CN117965492A - Recombinant acid phosphatase mutant and application thereof in synthesis of pyridoxal phosphate - Google Patents

Recombinant acid phosphatase mutant and application thereof in synthesis of pyridoxal phosphate Download PDF

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CN117965492A
CN117965492A CN202410059317.6A CN202410059317A CN117965492A CN 117965492 A CN117965492 A CN 117965492A CN 202410059317 A CN202410059317 A CN 202410059317A CN 117965492 A CN117965492 A CN 117965492A
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stapase
acid phosphatase
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pyridoxine
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吴静
王晶
刘立明
魏婉清
宋伟
闻建
周怡雯
陈修来
刘佳
高聪
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Jiangnan University
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Abstract

The invention discloses a recombinant acid phosphatase mutant and application thereof in pyridoxal phosphate synthesis, and belongs to the technical field of bioengineering. The acid phosphatase mutant is obtained by mutating one or more of amino acids at positions 57, 58, 59, 60, 91, 96 and 124 of acid phosphatase. Compared with the wild acid phosphatase StAPase, the mutant has 3.5 times higher specific enzyme activity and 4.87 times higher catalytic efficiency. Firstly, constructing recombinant acid phosphatase, taking pyridoxine hydrochloride as a substrate, and generating an intermediate product pyridoxine phosphate; and the pyridoxine phosphate is used as a substrate to produce the final product pyridoxal phosphate, the conversion rate of the intermediate product pyridoxine can reach 62%, the conversion rate of the speed limiting step of the path is greatly improved, the production capacity of a unit catalyst and the reaction efficiency of the catalyst are improved, and the cost of the reaction is reduced.

Description

Recombinant acid phosphatase mutant and application thereof in synthesis of pyridoxal phosphate
Technical Field
The invention relates to a recombinant acid phosphatase mutant and application thereof in pyridoxal phosphate synthesis, belonging to the technical field of bioengineering.
Background
Pyridoxal phosphate (Pyridoxal-5-phosphatemonohydrate, PLP), of formula C 8H10NO6 P, is the major active form of vitamin B6. Pyridoxal phosphate is a coenzyme for 160 enzymes and is involved in a very large number of metabolic reactions such as intracellular transamination, racemization, decarboxylation, etc. Pyridoxal phosphate has a wide range of applications in many fields: in the field of medicine, pyridoxal phosphate is widely applied to the synthesis of beta-carbonyl amide medicines, and the promotion of ammonia transfer to increase the content of dopamine in the body so as to treat parkinsonism and the like; in the food field pyridoxal phosphate is often used to evaluate the nutritional value of food products.
The natural pyridoxal phosphate in nature has very rare content, and the extraction process is complex and insufficient to meet the demands of people, so that the preparation of the pyridoxal phosphate with high purity has important significance. Pyridoxal phosphate is mainly chemically synthesized in the industrial field, pyridoxamine dihydrochloride is reacted with anhydrous phosphoric acid to generate pyridoxamine phosphate, and then manganese dioxide is used for oxidation to obtain pyridoxal phosphate. However, the method has the problems of excessive consumption of dangerous chemicals, high production cost, high environmental pollution cost, low product recovery efficiency and the like. In the metabolic pathway in the living body, pyridoxine kinase takes free ATP as a phosphate donor, pyridoxine is synthesized by taking pyridoxine as a substrate, pyridoxine oxidase is coupled, and an intermediate pyridoxine phosphate is oxidized into pyridoxal phosphate. However, this biosynthesis pathway uses ATP as a phosphate donor, and has a problem of excessive production cost. There is therefore a great need to develop a biological process for the efficient preparation of pyridoxal phosphate using low cost inorganic phosphoric acid as substrate.
Disclosure of Invention
The invention provides a recombinant acid phosphatase StAPase and a mutant thereof, wherein the amino acid sequence of the recombinant acid phosphatase StAPase is shown as SEQ ID NO.3, and the nucleotide sequence of the recombinant acid phosphatase is shown as SEQ ID NO. 4. The recombinant acid phosphatase StAPase is obtained by removing a signal peptide (the amino acid sequence of the signal peptide is shown as SEQ ID NO. 5) at the C-terminal of APase on the basis of acid phosphatase StAPase (the amino acid sequence is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO. 2) derived from Salmonella typhi.
The invention also provides a recombinant pyridoxine oxidase EcPNPO, the amino acid sequence of the recombinant pyridoxine oxidase EcPNPO is shown as SEQ ID NO.6, and the nucleotide sequence of the recombinant pyridoxine oxidase EcPNPO is shown as SEQ ID NO. 7. The recombinant pyridoxine oxidase EcPNPO is derived from escherichia coli (ESCHERICHIA COLI).
The invention also provides a recombinant acid phosphatase StAPase mutant, which is obtained by mutating one or more of 57 th, 58 th, 59 th, 60 th, 91 st, 96 th and 124 th amino acids of a recombinant acid phosphatase StAPase with an amino acid sequence shown as SEQ ID NO. 3.
The numbers of the 58 th, 91 st and 98 th mutation sites are numbered according to the sequence (the amino acid sequence is shown as SEQ ID NO. 3) obtained by removing the signal peptide from the amino acid sequence of the original source acid phosphatase StAPase of the recombinant acid phosphatase StAPase.
In one embodiment of the present invention, the mutant is any one of the following (a) to (g):
(a) An aspartic acid at position 57 of acid phosphatase StAPase with an amino acid sequence shown in SEQ ID NO.3 is mutated into asparagine, and the mutant is named D57N;
(b) The isoleucine at position 58 of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into threonine, and the mutant is named I58T;
(c) The serine at the 59 th position of the acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into threonine, and the mutant is named S59T;
(d) The valine at position 60 of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into leucine, and the mutant is named V60L;
(e) The leucine at position 91 of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into histidine, and the mutant is named L91H;
(f) The tyrosine at the 96 th position of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into tryptophan, and the mutant is named as Y96W;
(g) The aspartic acid at position 124 of acid phosphatase StAPase with the amino acid sequence shown in SEQ ID NO.3 is mutated into glutamic acid, and the mutant is named D124E.
In one embodiment of the invention, the nucleotide sequence encoding the recombinant acid phosphatase StAPase is shown in SEQ ID NO. 4.
The invention also provides a gene for encoding the recombinant acid phosphatase StAPase mutant.
The invention also provides a recombinant vector carrying the gene.
In one embodiment of the present invention, the recombinant vector uses pET-28a as an expression vector.
The invention also provides a microbial cell carrying the gene or the recombinant vector.
In one embodiment of the invention, the microbial cells use bacteria or fungi as expression hosts.
In one embodiment of the invention, the microbial cells have ESCHERICHIA COLI BL (DE 3) as expression hosts.
The invention also provides a recombinant escherichia coli which expresses the recombinant acid phosphatase StAPase mutant.
In one embodiment of the invention, the recombinant E.coli uses ESCHERICHIA COLI BL (DE 3) as the expression host and pET-28a as the expression vector.
The invention also provides a method for obtaining the recombinant acid phosphatase StAPase mutant, which comprises the following steps:
(1) Determining mutation sites based on the amino acid sequence of recombinant acid phosphatase StAPase; designing a mutation primer of site-directed mutagenesis, and carrying out site-directed mutagenesis by taking a carrier carrying a recombinant acid phosphatase StAPase gene as a template; constructing a plasmid vector containing the mutant;
(2) Transforming a plasmid vector comprising the mutant into a host cell;
(3) Positive clones were selected for fermentation culture and acid phosphatase StAPase was purified.
In one embodiment of the invention, the host cell is E.coli.
The invention also provides a preparation method of pyridoxine phosphate, which comprises the following steps:
adding the recombinant acid phosphatase StAPase mutant, the microbial cell or the recombinant escherichia coli into a reaction system containing pyridoxine hydrochloride and sodium pyrophosphate, and reacting to obtain the recombinant acid phosphatase StAPase mutant.
In one embodiment of the present invention, the microbial cells, or the recombinant E.coli, are added to the reaction system at a final concentration of 5 to 35g/L.
In one embodiment of the invention, the recombinant E.coli is added to the reaction system at a final concentration of 20g/L.
In one embodiment of the invention, the pyridoxine hydrochloride is present in the reaction system in a final concentration of: 10-40 g/L.
In one embodiment of the invention, the final concentration of sodium pyrophosphate in the reaction system is: 20-90 g/L.
In one embodiment of the invention, the reaction conditions are: reacting for 6-12 h at the pH of 3.5-5.0 and the temperature of 25 ℃.
The invention also provides application of the recombinant acid phosphatase StAPase mutant, the gene, the recombinant vector, the recombinant cell or the recombinant escherichia coli in preparing pyridoxine phosphate and pyridoxal phosphate or products containing pyridoxine phosphate and pyridoxal phosphate.
In one embodiment of the invention, the product is a chemical.
Advantageous effects
(1) The invention provides a recombinant acid phosphatase StAPase and a mutant thereof, which are used for catalyzing and producing pyridoxine phosphate and pyridoxal phosphate. Compared with the wild acid phosphatase StAPase, the specific activity is improved by 3.5 times, and the catalytic efficiency is improved by 4.87 times.
(2) The recombinant acid phosphatase StAPase mutant of the invention has higher pyridoxine phosphorylation activity, and the highest yield of pyridoxine phosphate is 30g/L, the yield is 62%, and compared with the wild acid phosphatase StAPase, the pyridoxine phosphate yield is improved by 60 times and the yield is improved by 60.2 times under the conditions of 25 ℃ and pH 4.0; after 9h of pyridoxine phosphorylation, the recombinant pyridoxine oxidase EcPNPO of the present invention was added and reacted at 37℃and pH 8.0 for 2h, the maximum yield of PLP was 14.55g/L, which was close to 50% compared to PNP production. The method of the invention improves the productivity of the unit catalyst and the reaction efficiency of the catalyst, reduces the cost of the reaction and accelerates the industrialization process of producing pyridoxal phosphate by the enzyme conversion method.
Drawings
Fig. 1: the reaction formula of the invention.
Fig. 2: SDS-PAGE of induced expression of acid phosphatase StAPase derived from Salmonella typhi and pyridoxine oxidase EcPNPO derived from E.coli; lane M refers to a low molecular weight protein Marker; the lane blank is the protein band of the empty competent cell E.coli BL21 (pET-28 a).
Fig. 3: an optimal mutant Q9 of acid phosphatase StAPase derived from salmonella typhi; lane M refers to the low molecular weight protein Marker.
Fig. 4: liquid phase detection patterns of reaction substrates and products; i: PN standard liquid phase detection, wherein the retention time of PN is 18.5min; ii: liquid phase detection of PNP standard, wherein the retention time of PNP is 4.4min; iii: PNP is produced by whole cell conversion PN for internal standard detection, and the retention time of PNP is 4.4min; iv: liquid phase detection of PLP standard substance, wherein the retention time is 6.8min; v: PLP was produced by whole cell conversion PN and subjected to internal standard detection, and the retention time of PLP was 6.8min.
Detailed Description
PET-28a (+) masses referred to in the examples below were purchased from Novagen (Madison, wis., U.S. A.), restriction enzymes, T4DNA ligase, KOD high fidelity enzymes, and the like from TaKaRa (Dalian, china). The standard pyridoxine hydrochloride is purchased from Hubei Huitian pharmaceutical industry Co., ltd, the pyridoxine phosphate and pyridoxal phosphate are purchased from Shanghai Nafu Biotechnology Co., ltd, and the rest reagents are all obtained by market purchase.
The following examples relate to the following media:
LB liquid medium: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, and sterilizing at 121deg.C for 20min.
LB solid medium: on the basis of LB liquid medium, 2% agar was added.
TB liquid medium: KH 2PO4 2.31g/L,K2HPO4·3H2 O16.42 g/L, yeast powder 24g/L, peptone 12g/L, glycerol 4g/L.
Preparing a reaction solution: 0.2mol/L pyridoxine hydrochloride and 0.2mol/L sodium pyrophosphate were dissolved in water and the pH of the system was adjusted to 4.0 using phosphoric acid, at which point the solution was yellowish.
The detection method involved in the following examples is as follows:
Detection of acid phosphatase StAPase phosphorylase activity:
The double-substrate pyridoxine hydrochloride and sodium pyrophosphate were dissolved in water at a concentration of 0.2mol/L in a ratio of 1:1, and the pH of the system was adjusted to 4.0 using phosphoric acid to prepare a yellowish reaction solution. Then adding 10 mu L of purified acid phosphatase pure enzyme, shaking and reacting for 4 hours at 25 ℃ and 220rpm, and centrifuging the reacted reaction liquid sample for 5-10 minutes at 3700 rpm. The supernatant was aspirated, diluted 30-fold and dissolved in an aqueous solution, filtered using a 0.22 μm water membrane, and analyzed using high performance liquid chromatography HPLC.
Definition of enzyme activity: 1U represents the amount of enzyme required for 1min to produce 1. Mu. Mol pyridoxine phosphate (PNP).
The specific enzyme activity is defined as: the micromole amount of pyridoxine phosphate (PNP) produced by 1g of protein at 1min at pH 4.0 and 25℃was defined as one enzyme activity unit, denoted U.g -1, using pyridoxine hydrochloride and sodium pyrophosphate as reaction substrates.
Detection of pyridoxine phosphate (PNP), pyridoxal phosphate (PLP) content:
Pyridoxal phosphate was produced by whole-cell conversion of pyridoxine hydrochloride and sodium pyrophosphate with ultrapure water as the aqueous phase, pyridoxine hydrochloride at 0.2mol/L and sodium pyrophosphate at 0.2mol/L as substrates. The whole cell concentration of the acid phosphatase is 20 g.L -1, after the reaction is carried out for 9 hours in a constant temperature shaking table with the pH of 4.0 and 220rpm at 25 ℃, the reaction solution is sampled, a small amount of the sampled sample is placed in an EP tube, the sample is centrifuged for 5 to 10 minutes under the condition of 3700rpm, the supernatant is sucked, diluted 30 times and dissolved in the aqueous solution, and the aqueous solution is subjected to HPLC analysis by a 0.22 mu m water film. The remaining reaction mixture was added with 20 g.L -1 of pyridoxine oxidase whole cells and reacted for 2 hours at 37℃in a constant temperature shaker at pH 8.0 and 220 rpm. After conversion, the sample reaction solution is centrifuged for 5 to 10 minutes at 3700rpm, the supernatant is sucked, diluted 30 times and dissolved in water solution, and the water film of 0.22 mu m is used for HPLC analysis.
The specific HPLC analysis method comprises the following steps:
C18 (5 μm,250×4.6mm) was used as a chromatographic column, suction-filtered, ultrasonically degassed methanol/sodium heptanesulfonate was used as a mobile phase, the sample injection amount was 10. Mu.L, the column temperature was 30deg.C, the wavelength of the ultraviolet detector was 319nm, the flow rate was 0.5mL/min, and the sample treatment time was 30min. Under this detection condition, the retention times of pyridoxine phosphate (PNP) and pyridoxal phosphate (PLP) were 4.8min and 6.8min, respectively.
Wherein: m PNP、mPLP represents the mass of PNP and PLP, g; m PN·HCl represents the initial mass of pyridoxine; 205.64, 249 and 247 represent the relative molecular masses of pyridoxine hydrochloride, pyridoxine phosphate and pyridoxal phosphate, respectively.
Example 1: construction of genetically engineered bacteria and expression of StAPase
(1) Construction of genetically engineered bacteria and expression of acid phosphatase StAPase:
The nucleotide sequence (shown in SEQ ID NO. 4) of the target protein acid phosphatase StAPase coding gene in Salmonella typhi Salmonella enterica subsp is artificially synthesized as a target gene fragment. The target gene fragment is connected between BamHI and HindIII of a vector pET-28a (+) to obtain a recombinant plasmid pET-28a (+) -StAPase, and the recombinant plasmid pET-28a (+) -StAPase is transformed into E.coli BL21 to obtain the genetically engineered bacterium E.coli BL21/pET-28a (+) -StAPase.
Inoculating engineering bacteria E.coli BL21/pET-28a (+) -StAPase into LB liquid culture medium, culturing at 37 ℃ and 220rpm for 12h to obtain seed liquid, inoculating the seed liquid into fresh TB liquid culture medium according to the inoculum size of 5% (v/v), culturing for 2h, adding IPTG with the final concentration of 0.2mM, culturing at 16 ℃ for 14h, and inducing to express recombinant protein, wherein OD 600 in fermentation broth is 2.3. Centrifuging at 6000r/min to collect thallus, and freeze preserving at-40deg.C.
(2) Construction of genetically engineered bacteria and expression of pyridoxine oxidase EcPNPO:
The nucleotide sequence (shown as SEQ ID NO. 7) encoded by pyridoxine oxidase EcPNPO, a target protein derived from Escherichia coli ESCHERICHIA COLI, was artificially synthesized as a target gene fragment. The target gene fragment is connected between BamHI and HindIII of a vector pET-28a (+) to obtain a recombinant plasmid pET-28a (+) -EcPNPO, and the recombinant plasmid pET-28a (+) -EcPNPO is transformed into E.coli BL21 to obtain the genetically engineered bacterium E.coli BL21/pET-28a (+) -EcPNPO.
Inoculating engineering bacteria E.coli BL21/pET-28a (+) -EcPNPO into LB liquid culture medium, culturing at 37 ℃ and 220rpm for 12h to obtain seed liquid, inoculating the seed liquid into fresh TB liquid culture medium according to the inoculum size of 5% (v/v), culturing at 37 ℃ and 220rpm for 2h, adding IPTG with the final concentration of 0.2mM, culturing at 16 ℃ for 14h, and inducing expression of recombinant target protein. 150mL of induced fermentation broth is taken, at the moment, OD 600 is about 2.5-3.0, the fermentation broth is centrifuged at 6000r/min to collect thalli, and the thalli are placed at-40 ℃ for freezing preservation.
Example 2: expression purification and phosphorylation Activity verification of acid phosphatase
The method comprises the following specific steps:
(1) Preparation of crude enzyme solution
Centrifuging the fermentation broth obtained in the step (1) in the example 1 at 6000rpm for 10min at 4 ℃ to obtain thalli. 10mL of the binding solution A (20 mM sodium phosphate, 0.5mM NaCl, 20mM imidazole, 1% glycerol, and pH was adjusted to 7.4 with HCl) was added to fully resuspend the cells, and then the centrifuge tube was placed in an ice bath and placed in an ultrasonic cytobreaker under the following conditions: the working time is 4s, the interval time is 4s, and the total time is 10min. And (3) centrifuging the obtained crushed liquid at a low temperature and a high speed for 30min at a temperature of 4 ℃ and at a speed of 12000rpm to obtain crude enzyme liquid. Filtering with 0.22 μm microporous membrane for use.
(2) Purification of acid phosphatase
A nickel ion affinity chromatography column was prepared, first, the column was flushed with ultra-pure water (about 6-12 column volumes) by pumping the column with a constant flow pump at 4℃and then the column environment was equilibrated with 10mL of binding solution A. When the effluent at the lower end of the column is consistent with the pH value of the low salt concentration buffer solution pumped into the column (about 5 column volumes of buffer solution are needed), the obtained membrane-passing crude enzyme solution is added into the column. The hybrid protein was washed with binding solution A to baseline equilibrium and eluted with eluent B (20 mM sodium phosphate, 0.5mM NaCl, 500mM imidazole). Collecting the eluent of absorption peak, and measuring enzyme activity to obtain the target protein reaching electrophoresis purity.
The purified StAPase pure enzyme solution was subjected to enzyme activity detection, and the result shows that the StAPase specific activity is 0.037 U.mg -1.
Example 3: preparation of pyridoxal phosphate by Whole cells
To a10 mL vial, 0.2g of whole cell E.coli BL21/pET-28a (+) -StAPase expressing acid phosphatase StAPase after induction culture, 0.41g of pyridoxine hydrochloride, 0.8g of sodium pyrophosphate were added, ultrapure water and phosphoric acid were added to a volume of 10mL, pH4.0 was adjusted, and the mixture was reacted in a shaking table at a constant temperature of 220rpm at 25℃for 9 hours. After conversion, the sample reaction solution is centrifuged for 5 to 10 minutes at 3700rpm, the supernatant is sucked, diluted 30 times and dissolved in water solution, and the water film of 0.22 mu m is used for HPLC analysis. The analysis results were: the yield of pyridoxine phosphate prepared by whole cell transformation of recombinant bacterium E.coli BL21/pET-28a (+) -StAPase is 0.5g/L, and the transformation rate is 1.03%. It can be seen that the catalytic efficiency of recombinant E.coli BL21/pET-28a (+) -StAPase for pyridoxine hydrochloride phosphorylation is not high.
Before the second reaction, the reaction solution 3700rpm of the phosphorylation step is centrifuged for 5min, and the recombinant E.coli BL21/pET-28a (+) -StAPase whole cells are separated. Then, 0.2g of recombinant E.coli BL21/pET-28a (+) -EcPNPO expressing pyridoxine oxidase obtained in the step (2) of example 1 after the induction culture, 0.02g of riboflavin sodium phosphate, and a proper amount of sodium hydroxide particles were added to the reaction mixture to adjust the reaction system to pH8.5, and the mixture was reacted in a shaking table at a constant temperature of 220rpm at 37℃for 2 hours to adjust the reaction pH to 8.5. After conversion, the sample reaction solution is centrifuged for 5 to 10 minutes at 3700rpm, the supernatant is sucked, diluted 30 times and dissolved in water solution, and the water film of 0.22 mu m is used for HPLC analysis. The analysis results were: the yield of pyridoxal phosphate prepared by whole cell transformation of recombinant bacterium E.coli BL21/pET-28a (+) -EcPNPO was 0.44g/L, the single step reaction conversion rate of PLP from PNP was 87.2%, and the total conversion rate of two steps of PLP production from pyridoxine hydrochloride (PNHCl) was 0.72%.
It can be seen that in the two-step reaction, the first pyridoxine phosphorylation step is the rate limiting step in the present reaction.
Example 4: construction and screening of Single mutation variants
The method comprises the following specific steps:
Using pET-28a (+) -StAPase constructed in example 1 as a template, primers for StAPase I58T、StAPaseL91H、StAPaseY96Q mutation site were designed, and mutant construction was performed by whole plasmid PCR as shown in Table 1.
TABLE 1 Single mutant primer sequences
Constructing a reaction PCR amplification system: 1.5. Mu.L of KOD high-fidelity enzyme, 1.5. Mu.L of 5 XKOD Buffer 5. Mu. L, dNTP. Mu. L, mgSO. Mu.L of two primers for each mutation site, 1.5. Mu.L of template (STAPASEWT) and 32. Mu.L of water; the reaction conditions are as follows: ①94℃5min;②98℃10s;③55℃30s;④72℃3min;;⑤ The ②～④ three steps are cycled 29 times; ⑥72℃5min;⑦ Preserving heat at 12 ℃. The reaction system is incubated for 3 hours at 37 ℃ to digest the plasmid template (the digestion system is that DpnI is 0.5 mu L, the reaction PCR product is 45 mu L and 10 xT Buffer is 5 mu L), and the digested product obtained after the digestion is introduced into competent cells of escherichia coli BL21 by a chemical conversion method, wherein the specific steps of the chemical conversion method are as follows: (1) 10. Mu.L of the homologous recombination product was introduced into 100. Mu.L of E.coli BL21 (DE 3) competent cells; (2) ice bath for 10min; (3) Carrying out heat shock on the mixture in a water bath at 42 ℃ for 1min for 30s, taking out the mixture, rapidly putting the mixture into ice, and standing the ice bath for 4min; (4) Adding 800 mu L of non-resistant LB culture medium, uniformly mixing, and culturing for 1h at 37 ℃ and 200 rpm; (5) centrifuging at 5000rpm for 2min to collect bacteria; (6) The supernatant was removed, and the remaining 100-200. Mu.L was applied to LB-resistant plates containing 0.05mg/mL kanamycin by pipetting, and incubated at 37℃for about 12 hours. (7) The monoclonal is selected and cultured in LB containing 0.05mg/mL kanamycin resistance for 12 hours at a constant temperature of 200rpm and 37 ℃, and then sent to a company for sequencing, and the positive transformant is obtained after the sequencing is correct.
Respectively preparing the genetically engineered bacteria E.coli BL21/pET-28a(+)-StAPaseI58T、E.coli BL21/pET-28a(+)-StAPaseL91H、E.coli BL21/pET-28a(+)-StAPaseY96W.
And then carrying out whole-cell transformation on the obtained genetically engineered bacteria according to the method of the example 3 to prepare pyridoxal phosphate, and screening out better mutants, wherein the result is shown in a table 2, engineering bacteria E.coli BL21/pET-28a(+)-StAPaseI58T、E.coli BL21/pET-28a(+)-StAPaseL91H、E.coli BL21/pET-28a(+)-StAPaseY96W have better pyridoxine hydrochloride phosphorylation effect, OD 600 after the mutant induction is 2.3-2.5, and PNP yield is 2.4-3 times that of wild E.coli BL21/pET-28a (+) -StAPase.
TABLE 2 wild-type and Single mutant PNP yields
EXAMPLE 5 construction and screening of multiple mutants
(1) Construction of double mutants:
The double mutant of this example was constructed by whole plasmid PCR based on the corresponding single mutant according to the primers in Table 4, for example, on mutant StAPase I58T using mutant primers L91H-R and L91HR-F (Table 1), and by whole plasmid PCR.
The preparation method of the genetically engineered bacteria is described in example 1, the primers are shown in Table 3, and the genetically engineered bacteria containing double mutants are prepared according to the method of example 4 ：E.coli BL21/pET-28a(+)-StAPaseI58T/L91H、E.coli BL21/pET-28a(+)-StAPaseI58T/Y96W、E.coli BL21/pET-28a(+)-StAPaseL91H/Y96W.
TABLE 3 double mutant primer sequences
(2) Screening of double mutants:
The mutant strain with correct sequence is inoculated to LB seed culture medium, cultured for 12h at 220rpm and 37 ℃, seed solution is respectively inoculated to fresh TB liquid culture medium according to the inoculum size of 5% (v/v), after 2h of culture at 220rpm and 37 ℃, lactose with 0.2mM of final concentration of IPTG is added for induction, the induction condition is 220rpm and 16 ℃ for 14h, and OD 600 is 2.3-2.5 after the mutant induction.
The obtained genetically engineered bacteria were then transformed into pyridoxine phosphate by whole cell transformation in the same manner as in example 3.
After the completion of the reaction, the yield of pyridoxine phosphate was measured by HPLC, and the results are shown in Table 4, and the double mutant-containing genetically engineered bacterium ：E.coli BL21/pET-28a(+)-StAPaseI58T/L91H、E.coli BL21/pET-28a(+)-StAPaseI58T/Y96W、E.coli BL21/pET-28a(+)-StAPaseL91H/Y96W had good phosphorylating effect on pyridoxine hydrochloride, among which the mutant E.coli BL21/pET-28a (+) -StAPase L91H/Y96W had the best effect.
TABLE 4 yield of double mutant PNPs
(3) Construction of the Tri-mutant variant:
Based on the mutant E.coli BL21/pET-28a (+) -StAPase L91H/Y96W, three-mutation variant construction is carried out by whole plasmid PCR by using mutation primers I58T-R and I58T-F (table 1), the specific embodiment is shown in the example 1, the primers are shown in the table 5, and the three-mutant E.coli BL21/pET-28a (+) -StAPase I58T/L91H/Y96W genetic engineering bacteria are prepared according to the method of the example 4. The whole cell transformation was performed to prepare pyridoxine phosphate according to the procedure of example 3; after the reaction is finished, the yield of PNP is measured by an HPLC method, the conversion rate of PNP prepared by the triple mutant E.coli BL21/pET-28a (+) -StAPase I58T/L91H/Y96W gene engineering bacteria is 13.4%, and the corresponding yield is 6.5g/L.
(4) Construction of tetramutant variants:
Based on the mutant E.coli BL21/pET-28a (+) -StAPase L91H/Y96W, four-mutation variant construction is carried out by whole plasmid PCR (table 5) by using mutation primers D124E-R and D124E-F, the specific embodiment is shown in the example 1, the primers are shown in the table 5, and the four-mutant E.coli BL21/pET-28a (+) -StAPase I58T/L91H/Y96W/D124E genetic engineering bacteria are prepared according to the method of the example 4. The whole cell transformation was performed to prepare pyridoxine phosphate according to the procedure of example 3; after the reaction is finished, the yield of PNP is measured by an HPLC method, the conversion rate of PNP prepared by the four mutant E.coli BL21/pET-28a (+) -StAPase I58T/L91H/Y96W/D124E genetic engineering bacteria is 19.6%, and the corresponding yield is 9.5g/L.
TABLE 5 four mutant mutation primer sequences
(5) Construction of seven-mutation variants:
Based on the mutant E.coli BL21/pET-28a (+) -StAPase I58T/L91H/Y96W/D124E, four-mutation variant construction is carried out by whole plasmid PCR by using mutation primers D57N/S59T/V60L-R and D57N/S59T/V60L-R (table 7), the specific embodiment is shown in the step (1) in the example 2, the used primers are shown in the table 6, and the seven-mutant E.coli BL21/pET-28a (+) -StAPase D57N/I58T/S59T/V60L/L91H/Y96W/D124E genetic engineering bacteria are prepared according to the method of the example 4. The whole cell transformation was performed to prepare pyridoxine phosphate according to the procedure of example 3; after the reaction is finished, the yield of PNP is measured by an HPLC method, the conversion rate of PNP prepared by the four mutant E.coli BL21/pET-28a (+) -StAPase D57N/I58T/S59T/V60L/L91H/Y96W/D124E genetic engineering bacteria is 62%, and the corresponding yield is 30g/L.
TABLE 6 seven mutant primer sequences
(6) Preparation of pyridoxal phosphate by mutant Whole cells
After the modification of the critical enzyme for the rate limiting step was completed, the actual yield of PLP produced by this route was determined. Pyridoxine phosphate was prepared whole cells using the wild type and each mutant according to the procedure of example 2. After the reaction, the reaction solution was centrifuged at 3700rpm for 5 to 10 minutes to remove the acid phosphatase from the reaction solution. The reaction mixture was adjusted to pH8.5 with an aqueous sodium hydroxide solution, FMN was added at a final concentration of 10mmol/L, ecPNPO whole cells were added at 37℃and 220rpm for 10 hours, and then the mixture was diluted 30-fold with a sample for liquid phase detection. The PNP and PLP data for all cells produced catalytically are shown in Table 7.
Table 7StAPase comparison of Whole cell transformation Effect of parent enzyme and mutant thereof
Example 6: performance assays of parent enzymes and mutants
(1) Measurement of enzyme Activity
The specific enzyme activities of the purified mutants were examined by the method of example 1-2, and the results are shown in Table 8.
Table 8StAPase comparison of enzyme activities of parent enzymes and mutants thereof
(2) Determination of kinetic parameters
For evaluation of beneficial mutants, the present invention measured the kinetic parameters of mutant parent Q0 (StAPase WT) and mutants Q1 to Q9 (with specific meanings as shown in table 9) at 25 ℃. k cat/Km is calculated by measuring the yield of pyridoxine phosphate produced by phosphorylation of pyridoxine hydrochloride at various concentrations at 25 ℃.
Kinetic parameters of the parent enzyme and mutants thereof in Table 9StAPase
The results show that the K m value of the optimal mutant is obviously reduced compared with that of the WT, which indicates that the affinity of the seven mutants to the substrate is increased; seven mutants StAPase D57N/I58T/S59T/V60L/L91H/Y96W/D124E had a catalytic efficiency k cat/Km(4.46±0.81)×10-2h-1·mM-1) that was 4.87-fold higher than WT (0.76±0.21) ×10 -2h-1·mM-1).
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A recombinant acid phosphatase StAPase mutant, wherein said mutant is a combination of one or more of the following (a) - (g):
(a) An aspartic acid at position 57 of acid phosphatase StAPase with an amino acid sequence shown in SEQ ID NO.3 is mutated into asparagine, and the mutant is named D57N;
(b) The isoleucine at position 58 of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into threonine, and the mutant is named I58T;
(c) The serine at the 59 th position of the acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into threonine, and the mutant is named S59T;
(d) The valine at position 60 of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into leucine, and the mutant is named V60L;
(e) The leucine at position 91 of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into histidine, and the mutant is named L91H;
(f) The tyrosine at the 96 th position of acid phosphatase StAPase with the amino acid sequence shown as SEQ ID NO.3 is mutated into tryptophan, and the mutant is named as Y96W;
(g) The aspartic acid at position 124 of acid phosphatase StAPase with the amino acid sequence shown in SEQ ID NO.3 is mutated into glutamic acid, and the mutant is named D124E.
2. A gene encoding the mutant of claim 1.
3. A recombinant vector carrying the gene of claim 2.
4. A microbial cell carrying the gene of claim 2, or the recombinant vector of claim 3.
5. Recombinant E.coli, characterized in that the mutant according to claim 1 is expressed using ESCHERICHIA COLI BL (DE 3) as host.
6. The recombinant escherichia coli of claim 5, wherein the expression vector of the recombinant escherichia coli includes, but is not limited to pET-28a.
7. A method for producing pyridoxal phosphate, characterized in that the mutant according to claim 1, or the microbial cell according to claim 4, or the recombinant escherichia coli according to claim 5 or 6 is added to a reaction system containing pyridoxine hydrochloride and sodium pyrophosphate for reaction.
8. The method of claim 7, wherein the mutant of claim 1 is added to the reaction system; in the reaction system, the final concentration of the mutant is 9-30g/L.
9. The method according to claim 7, wherein the microbial cell according to claim 4, or the recombinant escherichia coli according to claim 5 or 6 is added to the reaction system; in the reaction system, the final concentration of the microbial cells or the recombinant escherichia coli is 5-35g/L.
10. Use of a mutant according to claim 1, or a gene according to claim 2, or a recombinant vector according to claim 3, or a recombinant microbial cell according to claim 4, or a recombinant escherichia coli according to claim 5 or 6, for the preparation of pyridoxine phosphate, pyridoxal phosphate, or a product comprising pyridoxine phosphate and pyridoxal phosphate.
CN202410059317.6A 2024-01-15 2024-01-15 Recombinant acid phosphatase mutant and application thereof in synthesis of pyridoxal phosphate Pending CN117965492A (en)

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