Biological enzyme synthesis method of naringenin

Abstract

The invention provides a glycosyl hydrolase mutant and application thereof, the amino acid sequence of the mutant is compared with the amino acid sequence SEQ ID NO.1 of an original wild enzyme, in the amino acid sequence SEQ ID NO.1, six sites of E88K, G167Q, S252Y, G275V, L290P and M328L are respectively subjected to single mutation, pairwise combined mutation, three combined mutation, four combined mutation, five combined mutation or one of six combined mutation; the novel glycosyl hydrolase mutant industrial enzyme is used for synthesizing and preparing naringenin. The glycosyl hydrolase mutant enzyme constructed by the invention has the characteristics of low enzyme cost, short conversion time, simple process operation and the like, and has a wide prospect of large-scale industrial application.

Description

TECHNICAL FIELD

[0001] The present application relates to a brand new glycosyl hydrolase and its mutants, in particular to an industrial enzyme and its mutants for biosynthetic naringin, belonging to the field of bioengineering technology. BACKGROUND

[0002] Naringin belongs to dihydroflavonoids, mainly exists in grapefruit, tomato, grape and citrus fruits. Domestic and foreign pharmacological studies show that naringin has antibacterial, anti-inflammatory, antioxidant, anti-fibrosis, anti-cancer, anti-tumor, anti-virus, anti-arrhythmia, antitussive, prevention of atherosclerosis, immune regulation of fat metabolism, anti-aging, protection of liver function and estrogen-like activity, etc. It can be developed and applied in the fields of medicine, food, etc. Naringin has strong antibacterial effect on staphylococcus aureus, which can avoid bacterial infection. It can eliminate bacteria, have antipyretic, analgesic and anti-inflammatory effects. Naringin can eliminate free radicals by not contacting with air, has antioxidant effect, and can improve skin condition. The drug can restore the unbalanced immune state to a normal immune balance state, can effectively regulate the immune state and reduce the possibility of disease. Naringin can reduce the total cholesterol content in blood plasma, strengthen body metabolism, and reduce the occurrence of atherosclerosis and coronary atherosclerotic heart disease.

[0003] At present, naringin is mainly obtained by three methods, one is direct solvent extraction method, at present, the production method of naringin is mainly extracted from orange peel, but this method has problems of low production efficiency, high extraction cost and regional restriction, etc., and a large amount of organic solvent is used, which causes certain pollution to the environment, does not conform to the green development concept; the second is to convert by biological enzyme method, such as Li Xiaofeng et al. (CN201710391927.6) use aspergillus niger cells to catalyze naringin to prepare naringin, this method needs to use a large amount of aspergillus niger, and the reaction needs a long time, and finally needs to use ethyl acetate for purification to obtain high-purity naringin. Wang Xiuling et al. (CN201310689588.1) use streptococcus AUH-JLD109 to make great breakthrough in naringin biosynthesis, the conversion rate reaches more than 98%, but further separation and purification are needed on the preparation column; the third is that the research of cell reactor is more at present, and certain progress has been made, such as the team of Jiangnan University uses multi-pathway synergistic engineering strategy to synthesize naringin in saccharomyces cerevisiae (CN201810339593.7) Li et al., ACS Sustain. Chem. Eng. 2024, 12, 1, 59-71). That is, by optimizing the expression of the shikimic acid and aromatic amino acid synthesis pathway genes, then concentrated use of aromatic amino acids and the introduction of heterologous phosphoketolase pathway, carbon flux is redirected to the biosynthesis of naringenin overexpression endogenous malonyl-CoA synthesis pathway genes and the introduction of acetaldehyde dehydrogenase pathway to promote the synthesis of naringenin. Further use of subcellular organelle carbon flux regulation strategy to make naringenin production increased to 986.2 mg / L. Finally, through 5-L bioreactor fermentation, the yield of naringenin reached 3420.6 mg / L, although this method has certain advantages, but the process is complex, and the industrialization still needs a long time of exploration. Therefore, how to improve the catalytic activity of glycosyl hydrolase in the biotransformation of naringin is very important, and gradually becomes the research focus of people, but so far there is no report on the successful biosynthesis of naringenin by glycosyl hydrolase mutant enzyme. SUMMARY

[0004] The application discloses a glycosyl hydrolase and a mutant gene thereof derived from lemon peel Citrus sinensis and provides a construction method of an enzyme in-vitro heterologous expression system and a construction method of an enzyme mutant, and a method for preparing naringenin by using the enzyme and the mutant as a biological catalyst.

[0005] The amino acid sequence of the protein coded by the gene of the naringenin glycosyl hydrolase is shown in SEQ ID No. 1.

[0006] The gene sequence of the glycosyl hydrolase is obtained by full gene synthesis of Jiangsu Jinersi Biological Technology Co., Ltd., and EcoT14 and EcoT22 restriction endonuclease sites are added at both ends of the coding region. After the target gene fragment is cut by the restriction endonucleases EcoT14 and EcoT22, the same double enzyme cutting is carried out on the pET28a(+) vector, and then the connection, transformation and selection are carried out, and the positive plasmid Glycohydrolase-pET28a(+) obtained by screening is introduced into the BL21 (DE3) host bacteria, so that the in-vitro heterologous expression system of the glycosyl hydrolase is constructed.

[0007] The construction of the mutant of the glycosyl hydrolase is obtained by the technical means of directed evolution. Specifically, the mutant is obtained by using error-prone PCR, DNA rearrangement, semi-rational design and three-dimensional structure simulation and other directed evolution technologies. More specifically, the three-dimensional structure of the enzyme is simulated by the three-dimensional structure simulation technology. The three-dimensional structure of the carbonyl hydrolase is simulated by the homology modeling method, the possible one or more sites related to catalysis are predicted by using the energy minimum principle and molecular docking technology, and then the sites are subjected to saturation site mutation (NNK), and the mutant with significantly improved activity is screened from the sites.

[0008] The possible sites related to catalysis and substrate binding predicted by the three-dimensional structure simulation technology of the application are E88, G167, S252, G275, L290 and M328. The six sites are subjected to site-directed saturation mutation respectively.

[0009] wherein, The forward primer for mutation of the E88 site is CGCCATGCGCGCGCGGGCNNKAAAACCCTGGAATCGTTTTG, The reverse primer is CAAAACGATTCCAGGGTTTTMNNGCCCGCGCGCGCATGGCG. The forward primer for mutation of the G167 site is CGAAAAATATGGCGTGGCGNNKTGGGATTGCCCGGAAGCG, The reverse primer is CGCTTCCGGGCAATCCCAMNNCGCCACGCCATATTTTTCG. The forward primer for mutation of the S252 site is GCCGCCGCCGCCATGCGNNKGCGGGCTATAAAACCCTGGAAG, The reverse primer is CTTCCAGGGTTTTATAGCCCGCMNNCGCATGGCGGCGGCGGC. The forward primer for mutation of the G275 site is CGAAAGATATGGCGTGGCGNNKTGGGATTGCCAGGAAGCG, The reverse primer is CGCTTCCGGGCAATCCCAMNNCGCCACGCCATACTTTTCG. The forward primer for mutation of the L290 site is GCCGCCGCCGCCATGCGNNKGCGGGCTATAAAACCCTGGAAG, The reverse primer is CAAAACGATTCCAGGGTTTTMNNGCCCGCGCGCGCATGGCG. The forward primer for mutation of the M328 site is CGCCATGCGCGCGCGGGCNNKAAAACCCTGGAATCGTTTTG, The reverse primer is CTTCCAGGGTTTTATAGCCCGCMNNCGCATGGCGGCGGCGGC.

[0010] Then, high pressure liquid chromatography (HPLC) is used to screen the mutant. More specifically, when glutamic acid (E) at site 88 is mutated into lysine (K), the mutant enzyme activity is improved compared with the wild type enzyme; when glycine (G) at site 167 is mutated into glutamine (Q), the mutant enzyme activity is improved compared with the wild type enzyme; when serine (S) at site 252 is mutated into tyrosine (Y), the mutant enzyme activity is improved compared with the wild type enzyme; when glycine (G) at site 275 is mutated into valine (V), the mutant enzyme activity is improved compared with the wild type enzyme; when leucine (L) at site 290 is mutated into proline (P), the mutant enzyme activity is improved compared with the wild type enzyme; and when methionine (M) at site 328 is mutated into leucine, the catalytic activity of the mutant is improved compared with the wild type enzyme. When the above six sites are subjected to single mutation, two-site combined mutation, three-site combined mutation, four-site combined mutation, five-site combined mutation or six-site combined mutation, the catalytic activity of the mutant is further improved compared with the single mutant. According to the existing public knowledge, any gene can be connected into various expression vectors after operation or modification, transformed into suitable host cells, and induced under appropriate conditions to overexpress the target protein. Therefore, the expression vector of the glycosyl hydrolase and its mutant can be pET or pCW or pUC, and the expression host can be an E. coli strain, Pichia pastoris, Saccharomyces cerevisiae, Streptomyces strain, Bacillus subtilis strain, etc.

[0011] Advantages of the present application 1) The enzyme mutant and coenzyme regeneration system involved in the present application can convert 98% of the substrate into naringenin within 5h at 35℃, and the selectivity is >98%. 2) The reaction conditions are mild, there is almost no by-product, the energy cycle system is stable, and it has a broad industrial application prospect. DETAILED DESCRIPTION

[0012] The present application will be described in detail below with reference to the examples. The embodiments are for better understanding of the present application, but are not a limitation of the present application.

[0013] In the examples, the experimental methods not specified in the specific conditions are generally carried out according to the conventional conditions, such as the methods described in the Molecular Cloning Laboratory Guide (J. Sambrook, D. W. Russell, Huang Peitang, Wang Jiaxi, Zhu Houchu, etc. Translated from the third edition, Beijing: Science Press, 2002).

[0014] The glycosyl hydrolase mutant involved in the present application is produced in a recombinant microbial cell, which is one of an E. coli strain, a Bacillus subtilis strain, a Streptomyces strain, Saccharomyces cerevisiae or Pichia pastoris.

[0015] The mutant library construction and high-throughput screening method of Example 1: In order to improve the activity of wild-type glycosyl hydrolase, a random mutant library is constructed by error-prone PCR method with the recombinant expression vector pET28a(+)-RT-PLD as the DNA template, and the base mismatch rate of the mutant library is 0.5% by adjusting the Mg 2+ and Ca 2+ concentrations and dCTP and dTTP oligonucleotide concentrations in the error-prone PCR reaction system, that is, to ensure that a mutant has 1 to 3 amino acid mutations, and the specific process of constructing the mutant library is as follows.

[0016] The error-prone PCR reaction conditions are: pre-denaturation at 95℃ for 5 min; then denaturation at 94℃ for 30 s, annealing at 55℃ for 1 min, extension at 72℃ for 1.5 min, for a total of 30 cycles; and finally extension at 72℃ for 10 min.

[0017] The error-prone PCR product obtained above is cut and recovered for purification, linked with the prokaryotic expression vector pET28a(+), and then transformed into a recombinant genetically engineered bacterium, thereby obtaining a mutant library with large capacity.

[0018] Error-prone PCR reaction system and conditions: Error-prone PCR reaction system: 10× Buffer 5 μL 2 mmol / L dNTPS 5 μL 100 mmol / L dCTP 0.5 μL 100 mmol / L dTTP 0.5 μL 10 mmol / L CaCl2 5 mmol / L MgCl2 5 μL Primer T7 promoter 2 μL Primer T7 Terminator 2 μL Template pET-Glycohydrolase-wt 1 μL Taq DNA polymerase 1.5 μL ddH2O 22.5 μL Screening mutant library: The high-throughput screening method of the glycosyl hydrolase mutant library in the present application is the NADPH determination method, which has a maximum absorption peak at 283 nm, and 10 g / L naringin is used as the substrate for screening mutants. The smaller the absorbance value at 283 nm measured by the enzyme marker, the higher the enzyme activity, indicating that the ability to tolerate high-concentration substrate is stronger, which is a beneficial mutant.

[0019] About 20,000 clones are screened from the above mutant library, and 100 mutants with obvious numerical changes are obtained. Then, the 100 mutants are subjected to shake flask screening. The specific process is as follows: the 100 mutants are inoculated in 500 ml shake flasks containing 100 ml LB medium for fermentation and induction, and the activity of catalyzing 10 g / L naringin is determined by colorimetry to obtain the optimal mutant, which is named SEQ ID NO. 2. The sequencing result shows that the mutated sequence is: E88K, G167Q, S252Y, G275V, L290P, M328L.

[0020] Example 2: Mutant culture and screening: The glycosyl hydrolase gene fragment was synthesized by Jiangsu Jinerswai Biotechnology Co., Ltd. and recombined into a pUC18 vector. After double enzyme digestion with restriction enzymes EcoT14 and EcoT22 at 40°C for 4 h, 1% agarose gel electrophoresis separation and gel recovery were performed. Subsequently, the same double enzyme digestion was performed on the expression vector pET28a(+) at 16°C overnight. The ligation liquid was transformed into OmniMAX competent cells, and colony PCR screening and sequencing verification were performed, thereby obtaining a positive recombinant plasmid PLD-pET28a(+). The positive recombinant plasmid PLD-pET28a(+) was transformed into an expression host bacterium BL21(DE3), thereby obtaining a prokaryotic expression strain PLD-pET28a(+) / BL21(DE3) as a primary strain for subsequent directed evolution and fermentation.

[0021] After the plasmid obtained by the above mutation was transformed into a BL21(DE3) host bacterium, it was coated on LB solid medium containing 30 μg / ml kanamycin and incubated at 37°C overnight, and then a single colony was picked from the plate and placed in a 96-well plate for culture. The bacterial solution after overnight culture was transferred to a 96-well plate containing fresh LB medium, and after 4 h of 37°C, 220 rpm shaking culture, 0.1 mM IPTG was added for induction, and the culture was incubated at 30°C overnight. The bacterial cells were collected by centrifugation at 4°C, 5000 rpm for 10 min, and the crude enzyme solution or freeze-dried powder was prepared for use.

[0022] The substrate concentration was 10 g / L, 5 mM pH 7.0 sodium phosphate buffer, 5 mM sodium hexametaphosphate, 5 mM magnesium chloride, 10 mM calcium chloride, and the above prepared crude enzyme solution was added at a proportion of 10%, and the reaction was placed at 35°C, 220 rpm stirring. Samples were taken at 2 h and 5 h for HPLC detection.

[0023] The clones with significantly improved substrate conversion rate at both 2h and 5h were expanded and sequenced to verify the mutation. The sequencing results showed that the mutant enzyme activity of the clones with significantly improved substrate conversion rate at both 2h and 5h contained the following mutation sites: when glutamic acid (E) at site 88 was mutated to lysine (K), the mutant enzyme activity was improved relative to the wild-type enzyme; when glycine (G) at site 167 was mutated to glutamine (Q), the mutant enzyme activity was improved relative to the wild-type enzyme; when serine (S) at site 252 was mutated to tyrosine (Y), the mutant enzyme activity was improved relative to the wild-type enzyme; when glycine (G) at site 275 was mutated to valine (V), the mutant enzyme activity was improved relative to the wild-type enzyme; when leucine (L) at site 290 was mutated to proline (P), the mutant enzyme activity was improved relative to the wild-type enzyme; and when methionine (M) at site 328 was mutated to leucine, the catalytic activity of the mutant was improved relative to the wild-type enzyme. Subsequently, two-by-two combined mutations, three, four, five, or six combined mutations were performed on these sites, and activity detection found that the catalytic activity of some combined mutations was significantly improved compared to single-point mutations.

[0024] Example 3 Biocatalysis of double-site mutant: 5g of the substrate naringin was dissolved in 100ml of 5mM pH7.0 sodium phosphate buffer, and after the substrate was completely dissolved, 5mM pH7.0 sodium phosphate buffer, 5mM sodium hexametaphosphate, 5mM magnesium chloride, 10mM calcium chloride, and 0.2g of the glycosyl hydrolase mutant (G167Q, S252Y) freeze-dried powder were added. The reaction solution was placed in a 35°C constant-temperature water bath, and mechanically stirred. After 5h of reaction, HPLC detection was performed, and the substrate conversion rate was >80%. After purification, naringin with a purity of >90% was obtained.

[0025] Example 4 Biocatalysis of four-site mutant: 5g of the substrate naringin was dissolved in 100ml of 5mM pH7.0 sodium phosphate buffer, and after the substrate was completely dissolved, 5mM pH7.0 sodium phosphate buffer, 5mM sodium hexametaphosphate, 5mM magnesium chloride, 10mM calcium chloride, and 0.2g of the glycosyl hydrolase mutant (E88K, G167Q, S252Y, G275V) freeze-dried powder were added. The reaction solution was placed in a 35°C constant-temperature water bath, and mechanically stirred. After 5h of reaction, HPLC detection was performed, and the substrate conversion rate was >95%. After purification, phosphatidylserine with a purity of >95% was obtained.

[0026] Example 5 Biocatalysis of six-site mutant: 5g of the substrate naringin was dissolved in 100ml of 5mM pH7.0 sodium phosphate buffer, after the substrate was completely dissolved, 5mM pH7.0 sodium phosphate buffer, 5mM sodium hexametaphosphate, 5mM magnesium chloride, 10mM calcium chloride, 0.2g of glycosyl hydrolase mutant (E88K, G167Q, S252Y, G275V, L290P, M328L) freeze-dried powder were added. The reaction solution was placed in a 35℃ constant temperature water bath, and mechanically stirred. After 5h of reaction, HPLC detection was performed, and the substrate conversion rate was >99%. After purification, the purity of phosphatidylserine was greater than 98%.

[0027] Poly (ADP-ribose) glycohydrolase 1 [Citrus sinensis] GenBank: KAH9726564.1 SEQ ID NO.1 1 mesredlksi laylpvlvrs knlfwpskvv ealkemaqgp dhsrvnsgev lfvairdtrs 61 slsllqplap fasegyalff delisraeaa ewfgevlpal anlllqlpal leshyqnadd 121 ilgkygfktg lhllgsqegg mvflsqelig allacaffcl fpasnrganh lptinfdelf 181 aslyegysqk qenklkcivh yfkricscmp vgfvsferkv lpqdrhplft sypeadfwsk 241 svlplcafev hslgfiedqs analevdfan kyigggalhr gclqeeirfm inpeliagml 301 flpsmadnea ieivgaerfc dykgcvgmhc hfvlrvimqt kgiltvledv SEQ ID NO.2 1 mesredlksi laylpvlvrs knlfwpskvv ealkemaqgp dhsrvnsgev lfvairdtrs 61 slsllqplap fasegyalff delisrakaa ewfgevlpal anlllqlpal leshyqnadd 121 ilgkygfktg lhllgsqegg mvflsqelig allacaffcl fpasnrqanh lptinfdelf 181 aslyegysqk qenklkcivh yfkricscmp vgfvsferkv lpqdrhplft sypeadfwsk 241 svlplcafev hylgfiedqs analevdfan kyigvgalhr gclqeeirfm inpeliagmp 301 flpsmadnea ieivgaerfc dykgcvglhc hfvlrvimqt kgiltvledv

Claims

1. A glycosyl hydrolase mutant, characterized in that: The amino acid sequence of the mutant is compared with the wild enzyme amino acid sequence SEQ ID No. 1, wherein one of the following mutations is made at E88, G167, S252, G275, L290, M328 in the amino acid sequence SEQ ID No. 1: single mutation, two combined mutations, three or four combined mutations, five or six combined mutations.

2. The glycosyl hydrolase mutant of claim 1, wherein: The glutamic acid (E) at position E88, the glycine (G) at position G167, the serine (S) at position S252, the glycine (G) at position G275, the leucine at position L290, and the methionine at position M328 in the amino acid sequence SEQ ID No. 1 are mutated to lysine (K), glutamine (Q), tyrosine (Y), valine (V), proline (P), and leucine (L), respectively.

3. The glycosyl hydrolase mutant of claim 1, wherein: The glycosyl hydrolase mutant is produced in a recombinant microbial cell, which is one of Escherichia coli strain, Bacillus subtilis strain, Saccharomyces cerevisiae, or Pichia pastoris.

4. A method for preparing the glycosyl hydrolase mutant of claim 1, comprising the following steps: 1) using Glycohydrolase-pET28a(+) recombinant plasmid as a template, performing site-directed saturation mutation at E88, G167, S252, G275, L290, and M328, wherein, forward primer for mutation at E88: CGCCATGCGCGCGCGGGCNNKAAAACCCTGGAATCGTTTTG, reverse primer: CAAAACGATTCCAGGGTTTTMNNGCCCGCGCGCGCATGGCG; forward primer for mutation at G167: CGAAAAATATGGCGTGGCGNNKTGGGATTGCCCGGAAGCG, reverse primer: CGCTTCCGGGCAATCCCAMNNCGCCACGCCATATTTTTCG; forward primer for mutation at S252: GCCGCCGCCGCCATGCGNNKGCGGGCTATAAAACCCTGGAAG, reverse primer: CTTCCAGGGTTTTATAGCCCGCMNNCGCATGGCGGCGGCGGC; forward primer for mutation at G275: CGAAAGATATGGCGTGGCGNNKTGGGATTGCCAGGAAGCG, reverse primer: CGCTTCCGGGCAATCCCAMNNCGCCACGCCATACTTTTCG; forward primer for mutation at L290: GCCGCCGCCGCCATGCGNNKGCGGGCTATAAAACCCTGGAAG, reverse primer: CAAAACGATTCCAGGGTTTTMNNGCCCGCGCGCGCATGGCG. Forward primer for mutation at site M328: CGCCATGCGCGCGCGGGCNNKAAAACCCTGGAATCGTTTTG, Reverse primer: CTTCCAGGGTTTTATAGCCCGCMNNCGCATGGCGGCGGCGGC 2) Mutant culture: After transforming the above-mentioned mutant plasmid into BL21 (DE3) host bacteria, spread on LB solid medium containing 30 μg / ml kanamycin, 37°C inverted culture overnight, then pick single clone from the plate and place in 96-well plate for culture; the bacteria liquid after overnight culture is transferred to a 96-well plate containing fresh LB medium, 37°C, 220 rpm shaking culture for 4h, then induced by adding IPTG with a final concentration of 0.1 mM, 30°C culture overnight; 4°C, 5000 rpm centrifugation for 10 min to collect bacteria, then ultrasonic broken to obtain crude enzyme liquid, or prepared into freeze-dried powder; 3) Mutant screening: substrate concentration 10 g / L, 5 mM pH7.0 sodium phosphate buffer, 5 mM sodium hexametaphosphate, 5 mM magnesium chloride, 10 mM calcium chloride, add the above-mentioned prepared crude enzyme liquid at a proportion of 10%, put in 37°C, 220 rpm stirring reaction; sample at 2h and 10h for HPLC detection; sequencing results show that the mutant sites contained in the clone with significantly improved enzyme activity are as follows: glutamic acid (E) at site E88 is mutated to lysine (K), glycine (G) at site G167 is mutated to glutamine (Q), serine (S) at site S252 is mutated to tyrosine (Y), glycine (G) at site G275 is mutated to valine (V), leucine at site L290 is mutated to proline (P), methionine at site M328 is mutated to leucine (L), and the glycosyl hydrolase enzyme activity is increased by 563 times.

5. The application of the glycosyl hydrolase mutant industrial enzyme according to claim 1 in catalyzing the synthesis of naringenin.

6. The method for catalyzing the synthesis of L-phosphatidylserine by the glycosyl hydrolase mutant industrial enzyme according to claim 5, comprising the following steps: Dissolve 1 g of substrate naringin in 100 ml of 5 mM pH7.0 sodium phosphate buffer; After the substrate in step 1) is completely dissolved, add 0.2 g of ATP, 5 mM sodium hexametaphosphate, 5 mM magnesium chloride, 10 mM calcium chloride, and 0.2 g of glycosyl hydrolase mutant industrial enzyme freeze-dried powder; Put the reaction liquid prepared in step 2) in a 35°C constant temperature water bath, and mechanically stir the reaction; after 5h of reaction, perform HPLC detection, and the substrate conversion rate is >98%; after purification, naringenin is obtained, and the purity of the target product is greater than 98%.