.

In a ninth aspect, the present invention provides a method for preparing D-pantolactone, comprising the steps of: placing DL-pantolactone in a reaction system containing glucose and the enzyme combination according to the first aspect of the invention, and reacting for 20-40h at 30-40 ℃ and pH5.0-7.0 to obtain the D-pantolactone.

The tenth aspect of the invention provides an enzyme combination according to the first aspect of the invention, a recombinant engineering bacterium according to the third aspect, a recombinant engineering bacterium combination according to the fourth aspect or an enzyme reaction system according to the eighth aspect, and the application thereof in preparing D-pantolactone.

The detection method of each substance in the reaction process and after the reaction is finished comprises the following steps:

Instrument and working conditions:

Instrument: shimadzu LC-16 liquid chromatograph.

Chiral chromatographic column LuxTM 5: 5um Cellmlose-1 (Size 4.6X1250 mm), column temperature 30 ℃, wavelength 217nm, flow rate 1.0ml/min, acquisition time 30min, sample injection amount 10 μl, mobile phase n-hexane: ethanol=92:8.

Reversed phase chromatographic column: agilent chromatography column: eclipse XDB-C18,5 μm, 4.6X1250 mm. Mobile phase: 95%50mM NaH ₂PO₄ (pH 3.0 with phosphoric acid), 5% methanol, flow rate 1.0mL/min. Ultraviolet detection wavelength: 210nm, acquisition time: 30min, sample injection amount: 10 mu L.

The experimental steps are as follows: after the reaction is finished, firstly taking reaction solution to dilute by 100 times, filtering and then sampling, measuring the molar concentration of ketopantoic acid in the system by using a reversed phase chromatographic column, and calculating the ratio of ketopantoic acid to the molar concentration of ketopantoic acid at the end of the reaction/the molar concentration of DL-pantolactone added at the beginning of the reaction multiplied by 100 percent.

After the reaction, adjusting the pH to be=1.5 by using 2M sulfuric acid, heating to 80 ℃ for reaction for 1 hour to obtain a conversion solution, detecting the conversion solution by using a chiral chromatographic column by using high performance liquid chromatography, and respectively calculating an ee value and a D-pantolactone conversion rate. Extracting the conversion solution twice by using ethyl acetate, combining ethyl acetate extraction phases, and calculating an ee value by using the concentration of D-pantolactone and L-pantolactone in a chiral chromatographic column detection system, wherein the ee value has a calculation formula: ee value = (D-pantolactone concentration-L-pantolactone concentration)/(D-pantolactone concentration+l-pantolactone concentration) ×100%; conversion = concentration of D-pantolactone in the system after the end of the reaction/concentration of DL-pantolactone added initially to the reaction x 100%.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

1. the recombinant engineering bacteria can co-express the L-pantolactone dehydrogenase, the ketopantolactone reductase, the glucose dehydrogenase and the ketopantolactone reductase through induction culture, are used for efficiently preparing the D-pantolactone with high optical purity, and have the advantages of simplicity and convenience in operation, environment friendliness, suitability for industrial production and the like.

2. The invention also provides a method for synthesizing D-pantolactone by utilizing the enzyme induced by the recombinant engineering bacteria to catalyze DL-pantolactone or L-pantolactone efficiently, which can obviously improve the selectivity and the reaction efficiency of L-pantolactone dehydrogenase under the condition of high concentration substrate, obviously reduce the accumulation of intermediate product ketopantoic acid and improve the conversion rate of products. Has the advantages of simple operation, environmental protection, better cost, suitability for large-scale industrialized production, and the like.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1: construction of recombinant vectors

1. Acquisition and Synthesis of the Gene sequence of interest

A. The nucleotide sequence of the coding gene of the L-pantolactone dehydrogenase (GenBank: MBY 4014232.1) from the rhodococcus collecticus (Rhodococcus fascians) is subjected to codon optimization according to the codon preference of escherichia coli (E.coli) to obtain the nucleotide sequence of the L-pantolactone dehydrogenase, wherein the nucleotide sequence is shown as SEQ ID NO. 1; the nucleotide sequence was designated RfLpLDH by the commission of the manual synthesis by Suzhou gold only biotechnology Co.

B. The nucleotide sequence of the ketopantolactone reductase (GenBank: WLF 81497.1) encoding gene from the long spore lodycephem (Lodderomyces elongisporus) is subjected to codon optimization according to the codon preference of escherichia coli (E.coli) to obtain the nucleotide sequence of the ketopantolactone reductase, wherein the nucleotide sequence is shown as SEQ ID NO. 2; the nucleotide sequence was designated LeCPR by the commission of the manual synthesis by Suzhou gold only biotechnology Co.

C. The nucleotide sequence of glucose dehydrogenase (GenBank: ACB 59697.1) from Microbacterium (Exiguobacterium sibiricum) is subjected to codon optimization according to the codon preference of Escherichia coli (E.coli) to obtain the nucleotide sequence of the glucose, wherein the nucleotide sequence is shown as SEQ ID NO. 3; the nucleotide sequence was designated EsGDH by the commission of the manual synthesis by Suzhou gold only biotechnology Co.

D. The nucleotide sequence of the ketopantoate reductase (PanE, genBank: NP-414959) encoding gene from Escherichia coli (E.coli) is shown in SEQ ID NO. 4.

2. Construction of recombinant expression vectors

A. The RfLpLDH fragment was obtained by PCR amplification using the synthesized RfLpLDH DNA molecule as a template and primers RfLpLDH-F (SEQ ID NO: 5) and RfLpLDH-R (SEQ ID NO: 6). The pET21a plasmid (purchased from Hangzhou Baosier plasmid Strain preservation Co.) was double digested with NdeI and XhoI, and cleaned with a PCR cleaning kit to obtain a double digested linearized plasmid backbone. The NdeI and XhoI double enzyme tangential pET21a plasmid skeleton and RfLpLDH fragment were ligated using ClonExpress IIOne Step Cloning Kit (purchased from Nanjinouzan Biotechnology Co., ltd.) ligation kit, transformed into E.coli DH 5. Alpha. Competent cells (purchased from Takara), spread onto LB+Amp ^r solid plates, incubated overnight at 37℃to perform PCR verification of the grown transformants, and positive transformants were shaken overnight in LB+Amp ^r tubes, and plasmids were extracted using NdeI and XhoI double enzyme digestion verification to obtain the first recombinant vector pET21a-RfLpLDH.

B. The synthesized LeCPR DNA molecule is used as a template, primers LeCPR-F (SEQ ID NO: 7) and LeCPR-R (SEQ ID NO: 8) are used for PCR amplification to obtain LeCPR fragments, ndeI and XhoI are used for double digestion of pRSFDuet-1 plasmid (purchased from Hangzhou Baosier plasmid strain preservation company), and a PCR cleaning kit is used for cleaning to obtain a double digestion linearized plasmid skeleton. The pRSFDuet-1 plasmid skeleton and LeCPR fragment which are tangentially treated by NdeI and XhoI double enzyme are connected by using a ClonExpress IIOne Step Cloning Kit one-step connection kit, are transformed into competent cells of escherichia coli DH5 alpha, are coated on a solid plate of LB+Kan ^r, are cultured at 37 ℃ overnight, carry out PCR verification on the grown transformant, shake the positive transformant in a test tube of LB+Kan ^r overnight, extract plasmids and carry out double enzyme digestion verification by using NdeI and XhoI, and obtain recombinant plasmid pRSF-LeCPR.

Further utilizing restriction enzyme NcoI and HindIII to double-digest pRSF-LeCPR plasmid, and cleaning by using PCR cleaning kit to obtain NcoI/HindIII double-digested plasmid linearization skeleton; the synthesized EsGDH DNA molecule is used as a template, primers EsGDH-F (SEQ ID NO: 9) and EsGDH-R (SEQ ID NO: 10) are used for carrying out PCR amplification to obtain EsGDH fragments, a ClonExpress IIOne Step Cloning Kit one-step ligation kit is used for ligating a pRSF-LeCPR plasmid skeleton and EsGDH fragments which are tangentially subjected to NcoI/HindIII double enzyme digestion, the fragments are transformed into competent cells of escherichia coli DH5 alpha, the competent cells are coated on a solid plate of LB+Kan ^r, the solid plate is cultured at 37 ℃ overnight, PCR verification is carried out on the grown transformants, positive transformants are shaken in a test tube of LB+Kan ^r for overnight, and plasmids are extracted and verified by double enzyme digestion of NcoI and HindIII to obtain a second recombinant vector pRSF-EsGDH-LeCPR.

C. The genome of Escherichia coli MG1655 strain (purchased from Hangzhou Baozi plasmid strain preservation company) is used as a template, and the primers PanE-F (SEQ ID NO: 11) and PanE-R (SEQ ID NO: 12) are used for combination amplification to obtain a PanE gene fragment; double-enzyme digestion is carried out on pACYCDuet-1 plasmid by using NdeI and XhoI, a PCR cleaning kit is used for cleaning to obtain a double-enzyme digestion linearized plasmid skeleton, a ClonExpress IIOne Step Cloning Kit one-step method connection kit is used for connecting the NdeI and XhoI double-enzyme digestion linearized pACYCDuet-1 plasmid skeleton and panE fragments, the NdeI and XhoI double-enzyme digestion linearized pACYCDuet-1 plasmid skeleton and panE fragments are transformed into E coli DH5 alpha competent cells, the obtained product is coated on a solid flat plate of LB+Cm ^r, the obtained product is cultured overnight at 37 ℃, PCR verification is carried out on the obtained product, positive transformant is shaken overnight in a test tube of LB+Cm ^r, and the obtained plasmid is extracted by using NdeI and XhoI double-enzyme digestion verification to obtain recombinant plasmid pACYC-panE.

Further utilizing restriction enzyme NcoI and HindIII to double-digest pACYC-PanE plasmid, and cleaning by using a PCR cleaning kit to obtain an NcoI/HindIII double-digested plasmid linearization framework; the method comprises the steps of connecting a NcoI/HindIII double-enzyme tangential pACYC-panE plasmid skeleton and a EsGDH fragment obtained by PCR amplification by using a ClonExpress IIOne Step Cloning Kit one-step connecting kit, transforming into E.coli DH5 alpha competent cells, coating onto a solid plate of LB+Cm ^r, culturing overnight at 37 ℃, carrying out PCR verification on the grown transformant, shaking positive transformant in a test tube of LB+Cm ^r overnight, extracting plasmids, and carrying out double-enzyme cutting verification by using NcoI and HindIII to obtain a third recombinant vector pACYC-EsGDH-panE.

Example 2: construction of recombinant multienzyme expression Strain

The recombinant expression vectors constructed in example 1 were transformed into E.coli BL21 (DE 3) competent cells (purchased from Hangzhou Barceis plasmid strain collection) alone or in combination as shown in Table 1 to obtain different multi-enzyme recombinant expression strains. Wherein the P1 recombinant strain is a recombinant strain capable of coexpression of L-pantolactone dehydrogenase, ketopantolactone reductase and glucose dehydrogenase; the P2 recombinant strain is a recombinant strain capable of coexpressing L-pantolactone dehydrogenase, ketopantolactone reductase and glucose dehydrogenase and ketopantolactone reductase. The P3 recombinant strain is a recombinant strain capable of coexpression of ketopantoate reductase and glucose dehydrogenase.

TABLE 1 construction of recombinant multienzyme expression strains.

Example 3: culture of recombinant engineering bacteria and protein induction expression

Inoculating glycerol bacteria of each recombinant engineering bacterium prepared in the example 2 into 5mL of LB culture medium according to the proportion of 1%, and culturing overnight at 37 ℃ and 200rpm to obtain seed liquid; the seed solution was inoculated in 100mL of TB medium at an inoculum size of 2%, and was incubated at 37 ℃ for 4 hours at 200rpm, od=1.0, IPTG was added at a final concentration of 0.1mM, the incubation temperature was adjusted to 20 ℃, and the rotation speed was adjusted to 110rpm for further incubation for 24 hours, to obtain a fermentation broth, namely a crude enzyme solution (the wet weight of the cells in the crude enzyme solution was measured by sampling).

Wherein, the composition of the LB culture medium is as follows: 5g/L yeast powder, 10g/L tryptone and 10g/L sodium chloride. The composition of the TB medium is as follows: 12g/L peptone, 24g/L yeast powder, 4ml/L glycerin, 2.31g/L monopotassium phosphate and 12.54g/L dipotassium phosphate. The P1 recombinant strain is added with 100mg/L ampicillin and 50mg/L kanamycin when culturing and inducing protein expression; the P2 recombinant strain is added with 100mg/L ampicillin, 50mg/L kanamycin and 25mg/L chloramphenicol when culturing and inducing protein expression; the P3 recombinant strain was added with 25mg/L chloramphenicol at the time of culturing and inducing protein expression.

Example 4: d-pantolactone is synthesized by racemizing DL-pantolactone under the combination of single-strain fermentation and multienzyme catalysis

The racemization reaction system of DL-pantolactone is 1L, which comprises the following components: the recombinant strain P1 or P2 obtained in example 3 (containing 50g of wet cells in total according to wet weight conversion), crude enzyme solution of different concentrations of DL-pantolactone (0.5M, 0.75M, 1.0M, 1.5M), different concentrations of auxiliary substrate glucose (0.3M, 0.45M, 0.6M, 0.9M), water was added to a volume of 1L, and the mixture was stirred and dissolved, then added to a three-necked flask, and the temperature was raised to 35℃and stirred at 600rpm for 36 hours, and the pH of the reaction solution was adjusted to 6.5 with ammonia water. After the reaction is finished, sampling and measuring the amount of ketopantoic acid in a reaction system, then adjusting the acid to pH=1.5 by using 2M sulfuric acid, heating to 80 ℃ for reaction for 1 hour to obtain a conversion solution, performing high performance liquid chromatography detection on the conversion solution, and respectively calculating an ee value and a D-pantolactone conversion rate. The reaction results are shown in Table 2.

As can be seen from Table 2, when the crude enzyme liquid obtained by fermentation of P1 strain which co-expresses L-pantolactone dehydrogenase, ketopantolactone reductase and glucose dehydrogenase is used to catalyze the racemization of DL-pantolactone, all L-pantolactone in the system can be converted when the substrate concentration is 0.5M to 1.5M, and the ee value is more than 99%; when the substrate concentration is 0.5M-0.75M, L-pantolactone in the system can be completely converted into D-lactone, the conversion rate is more than 99%, but when the substrate concentration is 1.0M and 1.5M, obvious ketopantoic acid accumulation occurs in the catalytic end reaction system, so that the conversion rate of D-pantolactone in the conversion solution is only 95.01% and 89.23%, respectively.

When crude enzyme liquid which can co-express L-pantolactone dehydrogenase, ketopantolactone reductase, glucose dehydrogenase and ketopantolactone reductase and is fermented by P2 strain is used for catalyzing DL-pantolactone to despinpoint, when the concentration of a substrate is 0.5M-1.5M, all L-pantolactone in a system can be converted, and the ee value is more than 99%; when the substrate concentration is 1.0M and 1.5M, the ketopantoic acid accumulated in the catalytic end reaction system can be obviously reduced, and the conversion rate of D-pantolactone in the corresponding conversion solution is increased to 98.82% and 95.51%.

Example 5: d-pantolactone is synthesized by racemizing DL-pantolactone under the combined catalysis of double-strain fermentation multienzyme

The racemization reaction system of DL-pantolactone is 1L, which comprises the following components: the crude enzyme solution of the recombinant strain P1 obtained in example 3 (containing 50g of wet cells in total according to wet weight), the crude enzyme solution of the recombinant strain P3 obtained in example 3 (containing 10g of wet cells in total according to wet weight), DL-pantolactone (0.5M, 0.75M, 1.0M, 1.5M) at different concentrations, and glucose as a co-substrate (0.3M, 0.45M, 0.6M, 0.9M) at different concentrations were added to a volume of 1L, and after dissolving by stirring, they were added to a three-necked flask, heated to 35℃and stirred at 600rpm for 36 hours, and the pH of the reaction solution was adjusted to 6.5 by ammonia. After the reaction is finished, sampling and measuring the amount of ketopantoic acid in a reaction system, then adjusting the acid to pH=1.5 by using 2M sulfuric acid, heating to 80 ℃ for reaction for 1 hour to obtain a conversion solution, performing high performance liquid chromatography detection on the conversion solution, and respectively calculating an ee value and a D-pantolactone conversion rate. The reaction results are shown in Table 2.

As can be seen from Table 2, when the crude enzyme liquid fermented by the P1 strain capable of coexpression of L-pantolactone dehydrogenase, ketopantolactone reductase and glucose dehydrogenase and the crude enzyme liquid fermented by the P3 strain capable of coexpression of ketopantolactone reductase and glucose dehydrogenase together catalyze the racemization of DL-pantolactone, all L-pantolactone in the system can be converted when the substrate concentration is 0.5M to 1.5M, and the ee value is > 99%; when the substrate concentration is 1.0M and 1.5M, the accumulation of ketopantoic acid is hardly detected in the catalytic end reaction system, so that the conversion rate of the conversion solution D-pantolactone can be respectively improved to 99.02 percent and 98.83 percent.

Table 2. Comparative ee value and conversion of D-pantolactone by combination of multiple enzymes to catalyze the racemization of DL-pantolactone.

Remarks: and/represents undetected.

Sequence listing

SEQ ID NO.1 nucleotide sequence of L-pantolactone dehydrogenase (GenBank: MBY 4014232.1) encoding gene derived from Rhodococcus collecticus (Rhodococcus fascians), codon optimization according to codon preference of E.coli (E.coli), artificial synthesis

ATGGCGAAAAGCGCGTGGTTTGAAACCGTGGCGGAAGCGCAGCGCCGCGCGAAAA

AACGCCTGCCGAAAAGCGTGTATGCGGCGCTGGTGGCGGGCAGCGAACGCGGCAT

TACCATTGATGATAACATGGCGGCGTTTGGCGAACTGGGCTTTGCGCCGCATGTGGC

GGGCCTGAGCGATAAACGCGATCTGAGCACCACCGTGATGGGTCAGCCGCTGAGCT

TTCCGGTGATGATTAGTCCAACCGGCGTGCAAGCCGTTCATCCGGATGGTGAAGTG

GCGGTGGCGCGCGCGGCCGCGGCCCGCGGCATTCCGATTGGCCTGAGCAGCTTTGC

GAGCAAAAGCGTGGAAGAAGTGGCGGCCGCGAACCCGCAGACCTTTTTTCAGATG

TATTGGGTGGGCAGCCGCGAAATTCTGCTGCAGCGCATGGAACGCGCCCGCGCGGC

CGGCGCGGTGGGCCTGATTATGACCCTGGATTGGAGCTTTAGCAACGGCCGCGATT

GGGGCAGCCCGAGCATTCCGGAAAAAATGGATCTGAAAGCGATGGTGCAGTTTGC

GCCGGAAGGCGTGATGCGCCCGAAGTGGCTGTGGGAATTTGCGAAAACCGGCAAA

ATTCCGGATCTGACCACCCCGAACCTGACCCCGCCGACCGGCGGCCCGGCGCCGAC

CTTTTTTGGCGCGTATGGCGAATGGATGGGCACCCCGCTGCCGACCTGGGAAGATG

TGGCGTGGCTGCGCGAACAGTGGGGCGGCCCGTTTATGCTGAAGGGCGTTATGCGC

GTTGATGATGCGAAACGCGCGGTGGATGCGGGCGTGACCGCGATTAGCGTGAGCAA

CCATGGCGGCAACAACCTGGATGGCACCCCGGCGCCGATTCGCGCGCTGCCGGCGA

TTGCGGAAGCGGTGGGCGATCAAGTGGAAATTACCCTGGATGGCGGCATTCGCCGC

GGCAGCGATGTGGTTAAGGCGCTGGCCCTGGGTGCCCGCGCCGTGTTAATTGGTCG

CGCGTACCTGTGGGGCCTGAGTGCGGGCGGCCAAGCGGGCGTGGAAAACGTGCTG

GATATTCTGCGCGGCGGCGTGGATAGCGCGGTGCTGGGCCTGGGCCATACGAGCGT

GCATGATCTGAGCCCGAGCGATGTGGTGATGCCGGCGGGCTTTGATCGCAAACTGG

GCGTGTAA

Amino acid sequence of L-pantolactone dehydrogenase:

MAKSAWFETVAEAQRRAKKRLPKSVYAALVAGSERGITIDDNMAAFGELGFAPHVAGL

SDKRDLSTTVMGQPLSFPVMISPTGVQAVHPDGEVAVARAAAARGIPIGLSSFASKSVEE

VAAANPQTFFQMYWVGSREILLQRMERARAAGAVGLIMTLDWSFSNGRDWGSPSIPE

KMDLKAMVQFAPEGVMRPKWLWEFAKTGKIPDLTTPNLTPPTGGPAPTFFGAYGEWM

GTPLPTWEDVAWLREQWGGPFMLKGVMRVDDAKRAVDAGVTAISVSNHGGNNLDG

TPAPIRALPAIAEAVGDQVEITLDGGIRRGSDVVKALALGARAVLIGRAYLWGLSAGGQAGVENVLDILRGGVDSAVLGLGHTSVHDLSPSDVVMPAGFDRKLGV(SEQ ID NO:5)SEQ ID NO:2 Nucleotide sequence of ketopantolactone reductase (GenBank: WLF 81497.1) encoding gene derived from Luoder long spore yeast (Lodderomyces elongisporus), codon optimization according to codon preference of Escherichia coli (E.coli), artificial synthesis

ATGACCTATAGTCAGAGCCATCCGGTGCAGCTGCATCCGCAGTTTAAAACCAAAAG

CGGTCAGCCGCTGAGCATGGGCACCGGCACCGGCACCAAATGGAAAAAAGATCAA

GATAAAAGCGATATTAACGAAGAACTGGTGGAACAGCTGCTGTTAAGCCTGAAACT

GGGCTATCGCCATATTGATACCGCGGAAGTGTATAACACCCATAAAGAAGTGGGCGA

AGCGCTGAAACGCACCGATATTCCGCGCGAAGATCTGTGGATTACCACCAAATATAA

CGGCGGCTGGGGTCAGCATAAAGGCAGCGAAAGCCCGGAAAAAAGCATTGAAAGC

GCGCTGAAAGATCTGGGCGTGGAATATATTGATCTGTTTCTGATTCATAGCCCGTTTT

TTAGCACCGATGTGAGCAACGGCTATACCCTGGAAGATGTGTGGAAAGTGCTGATT

GAAGCGAAAAAACAAGGCAAAGTGCGCGAAATTGGCATTAGCAACGCGGCGAAA

GTGCATCTGGAACGCCTGTATAAAGTGAGCCCGAGCCCGGAATTTTATCCGGTGGTG

AATCAGATTGAATTTCATGCGTTTCTGCAGAATCAGAGCACCGATATTGTGAAATATA

GCCAAGAACAAGGCATTCTGGTGGAAGCGTTTAGCCCGCTGGCGCCGCTGGCGCG

CGTGAAAGATGGCGCGCTGGGCGGCCTGCTGAAAGAACTGAGCAGCAAATATAGC

AAAACCGATGCGCAGCTGTTACTGCGCTATGTGCTGCAGAAAGGCGTGCTGCCGAT

TACCACGAGCAGCAAAGAAAGCCGCATTAAAGAAAGCCTGGATCTGTTTGATTTTG

AACTGACCGCGGAAGAAGTGCAAGAAATTGATAAAATTGGCAAAGAAAACCCGTA

TCGCGCGTTTTTTCATGAACAGTTTAAAGATCTGTAA

Amino acid sequence of ketopantolactone reductase:

MTYSQSHPVQLHPQFKTKSGQPLSMGTGTGTKWKKDQDKSDINEELVEQLLLSLKLG

YRHIDTAEVYNTHKEVGEALKRTDIPREDLWITTKYNGGWGQHKGSESPEKSIESALK

DLGVEYIDLFLIHSPFFSTDVSNGYTLEDVWKVLIEAKKQGKVREIGISNAAKVHLERL

YKVSPSPEFYPVVNQIEFHAFLQNQSTDIVKYSQEQGILVEAFSPLAPLARVKDGALGG

LLKELSSKYSKTDAQLLLRYVLQKGVLPITTSSKESRIKESLDLFDFELTAEEVQEIDKIGKENPYRAFFHEQFKD(SEQ ID NO:6)

SEQ ID NO. 3A nucleotide sequence encoding glucose dehydrogenase (GenBank: ACB 59697.1) derived from Microbacterium (Exiguobacterium sibiricum), codon optimization according to the codon preference of E.coli, and artificial synthesis

ATGGGCTATAACAGCCTGAAAGGCAAAGTGGCGATTGTGACCGGCGGCAGCATGGG

CATTGGCGAAGCGATTATTCGCCGCTATGCGGAAGAAGGCATGCGCGTGGTGATTAA

CTATCGCAGCCATCCGGAAGAAGCGAAAAAAATTGCGGAAGATATTAAACAAGCGG

GCGGCGAAGCGCTGACCGTTCAAGGCGATGTGAGCAAAGAAGAAGATATGATTAA

CCTGGTGAAACAGACCGTGGATCATTTTGGTCAGCTGGATGTGTTTGTGAACAACG

CGGGCGTGGAAATGCCGAGCCCGAGCCATGAAATGAGCCTGGAAGATTGGCAGAA

AGTGATTGATGTGAACCTGACCGGCGCGTTTCTGGGCGCGCGCGAGGCCCTGAAAT

ATTTTGTGGAACATAACGTGAAAGGCAACATTATTAACATGAGCAGCGTGCATGAAA

TTATTCCGTGGCCGACCTTTGTGCATTATGCGGCGAGCAAAGGCGGCGTGAAACTG

ATGACGCAGACCCTGGCGATGGAATATGCGCCGAAAGGCATTCGCATTAACGCGATT

GGCCCGGGCGCGATTAACACCCCGATTAACGCGGAAAAATTTGAAGATCCGAAACA

GCGCGCGGATGTGGAAAGCATGATTCCGATGGGCAACATTGGCAAACCGGAAGAA

ATTAGCGCGGTGGCGGCGTGGCTGGCGAGCGATGAAGCGAGCTATGTGACCGGCAT

TACCCTGTTTGCGGATGGCGGCATGACCCTGTATCCGAGCTTTCAAGCGGGCCGCG

GCTAA

Amino acid sequence of glucose dehydrogenase:

MGYNSLKGKVAIVTGGSMGIGEAIIRRYAEEGMRVVINYRSHPEEAKKIAEDIKQAGGE

ALTVQGDVSKEEDMINLVKQTVDHFGQLDVFVNNAGVEMPSPSHEMSLEDWQKVID

VNLTGAFLGAREALKYFVEHNVKGNIINMSSVHEIIPWPTFVHYAASKGGVKLMTQTL

AMEYAPKGIRINAIGPGAINTPINAEKFEDPKQRADVESMIPMGNIGKPEEISAVAAWLASDEASYVTGITLFADGGMTLYPSFQAGRG(SEQ ID NO:7)

SEQ ID NO.4 nucleotide sequence of ketopantoate reductase (PanE, genBank: NP-414959) encoding gene from Escherichia coli (E.coli), obtained by PCR amplification

ATGAAAATTACCGTATTGGGATGCGGTGCCTTAGGGCAATTATGGCTTACAGCACTTT

GCAAACAGGGTCATGAAGTTCAGGGCTGGCTGCGCGTACCGCAACCTTATTGTAGC

GTGAATCTGGTTGAGACAGATGGTTCGATATTTAACGAATCGCTGACCGCCAACGAT

CCCGATTTTCTCGCCACCAGCGATCTGCTCCTGGTGACGCTGAAAGCATGGCAGGT

TTCCGATGCCGTCAAAAGCCTCGCGTCCACACTGCCTGTAACTACGCCAATACTGTT

AATTCACAACGGCATGGGCACCATCGAAGAGTTGCAAAACATTCAGCAGCCATTAC

TGATGGGCACCACCACCCATGCAGCCCGCCGCGACGGCAATGTCATTATTCATGTGG

CAAACGGTATCACGCATATTGGCCCGGCACGGCAACAGGACGGGGATTACAGTTAT

CTGGCGGATATTTTGCAAACCGTGTTGCCTGACGTTGCCTGGCATAACAATATTCGC

GCCGAGCTGTGGCGCAAGCTGGCAGTCAACTGCGTGATTAATCCACTGACTGCCAT

CTGGAATTGCCCGAACGGTGAATTACGTCATCATCCGCAAGAAATTATGCAGATATG

CGAAGAAGTCGCGGCGGTGATCGAACGCGAAGGGCATCATACTTCAGCAGAAGATT

TGCGTGATTACGTGATGCAGGTGATTGATGCCACAGCGGAAAATATCTCGTCGATGT

TGCAGGATATCCGCGCGCTGCGCCACACTGAAATCGACTATATCAATGGTTTTCTCTT

ACGCCGCGCCCGCGCGCATGGGATTGCCGTACCGGAAAACACCCGCCTGTTTGAAA

TGGTAAAAAGAAAGGAGAGTGAATATGAGCGCATCGGCACTGGTTTGCCTCGCCCC

TGGTAG

Amino acid sequence of ketopantoate reductase

MKITVLGCGALGQLWLTALCKQGHEVQGWLRVPQPYCSVNLVETDGSIFNESLTANDP

DFLATSDLLLVTLKAWQVSDAVKSLASTLPVTTPILLIHNGMGTIEELQNIQQPLLMGTT

THAARRDGNVIIHVANGITHIGPARQQDGDYSYLADILQTVLPDVAWHNNIRAELWRK

LAVNCVINPLTAIWNCPNGELRHHPQEIMQICEEVAAVIEREGHHTSAEDLRDYVMQVI

DATAENISSMLQDIRALRHTEIDYINGFLLRRARAHGIAVPENTRLFEMVKRKESEYERIGTGLPRPW(SEQ ID NO:8)

Primer sequence listing

While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

Claims (12)

1. An enzyme combination comprising an L-pantolactone dehydrogenase, a ketopantolactone reductase, a glucose dehydrogenase and a ketopantolactone reductase.

2. The enzyme combination according to claim 1, wherein the L-pantoate dehydrogenase is selected from rhodococcus collecticus (Rhodococcus fascians), the ketopantoate reductase is selected from rhodotorula longa (Lodderomyces elongisporus), the glucose dehydrogenase is selected from microbacterium (Exiguobacterium sibiricum), and the ketopantoate reductase is selected from escherichia coli (e.coli);

Preferably, the amino acid sequence of the L-pantolactone dehydrogenase is shown as SEQ ID NO. 5, the amino acid sequence of the ketopantolactone reductase is shown as SEQ ID NO. 6, the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO. 7, and the amino acid sequence of the ketopantolactone reductase is shown as SEQ ID NO. 8.

3. A recombinant vector comprising a nucleotide sequence encoding the enzyme combination of claim 1 or 2;

preferably, the nucleotide sequence for encoding the L-pantolactone dehydrogenase is shown as SEQ ID NO. 1, the nucleotide sequence for encoding the ketopantolactone reductase is shown as SEQ ID NO. 2, the nucleotide sequence for encoding the glucose dehydrogenase is shown as SEQ ID NO. 3, and the nucleotide sequence for encoding the ketopantolactone reductase is shown as SEQ ID NO. 4.

4. The recombinant vector of claim 3, wherein the nucleotide sequences encoding the enzyme combination are on the same vector or separate vectors;

Preferably, the separate vectors are a first vector comprising a nucleotide sequence encoding an L-pantolactone dehydrogenase, a second vector comprising a nucleotide sequence encoding a ketopantolactone reductase and a glucose dehydrogenase, and a third vector comprising a nucleotide sequence encoding a glucose dehydrogenase and a ketopantolactone reductase;

More preferably, the backbone plasmid of the first vector is pET21a, the backbone plasmid of the second vector is pRSFDuet-1, and the backbone plasmid of the third vector is pACYCDuet-1.

5. A recombinant engineering bacterium, characterized in that it comprises the enzyme combination according to claim 1 or 2, or the recombinant vector according to claim 3 or 4; preferably, the recombinant engineering bacteria comprise a first vector, a second vector and a third vector, wherein the first vector comprises a nucleotide sequence for encoding L-pantolactone dehydrogenase, the second vector comprises a nucleotide sequence for encoding ketopantolactone reductase and glucose dehydrogenase, and the third vector comprises a nucleotide sequence for encoding glucose dehydrogenase and ketopantolactone reductase.

6. The recombinant engineering bacteria combination is characterized by comprising a first recombinant engineering bacteria and a second recombinant engineering bacteria, wherein the first recombinant engineering bacteria comprise a first carrier and a second carrier, and the second recombinant engineering bacteria comprise a third carrier; wherein the first vector comprises a nucleotide sequence encoding an L-pantolactone dehydrogenase, the second vector comprises a nucleotide sequence encoding a ketopantolactone reductase and a glucose dehydrogenase, and the third vector comprises a nucleotide sequence encoding a glucose dehydrogenase and a ketopantolactone reductase.

7. A method of constructing a recombinant engineering bacterium comprising introducing the recombinant vector of claim 3 or 4 into a host cell; preferably, the host cell is selected from the group consisting of E.coli; more preferably, the host cell is Escherichia coli BL21 (DE 3).

8. A method for culturing recombinant engineering bacteria or recombinant engineering bacteria combination is characterized in that,

The method comprises the steps of fermenting and culturing the recombinant engineering bacteria of claim 5 or the recombinant engineering bacteria of claim 6 in a culture medium;

Preferably, the method further comprises seed culture to obtain seed liquid before fermentation culture, and IPTG is added for induction after fermentation culture; preferably, the culture medium for fermentation culture is TB, and the culture medium for seed culture is LB;

More preferably, the recombinant engineering bacteria or the recombinant engineering bacteria combination are inoculated into LB culture medium according to 0.1-5% of inoculation amount, and the seed solution is inoculated into TB culture medium according to 1-10% of inoculation amount.

9. A method for preparing the enzyme combination according to claim 1, characterized in that it comprises the following steps:

(1) Culturing a recombinant engineering bacterium or a combination of recombinant engineering bacteria using the method of claim 8;

(2) And collecting thalli to obtain bacterial suspension.

10. An enzyme reaction system comprising the enzyme combination of claim 1; preferably, one or more of DL-pantolactone and glucose are also included.

11. A process for the preparation of D-pantolactone, characterized in that it comprises the steps of: placing DL-pantolactone in a reaction system containing glucose and the enzyme combination as defined in claim 1 or 2, and reacting at 30-40 ℃ and pH5.0-7.0 for 20-40h to obtain the D-pantolactone.

12. Use of the enzyme combination according to claim 1 or 2, the recombinant engineering bacterium according to claim 5, the recombinant engineering bacterium combination according to claim 6 or the enzyme reaction system according to claim 10 for the preparation of D-pantolactone.