CN112921023B

CN112921023B - Recombinant aspartate lyase and method for preparing R-3-aminobutyric acid with high repeated utilization rate

Info

Publication number: CN112921023B
Application number: CN202110340692.4A
Authority: CN
Inventors: 杨卫华; 张利坤; 谈聪; 唐云平; 李业; 彭汶铎; 钱敏帆; 严燕兵; 倪建洲; 吴怀春
Original assignee: Changxing Pharmaceutical Co ltd
Current assignee: Changxing Pharmaceutical Co ltd
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2022-11-11
Anticipated expiration: 2041-03-30
Also published as: CN112921023A

Abstract

The invention discloses a recombinant aspartate lyase and a method for preparing R-3-aminobutyric acid with high repeated utilization rate, wherein a section of polypeptide chain which is provided with a plurality of amino groups and is polar amino acid residue is respectively added at two ends of an original gene through genetic engineering, enzyme liquid is obtained through biological fermentation based on the sequence, and the immobilized enzyme is obtained through immobilization with resin. The immobilized enzyme reduces the binding probability of the covalent binding of the immobilized carrier and the protein to the enzyme activity center, thereby reducing the damage to the enzyme activity after immobilization, improving the recovery rate of the enzyme activity to more than 50 percent at most, improving the concentration of a substrate which can be used for conversion due to the increase of the number of polar amino acids, greatly reducing the cost of the enzyme in the industrial enzyme fermentation engineering and saving the production process.

Description

Recombinant aspartate lyase and method for preparing R-3-aminobutyric acid with high repeated utilization rate

Technical Field

The invention relates to the field of enzyme engineering and biocatalysis, in particular to a recombinant aspartate lyase and a method for preparing R-3-aminobutyric acid with high repeated utilization rate.

Background

Chinese patent ZL 201810198044.8 (published: 2018, 08.07) discloses a preparation method of R-3-aminobutyric acid, which takes crotonic acid and ammonium salt with higher concentration as substrates, adds salt containing magnesium ions, and adds recombinant aspartase under alkaline conditions for biocatalysis to obtain the conversion rate of more than 98%. Wherein the recombinant aspartase is obtained based on the gene of Bacillus recombinant aspartase.

The enzyme can be used for resin as a carrier for immobilization, and the method is mainly a method for covalently bonding the enzyme on the carrier, namely covalently bonding an inactive site functional group on an enzyme molecule and a surface reactive group of the carrier. The advantages are that: the enzyme is firmly combined with the carrier, and the stability is good; but has the following disadvantages: the use of relatively strong reaction conditions may cause changes in the higher structure of the enzyme protein, and the organic ratio may destroy the active center, so that immobilized enzymes having a high specific activity may not be obtained.

After most of the enzyme is immobilized on the resin, the activity of the enzyme is greatly affected. After the recombinant aspartase is immobilized by using resin, the recovery rate of enzyme activity is only 10%, the immobilized enzyme can hardly be prepared, and the carrier is inactivated and denatured after being combined with enzyme protein.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a recombinant aspartate lyase which is used for resin carrier immobilization and has high repeated utilization rate and a coding gene thereof; the invention also provides a recombinant expression vector of the gene segment and a corresponding host cell; it is still another object of the present invention to provide a method for preparing R-3-aminobutyric acid with high recycling rate based on the aforementioned recombinant aspartic lyase-immobilized enzyme.

The technical scheme is as follows: a recombinant aspartate lyase has an amino acid sequence formed by sequentially connecting a first sequence SEQ ID NO.1 and a second sequence in series, wherein the lengths of the first sequence and the second sequence are respectively 2-5% of the SEQ ID NO.1, and any amino acid is selected from any one of C cysteine, G glycine, H histidine, K lysine, N aspartic acid, Q glutamine, R arginine, S serine, T threonine and Y tyrosine.

The sequence of SEQ ID NO.1 is the same as that disclosed in ZL 201810198044.8, and the invention can perform enzyme catalytic reaction under a high-concentration substrate to obtain R-3-aminobutyric acid with chiral purity of not less than 99.95%, impurity purity of not less than 99%, content of not less than 99% and product yield of not less than 85%. The principle of the enzyme-catalyzed reaction and the method for preparing the enzyme are described in the specification of ZL 201810198044.8. On the basis of the prior art, the invention further discusses how to improve the repeated utilization rate of the enzyme, and the repeated utilization rate is obviously improved and the higher enzyme catalytic conversion rate is maintained by respectively adding two hydrophilic sequences at the 3 'end and the 5' end.

It is noted that the length of each of the two hydrophilic sequences is 2-5% of that of SEQ ID No.1, and the amino acids of the two sequences are selected from any one of cysteine, glycine, histidine, lysine, aspartic acid, glutamine, arginine, serine, threonine and tyrosine, while the other hydrophilic amino acids constitute insufficient enzyme stability or significantly reduce the conversion rate. Preferably, arginine and lysine in the two hydrophilic sequences have a high content of not less than 20% of any hydrophilic sequence. Specifically, the proportion of arginine in the first sequence is not less than 10%, the proportion of lysine is not less than 10%, and the proportion of histidine is not less than 9%. The arginine content in the second sequence is not less than 20%, and the lysine content is not less than 15%.

Specifically, the first sequence is SEQ ID NO.2, and the amino acid sequence is TCRKSYHKQGNRYQTYSRCKH; the second sequence is SEQ ID NO.3, and the amino acid sequence is CHTSYGRYRKQRK.

The enzyme provided by the invention is not only suitable for a traditional enzyme catalysis system, but also suitable for enzyme catalysis reaction with high substrate concentration. Due to high salt concentration caused by high substrate concentration, electrostatic interaction between enzyme amino acid residues is interfered, an essential water molecule layer on the surface of the protein is reduced, hydrophobic interaction is increased, and enzyme denaturation, aggregation and sedimentation are accelerated. The invention provides a method for obtaining the first sequence and the second sequence which are positioned at two ends of an active catalytic fragment, which can effectively shield high salt, increase and stabilize a hydration layer on the surface of protein and prevent the protein from spreading under the high salt condition. Based on the structural model of aspartate lyase, the number of polar amino acids is increased on the surface of the enzyme by introducing site-directed mutagenesis, so that an enzyme mutant with improved stability is obtained, and the mutant still ensures extremely high stability under the enzyme catalysis system with high salt concentration (namely high substrate concentration).

The invention also provides a separated gene segment for expressing the aspartate lyase, which comprises a nucleotide sequence shown as SEQ ID NO.4 and an amino acid sequence formed by sequentially connecting SEQ ID NO.2, SEQ ID NO.1 and SEQ ID NO.3 in series.

The invention also provides a recombinant expression vector which comprises the gene segment of the SEQ ID NO.4, and any one of pET, pGEX and pMAL is selected as a vector, preferably pET-28 plasmid.

Correspondingly, the invention also provides a host cell prepared by using the recombinant expression vector. The host cell is selected from any one of Escherichia coli, bacillus subtilis and Pichia pastoris, and preferably Escherichia coli is used as the host cell.

Based on the amino acid sequence and the gene fragment, the method for preparing the R-3-aminobutyric acid with high recycling rate, provided by the invention, comprises the following steps:

(1) Breaking the cell wall of a strain expressing the recombinant aspartic lyase of claim 1 to prepare a crude enzyme solution, centrifuging to obtain an enzyme clear solution, and mixing every 1000U of the enzyme clear solution with 1-10g of a carrier to obtain an immobilized enzyme;

(2) Crotonic acid and ammonium salt with the concentration of 100 to 400g/L are used as substrates, the immobilized enzyme is added, and biological enzyme catalysis is carried out under the condition that the pH value is 8.5-9.5, so that the conversion rate is not lower than 98%;

(3) And (3) recovering the immobilized enzyme, and repeatedly using the immobilized enzyme for the biological enzyme catalytic reaction in the step (2).

For the step (1), on the basis of the protein sequence of the reporter enzyme in the patent ZL 201810198044.8, a second sequence CHTSYGRYRKQRK is added at the N end, a first sequence TCRKSYHKQGNRYQTYSRCKH is added at the C end, and the gene is synthesized after codon optimization. Carrying out double enzyme digestion and purification on the synthetic gene and a vector, then connecting a target fragment and the vector according to a proper proportion to form a recombinant plasmid, adding competent cells to convert to form a recombinant strain, coating the recombinant strain on a plate with corresponding resistance to culture, then carrying out fermentation culture and cell disruption treatment in sequence to obtain crude enzyme liquid, centrifuging to obtain enzyme clear liquid, and mixing the enzyme clear liquid with 1-10g vector per 1000U to obtain the immobilized enzyme.

In the step (2), in the bio-enzyme catalytic reaction, the molar ratio of ammonium ions in the ammonium salt to crotonic acid is 0.2 to 0.4, preferably 0.3. The ammonium salt is selected from any one or a combination of ammonium sulfate, ammonium chloride, ammonium acetate and ammonium formate, and preferably ammonium acetate.

Before the bio-enzyme catalysis reaction, ammonia water is added to adjust the pH value to 6.5-7.5, so that the reaction liquid is in a neutral condition, and the enzyme can be repeatedly utilized for many times. After the immobilized enzyme is added, the catalytic reaction is maintained under neutral conditions, preferably the pH is 5.5 to 8.5, and more preferably 6.5 to 7.5.

Preferably, the carrier is an epoxy resin and/or an amino resin. More preferably, the immobilized enzyme and the carrier can be repeatedly utilized for 20 times after being immobilized, and the relative enzyme activity of not less than 50% is still retained, or the enzyme activity recovery rate of the immobilized enzyme is not less than 30%.

More preferably, the carrier is amino resin, the immobilized enzyme and the carrier can be repeatedly utilized for 20 times after being immobilized, the relative enzyme activity of the immobilized enzyme can be retained by not less than 65%, or the enzyme activity recovery rate of the immobilized enzyme is not less than 40%.

The recombinant aspartic lyase has a conversion rate of not less than 98% when being used for carrying out biocatalytic reaction, can still keep better stability even under the reaction condition of high substrate concentration, and can still maintain the conversion rate of more than 98% after being repeatedly used for 20 times through resin preparation immobilized enzyme.

The immobilized enzyme provided by the invention can reduce the inhibition effect of high-concentration substrates and products on the efficiency of an enzyme catalysis system. There are roughly two cases: 1. in the case of allosteric inhibition, the inhibitor binds to a specific site on the enzyme surface, which in turn causes a change in the overall structure of the enzyme, resulting in a decrease in the catalytic efficiency of the active center. By enzyme immobilization, it is possible to prevent the substrate or product from binding to a specific site on the enzyme surface, and to release allosteric inhibition; 2. if the substrate or product binds directly to the active center to produce an inhibitory effect, enzyme immobilization may cause slight deformation of the active pocket that would affect the interaction of the inhibitor with the enzyme, but not the binding of the substrate to the enzyme. This is more common in cases where the substrate molecule is larger than the inhibitor molecule.

According to the invention, through gene engineering, a section of polypeptide chain which is provided with a plurality of amino groups and is polar amino acid residue is respectively added at two ends of an original gene, and the probability of the covalent binding of an immobilized carrier and protein to the enzyme activity center is reduced, so that the damage to the enzyme activity after immobilization is reduced, the enzyme activity recovery rate can be improved to more than 50% to the maximum extent, and the concentration of a substrate which can be used for conversion is improved due to the increase of the number of polar amino acids, so that the cost of the enzyme is greatly reduced in the industrial enzyme fermentation engineering, and the production process is saved.

Drawings

FIG. 1 is a graph of the amino acid hydrophilicity profile of the first sequence of example 1;

FIG. 2 is the amino acid hydrophilicity profile of the second sequence of example 1;

FIG. 3 is a graph showing a comparison of the amounts of products formed with the reaction time between the immobilized enzyme A and the control group in the test example;

FIG. 4 is a graph showing a comparison of relative enzyme activities of the immobilized enzyme A and the control group in the test example after 20 times of repeated use.

Detailed Description

The invention is further illustrated with reference to the following figures and examples.

Example 1

1. Construction of recombinant aspartic acid lyase expression Strain

A recombinant aspartate lyase has an amino acid sequence formed by sequentially connecting SEQ ID NO.2, SEQ ID NO.1 and SEQ ID NO.3 in series, wherein the amino acid sequence of SEQ ID NO.2 is TCRKSYHKQGNRYQTYSRCKH; the amino acid sequence of SEQ ID NO.3 is CHTSYGRYRKQRK. The nucleotide coding sequence of the whole amino acid sequence is shown in SEQ ID NO. 4.

The hydrophilicity profile of the sequence SEQ ID NO.2 is shown in FIG. 1, with the ordinate indicating the magnitude of the hydrophobicity (positive values for hydrophobicity and negative values for hydrophilicity); FIG. 2 shows the hydrophilicity profile of SEQ ID NO.3, showing a larger proportion of polar amino acid residues and higher salt tolerance.

Entrusts Shanghai Jieli bioengineering GmbH to synthesize gene, double enzyme digestion and purification are carried out on the synthesized gene and vector, then the target fragment and pET-28 vector are connected according to a proper proportion to form recombinant plasmid, BL21 (DE 3) competent cell is added to be transformed to form recombinant strain, the recombinant strain is coated on a flat plate with corresponding resistance to be cultured, and the recombinant aspartate lyase expression strain BL21 (DE 3)/pET-28 a (+) -Asp is obtained.

2. Preparation of enzyme solution

Inoculating the recombinant bacteria to kanamycin LB slant culture with the concentration of 50 mug/mL, and culturing 12h at 37 ℃ to obtain slant bacteria; the slant thallus cultured by washing with sterile water is inoculated into a 500 mL triangular flask filled with 100mL kanamycin LB fermentation medium with the final concentration of 50 mug/mL, cultured for 6 to 8 hours at 37 ℃ and 150 r/min, and then induced and cultured for 10 to 12 hours at 25 ℃ and 150 r/min. The fermentation broth was centrifuged (5000 r/min,10 min) to collect the precipitated cells. Suspending thallus with 100 mmol/L, pH 7.0.0 phosphate buffer solution to make thallus final concentration 20% (m/V), ultrasonicating for 20 min, centrifuging at 9000 r/min at 4 deg.C for 10 min, and retaining supernatant to obtain crude enzyme solution.

Adding 0.1-0.3g/L chitosan into the crude enzyme solution for flocculation, and then performing high-speed centrifugation to obtain an enzyme clear solution.

3. Carrier resin activation

After 25g of amino resin LX-1000HAA (New science and technology materials Co., ltd., xian blue) carrier is washed twice, 100mL of water and 0.2% (v/v) of glutaraldehyde and 150 r/min are added, and after activation for 1h at 25 ℃, the carrier is washed twice for standby and is filtered and dried for standby.

4. Preparation of immobilized enzymes

Weighing the required activated resin, adding 8g of immobilized resin (the amount of the enzyme solution after treatment is about 15-25g according to the general experience of 8g of resin per 1000U of the flocculated enzyme solution), wherein the ratio of the mass of the resin to the total mass of the enzyme solution is 1:5, and PBS solution is not added enough, fixing at 25 ℃ (shaking or stirring at 100-200 rpm) for 24h, then carrying out suction filtration and washing, refrigerating and storing in 0.1M PBS solution with pH7.4 (the PBS soaked resin can be used for ensuring that the storage is not dry, and chloramphenicol with the amount of 34mg/L can be added for preventing mixed bacteria from breeding), and obtaining the immobilized enzyme A.

If the enzyme solution disclosed by ZL 201810198044.8 is adopted to be fixed with LX-1000HAA, the recovery rate of the enzyme activity is 10.5%. The immobilized enzyme A is repeatedly used for 20 times, the relative enzyme activity of not less than 65 percent can be kept, the enzyme activity recovery rate of the immobilized enzyme is 50.5 percent, and the conversion rate is more than 98 percent.

Example 2

This example was carried out in the same manner as in example 1 except that the amino resin LX-1000HAA was replaced with an epoxy resin LX-1000EP (Sean blue, advanced science and technology materials Co., ltd.) to obtain an immobilized enzyme B.

If the enzyme solution disclosed by ZL 201810198044.8 is adopted to be fixed with LX-1000EP, the recovery rate of the enzyme activity is 4.8%. The immobilized enzyme B of the embodiment is repeatedly utilized for 20 times after being immobilized with the carrier, and the conversion rate is more than 98%.

Example 3

This example was modified as appropriate based on example 1 to obtain immobilized enzyme C by replacing amino resin LX-1000HAA with epoxy resin LX-103B (Sean blue, seisan, seisakusho, seisan Co., ltd.).

If the enzyme solution disclosed by ZL 201810198044.8 is adopted to be fixed with LX-103B, the recovery rate of the enzyme activity is 5.3%. The immobilized enzyme C of the embodiment is repeatedly utilized for 20 times after being immobilized with the carrier, and the conversion rate is more than 98%.

Example 4 biocatalytic reaction of 1000L System

250kg of crotonic acid, 2.4kg of magnesium sulfate and 58kg of ammonium acetate were added to 1000L of the catalyst system, the pH was adjusted to 7.0 with ammonia water, and 10kg of the immobilized aspartic acid bacterium obtained in example 1 was added under stirring at 40 ℃ to start the reaction. After the reaction is finished for 12 hours, the concentration of the R-3-aminobutyric acid in the solution can reach 294g/L through detection, and the conversion rate reaches more than 98%.

After the reaction is finished, the reaction liquid is filtered to retain the immobilized enzyme, and the clear liquid is subjected to nanofiltration membrane to remove salt and most of pigment. Microfiltration adopts a microfiltration membrane for separating substances with the intercepted relative molecular mass of more than 3000-5000 daltons, and nanofiltration adopts a sodium filtration membrane for separating substances with the intercepted relative molecular mass of more than 120-200 daltons. The obtained nanofiltration clear liquid is concentrated, crystallized, centrifuged and dried to obtain 255kg of finished products.

The chiral purity of the finished product R-3-aminobutyric acid is 99.98 percent, the impurity purity is 99.54 percent, the content is 99.7 percent, and the product yield is 88 percent.

The reaction is repeated for 50 times, the reaction time is controlled within 12 hours, the conversion rate reaches more than 98 percent, and the obtained product meets the quality standard.

Example 5 15000L System biocatalytic reactions

3900kg of crotonic acid, 36kg of magnesium sulfate and 870kg of ammonium acetate are added into a 15000L catalytic system, the pH is adjusted to 6.8 by ammonia water, stirring is carried out at 42 ℃, and 170kg of aspartic acid immobilized enzyme is added to start the reaction. After the reaction is finished for 12 hours, the concentration of the R-3-aminobutyric acid in the solution can reach 307g/L through detection, and the conversion rate reaches more than 98%.

After the reaction is finished, the reaction liquid is filtered to retain the immobilized enzyme, and the clear liquid is subjected to nanofiltration membrane to remove salt and most of pigments. Microfiltration adopts a microfiltration membrane for separating substances with the intercepted relative molecular mass of more than 3000-5000 daltons, and nanofiltration adopts a sodium filtration membrane for separating substances with the intercepted relative molecular mass of more than 120-200 daltons. The obtained nanofiltration clear liquid is concentrated, crystallized, centrifuged and dried to obtain 4154kg of finished product.

The chiral purity of the finished product R-3-aminobutyric acid is 99.99 percent, the impurity purity is 99.60 percent, the content is 99.6 percent, and the product yield is 90.2 percent.

Test examples

In this example, the immobilized enzyme was prepared by the method of example 1 using the modified gene strain (experimental group) of the present invention and the strain disclosed in ZL 201810198044.8 (control group), then 25g of crotonic acid, 024g of magnesium sulfate, and 5.8g of ammonium acetate were added to 100mL of the catalyst system, the pH was adjusted to 7.0 with ammonia water, the mixture was stirred at 40 ℃, and the immobilized enzymes a to C obtained in example 1~3 and 1g of the immobilized enzyme obtained in the control group were added, respectively, and the reaction was started. And after the reaction is finished for 16h, detecting the concentration of the R-3-aminobutyric acid in the two groups of solutions.

As shown in FIG. 3, the product yield over time indicates that the concentration of the immobilized enzyme A reaches 285g/L after reacting for 16h, while the concentration of the immobilized enzyme A in the control group is only one fifth of that in the experimental group, and the subsequent reaction rate is slow, so that the immobilized enzyme A can hardly react normally, which indicates that the immobilization effect of the protogenic enzyme is unsatisfactory. As can be seen from FIG. 4, the enzyme activity of the immobilized enzyme used for the first time is 100%, after 20 batches of reactions are carried out repeatedly by using the genetically modified immobilized enzyme according to the process, 88% of the relative enzyme activity is still kept in 10 batches before the reaction, more than 72% of the relative enzyme activity is still kept in 15 batches after the reaction, and more than 65% of the relative enzyme activity is still kept in 20 batches after the reaction.

Sequence listing

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Claims

1. A recombinant aspartate lyase characterized by: the lyase has an amino acid sequence formed by sequentially connecting a first sequence, SEQ ID No.1 and a second sequence in series, wherein the length of the first sequence and the length of the second sequence are respectively 2-5% of that of the SEQ ID No.1, and any amino acid is selected from any one of C cysteine, G glycine, H histidine, K lysine, N aspartic acid, Q glutamine, R arginine, S serine, T threonine and Y tyrosine; wherein, the first sequence is SEQ ID NO.2, and the second sequence is SEQ ID NO.3.

2. A recombinant expression vector comprising the gene segment of claim 1.

3. A host cell produced using the recombinant expression vector of claim 2.

4. The separated gene segment comprises a nucleotide sequence shown as SEQ ID NO.4 and expresses an amino acid sequence formed by sequentially connecting SEQ ID NO.2, SEQ ID NO.1 and SEQ ID NO.3 in series.

5. A method for preparing R-3-aminobutyric acid with high repeated utilization rate is characterized by comprising the following steps:

(2) Crotonic acid and ammonium salt with the concentration of 100-400g/L are used as substrates, the immobilized enzyme is added, biological enzyme catalysis is carried out under the condition that the pH value is 8.5-9.5, and the conversion rate is not lower than 98%;

6. The method for preparing R-3-aminobutyric acid with high recycling rate according to claim 5, wherein: the carrier is epoxy resin and/or amino resin.

7. The method for preparing R-3-aminobutyric acid with high recycling rate according to claim 5, wherein: the immobilized enzyme and the carrier can be reused for 20 times after being immobilized, and the relative enzyme activity of not less than 50 percent is still kept.

8. The method for preparing R-3-aminobutyric acid with high recycling rate according to claim 7, wherein: the immobilized enzyme and the amino resin can be repeatedly utilized for 20 times after being immobilized, the relative enzyme activity of not less than 65 percent is still kept, or the enzyme activity recovery rate of the immobilized enzyme is not less than 40 percent.