CN115786295A - L-pantolactone dehydrogenase, coding gene and application - Google Patents

Abstract

The invention relates to L-pantolactone dehydrogenase, a coding gene, a carrier containing the coding gene, a gene engineering bacterium and application thereof in preparing ketopantolactone under the catalysis of microorganisms. The amino acid sequence of the L-pantolactone dehydrogenase is shown as SEQ ID NO.1, and the coding gene is shown as SEQ ID NO. 2. The L-pantolactone dehydrogenase derived from pseudomona sp.73-21 excavated by the invention has strict L-stereoselectivity, the recombinant Escherichia coli constructed by the invention has good solubility for expressing the L-pantolactone dehydrogenase, and the problem that a large amount of inclusion bodies are easily formed by heterologous expression of the L-pantolactone dehydrogenase is solved. The L-pantolactone dehydrogenase provided by the invention has application value in preparing ketopantolactone/D-pantolactone by biocatalysis.

Description

L-pantolactone dehydrogenase, coding gene and application

(I) technical field

The invention relates to L-pantolactone dehydrogenase, a coding gene, a vector containing the coding gene, a genetic engineering bacterium and application thereof in preparing ketopantolactone/D-pantolactone under the catalysis of microorganisms.

(II) background of the invention

Calcium pantothenate is a water-soluble vitamin B group and is also one of the essential nutrients for normal growth of organisms. Calcium pantothenate is available in three forms, namely, D-calcium pantothenate (D-pantothenate), DL-calcium pantothenate (DL-pantothenate) and L-calcium pantothenate (L-pantothenate), of which only D-calcium pantothenate is biologically active. Pantothenic acid has multiple physiological functions as coenzyme A precursor. Calcium pantothenate is widely used in the industries of feed, medicine, food and the like due to its unique biochemical function. At present, calcium D-pantothenate is synthesized by beta-calcium aminopropionate and D-pantoic acid lactone.

D- (-) -pantoic acid lactone, also called (R) -pantoic acid lactone, is gamma-lactone of D- (-) -pantoic acid and is a key chiral intermediate for synthesizing D- (+) -pantothenic acid. The industrial synthesis of D-pantoic acid lactone is a technical route combining a chemical method and a chemical method-a hydrolytic enzyme resolution method. The generation ways of the intermediate mixture DL-pantoic acid lactone in the two ways are consistent, and DL-pantoic acid lactone is synthesized by a chemical method starting from isobutyraldehyde and formaldehyde as starting materials; the industrial complete conversion of DL-pantolactone to D-pantolactone includes chemical and hydrolytic enzyme resolution. The chemical synthesis method has many limitations due to many environmental pollution problems, the use of dangerous reagents and other factors. The hydrolase splitting method selectively hydrolyzes D-pantoic acid lactone in the mixed pantoic acid lactone to generate D-pantoic acid, then the D-pantoic acid and L-pantoic acid lactone are separated, the separated D-pantoic acid is acidified to form ring to form D-pantoic acid lactone, and the L-pantoic acid lactone is racemized and recycled. Therefore, although the process is mature, the chiral resolution method catalyzed by hydrolase still has the problems of long steps, high acid and base consumption and the like. In view of this, the development of a more direct, efficient and environment-friendly asymmetric synthesis method of D-pantolactone to replace the existing chiral resolution technology has important application value.

The first method is to take DL-pantolactone as a substrate, utilize L-pantolactone dehydrogenase with specific stereoselectivity to catalyze L-pantolactone to dehydrogenate to generate ketopantolactone, and then the ketopantolactone is asymmetrically generated to generate the D-pantolactone under the catalysis of the D-ketopantolactone reductase; the second approach is also to catalyze the dehydrogenation of L-pantolactone with L-pantolactone dehydrogenase to produce ketopantolactone, then the spontaneous hydrolysis of ketopantolactone to form ketopantoate, then the D-pantoate is produced under the action of D-ketopantoate reductase, and then the ring closure of D-pantoate is carried out under the action of acid to form D-pantolactone. The process is simpler and more convenient by the first path, compared with the existing hydrolase catalysis path, the process is simpler, the substrate of the mixed rotation directly obtains an optical pure product by biological catalysis, a racemization step is not needed, and a separation step of lactone and acid is not needed; a coenzyme circulating system is constructed in the genetic engineering bacteria, and the coenzyme does not need to be added; the genetic engineering bacteria are used as the whole cell catalyst, and the separation and purification steps of enzyme are not needed. Therefore, the method for asymmetrically synthesizing D-pantolactone by using oxidoreductase is a very promising substitute for biological hydrolysis enzyme method. Dehydrogenation of L-pantolactone in a redox process is one of its key steps, and L-pantolactone dehydrogenase is a key enzyme catalyzing the reaction.

The number of known L-pantolactone dehydrogenases is small, and the L-pantolactone dehydrogenases derived from Rhodococcus erythropolis and Nocardia asteroides have been studied more. L-pantolactone dehydrogenase derived from Rhodococcus erythropolis is less soluble expressed in an E.coli system, limiting the catalytic ability of the enzyme on a substrate. A gene engineering bacterium AKU2103 which enhances the expression of an L-pantolactone dehydrogenase gene of rhodococcus erythropolis in the same rhodococcus erythropolis is used as a biocatalyst to catalyze the dehydrogenation reaction of 0.768M of L-pantolactone for 144h, and the conversion rate of the reaction is 91.9%. Although L-pantoate lactone dehydrogenase derived from nocardia asteroides has more detailed enzymatic property research, the protein is easy to form inclusion bodies when being expressed in escherichia coli, and the enzyme activity is low, so that the further application of the protein in biocatalysis is prevented. The lack of L-pantolactone dehydrogenase having excellent catalytic performance limits the use of the redox enzyme method for the asymmetric synthesis of D-pantolactone.

Disclosure of the invention

The invention aims to provide a novel L-pantolactone dehydrogenase, a coding gene thereof, a carrier containing the coding gene, a genetic engineering bacterium and application thereof in preparing keto-pantolactone/D-pantolactone under the catalysis of microorganisms.

The technical scheme adopted by the invention is as follows:

an L-pantolactone dehydrogenase whose amino acid sequence is shown in SEQ ID NO. 1.

Through a large amount of previous screening work, the invention discovers a protein derived from a microorganism Pseudonocardia sp.73-21, which has L-pantolactone dehydrogenase activity, and the prior art does not discover that the protein has the function, and the protein can catalyze L-pantolactone to generate ketopantolactone. The protein with the L-pantolactone dehydrogenase activity has good solubility, so that the protein can be fully dissolved in a catalytic reaction liquid, and the catalytic activity can be fully exerted when the protein is applied to catalyzing relevant reactions. The discovery of the protein has important practical significance for the fermentation production of L-pantolactone dehydrogenase in microorganisms and the production of related catalytic products thereof.

Without altering the biochemical properties of the L-pantolactone dehydrogenase, it is within the scope of the present invention that certain modifications of its amino acid sequence, for example the introduction of deletions, additions and/or substitutions of one or several amino acid residues, are possible for the skilled worker.

The invention also relates to a gene coding the L-pantolactone dehydrogenase.

Preferably, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

The invention also relates to a recombinant vector and a genetic engineering bacterium containing the L-pantolactone dehydrogenase coding gene.

The invention also relates to the expression of a recombinant vector containing the L-pantolactone dehydrogenase coding gene in Escherichia coli. Specifically, the L-pantolactone dehydrogenase is prepared by the following method: inoculating recombinant engineering bacteria containing a vector of an L-pantolactone dehydrogenase encoding gene into an LB liquid culture medium containing kanamycin with the final concentration of 50 mug/mL, and culturing at 37 ℃ for 10 hours to obtain a seed solution; the seed solution was inoculated into a fresh LB liquid medium containing kanamycin to a final concentration of 50. Mu.g/mL in an inoculum size of 1.0% by volume, and cultured at 37 ℃ and 180rpm for 2 hours (OD) ₆₀₀ = 0.4-0.6), adding Isopropyl thiogalactoside (IPTG) with a final concentration of 0.1mM into the culture solution, culturing at 20 ℃ for 12h, and centrifuging at 4 ℃ and 8000rpm for 10min to obtain wet thalli containing L-pantolactone dehydrogenase.

Further, the solubility of the recombinant Escherichia coli expressing L-pantolactone for dehydrogenation is studied and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Wet cells containing L-pantolactone dehydrogenase were suspended in a pH 7.0, 50mM PB buffer at a weight of 10g/L, and sonicated on an ice-water mixture for 30min under the sonication conditions: the amplitude is 50%, the crushing is carried out for 1s, the suspension is carried out for 2s, the crushing mixed solution is taken, the centrifugation is carried out for 10min at 8000rpm and 4 ℃, the supernatant is collected, and the equal volume of PB buffer solution is added into the sediment for carrying out the resuspension. 60 μ L of the supernatant and the pellet were separately added to a 1.5mL EP tube, and 20 μ L of 4 XProtein loading buffer was added and mixed. After the protein sample is boiled for 10min, 10 μ L of the sample is loaded into protein pre-fabricated gel (Kinsrui, nanjing) and subjected to 180V electrophoresis for 30min. After being dyed and decolored by a protein dyeing instrument (eStain L1, kisry), the expression of L-pantolactone dehydrogenation is analyzed by a protein imaging system. The L-pantolactone dehydrogenase from Pseudonocardia sp.73-21 is very soluble in E.coli and almost completely dissolved in the supernatant.

The invention also relates to the substrate specificity of the L-pantolactone dehydrogenase. The L-pantolactone dehydrogenase can specifically catalyze L-pantolactone or L-isomer in racemate to generate ketopantolactone. The substrates D-pantolactone, D-pantoate and L-pantoate cannot be recognized, indicating that the L-pantolactone dehydrogenase derived from Pseudonocardia sp.73-21 has strict L-selectivity.

The invention has the following beneficial effects: the L-pantolactone dehydrogenase derived from pseudomona sp.73-21 excavated by the invention has strict L-stereoselectivity, the recombinant Escherichia coli constructed by the invention has good solubility for expressing the L-pantolactone dehydrogenase, and the problem that a large amount of inclusion bodies are easily formed by heterologous expression of the L-pantolactone dehydrogenase is solved. The L-pantolactone dehydrogenase provided by the invention has application value in preparing ketopantolactone/D-pantolactone by biocatalysis.

Description of the drawings

FIG. 1 is a gas phase diagram of PseLPLDH catalyzed substrate L-pantolactone to produce product ketopantolactone in accordance with an embodiment of the present invention.

FIG. 2 is a gas phase diagram of the retention time of L-pantolactone in accordance with an embodiment of the present invention;

FIG. 3 is a gas phase diagram of retention time of ketopantoate lactone in accordance with an embodiment of the present invention;

FIG. 4 is a calibration curve for L-pantolactone of an embodiment of the present invention;

FIG. 5 is a calibration curve of ketopantolactone in accordance with one embodiment of the present invention;

FIG. 6 is a graph showing a comparison of the solubility of L-pantolactone dehydrogenase of different sequences in one embodiment of the present invention.

(V) detailed description of the preferred embodiments

The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples:

example 1: construction of bacterial cells

The gene SEQ ID NO 2 after codon optimization is constructed on an expression vector pET 28a, and a 6-chi-is label is added at the C end to facilitate subsequent protein expression. The construction process is as follows:

1. f-1 and R-1 in Table 1 are taken as primers, and PseLPLDH is taken as a template to expand a gene fragment 1;

2. f-2 and R-2 in the table 1 are used as primers, and pET 28a is used as a template to amplify a vector fragment 2;

3. purifying the fragment 1 and the fragment 2, and digesting the purified fragment 1 and the purified fragment 2 by using NcoI and XhoI;

4. connecting the purified fragment 1 and the fragment 2 by using T4 ligase, and transforming the Escherichia coli BL21 competent cells by using an enzyme-linked product;

5. single colonies were picked and grown in liquid medium containing 50. Mu.g/. Mu.l kanamycin and sent for sequencing.

Table 1: amplification primers

Example 2: substrate catalysis

A single colony of the engineered strain constructed in example 1 was picked and inoculated into LB medium containing 50. Mu.g/. Mu.L kanamycin, and cultured overnight on a shaker at 37 ℃ and 180 rpm. Inoculating the seed solution to 100mL LB liquid medium containing 50. Mu.g/. Mu.L kanamycin at an inoculation amount of 1%, culturing at 37 deg.C for 2-2.5h in a shaker at 180rpm, and adjusting OD of the bacterial solution ₆₀₀ Up to between 0.6 and 0.8. To the medium was added Isopropyl thiogalactoside (IPTG) at a final concentration of 0.1mM at 28 ℃ for 12h at 180 rpm. Then, the cells were centrifuged at 8000rpm for 10min at 4 ℃ to obtain wet cells containing the L-pantolactone dehydrogenase-active protein PseLPLDH.

Two portions of PB buffer (0.2M Na) at pH 7.0 and 50mM were prepared ₂ HPO ₄ ，0.2M NaH ₂ PO ₄ ) The reaction medium was used to resuspend 10g/L of wet cells, one portion was added with 1mM substrate L-pantolactone and the other portion was added with 1mM substrate D-pantolactone, and the two samples were reacted at 30 ℃ for 30min in a thermostatted shaker at 800 rpm. 200uL of the reaction mixture was quenched with 6M HCl of the same volume, extracted 3 times with 200uL of ethyl acetate, and the ethyl acetate phases were combined. And centrifuging the extract, absorbing the upper organic phase into a centrifuge tube, adding anhydrous sodium sulfate to remove water, centrifuging again, taking the supernatant, and transferring into a gas phase sample bottle for gas chromatography detection. GC is adopted to detect the concentration and the conversion rate of L-pantolactone and L-pantolactone. Gas phaseDetection conditions are as follows: column agilent cyclossil-B (30 m × 0.25mm,0.25 μm), carrier gas: helium, flow rate: 0.5mL/min, injection port, detector temperature: 250 ℃; sample injection amount: 1 mu L of the solution; the split ratio is as follows: 30; the procedure is as follows: heating to 140 deg.C at 100 deg.C and 10 deg.C/min, and maintaining for 5min. The retention times of L-pantolactone and ketopantolactone are respectively as follows: 9.2min and 6.6min. As shown in FIGS. 1 to 5, the gas phase patterns of the retention times of L-pantolactone and ketopantolactone, respectively, and the respective standard curves are shown. As a result, it was confirmed that SEQ ID NO 2 of the strain constructed in this example can encode a protein having L-pantolactone dehydrogenase activity, thereby catalyzing the reaction of L-pantolactone. After the reaction is carried out for 30min, the conversion rate of the substrate L-pantolactone reaches more than 40 percent.

Example 3: protein induced expression

Wet bacteria after expression of the protein having L-pantolactone dehydrogenase activity of example 2 by PseLPLDH and L-pantolactone dehydrogenase NsLPLDH derived from Nocardia asteroides in Escherichia coli BL21 (DE 3) were washed twice with 0.9g/mL physiological saline. Adding the wet thallus into 100mM PB buffer solution with the pH value of 7.0 according to the amount of 10g/L of the total wet thallus, resuspending the mixture, and carrying out ultrasonic crushing on an ice-water mixture for 5min under the ultrasonic crushing condition: amplitude 10%, crushing for 1s, pausing for 2s, taking the crushed mixture, centrifuging at 8000rpm for 10min at 4 ℃, collecting the supernatant (superntant) and the precipitate (segment), and identifying the size and soluble expression of the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As a result, as shown in FIG. 6, it was revealed that the protein expressing L-pantolactone dehydrogenase activity of example 1 was very soluble and almost completely dissolved in the supernatant; the L-pantolactone dehydrogenase from Nocardia asteroides is poorly soluble and is mostly distributed in the precipitate.

Example 4: investigation of substrate specificity of L-pantolactone dehydrogenase

The wet cells of example 2, in which PseLPLDH expressing a protein having L-pantolactone dehydrogenase activity was expressed in Escherichia coli BL21 (DE 3), were used as biocatalysts. The substrate specificity of L-pantolactone dehydrogenase derived from Pseudomonas sp.73-21 was examined by whole-cell catalysis using 10mM of D-pantolactone, L-pantolactone and DL-pantolactone as substrates, respectively. The reaction system for catalyzing L-pantolactone dehydrogenation by L-pantolactone dehydrogenase is 1mL, and comprises the following components: 10g/L wet cells, 10mM substrate and 200mM phosphate buffer (pH 7.0). The reaction solution was put into an EP tube, and the reaction conditions were maintained at 30 ℃ and 1200rpm on a constant temperature shaker. After 30min of reaction, 200. Mu.L of the reaction solution was added with 6M hydrochloric acid of the same volume and added with a proper amount of anhydrous sodium sulfate. The mixture was extracted three times with 200. Mu.L of ethyl acetate. And centrifuging the extract, absorbing the upper organic phase into a centrifuge tube, adding anhydrous sodium sulfate to remove water, centrifuging again, taking the supernatant, and transferring into a gas phase sample bottle for gas chromatography detection. The results of substrate specificity are shown in Table 2, and as a result, it was found that the L-pantoate dehydrogenase-active protein derived from Pseudomonas sp.73-21 can catalyze neither D-pantolactone nor D-pantoic acid nor L-pantolactone. The above results show that the L-pantoic acid dehydrogenase-active protein derived from Pseudomonas sp.73-21 acts exclusively on the dehydrogenation of L-pantoic acid lactone.

Table 2: substrate specificity of L-pantoate dehydrogenase-active protein derived from Pseudonocardia sp.73-21

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and do not limit the protection scope of the present invention, and those skilled in the art can make simple modifications or equivalent substitutions on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (6)

1. An L-pantolactone dehydrogenase whose amino acid sequence is shown in SEQ ID NO. 1.

2. A gene encoding the L-pantolactone dehydrogenase according to claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.

4. A recombinant vector comprising the gene encoding L-pantolactone dehydrogenase according to claim 2.

5. A genetically engineered bacterium containing the gene encoding L-pantolactone dehydrogenase according to claim 2.

6. Use of the L-pantolactone dehydrogenase of claim 1 in the microbial catalysis of L-pantolactone for the preparation of ketopantolactone.

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