A catalase EcCAT mutant and its application

Abstract

This invention belongs to the field of enzyme engineering technology, specifically relating to an EcCAT mutant of catalase and its applications. The mutant is obtained by mutating at least one amino acid from the following positions in SEQ ID NO:1: Ile (126th position), Ser (167th position), Val (199th position), Gly (200th position), Asn (201st position), Phe (206th position), Ala (211th position), Phe (214th position), Gly (273rd position), Ala (389th position), Phe (391st position), Leu (407th position), and Gly (410th position). Compared to wild-type catalase, the EcCAT mutant of this invention exhibits higher catalytic efficiency. When used in combination with pyridoxine 5'-phosphate oxidase, the whole-cell catalytic synthesis of pyridoxal 5'-phosphate can be completed in just 5 hours, with a conversion rate exceeding 99%.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a catalase EcCAT mutant and its application in the synthesis of pyridoxal 5'-phosphate by combining it with pyridoxine 5'-phosphate oxidase. Background Technology

[0002] Catalase (CAT, EC 1.11.1.6) is a key antioxidant enzyme widely found in animals, plants, and microorganisms. Its core biological function is to catalyze the decomposition of hydrogen peroxide (H₂O₂) into water and oxygen (2H₂O₂ → 2H₂O + O₂), thereby eliminating reactive oxygen species generated during cellular metabolism and protecting cells from oxidative damage. Due to its highly efficient and environmentally friendly catalytic properties, catalase has demonstrated significant application value in various fields, including food processing (such as milk pasteurization and cake baking), textile industry (such as removing residual H₂O₂ after fabric bleaching), papermaking industry (such as chlorine-free bleaching processes), medical diagnostics, and environmental protection.

[0003] Currently, the application of catalase is expanding from a single "residual H2O2 scavenger" to a "synergistic enhancer of oxidase systems." In many biocatalytic and biosensing systems, oxidases (such as glucose oxidase, cholesterol oxidase, and choline oxidase) inevitably produce an equimolar amount of hydrogen peroxide as a byproduct when catalyzing the oxidation of substrates. For example, in the study of vitamin B6 metabolism and synthesis, pyridoxine 5'-phosphate oxidase (PNPO) is used as a byproduct. x The enzyme is responsible for catalyzing the oxidation of pyridoxine 5'-phosphate (PNP) to pyridoxal 5'-phosphate (PLP), a reaction that generates H2O2. Accumulated H2O2 in the reaction system can lead to a series of negative effects; in some whole-cell catalytic systems, H2O2 accumulation may even be toxic to the growth and metabolism of host cells. Therefore, timely and efficient removal of H2O2 generated during the reaction has become a key technical bottleneck for improving the catalytic efficiency of oxidases, extending enzyme lifespan, and ensuring the stability of the reaction process.

[0004] Combining catalase with 5'-pyridoxine oxidase or other industrial oxidases to construct a "dual-enzyme synergistic catalytic system" holds promise for solving the aforementioned problems. This synergistic effect can not only significantly improve the catalytic efficiency of 5'-pyridoxine oxidase (manifested as increased conversion rate and shortened reaction time), but also effectively protect the conformational stability of the oxidase, extend its service life, and thus reduce production costs.

[0005] However, naturally derived catalases often fail to meet the stringent requirements of industrial applications due to their low enzyme activity and poor thermal stability. Summary of the Invention

[0006] The purpose of this invention is to provide an EcCAT mutant of catalase and its application in the synthesis of pyridoxal 5'-phosphate, so as to solve the problems of low enzyme activity, insufficient catalytic efficiency and poor thermal stability of wild-type catalase.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a catalase EcCAT mutant, which is obtained by mutating at least one amino acid from the following positions: Ile at position 126, Ser at position 167, Val at position 199, Gly at position 200, Asn at position 201, Phe at position 206, Ala at position 211, Phe at position 214, Gly at position 273, Ala at position 389, Phe at position 391, Leu at position 407, and Gly at position 410, based on SEQ ID NO:1.

[0009] Preferably, the mutant is based on SEQ ID NO:1, with its 126th position Ile mutated to Phe; or

[0010] Mutate its 167th Ser bit to Asp; or

[0011] Mutate its 167th Ser position to Cys; or

[0012] Transform its 199th Val position into Ala; or

[0013] Mutate its 200th Gly to Gly (synonymous mutation); or

[0014] Mutate its 201st Asn to Leu; or

[0015] Transform its 206th Phe into Asn; or

[0016] Mutate its 211th Ala position to Arg; or

[0017] Mutate its 214th Phe to Phe (synonymous mutation); or

[0018] Mutate its 273rd Gly position to Val; or

[0019] Mutate its 389th Ala position to Gly; or

[0020] Transform its 391st Phe into Met; or

[0021] Transform its 407th position, Leu, into Cys; or

[0022] Transform its 407th position, Leu, into Val; or

[0023] The 410th position of Gly was mutated to Phe.

[0024] More preferably, the mutant is based on SEQ ID NO:1, with its 199th position Val mutated to Ala and its 407th position Leu mutated to Val; or

[0025] Mutate its 199th Val position to Ala and its 201st Asn position to Leu; or

[0026] Its 199th Val was mutated to Ala, and its 273rd Gly was mutated to Val.

[0027] More preferably, the mutant is based on SEQ ID NO:1, with its 199th position Val mutated to Ala, its 407th position Leu mutated to Val, and its 201st position Asn mutated to Leu; or

[0028] Its 199th Val was mutated to Ala, its 407th Leu was mutated to Val, its 201st Asn was mutated to Leu, and its 273rd Gly was mutated to Val.

[0029] The present invention also provides a gene encoding any of the above-mentioned catalase EcCAT mutants and a recombinant expression vector carrying the gene.

[0030] Furthermore, the recombinant expression vector uses pET-29a(+) as the original expression vector.

[0031] The present invention also provides recombinant microorganisms comprising the recombinant expression vector.

[0032] Preferably, the host bacterium of the recombinant microorganism is Escherichia coli, preferably Escherichia coli BL21(DE3).

[0033] The present invention also provides the use of any of the above-mentioned catalase EcCAT mutants in the production of pyridoxal 5'-phosphate in combination with pyridoxine oxidase.

[0034] The present invention also provides a method for producing pyridoxal 5'-phosphate, comprising the following steps:

[0035] The above-mentioned catalase EcCAT mutant or its recombinant microorganism and pyridoxine 5'-phosphate oxidase were added to a reaction system containing pyridoxine phosphate (PNP) and flavin mononucleotide (FMN); the oxidation reaction was carried out under the conditions of pH 7.0-11.0 and temperature 36-40℃ to generate pyridoxal 5'-phosphate.

[0036] Preferably, in the reaction system, the concentration of pyridoxine phosphate is 5-20 g / L, the concentration of the recombinant microorganism with catalase EcCAT mutant is 1-25 g / L, and the concentration of flavin mononucleotide is 1-10 mg / L.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention utilizes directed evolution technology to modify the wild-type catalase EcCAT (amino acid sequence shown in SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO:2) derived from *Escherichia coli* (NCBI protein sequence number YP_025308). The resulting catalase mutant exhibits significantly enhanced enzyme activity and thermostability, with its activity reaching up to 2.18 times that of the wild-type catalase. The mutant described in this invention can be used in combination with doxorubicin 5'-phosphate oxidase for whole-cell catalysis, instantly relieving product inhibition. A conversion rate of over 99% can be achieved in just 5 hours, with advantages such as mild reaction conditions. This invention has significant research value and broad market prospects for constructing efficient and stable biocatalytic platforms and promoting the green biomanufacturing of high-value-added products such as vitamin B6 and its derivatives. Attached Figure Description

[0039] Figure 1 This is a map of the catalase EcCAT expression vector.

[0040] Figure 2 This is an SDS-PAGE image of catalase-induced expression; where M is the protein marker and the catalase size is 84.2 kDa. Detailed Implementation

[0041] The following examples are provided to further illustrate the present invention, but do not limit the invention in any way. Processes and methods not described in detail in the following examples are conventional methods known in the art, and the reagents used in the examples are commercially available or prepared by methods well known to those skilled in the art. The following examples all achieve the objectives of the present invention.

[0042] The culture media involved in the following examples are as follows:

[0043] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121°C for 20 min.

[0044] LB solid medium: LB liquid medium with 2% agar added.

[0045] TB liquid culture medium: KH2PO4 2.31g / L, K2HPO4·3H2O 16.42g / L, yeast extract 24g / L, peptone 12g / L, glycerol 4g / L.

[0046] Example 1: Preparation of genetically engineered strains containing the wild-type catalase EcCAT gene

[0047] The catalase mutant of the present invention was obtained by modifying the amino acid sequence of wild-type catalase from Escherichia coli (NCBI protein sequence number YP_025308) using directed evolution technology. The amino acid sequence of the wild-type catalase is shown in SEQ ID NO:1, with a total length of 753 amino acids, and the nucleotide sequence is shown in SEQ ID NO:2, with a total length of 2262 bases.

[0048] After designing primers, the gene for wild-type catalase from *E. coli* was cloned into the pET29a vector using homologous recombination, constructing the recombinant vector pET29a-EcCAT. The plasmid map is shown below. Figure 1 As shown in the figure; subsequently, the recombinant vector was transformed into E. coli BL21 (DE3) competent cells, plated on LB solid selective medium plates containing kanamycin (Kana), and incubated upside down for 12-16 h. Single colonies were picked for colony PCR identification, and the correctly identified positive transformants were the genetically engineered strains containing the wild-type catalase EcCAT gene.

[0049] Example 2: Construction of a point mutation library of the catalase EcCAT gene

[0050] (1) Based on the spatial structure of catalase EcCAT and the spatial position of cofactor binding, 29 amino acid sites (R125, I126, V127, H128, R165, S167, G184, F185, A186, V199, G200, N201, F206, A211, F214, G273, I274, H275, A389, A390, F391, L407, G410, R411, S414, Y415, T418, Q419, R422) were selected for saturation mutation.

[0051] (2) Construct engineered strains of catalase EcCAT gene mutants expressing 29 catalase EcCAT mutants respectively.

[0052] The catalase EcCAT mutant was obtained by designing degenerate primers to construct a mutant library and screening. Four forward mutagenesis primers were designed using the Tang method to uniformly and without redundancy introduce twenty natural amino acids into the mutant library. The designed degenerate primer sequences are shown below, and the specific construction method is as follows:

[0053] Using the recombinant vector pET29a-EcCAT constructed in Example 1 as a template, two rounds of PCR reactions were performed using primers corresponding to each mutant. The PCR reaction system for the first round is shown in Table 1, the PCR reaction system for the second round is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0054] Primer names and sequences for constructing the EcCAT gene unit point mutation library:

[0055] R125 mutant library:

[0056] R125-NDT-F1: GAGCGCATTCCGGAANDTATTGTTCATGCACGC;

[0057] R125-VMA-F2: GAGCGCATTCCGGAAVMAATTGTTCATGCACGC;

[0058] R125-ATG-F3:GAGCGCATTCCGGAAATGATTGTTCATGCACGC;

[0059] R125-TGG-F4: GAGCGCATTCCGGAATGGATTGTTCATGCACGC;

[0060] R1:GTGCAGAGTTTCAGGTTGCAGAGAAACATAATC.

[0061] I126 mutation library:

[0062] I126-NDT-F1:CGCATTCCGGAACGTNDTGTTCATGCACGCGGA;

[0063] I126-VMA-F2: CGCATTCCGGAACGTVMAGTTCATGCACGCGGA;

[0064] I126-ATG-F3: CGCATTCCGGAACGTATGGTTCATGCACGCGGA;

[0065] I126-TGG-F4: CGCATTCCGGAACGTTGGGTTCATGCACGCGGA;

[0066] R1: GTGCAGAGTTTCAGGTTGCAGAGAAACATAATC.

[0067] V127 mutant library:

[0068] V127 - NDT - F1: ATTCCGGAACGTATTNDTCATGCACGCGGATCA;

[0069] V127 - VMA - F2: ATTCCGGAACGTATTVMACATGCACGCGGATCA;

[0070] V127 - ATG - F3: ATTCCGGAACGTATTATGCATGCACGCGGATCA;

[0071] V127 - TGG - F4: ATTCCGGAACGTATTTGGCATGCACGCGGATCA;

[0072] R1: GTGCAGAGTTTCAGGTTGCAGAGAAACATAATC.

[0073] H128 mutant library:

[0074] H128 - NDT - F1: CCGGAACGTATTGTTNDTGCACGCGGATCAGCC;

[0075] H128 - VMA - F2: CCGGAACGTATTGTTVMAGCACGCGGATCAGCC;

[0076] H128 - ATG - F3: CCGGAACGTATTGTTATGGCACGCGGATCAGCC;

[0077] H128 - TGG - F4: CCGGAACGTATTGTTTGGGCACGCGGATCAGCC;

[0078] R1: GTGCAGAGTTTCAGGTTGCAGAGAAACATAATC.

[0079] R165 mutant library:

[0080] R165 - NDT - F1: ACCCCAGTATTTGTANDTTTCTCTACCGTTCAG;

[0081] R165 - VMA - F2: ACCCCAGTATTTGTAVMATTCTCTACCGTTCAG;

[0082] R165 - ATG - F3: ACCCCAGTATTTGTAATGTTCTCTACCGTTCAG;

[0083] R165 - TGG - F4: ACCCCAGTATTTGTATGGTTCTCTACCGTTCAG;

[0084] R1: GTGCAGAGTTTCAGGTTGCAGAGAAACATAATC.

[0085] S167 Mutation Library:

[0086] S167 - NDT - F1: GTATTTGTACGTTTCNDTACCGTTCAGGGTGGT;

[0087] S167 - VMA - F2: GTATTTGTACGTTTCVMAACCGTTCAGGGTGGT;

[0088] S167 - ATG - F3: GTATTTGTACGTTTCATGACCGTTCAGGGTGGT;

[0089] S167 - TGG - F4: GTATTTGTACGTTTCTGGACCGTTCAGGGTGGT;

[0090] R1: GTGCAGAGTTTCAGGTTGCAGAGAAACATAATC.

[0091] G184 Mutation Library:

[0092] G184 - NDT - F1: GTGCGTGATATCCGTNDTTTTGCCACCAAGTTC;

[0093] G184 - VMA - F2: GTGCGTGATATCCGTVMATTTGCCACCAAGTTC;

[0094] G184 - ATG - F3: GTGCGTGATATCCGTATGTTTGCCACCAAGTTC;

[0095] G184 - TGG - F4: GTGCGTGATATCCGTTGGTTTGCCACCAAGTTC;

[0096] R2: CGGGTCACGTCCGGTGAGTTTTTGTGCTTCATC.

[0097] F185 Mutation Library:

[0098] F185-NDT-F1: CGTGATATCCGTGGCNDTGCCACCAAGTTCTAT;

[0099] F185-VMA-F2: CGTGATATCCGTGGCVMAGCCACCAAGTTCTAT;

[0100] F185-ATG-F3: CGTGATATCCGTGGCATGGCCACCAAGTTCTAT;

[0101] F185-TGG-F4: CGTGATATCCGTGGCTGGGCCACCAAGTTCTAT;

[0102] R2: CGGGT CACGTCCGGTGAGTTTTTGTGCTTCATC.

[0103] A186 Mutation Library:

[0104] A186-NDT-F1: GATATCCGTGGCTTTNDTACCAAGTTCTATACC;

[0105] A186-VMA-F2: GATATCCGTGGCTTTVMAACCAAGTTCTATACC;

[0106] A186-ATG-F3: GATATCCGTGGCTTTATGACCAAGTTCTATACC;

[0107] A186-TGG-F4: GATATCCGTGGCTTTTGGACCAAGTTCTATACC;

[0108] R2: CGGGT CACGTCCGGTGAGTTTTTGTGCTTCATC.

[0109] V199 Mutation Library:

[0110] V199-NDT-F1: GGTATTTTTGACCTCNDTGGCAATAACACGCCA;

[0111] V199-VMA-F2: GGTATTTTTGACCTCVMAGGCAATAACACGCCA;

[0112] V199-ATG-F3: GGTATTTTTGACCTCATGGGCAATAACACGCCA;

[0113] V199 - TGG - F4: GGTATTTTTGACCTCTGGGGCAATAACACGCCA;

[0114] R2: CGGGTCACGTCCGGTGAGTTTTTGTGCTTCATC.

[0115] G200 mutant library:

[0116] G200 - NDT - F1: ATTTTTGACCTCGTTNDTAATAACACGCCAATC;

[0117] G200 - VMA - F2: ATTTTTGACCTCGTTVMAAATAACACGCCAATC;

[0118] G200 - ATG - F3: ATTTTTGACCTCGTTATGAATAACACGCCAATC;

[0119] G200 - TGG - F4: ATTTTTGACCTCGTTTGGAATAACACGCCAATC;

[0120] R2: CGGGTCACGTCCGGTGAGTTTTTGTGCTTCATC.

[0121] N201 mutant library:

[0122] N201 - NDT - F1: TTTGACCTCGTTGGCNDTAACACGCCAATCTTC;

[0123] N201 - VMA - F2: TTTGACCTCGTTGGCVMAAACACGCCAATCTTC;

[0124] N201 - ATG - F3: TTTGACCTCGTTGGCATGAACACGCCAATCTTC;

[0125] N201 - TGG - F4: TTTGACCTCGTTGGCTGGAACACGCCAATCTTC;

[0126] R2: CGGGTCACGTCCGGTGAGTTTTTGTGCTTCATC.

[0127] F206 mutant library:

[0128] F206-NDT-F1: AATAACACGCCAATCNDTTTTATCCAGGATGCG;

[0129] F206-VMA-F2: AATAACACGCCAATCVMATTTATCCAGGATGCG;

[0130] F206-ATG-F3: AATAACACGCCAATCATGTTTATCCAGGATGCG;

[0131] F206-TGG-F4: AATAACACGCCAATCTGGTTTATCCAGGATGCG;

[0132] R2: CGGGTCACGTCCGGTGAGTTTTTGTGCTTCATC.

[0133] A211 Mutation Library:

[0134] A211-NDT-F1: TTCTTTATCCAGGATNDTCATAAATTCCCCGAT;

[0135] A211-VMA-F2: TTCTTTATCCAGGATVMACATAAATTCCCCGAT;

[0136] A211-ATG-F3: TTCTTTATCCAGGATATGCATAAATTCCCCGAT;

[0137] A211-TGG-F4: TTCTTTATCCAGGATTGGCATAAATTCCCCGAT;

[0138] R2: CGGGTCACGTCCGGTGAGTTTTTGTGCTTCATC.

[0139] F214 Mutation Library:

[0140] F214-NDT-F1: CAGGATGCGCATAAANDTCCCGATTTTGTTCAT;

[0141] F214-VMA-F2: CAGGATGCGCATAAAVMACCCGATTTTGTTCAT;

[0142] F214-ATG-F3: CAGGATGCGCATAAAATGCCCGATTTTGTTCAT;

[0143] F214-TGG-F4: CAGGATGCGCATAAATGGCCCGATTTTGTTCAT;

[0144] R2: CGGGTCACGTCCGGTGAGTTTTTGTGCTTCATC.

[0145] G273 mutation library:

[0146] G273-NDT-F1: ACCATGGAAGGCTTCNDTATTCACACCTTCCGC;

[0147] G273-VMA-F2: ACCATGGAAGGCTTCVMAATTCACACCTTCCGC;

[0148] G273-ATG-F3: ACCATGGAAGGCTTCATGATTCACACCTTCCGC;

[0149] G273-TGG-F4: ACCATGGAAGGCTTCTGGATTCACACCTTCCGC;

[0150] R3: GTTGCGATTGAGCACCATTTTGCCGACACGCTG.

[0151] I274 mutation library:

[0152] I274-NDT-F1: ATGGAAGGCTTCGGTNDTCACACCTTCCGCCTG;

[0153] I274-VMA-F2: ATGGAAGGCTTCGGTVMACACACCTTCCGCCTG;

[0154] I274-ATG-F3: ATGGAAGGCTTCGGTATGCACACCTTCCGCCTG;

[0155] I274-TGG-F4: ATGGAAGGCTTCGGTTGGCACACCTTCCGCCTG;

[0156] R3: GTTGCGATTGAGCACCATTTTGCCGACACGCTG.

[0157] H275 mutation library:

[0158] H275-NDT-F1: GAAGGCTTCGGTATTNDTACCTTCCGCCTGATT;

[0159] H275-VMA-F2: GAAGGCTTCGGTATTVMAACCTTCCGCCTGATT;

[0160] H275-ATG-F3: GAAGGCTTCGGTATTATGACCTTCCGCCTGATT;

[0161] H275-TGG-F4: GAAGGCTTCGGTATTTGGACCTTCCGCCTGATT;

[0162] R3: GTTGCGATTGAGCACCATTTTGCCGACACGCTG.

[0163] A389 mutant library:

[0164] A389-NDT-F1: GCTGAAAACGAACAGNDTGCTTTCCATCCTGGG;

[0165] A389-VMA-F2: GCTGAAAACGAACAGVMAGCTTTCCATCCTGGG;

[0166] A389-ATG-F3: GCTGAAAACGAACAGATGGCTTTCCATCCTGGG;

[0167] A389-TGG-F4: GCTGAAAACGAACAGTGGGCTTTCCATCCTGGG;

[0168] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0169] A390 mutant library:

[0170] A390-NDT-F1: GAAAACGAACAGGCGNDTTTCCATCCTGGGCAT;

[0171] A390-VMA-F2: GAAAACGAACAGGCGVMATTCCATCCTGGGCAT;

[0172] A390-ATG-F3: GAAAACGAACAGGCGATGTTCCATCCTGGGCAT;

[0173] A390-TGG-F4: GAAAACGAACAGGCGTGGTTCCATCCTGGGCAT;

[0174] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG。

[0175] F391 Mutation Library:

[0176] F391-NDT-F1: AACGAACAGGCGGCTNDTCATCCTGGGCATATC;

[0177] F391-VMA-F2: AACGAACAGGCGGCTVMACATCCTGGGCATATC;

[0178] F391-ATG-F3: AACGAACAGGCGGCTATGCATCCTGGGCATATC;

[0179] F391-TGG-F4: AACGAACAGGCGGCTTGGCATCCTGGGCATATC;

[0180] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG。

[0181] L407 Mutation Library:

[0182] L407-NDT-F1: TTCACCAACGATCCGNDTTTGCAGGGACGTTTG;

[0183] L407-VMA-F2: TTCACCAACGATCCGVMATTGCAGGGACGTTTG;

[0184] L407-ATG-F3: TTCACCAACGATCCGATGTTGCAGGGACGTTTG;

[0185] L407-TGG-F4: TTCACCAACGATCCGTGGTTGCAGGGACGTTTG;

[0186] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG。

[0187] G410 Mutation Library:

[0188] G410-NDT-F1: GATCCGCTGTTGCAGNDTCGTTTGTTCTCCTAT;

[0189] G410-VMA-F2: GATCCGCTGTTGCAGVMACGTTTGTTCTCCTAT;

[0190] G410 - ATG - F3: GATCCGCTGTTGCAGATGCGTTTGTTCTCCTAT;

[0191] G410 - TGG - F4: GATCCGCTGTTGCAGTGGCGTTTGTTCTCCTAT;

[0192] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0193] R411 mutant library:

[0194] R411 - NDT - F1: CCGCTGTTGCAGGGANDTTTGTTCTCCTATACC;

[0195] R411 - VMA - F2: CCGCTGTTGCAGGGAVMATTGTTCTCCTATACC;

[0196] R411 - ATG - F3: CCGCTGTTGCAGGGAATGTTGTTCTCCTATACC;

[0197] R411 - TGG - F4: CCGCTGTTGCAGGGATGGTTGTTCTCCTATACC;

[0198] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0199] S414 mutant library:

[0200] S414 - NDT - F1: CAGGGACGTTTGTTCNDTTATACCGATACACAA;

[0201] S414 - VMA - F2: CAGGGACGTTTGTTCVMATATACCGATACACAA;

[0202] S414 - ATG - F3: CAGGGACGTTTGTTCATGTATACCGATACACAA;

[0203] S414 - TGG - F4: CAGGGACGTTTGTTCTGGTATACCGATACACAA;

[0204] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0205] Y415 mutant library:

[0206] Y415 - NDT - F1: GGACGTTTGTTCTCCNDTACCGATACACAAATC;

[0207] Y415 - VMA - F2: GGACGTTTGTTCTCCVMAACCGATACACAAATC;

[0208] Y415 - ATG - F3: GGACGTTTGTTCTCCATGACCGATACACAAATC;

[0209] Y415 - TGG - F4: GGACGTTTGTTCTCCTGGACCGATACACAAATC;

[0210] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0211] T418 mutation library:

[0212] T418 - NDT - F1: TTCTCCTATACCGATNDTCAAATCAGTCGTCTT;

[0213] T418 - VMA - F2: TTCTCCTATACCGATVMACAAATCAGTCGTCTT;

[0214] T418 - ATG - F3: TTCTCCTATACCGATATGCAAATCAGTCGTCTT;

[0215] T418 - TGG - F4: TTCTCCTATACCGATTGGCAAATCAGTCGTCTT;

[0216] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0217] Q419 mutation library:

[0218] Q419 - NDT - F1: TCCTATACCGATACANDTATCAGTCGTCTTGGT;

[0219] Q419 - VMA - F2: TCCTATACCGATACAVMAATCAGTCGTCTTGGT;

[0220] Q419 - ATG - F3: TCCTATACCGATACAATGATCAGTCGTCTTGGT;

[0221] Q419-TGG-F4:TCCTATACCGATACATGGATCAGTCGTCTTGGT;

[0222] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0223] R422 mutant library:

[0224] R422-NDT-F1: GATACACAAATCAGTNDTCTTGGTGGGCCGAAT;

[0225] R422-VMA-F2: GATACACAAATCAGTVMACTTGGTGGGCCGAAT;

[0226] R422-ATG-F3: GATACACAAATCAGTATGCTTGGTGGGCCGAAT;

[0227] R422-TGG-F4: GATACACAAATCAGTTGGCTTGGTGGGCCGAAT;

[0228] R4: AGCCAGAACAGACGCGGATGGGAATAATATTCG.

[0229] Table 1. First-round PCR reaction system

[0230]

[0231] Table 2 Second round PCR reaction system

[0232]

[0233] Table 3 PCR reaction procedure

[0234]

[0235] After completing two rounds of PCR reactions, add 2 μL of Dpn I enzyme to each reaction system, digest at 37°C for 3 hours, and take 1 μL to electroporate into E. coli BL21(DE3) competent cells. Incubate in an inverted incubator at 37°C for 12-16 hours. Once single clones have grown, the construction of the mutant library is complete.

[0236] Example 3: High-throughput screening of a catalase EcCAT gene mutant library and acquisition of engineered strains with single-point mutants.

[0237] In this embodiment, the high-throughput screening method for the EcCAT gene mutant library of catalase was ultraviolet spectrophotometry. Hydrogen peroxide, under the action of catalase, generates water and oxygen; during the reaction, the absorbance gradually decreases at 240 nm and bubbles are generated.

[0238] The specific methods and operating steps are as follows:

[0239] (1) Preparation of bacterial cells

[0240] The single colonies grown in Example 2 were picked up with a toothpick and cultured in a 96-well plate, which was designated as the mother plate. The plates were cultured at 37°C and 1200 rpm for 12 h with shaking. 120 μL of the overnight culture was transferred to a 96-well glycerol plate, and 80 μL of 50% glycerol was added and thoroughly mixed by pipetting. The plate was then capped and stored at -80°C. Simultaneously, 800 μL of TB+IPTG+Kana medium (0.1 M IPTG, 50 μg / mL Kana) was added to the 96-well plate, and the plates were cultured at 25°C and 1200 rpm for 12 h for protein expression.

[0241] (2) Whole-cell catalytic reaction

[0242] The reaction solution consisted of 15 mmol hydrogen peroxide solution and 50 mM pH 8.0 potassium phosphate buffer. Immediately afterwards, 50 μL of the bacterial culture induced in step (1) was added to a 96-well microplate, and the change in absorbance at 240 nm over 60 seconds was measured. Mutant strains with relatively high changes (i.e., relatively high enzyme activity) were screened for further selection.

[0243] (3) Obtaining single point mutant engineered strains

[0244] OD 240 Mutant strains with a value higher than that of wild-type strains under the nm index were sent for sequencing. Transformants that were correctly sequenced were identified as engineered strains of the EcCAT gene mutant of catalase.

[0245] Example 4: Preparation of engineered bacteria with a two-site mutant of the catalase EcCAT gene

[0246] The double mutant in this embodiment is constructed by using full plasmid PCR based on the corresponding single mutant and the primers in Table 4. For example, based on mutant V199A, the double mutant V199A / N201L is constructed by using mutant primers N201L-F and N201L-R (Table 4). The PCR reaction system and procedure are shown in Tables 5 and 3.

[0247] Table 4 Primer sequences used for multisite catalase mutants

[0248]

[0249] Table 5 PCR reaction system

[0250]

[0251] Three double mutants were obtained using the above method and named mutant V199A / N201L, mutant V199A / G273V, and mutant V199A / L407V, respectively.

[0252] Example 5: Preparation of engineered bacteria containing three-site and four-site mutants of the catalase EcCAT gene

[0253] Based on the mutant V199A / L407V, a triple mutant was constructed by whole plasmid PCR using the mutant primers N201L-F and N201L-R (Table 4). For specific methods, please refer to Example 2. The primers used are shown in Table 4.

[0254] The above method yields three mutants, which are named mutants V199A / L407V / N201L.

[0255] Based on the mutant V199A / L407V / N201L, a quadratic mutant was constructed by whole plasmid PCR using the mutant primers G273V-F and G273V-R (Table 4). For specific methods, please refer to Example 2. The primers used are shown in Table 4.

[0256] The above method yields four mutants, which are named mutants V199A / L407V / N201L / G273V.

[0257] Example 6: Obtaining crude catalase solution and determining its activity

[0258] (1) Obtaining crude enzyme solution

[0259] Recombinant bacterial transformants containing the wild-type catalase EcCAT gene or the catalase EcCAT mutants obtained in Examples 3-5 were selected and added to 5 mL LB liquid medium containing 50 μg / mL kanamycin. The mixture was incubated overnight at 37°C and 220 rpm for 12-16 hours with shaking. Then, 1% (v / v) of the inoculum was added to LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 5 hours. IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 25°C and 220 rpm for 18 hours. The cells were then collected by centrifugation at 4°C and 7000 rpm for 10 minutes. The collected cells were then rinsed with PBS buffer (50 mM, pH 7). 4) Resuspend the cells to obtain whole cells containing catalase (EcCAT). Then, sonicate the bacterial cells under ice bath conditions to obtain a sonicated sample. Centrifuge the sonicated sample at 4℃ and 7000 rpm for 10 min, and collect the supernatant (i.e., crude enzyme solution). Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on the crude enzyme solution. The results are as follows: Figure 2 As shown.

[0260] (2) Detection of catalase EcCAT enzyme activity

[0261] Working solution: Add 50 μL of reagent II to 13 mL of reagent I and mix thoroughly (approximately 13 mL). (Incubate in a water bath at 25°C for 10 min before use.)

[0262] Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 240 nm, and zero it with distilled water.

[0263] The enzyme reaction system is 1.035 mL. Take 1 mL of the detection working solution into a quartz cuvette, add 35 μL of crude enzyme solution, mix well for 5 s, and immediately measure the initial absorbance value A1 at 242 nm and the absorbance value A2 after 1 min at room temperature. Calculate ΔA = A1 - A2.

[0264] Enzyme activity is defined as the ability of 10,000 bacteria or cells to catalyze the degradation of 1 μmol of hydrogen peroxide per minute in a reaction system, which is defined as one unit of enzyme activity.

[0265] Table 6 shows the enzyme activity detection results of single-site mutants with higher enzyme activity than wild-type catalase, and Table 7 shows the enzyme activity detection results of multi-site catalase mutants.

[0266] Table 6. Enzyme activity detection results of wild-type catalase and single-site catalase mutants.

[0267]

[0268] Table 7. Enzyme activity detection results of multi-site catalase mutants

[0269]

[0270] Example 7 Thermal Stability Analysis

[0271] The catalase activity of the multisite mutants V199A / L407V and V199A / L407V / N201L and the wild-type enzyme solution was measured after treatment at 50℃, 60℃ and 70℃ for 0.5h, respectively. The enzyme activity of the untreated sample was taken as 100%, and the enzyme activity reduction rate was calculated.

[0272] The results are shown in Table 8. The thermostability of mutants V199A / L407V and V199A / L407V / N201L, which have higher enzyme activity, is higher than that of wild type.

[0273] Table 8. Results of enzyme activity thermostability assays for multi-site catalase mutants and wild-type enzymes.

[0274]

[0275] Example 8: Application of catalase mutant

[0276] In a 100 mL reaction system, 1.263 g of pyridoxine phosphate, 0.4 mg of FMN, 25 g / L of wetted catalase mutant V199A / L407V / N201L, and wetted 5'-pyridoxine phosphate oxidase mutant A68K / N147A / W174D with a total enzyme activity of 400 U were added sequentially. The pH was adjusted to 9.0, and the volume was brought to 1 L. The reaction was started at 40 °C with stirring at 220 rpm. After 5 h of reaction, the conversion rate was 99.42%.

[0277] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A catalase EcCAT mutant, characterized in that, The mutant is obtained by mutating at least one of the following amino acids from SEQ ID NO:1: Ile at position 126, Ser at position 167, Val at position 199, Gly at position 200, Asn at position 201, Phe at position 206, Ala at position 211, Phe at position 214, Gly at position 273, Ala at position 389, Phe at position 391, Leu at position 407, and Gly at position 410.

2. The EcCAT mutant of catalase according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Transform its 126th Ile into Phe; or Mutate its 167th Ser bit to Asp; or Mutate its 167th Ser position to Cys; or Transform its 199th Val position into Ala; or Mutate its 200th Gly to Gly (synonymous mutation); or Mutate its 201st Asn to Leu; or Transform its 206th Phe into Asn; or Mutate its 211th Ala position to Arg; or Mutate its 214th Phe to Phe (synonymous mutation); or Mutate its 273rd Gly position to Val; or Mutate its 389th Ala position to Gly; or Transform its 391st Phe into Met; or Transform its 407th position, Leu, into Cys; or Transform its 407th position, Leu, into Val; or The 410th position of Gly was mutated to Phe.

3. The catalase EcCAT mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Transform its 199th position Val into Ala, and its 407th position Leu into Val; or Mutate its 199th Val position to Ala and its 201st Asn position to Leu; or Its 199th Val was mutated to Ala, and its 273rd Gly was mutated to Val.

4. The catalase EcCAT mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Mutate its 199th position Val to Ala, its 407th position Leu to Val, and its 201st position Asn to Leu; or Its 199th Val was mutated to Ala, its 407th Leu was mutated to Val, its 201st Asn was mutated to Leu, and its 273rd Gly was mutated to Val.

5. A gene encoding the EcCAT mutant of catalase as described in any one of claims 1-4.

6. A recombinant expression vector carrying the gene of claim 5.

7. Recombinant microorganisms comprising the recombinant expression vector as described in claim 6.

8. The use of the catalase EcCAT mutant according to any one of claims 1-4 in the synthesis of pyridoxal 5'-phosphate in combination with pyridoxine oxidase.

9. A method for producing pyridoxal 5'-phosphate, characterized in that, Includes the following steps: The catalase EcCAT mutant of any one of claims 1-4 or the recombinant microorganism of claim 7, along with pyridoxine 5'-phosphate oxidase, is added to a reaction system containing pyridoxine phosphate and flavin mononucleotide, and an oxidation reaction is carried out under the conditions of pH 7.0-11.0 and temperature 36-40°C to generate pyridoxal 5'-phosphate.

10. The method according to claim 9, characterized in that, In the reaction system, the concentration of pyridoxine phosphate is 5-20 g / L, the concentration of recombinant microorganisms with catalase EcCAT mutant is 1-25 g / L, and the concentration of flavin mononucleotide is 1-10 mg / L.

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