CN113930376A

CN113930376A - Engineering bacterium for catalytic production of D-p-hydroxyphenylglycine, high-density culture method and catalytic production method

Info

Publication number: CN113930376A
Application number: CN202111181539.8A
Authority: CN
Inventors: 史大华; 陈科奇; 李婷婷; 武文龙; 杨顺; 苏佳斌; 朱海; 郑梦泽; 刘劲齐; 娄运
Original assignee: Jiangsu Ocean University
Current assignee: Jiangsu Ocean University
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-01-14

Abstract

The invention relates to the technical field of biological catalysis, in particular to an engineering bacterium for catalytically producing D-p-hydroxyphenylglycine, a high-density culture method and a catalytic production method. The engineering bacteria of the invention are constructed by respectively constructing DHD gene and DCB gene from Agrobacterium tumefaciens on pBAD carrier, and transforming the pBAD carrier successfully constructed into escherichia coli BL21(DE3) to obtain DHD engineering bacteria and DCB engineering bacteria for catalytically producing D-p-hydroxyphenylglycine. The engineering bacteria can be cultured at high density, the obtained engineering bacteria have high activity and large bacteria quantity, and the yield of more than 95 percent can be reached within 48 hours by simply crushing without purification, so that the problems existing in the current industry can be well solved.

Description

Engineering bacterium for catalytic production of D-p-hydroxyphenylglycine, high-density culture method and catalytic production method

Technical Field

The invention relates to the technical field of biological catalysis, in particular to an engineering bacterium for catalytically producing D-p-hydroxyphenylglycine, a high-density culture method and a catalytic production method.

Background

D-p-hydroxyphenylglycine (D-HPG) is an essential medical intermediate which can be used for semi-synthesizing important compounds such as penicillin amoxicillin, cephalosporins, cefoperazone and the like. However, D-HPG can be obtained by artificial synthesis, and the synthetic methods are roughly divided into two types: one is to synthesize a racemate D, L-p-hydroxyphenylglycine (D, L-HPG) by a chemical method, and then obtain the D-HPG by splitting. The method for obtaining D-HPG by splitting is gradually eliminated due to the defects of expensive raw materials, low yield, long reaction time and large amount of waste; the other is the method for synthesizing D-HPG selectively by using biological enzyme catalysis, which has high selectivity and little pollution, but has certain technical difficulty in large-scale industrial production. Therefore, the preparation of D-HPG using a bio-enzymatic method is becoming a popular area of social interest.

The principle of the bio-enzyme method is shown in the following reaction chemical formula, D, L-p-hydroxyphenylhydantoin (D, L-HPH) is catalyzed by D-hydantoinase (DHD) to generate an intermediate product D-carbamoyl-p-hydroxyphenylglycine (D-CpHPG), and the carbamoyl is removed by the action of D-carbamoyl hydrolase (DCB) to obtain a product D-p-hydroxyphenylglycine (D-HPG). This biocatalytic process for the hydantoinase, carbamoylase, is currently the predominant method for the commercial production of D-HPG.

At present, there are many research reports on the production of D-p-hydroxyphenylglycine by a biological enzyme method.

There are methods in which two enzymes are separately constructed and then reacted, but there are still disadvantages in the methods in which two enzymes are separately constructed and used for the reaction. The current research on the separate high-density fermentation processes of two enzymes is less, so that the amount of the enzymes for industrial production has a huge gap, and as described in patent CN 112080532A, a large amount of purified enzymes is added, and the total reaction time exceeds 100 hours, which is undoubtedly not beneficial to the industrial production.

Another method is to construct two enzymes on the same vector for co-expression, and the research in the direction is rapidly developed in recent years. In 2007, an example of producing L-amino acid by combining two enzymes is published in patent CN 101215533B, and in 2008, Yangqiu Liu et al carries out new construction, optimizes a new RBS region and improves the efficiency of co-expression of two enzymes; in 2019, Li Fa Bin et al used a Bacillus subtilis system for expression instead of the traditional E.coli expression system. Compared with the separate construction of two enzymes, the double-enzyme co-expression has the advantages of one-step reaction, convenience and rapidness, but because the activities of the two enzymes are different, the co-expression cannot accurately control the expression quantity of the two enzymes, and the optimal fermentation conditions of the two enzymes with different properties are different, so that the method has great limitation on how to obtain a large amount of industrial enzymes, and therefore, the method only exists in a laboratory stage.

Disclosure of Invention

In order to solve the problems, the invention provides the engineering bacteria for catalytically producing the D-p-hydroxyphenylglycine, the high-density culture method and the catalytic production method, compared with the common shake flask or basic culture medium, the fermentation of the two engineering bacteria has the advantages of obviously improved yield and obviously improved enzyme activity, and the invention is a one-pot method, has simple reaction, controls the reaction time within 48 hours and is undoubtedly obviously improved. The two engineering bacteria reconstructed by the invention have high activity and large bacteria quantity, and the yield of more than 95 percent can be reached within 48 hours only by simple crushing without purification according to fixed process conditions, thereby well solving the problems existing in the current industry.

An engineering bacterium for catalytically producing D-p-hydroxyphenylglycine is characterized in that DHD genes and DCB genes from Agrobacterium tumefaciens are respectively constructed on pBAD vectors, and the successfully constructed pBAD vectors are transformed into escherichia coli to obtain the DHD engineering bacterium and DCB engineering bacterium for catalytically producing the D-p-hydroxyphenylglycine.

Preferably, the E.coli is selected from the strain BL21(DE 3).

The high-density fermentation culture medium of the engineering bacteria comprises the following components: 25-40 g/L of yeast extract; 10-15 g/L of soybean peptone; 5-10 g/L of glucose; KH (Perkin Elmer)₂PO₄And K₂HPO₄15-30 g/L of said KH₂PO₄And K₂HPO₄The molar ratio of (A) to (B) is 1: 3-5.

Preferably, the high-density fermentation medium is: 30g/L of yeast extract; soybean peptone 10 g/L; 10g/L of glucose; KH (Perkin Elmer)₂PO₄And K₂HPO₄30 g/L。

Preferably, said KH₂PO₄And K₂HPO₄In a molar ratio of 1: 4.

The high-density culture method of the engineering bacteria, which comprises the following steps:

s1, preparing seed liquid: inoculating the preserved engineering bacterium strain in an LB liquid culture medium, and carrying out overnight culture at 37 +/-1 ℃ to prepare a seed solution;

s2, fermentation culture: transferring the seed solution grown in the step S1 into any one of the fermentation culture media for culture through aseptic operation, wherein the inoculation amount is 1% -3%, L-arabinose with the final concentration of 2-4 g/L is added for induced expression when the seed solution is cultured at 37 +/-3 ℃ to the logarithmic phase, and the induced expression time of the DCB engineering bacteria is 16-32 h; the induced expression time of the DHD engineering bacteria is 15-17 h;

and S3, after the fermentation is finished, centrifuging the fermentation liquor to obtain DHD or DCB bacterial sludge.

Preferably, the pH value of the S2 is controlled to be maintained at 6-8 in the fermentation culture process.

Preferably, the pH of the S2 is controlled to be maintained at 7.0 during the fermentation culture process.

A method for catalytically producing D-p-hydroxyphenylglycine comprises the following steps:

A. respectively using 1 XPBS buffer solution with the volume being 10 times that of the DHD and DCB bacterial sludge obtained in any one of the previous steps to carry out heavy suspension, and respectively crushing to obtain a DHD crude enzyme solution and a DCB crude enzyme solution;

adding the DHD crude enzyme solution and the DCB crude enzyme solution into a substrate with the final concentration of 5g/L, wherein the addition amount W/V (g/ml) of the DHD crude enzyme solution is 1-2%, and the mass ratio of the DHD crude enzyme solution to the DCB crude enzyme solution is 1: 7 to 9. The substrate is referred to herein as D, L-p-hydroxyphenylhydantoin.

Advantageous effects

The engineering bacteria obtained by the invention have high activity, large bacterial quantity and strong soluble expression, and the engineering bacteria do not need to be purified, only need to be simply crushed, can reach high yield within 48 hours according to fixed process conditions, and can well solve the problems existing in the current industry. The invention provides an important basis for industrial application of enzymatic synthesis of D-p-hydroxyphenylglycine, is beneficial to industrial large-scale popularization, and greatly improves the economic value and the social value.

Drawings

FIG. 1 shows the construction of engineering bacteria DHD and DCB.

FIG. 2 is a SDS-PAGE image of protein expression.

FIG. 3 Standard HPLC plots of substrate, intermediate and product.

FIG. 4 HPLC chromatogram after reaction of example 20.

Detailed Description

The following will clearly and completely describe the technical solutions in the specific embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Experimental Material and bacterial Gene

D-hydantoinase gene, D-carbamoyl hydrolase gene (GenB ank: X91070.1) and related primers from Agrobacterium tumefaciens were synthesized by Anhui general biology, Inc.;

strains of e.coli TOP10, e.coli BL21(DE3) and expression vector pBAD: both were deposited in the laboratory where the authors were located.

Restriction enzymes NcoI and HindIII, agarose and standard nucleic acid relative molecular mass, plasmid miniprep kit: all purchased from Yugong Biotechnology, Inc.;

ampicillin (Amp) was purchased from shanghai bioengineering gmbh;

yeast extract and peptone: both from Oxiod corporation, uk;

DNA gel recovery kit: from Jiangsu Yugong Life technologies Ltd

Other reagents: all are domestic analytically pure and purchased from Shanghai national drug group chemical reagents, Inc.

LB liquid medium: laboratory preparation, adding 10g of tryptone, 5g of yeast extract and 10g of NaCl into 950ml of deionized water, shaking the container until the solute is dissolved, fixing the volume to 1L by using the deionized water, and performing steam sterilization at 121 ℃ and high pressure for 20 min.

Examples characterization of enzymatic Properties

Definition of enzyme activity:

DHD: the amount of enzyme consumed to catalytically consume 1. mu. mol of substrate per minute at a given temperature and under a given condition was defined as 1U.

DCB: the amount of enzyme consumed to catalytically produce 1. mu. mol of product per minute at a given temperature and under given conditions was defined as 1U.

S1, the collected DHD cells were resuspended in PBS buffer at a concentration of 0.1g/mL, and then disrupted under high pressure. 100 mu L of crushed crude enzyme solution, 5mmol/L of D, L-HPH aqueous solution and 2ml of reaction system are put on a constant temperature shaking bed at 37 ℃ for shaking reaction for 60min, and 10 mu L of 6mol/L HCl is added to stop the reaction. The supernatant was subjected to HPLC to quantitatively determine the content of D, L-HPH.

S2, the collected cells of DCB were resuspended in PBS buffer at a concentration of 0.1g/mL, and then disrupted under high pressure. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. The supernatant was subjected to HPLC to quantitatively determine the content of D-HPG.

Example 1 construction and expression of DHD and DCB engineering bacteria

(first) construction As shown in FIG. 1, plasmid pBAD was digested simultaneously with restriction enzymes Nco I and Hind III, respectively, and the corresponding gene fragments were recovered with a DNA gel recovery kit. Then using PCR amplification to obtain D-hydantoinase gene and D-carbamyl hydrolase gene, and using DNA gel recovery kit to recover correspondent gene fragment. The DHD gene and DCB gene and their PCR primer sequences are shown below:

the DHD gene reaction system is as follows:

the DHD gene amplification process comprises the following steps:

the DCB gene reaction system is as follows:

the DCB gene amplification process comprises the following steps:

the DHD gene and DCB gene PCR products were ligated to pBAD enzyme fragments at 50 ℃ for 30min using 2 XHi-Fusion, respectively, to obtain ligation products. The ligation products were transformed into e.coli TOP10 and spread on Amp-resistant LB plates, positive clones were picked, and the correct recombinant plasmid was sent to sequencing companies for gene sequencing by colony PCR.

② active expression: the obtained recombinant plasmids pBAD-DHD and pBAD-DCB are transformed into an allelopathic bacterium of an escherichia coli expression strain E.coli BL21(DE3) by a heat shock method to obtain DE3-pBAD-DHD and DE 3-pBAD-DCB. DE3-pBAD-DHD and DE3-pBAD-DCB were inoculated into 10ml of LB medium containing Amp at a final concentration of 50. mu.g/ml, respectively, and left overnight at 37 ℃. The obtained expression strains are respectively inoculated into 500ml of LB liquid culture medium containing Amp with the final concentration of 50 mu g/ml and the formula according to the inoculation amount of 1 percent for amplification culture,37 ℃ at 220r/min to OD₆₀₀When the value is 0.6, adding Lara sugar (namely L-arabinose) with the final concentration of 2g/L, carrying out induced expression at 16 ℃, centrifuging after 16h, collecting thalli, crushing the thalli under high pressure, and verifying the molecular size of the protein through SDS-PAGE electrophoresis, wherein the molecular weight of the target protein is confirmed to be about 50KD and is consistent with the theoretical expected value. The protein molecular weight of DCB can not be observed visually, and the enzyme activity is measured by an HPLC method.

③ measuring the DCB enzyme activity: the collected cells of DCB were resuspended in PBS buffer at a concentration of 0.1g/mL, and then disrupted under high pressure. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. Taking supernatant, and quantitatively determining the content of D and HPG by HPLC to obtain the enzyme activity of 0.112U/g and obtain 9.2g of thalli.

Example 2 construction and expression of tagged DHD and DCB engineering bacteria

The construction process includes double enzyme digestion of plasmid pBAD-MBP containing MBP label (maltose binding protein) with restriction endonucleases NcoI and Hind III separately and recovering corresponding gene segment with DNA glue recovering kit.

Then, the D-carbamyl hydrolase DCB is obtained by PCR amplification, and the corresponding gene fragment is recovered by a DNA gel recovery kit. The DCB gene with the MBP label and the PCR primer sequence are respectively shown as follows:

the MBP-DCB gene reaction system is as follows:

the MBP-DCB gene amplification process comprises the following steps:

the MBP-DCB gene PCR product was ligated with pBAD-MBP restriction fragment using 2 XHi-Fusion at 50 ℃ for 30 min. The ligation product was transformed into e.coli TOP10 and spread on Amp resistant LB plates, positive clones were picked, and the correct recombinant plasmid was confirmed by colony PCR and sent to sequencing company for gene sequencing.

② active expression: the obtained recombinant plasmid pBAD-MBP-DCB is transformed into an allelopathic bacterium of an escherichia coli expression strain E.coli BL21(DE3) by adopting a heat shock method to obtain DE 3-pBAD-MBP-DCB. DE3-pBAD-MBP-DCB was inoculated into 10ml of LB medium containing Amp at a final concentration of 50. mu.g/ml, respectively, overnight at 37 ℃. Inoculating the obtained expression strain into LB liquid culture medium containing Amp with final concentration of 50 μ g/ml according to 1% inoculation amount, performing amplification culture at 37 deg.C and 220r/min to OD₆₀₀When the value is 0.6, adding Lara sugar with the final concentration of 2g/L, carrying out induced expression at 16 ℃, centrifuging for 16h, collecting thalli, carrying out high-pressure crushing on the thalli, and carrying out size verification through SDS-PAGE electrophoresis to obtain MBP-DCB, wherein the protein molecular weight of the MBP-DCB is about 75KD and is consistent with the theoretical expected value.

③ measuring the DCB enzyme activity: the collected cells were resuspended in PBS buffer at a concentration of 0.1g/mL, and then disrupted under high pressure. 100 mu.l of crushed crude enzyme solution and 5mmol/L of D-CpHPG substrate are dissolved in water, and in a reaction system of 2ml, the mixture is shaken on a constant temperature shaking bed at 37 ℃ for 60min, and 10 mu.l of 6mol/LHCl is added to terminate the reaction. The supernatant was subjected to quantitative determination of the D-HPG content by HPLC, and no enzyme activity was observed.

As can be seen from examples 1 and 2, although the amount of protein expression is increased after addition of the tag, the activity is lost, and it is possible to select a tag without addition because folding of the protein is affected and because industrial production does not require purification for cost.

The SDS-PAGE patterns of examples 1 and 2 are shown in FIG. 2, wherein A is the SDS-PAGE pattern of the DHD protein without the tag; SDS-PAGE electrophoresis picture of DCB protein without label; c: SDS-PAGE electrophoresis chart of DCB protein containing MBP label. As can be seen from the figure, the DHD protein without the tag can be obviously observed, which shows successful expression, while the DCB without the tag can not observe the band, and the DCB protein with the MBP tag is obviously expressed.

Example 3

The plasmid obtained in example 1 was transformed into a competent cell of E.coli expression strain E.coli BL21(DE3) to obtain engineered bacteria DCB, which was inoculated into 10ml LB medium containing Amp at a final concentration of 50. mu.g/ml and incubated overnight at 37 ℃ to obtain a seed solution.

Inoculating the seed solution to 500ml LB liquid culture medium with final concentration of 50 μ g/ml Amp antibiotic and 5g/L glucose according to 1% volume inoculation amount, performing amplification culture at 37 deg.C and 220r/min to OD₆₀₀When the value is 0.6, Lara sugar with a final concentration of 2g/L is added, expression is induced at 16 ℃, the cells are collected by centrifugation after 16h, and the collected cells are resuspended in PBS buffer at a concentration of 0.1g/mL and then disrupted under high pressure. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. And taking the supernatant, and quantitatively determining the content of D-HPG by using HPLC, wherein the enzyme activity is 0.357U/g, so as to obtain 12.4g of thalli.

Example 4

The seed solution was prepared as in example 3, and then the seed solution was inoculated in a final concentration of 50. mu.g/ml Amp antibiotic, 4ml/L glycerol, 500ml LB liquid medium at 1% inoculum size, and the medium was subjected to scale-up culture at 37 ℃ at 220r/min to OD₆₀₀When the value is 0.6, adding final concentration of 2g/L Lara sugar, 16 degrees C induced expression, after 16h centrifugal collection of bacterial cells, the collected bacterial cells, according to the concentration of 0.1g/mL PBS buffer solution for heavy suspension, then high pressure crushing. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. Taking supernatant, and quantitatively determining the content of D-HPG by HPLC, wherein the enzyme activity is 0.252U/g to obtain thallus 11.5g。

From these results, it was found that the enzyme activity and the amount of the cells were decreased when 5g/L of glucose was changed to 4ml/L of glycerol.

Example 5

The seed solution was prepared in the same manner as in example 3, and then the seed solution was inoculated in an inoculum size of 1% into 500ml of LB liquid medium having a final concentration of 50. mu.g/ml Amp antibiotic and 5g/L glucose, except that the nitrogen source in the LB liquid medium was changed to soybean peptone, and the other conditions were the same as in example 3. And taking the supernatant, and quantitatively determining the content of D and HPG by using HPLC, wherein the enzyme activity is 0.535U/g, and obtaining 12.8g of thalli.

From this, it was found that, when the nitrogen source in the LB medium was changed to soybean peptone, that is, when the soybean peptone was changed to tryptone, both the enzyme activity and the cell mass were improved.

Example 6

Seed solutions were prepared as in example 3. Inoculating the seed solution into 500ml LB liquid culture medium with final concentration of 50 μ g/ml Am p antibiotic and 5g/L glucose according to 1% inoculum size, changing nitrogen source to ammonium chloride, performing amplification culture at 37 deg.C and 220r/min to OD₆₀₀When the value is 0.6, adding final concentration of 2g/L Lara sugar, 16 degrees C induced expression, after 16h centrifugal collection of bacterial body, the collected bacterial body, according to the concentration of 0.1g/mL PB S buffer solution for heavy suspension, then high pressure crushing. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. The supernatant was subjected to HPLC to quantitatively determine the content of D-HPG, and the enzyme activity was 0.203U/g, to obtain 9.1g of cells.

From this result, it was found that the enzyme activity and the cell mass were decreased when the nitrogen source was changed to ammonium chloride.

Example 7

Seed solutions were prepared as in example 3. Inoculating the seed liquid into 500ml LB liquid culture medium with a final concentration of 50 mug/ml Amp antibiotic and 5g/L glucose according to the inoculation amount of 1%, changing the nitrogen source into soybean peptone, changing the inorganic salt from NaCl into 10g/L with the molar ratio of 1:4 KH₂PO₄And K₂HPO₄Amplification culture at 37 deg.C and 220r/min to OD₆₀₀When the value is 0.6, adding final concentration of 2g/L Lara sugar, 16 degrees C induced expression, after 16h centrifugal collection of bacterial cells, the collected bacterial cells, according to the concentration of 0.1g/mL PBS buffer solution for heavy suspension, then high pressure crushing. Mu.l of the crushed crude enzyme solution and 5mmol/L of substrate D-CpHPG were dissolved in water, and the mixture was shaken on a shaker at 37 ℃ for 60min in 2ml of a reaction system, and then 10. mu.l of 6mol/L HCl was added to terminate the reaction. And taking the supernatant, and quantitatively determining the content of D-HPG by using HPLC, wherein the enzyme activity is 0.842U/g, and obtaining 13.8g of thalli.

Compared with the example 5, the embodiment shows that after the inorganic salt is replaced, the existing inorganic salt can provide a stable pH buffer environment besides providing osmotic pressure, so that the growth of bacteria is more stable, and the enzyme activity and the thallus quantity are greatly improved.

Example 8

Based on the components of the medium determined in examples 3 to 7, the amounts of the components added were optimized in order to improve the fermentation results. First, PB experiment confirms that the content of yeast extract and inorganic salt has more obvious effect on enzyme activity. When the content of the yeast extract reaches 24g/L and the content of the inorganic salt reaches 15g/L, the enzyme activity can reach 1.263U/g and the thallus is 17.3g, and compared with the content of the original culture medium, the change of the component obviously improves the enzyme activity of DCB. In order to further determine the optimal component content of the culture medium, 4-factor 2-level orthogonal experiments are designed, 12 experiments are designed in total, each component is optimized, the enzyme activity of the component is detected, and finally, the optimal scheme is determined according to calculation, wherein the specific optimization process is as follows:

example 9

Through the optimization process of example 8, the optimal medium conditions were determined, as follows:

seed solutions were prepared as in example 3. Inoculating the seed liquid according to the inoculation amount of 1% to Amp antibiotic with the final concentration of 50 mug/ml, 10g/L glucose, 36g/L yeast extract, 10g/L soybean peptone and inorganic salt, wherein the molar ratio is 1:4 KH₂PO₄And K₂HPO₄In 500ml of liquid medium, the mixture is subjected to scale-up culture at 37 ℃ and 220r/min to OD₆₀₀When the value is 0.6, adding final concentration of 2g/L Lara sugar, 16 degrees C induced expression, after 16h centrifugal collection of bacterial cells, the collected bacterial cells, according to the concentration of 0.1g/mL PBS buffer solution for heavy suspension, then high pressure crushing. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. And (3) taking the supernatant, and quantitatively determining the content of D-HPG by using HPLC, wherein the enzyme activity is 2.734U/g, so that 17.9g of thalli are obtained.

In the embodiment, the culture medium increases the use amount of the yeast extract and the inorganic salt, and the enzyme activity is increased by 1.16 times. Compared with the original LB culture medium in the embodiment 1, the enzyme activity is improved by 23.4 times, the bacterial quantity is improved by about 1 time, and the unexpected technical effect is achieved.

Example 10

An optimal medium protocol has been obtained by the above example, followed by optimization of the culture conditions. Firstly, the temperature condition of bacterial culture is firstly cultured at 16 ℃, the highest culture temperature of the escherichia coli is 37 ℃, and the temperature is optimized in the interval.

Seed solutions were prepared as in example 3. Inoculating the seed solution into 500ml of the culture medium of example 9 at an inoculation amount of 1%, and performing amplification culture at 16 deg.C and 220r/min to OD₆₀₀When the value is 0.6, adding final concentration of 2g/L Lara sugar, 37 degrees C induced expression, after 16h centrifugal collection of bacterial cells, the collected bacterial cells, according to the concentration of 0.1g/mL PBS buffer solution for heavy suspension, then high pressure crushing. 100. mu.l of the crushed crude enzyme solution, 5mmol/L of D-CpHPG dissolved in water, 2ml of the reaction system, were placed in a reaction vessel of 37Shaking the mixture on a constant temperature shaking bed to react for 60min, and adding 10. mu.l of 6mol/L HCl to terminate the reaction. And taking the supernatant, and quantitatively determining the content of D-HPG by using HPLC, wherein the enzyme activity is 5.514U/g, so that 19.4g of thalli are obtained.

Example 11

The experiment was performed as in example 10, except that the temperature for inducing expression was changed to 27 ℃. And (3) taking the supernatant, and quantitatively determining the content of D-HPG by using HPLC, wherein the enzyme activity is 2.573U/g, so that 17.6g of thalli are obtained.

The medium components of example 10 and example 11 are the same as example 9, except that the temperature for inducing expression is changed, and it can be seen by comparison that when the induction temperature is increased to 27 ℃, the enzyme activity and the bacterial quantity are slightly reduced, and the whole change is not large, but when the induction temperature is increased to 37 ℃, the enzyme activity and the bacterial quantity are obviously increased, and especially the enzyme activity is increased nearly twice.

Example 12

Examples 2-11 were all fermented by shake flask followed by fermentation in a fermenter. When the shake flask is used for fermentation, the pH value can not be maintained in the fermentation process, and the pH condition in the fermentation process can be controlled by using the fermentation tank. First, a preliminary attempt was made to maintain the original pH of the culture medium, i.e., to control the pH to 6 during the culture.

Seed solutions were prepared as in example 3. The seed solution was inoculated to 500ml of the medium of example 9 in an inoculum size of 1%, and the medium was subjected to scale-up in a 1L fermenter at 37 ℃ and 220r/min with a pH of 6, and when the carbon source had been consumed, the medium was in the logarithmic phase at the time of the logarithmic phase, OD₆₀₀About 8.4, add final concentration of 4g/L Lara sugar, 37 degrees C induced expression, 16h later centrifugal collection of bacterial body, the collection of the bacterial body, according to the concentration of 0.1g/mL PBS buffer solution heavy suspension, then high pressure crushing. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. The supernatant was subjected to HPLC to quantitatively determine the content of D-HPG, and the enzyme activity was 5.210U/g, to obtain 23.5g of the cells.

It was found that there was a significant increase in enzyme activity when the pH was stably controlled during the culture.

Example 13

Next, pH was changed during the culture under otherwise unchanged conditions, and the pH conditions were optimized by measuring the enzyme activity. The experimental procedure is as shown in example 12, with specific results as given in the table below;

experimental number	pH	Enzyme activity U/g	Amount of bacteria g
				1	5	0.443(±0.03)	8.6(±1.53)
2	6	5.210(±0.08)	23.5(±2.68)
				3	7	6.211(±0.16)	23.0(±2.31)
4	8	5.283(±0.32)	23.4(±0.71)
				5	9	0.497(±0.08)	7.4(±0.69)

It can be seen that the specific activity and the total enzyme activity are highest when the pH is maintained at 7.

Example 14

The above examples finally determined that the fermentation medium of DCB (example 9) was 10g/L glucose, 36g/L yeast extract, 12g/L soy peptone, inorganic salts 30g/L in a molar ratio of 1:4 KH₂PO₄And K₂HPO₄On this basis, the culture was confirmed by scale-up culture in a 5L system. Respectively fermenting for different times to determine proper fermentation time, wherein the specific conditions are as follows:

experimental number	Fermentation time h	Enzyme activity U/g	Amount of bacteria g
				1	8	6.26±(0.14)	85.1±(3.15)
2	16	7.68±(0.06)	109.2±(4.04)
				3	24	9.04±(0.09)	140.1±(5.18)
4	32	9.13±(0.13)	140.0±(5.17)
				5	40	8.42±(0.03)	145.6±(5.37)

The fermentation time herein refers to the expression time after induction.

Example 15

From example 14, it can be seen that the amount of bacteria in DCB fermentation is almost unchanged after 24h, and the enzyme activity is also less changed, so the fermentation time is selected to be 24 h.

Seed solutions were prepared as in example 3. The seed solution was inoculated to 3L of the medium of example 9 in an inoculum size of 1%, and the medium was subjected to scale-up in a 5L fermenter at 37 ℃ and 220r/min with pH 7 adjusted to the logarithmic growth phase OD after the carbon source had been consumed₆₀₀About 8.4, add final concentration of 4g/L Lara sugar, 37 degrees C induced expression, 24h later centrifugal collection of bacterial body, the collection of the bacterial body, according to the concentration of 0.1g/mL PBS buffer solution heavy suspension, then high pressure crushing. 100 mu.l of the crushed crude enzyme solution and 5mmol/L of D-CpHPG are dissolved in water, and the solution is shaken on a rocking bed at the constant temperature of 37 ℃ in a 2ml reaction system for 60min, and 10 mu.l of 6mol/L HCl is added to stop the reaction. Taking supernatant, and quantitatively determining the content of D-HPG by HPLC, wherein the enzyme activity is 9.04U/g, and 140.1g of thalli are obtained.

Example 16

Based on the previous media formulation and culture conditions of DCB, we tried fermentation of DHD using this condition and tried different fermentation times, here referred to as expression time after induction.

Experimental number	Fermentation time h	Enzyme activity U/g	Amount of bacteria g
				1	8	7.76±(3.90)	94.3±(7.07)
2	16	15.18±(5.00)	159.6±(15.9)
				3	24	18.06±(0.09)	156.9±(6.89)
4	32	16.55±(3.16)	160.3±(4.95)

Example 17

As can be seen from the above example 16, after 16h, the enzyme activity and the bacterial load of DHD begin to decrease, so the fermentation time of DHD is selected to be 16h, and the specific implementation conditions are as follows:

the plasmid obtained in example 1 was transformed into a competent cell of E.coli expression strain E.coli BL21(DE3) to obtain engineered bacterium DHD, which was inoculated into 10ml LB medium containing Amp at a final concentration of 50. mu.g/ml and incubated overnight at 37 ℃ to obtain a seed solution. The seed solution was inoculated to 3L of the medium of example 9 in an inoculum size of 1%, and the medium was subjected to scale-up in a 5L fermenter at 37 ℃ and 220r/min with pH 7 adjusted to the logarithmic growth phase OD after the carbon source had been consumed₆₀₀About 8.0, adding final concentration of 4g/L Lara sugar, 37 degrees C induced expression, 16h later centrifugal collection of bacterial body, the collection of the bacterial body, according to the concentration of 0.1g/mL PBS buffer solution heavy suspension, then high pressure crushing. 100 mu L of the crushed crude enzyme solution and 5mmol/L of D, L-HPH substrate are dissolved in water, the mixture is shaken on a constant temperature shaking bed at 37 ℃ in 2ml of reaction system for 60min, and 10 mu L of 6mol/L HCl is added to stop the reaction. Taking supernatant, and quantitatively determining the content of D, L-HPH by HPLC, wherein the enzyme activity is 18.01U/g, and obtaining 160.3g of thalli.

Compared with the common LB culture medium, the enzyme activity and the yield of DHD are also obviously improved, and the DHD culture medium can meet the requirement of industrial production.

Example 18

The DHD collected by fermentation in example 15 and the cells of DCB in example 14 were resuspended in 1 × PBS buffer, and then disrupted under high pressure. The concentration of the substrate D, L-p-hydroxyphenylhydantoin is 5g/L, the dosage of DHD is 0.1g, the dosage of DCB is 0.2g, the substrate D, L-p-hydroxyphenylhydantoin is put into a 10ml reaction system, the mixture is shaken on a constant temperature shaking table at 37 ℃ for 48h, the yield is verified by an HPLC method, and the yield is 61.7%.

Example 19

The DHD collected by fermentation in example 15 and the cells of DCB in example 14 were resuspended in 1 × PBS buffer, and then disrupted under high pressure. The concentration of the substrate D, L-p-hydroxyphenylhydantoin is 5g/L, the dosage of DHD is 0.1g, the dosage of DCB is 0.8g, the substrate D, L-p-hydroxyphenylhydantoin is put into a 10ml reaction system, the mixture is shaken on a constant temperature shaking table at 37 ℃ for 48h, the yield is verified by an HPLC method, and the yield is 76.3%.

Example 20

The DHD collected by fermentation in example 15 and the cells of DCB in example 14 were resuspended in 1 × PBS buffer, and then disrupted under high pressure. The concentration of the substrate D, L-p-hydroxyphenylhydantoin is 5g/L, the dosage of DHD is 0.2g, the dosage of DCB is 1.6g, the mixture is put into a 10ml reaction system and shaken on a constant temperature shaking table at 37 ℃ for 48 h. FIG. 3 shows a comparison graph of three substance standards, D, L-HPH, D-cPHPPG and D-HPG, in which the peak appearance time 3.895 is the substrate D, L-HPH; the peak-out time 4.340 is intermediate D-cPHPG; the product D-HPG appeared at a time of peak 8.428. The final product of this example was obtained in the form shown in FIG. 4, and the yield was confirmed by HPLC, and the substrate consumption was found to be complete, and the yield of the product D-HPG (at the time of peak 3.786) was 99.3%.

According to the invention, high-density fermentation research is carried out on D-hydantoinase (DHD) and D-carbamyl hydrolase (DCB) to obtain a large amount of industrial enzymes, and a process for catalytic reaction by using the two enzymes is optimized, finally, the amount of the bacteria obtained by DHD is 160.3g, the amount of the bacteria obtained by DC B is 140.1g, the enzyme activity of DHD is 18.01U/g, and the enzyme activity of DCB is 9.04U/g. The yield is 99.3%, 5g/L of substrate can be catalyzed, and the requirement of industrial production can be met preliminarily.

The invention discloses a high-density fermentation process of two enzymes, namely D-hydantoinase (DHD) and D-carbamyl hydrolase (DCB), and discusses a method for producing D-p-hydroxyphenylglycine by the two enzymes. The synthetic method has the advantages of easily obtained raw materials, simple method, mild chemical reaction conditions, sufficient obtained enzymes, simple reaction conditions, simple crushing in the whole production process, appropriate enzyme dosage, feasibility, low pollution and guiding significance for industrial application of enzyme-catalyzed production of D-p-hydroxyphenylglycine.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

SEQUENCE LISTING

<110> university of oceanic Jiangsu

<120> engineering bacteria for catalytic production of D-p-hydroxyphenylglycine, high-density culture method and catalytic production

Method

<130> 20211011

<160> 9

<170> PatentIn version 3.3

<210> 1

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<213> Artificial Sequence (Artificial Sequence)

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atggatatca tcatcaagaa cggtaccatt gttaccgcag atggcattag tccggcagat 60

ctgggtatta aggatggcaa aattgcacag attggtggta cctttggtcc ggcaggtcgt 120

accattgatg caagcggtcg ctatgttttt ccgggtggta ttgatgtgca tacccatgtg 180

gaaaccgtta gttttaatac ccagagcgcc gatacctttg caaccgcaac cgttgccgcc 240

gcatgtggtg gtaccaccac cattgttgat ttttgccagc aggatcgtgg ccatagtctg 300

cgcgaagcag ttgcaaaatg ggatggcatg gcaggcggca aaagcgccat tgattatggt 360

tatcatatta ttgtgctgga tccgaccgat agtgttattg aagaactgga agtgctgccg 420

gatctgggca ttaccagttt taaagtgttt atggcatacc gcggcatgaa tatgattgat 480

gatgtgaccc tgctgcgtac cctggataaa gcagcaaaaa ccggtagtct ggttatggtg 540

catgcagaaa atggtgacgc cgcagattat ctgcgtgata aatttgtggc agatggcaaa 600

accgcaccga tctatcatgc cctgagtcgt ccgccgcgtg ttgaagcaga agcaaccgcc 660

cgcgcactgg ccctggctga aattgtgaat gccccgatct atattgtgca tctgacctgc 720

gaagaaagtt ttgatgaact gatgcgcgca aaagcacgcg gtgtgcatgc actggcagaa 780

acctgtaccc agtatctgta tctgaccaaa gatgatctgg aacgcccgga ttttgaaggt 840

gcaaaatatg tttttacccc gccgccgcgt accaaaaaag atcaggaaat tctgtggaat 900

gccctgcgca atggtgtgct ggaaaccgtt tcaagcgatc attgcagctg gctgtttgaa 960

ggtcataaag atcgcggccg taatgatttt cgcgccattc cgaatggcgc accgggcgtt 1020

gaagaacgtc tgatgatggt gtatcagggc gttaatgaag gccgcattag cctgacccag 1080

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attgaacaga gtgccatgca taatgcaatg gattatagca gttatgaagg ccataaaatt 1260

aagggtgtgc cgaaaaccgt tctgctgcgt ggtaaagtga ttgttgatga aggtacctat 1320

gttggtgcac cgaccgatgg tcagtttcgc aaacgccgta aatataaaca gtaa 1374

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aattttattg tttttccgga actggcactg accacctttt tcccgcgttg gcattttacc 180

gatgaagccg aactggatag cttttatgaa accgaaatgc cgggcccggt ggttcgcccg 240

ctgtttgaaa ccgcagccga actgggtatt ggttttaatc tgggttatgc agaactggtg 300

gttgaaggtg gtgttaaacg tcgttttaat accagcattc tggttgataa aagtggtaaa 360

attgtgggta aataccgcaa aattcatctg ccgggccata aagaatatga agcctatcgc 420

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gatgttgatg cagcaaaaat gggtatgttt atttgtaatg accgccgttg gccggaaacc 540

tggcgtgtga tgggtctgaa aggtgcagaa attatttgcg gtggctataa taccccgacc 600

cataatccgc cggtgccgca gcatgatcat ctgaccagct ttcatcatct gctgagcatg 660

caggcaggca gttatcagaa tggtgcctgg agtgccgcag caggcaaagt tggcatggaa 720

gaaggctgta tgctgctggg ccatagctgc attgtggcac cgaccggcga aattgttgca 780

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aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc 480

acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac 540

gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt 600

gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc 660

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aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggtgaa agatccgcgt 960

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agccgcggtg ttaattttat tgtttttccg gaactggcac tgaccacctt tttcccgcgt 1320

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gtggttcgcc cgctgtttga aaccgcagcc gaactgggta ttggttttaa tctgggttat 1440

gcagaactgg tggttgaagg tggtgttaaa cgtcgtttta ataccagcat tctggttgat 1500

aaaagtggta aaattgtggg taaataccgc aaaattcatc tgccgggcca taaagaatat 1560

gaagcctatc gcccgtttca gcatctggaa aaacgctatt ttgaaccggg cgatctgggc 1620

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tggccggaaa cctggcgtgt gatgggtctg aaaggtgcag aaattatttg cggtggctat 1740

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Claims

1. An engineering bacterium for catalytically producing D-p-hydroxyphenylglycine is characterized in that DHD genes and DCB genes from Agrobacterium tumefaciens are respectively constructed on pBAD vectors, and the successfully constructed pBAD vectors are transformed into escherichia coli to obtain the DHD engineering bacterium and DCB engineering bacterium for catalytically producing the D-p-hydroxyphenylglycine.

2. The engineered bacterium of claim 1, wherein said E.coli is selected from strain BL21(DE 3).

3. The high-density fermentation medium of an engineered bacterium according to any one of claims 1 to 2,

the high-density fermentation culture medium of the DHD engineering bacteria and the DCB engineering bacteria comprises the following components: 25-40 g/L of yeast extract; 10-15 g/L of soybean peptone; 5-10 g/L of glucose; KH (Perkin Elmer)₂PO₄And K₂HPO₄15-30 g/L of said KH₂PO₄And K₂HPO₄The molar ratio of (A) to (B) is 1: 3-5.

4. The high-density fermentation medium of engineering bacteria according to claim 3, wherein the high-density fermentation medium is: 30g/L of yeast extract; soybean peptone 10 g/L; 10g/L of glucose; KH (Perkin Elmer)₂PO₄And K₂HPO₄30 g/L。

5. The high-density fermentation medium of engineering bacteria according to claim 4, wherein said KH is selected from the group consisting of KH, and KH₂PO₄And K₂HPO₄In a molar ratio of 1: 4.

6. The method for culturing the engineered bacteria of any one of claims 1 to 2 at high density, comprising the steps of:

s1, preparing seed liquid: inoculating the preserved engineering bacteria strain of any one of claims 1-2 into LB liquid culture medium, and culturing overnight at 37 +/-1 ℃ to prepare a seed solution;

s2, fermentation culture: transferring the seed solution grown in the step S1 into the fermentation culture medium of any one of claims 3-5 for culture through aseptic operation, wherein the inoculation amount is 1% -3%, L-arabinose with the final concentration of 2-4 g/L is added for induced expression when the seed solution is cultured at 37 +/-3 ℃ to the logarithmic phase, and the time for induced expression of the DCB engineering bacteria is 16-32 h; the induced expression time of the DHD engineering bacteria is 15-17 h;

7. The high-density culture method according to claim 6, wherein the pH of the S2 is controlled to 6-8 during the fermentation culture.

8. The method according to claim 7, wherein the pH of the S2 fermentation medium is maintained at 7.0.

9. A method for catalytically producing D-p-hydroxyphenylglycine is characterized by comprising the following steps:

A. respectively using 10-fold volume of 1 XPBS buffer solution to carry out heavy suspension on the DHD and DCB bacterial sludge obtained in any one of claims 6 to 8, and respectively crushing to obtain a DHD crude enzyme solution and a DCB crude enzyme solution;

adding the DHD crude enzyme solution and the DCB crude enzyme solution into D, L-p-hydroxyphenylhydantoin with the final concentration of 5g/L, wherein the addition amount W/V (g/ml) of the DHD crude enzyme solution is 1-2%, and the mass ratio of the DHD crude enzyme solution to the DCB crude enzyme solution is 1: 7 to 9.