A tetrahydropyrimidine hydroxylase mutant and its application

Abstract

The invention discloses a tetrahydropyrimidine hydroxylase mutant and its application in the preparation of 5-hydroxytetrahydropyrimidine, belonging to the field of synthetic biotechnology. The amino acid sequence of the tetrahydropyrimidine hydroxylase mutant is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2. Compared with the original hydroxylase, the mutant enzyme activity is enhanced, and the efficiency of catalyzing the conversion of tetrahydropyrimidine into 5-hydroxytetrahydropyrimidine is increased by 45.8%. The tetrahydropyrimidine hydroxylase mutant is used to construct a recombinant expression strain, and it is applied in the preparation of 5-hydroxytetrahydropyrimidine, the synthesis efficiency of 5-hydroxytetrahydropyrimidine is improved, the reaction system tetrahydropyrimidine residue is small, 5-hydroxytetrahydropyrimidine is easy to separate and extract, and the preparation cost of 5-hydroxytetrahydropyrimidine is low, which is conducive to the promotion and application of 5-hydroxytetrahydropyrimidine.

Description

Technical Field

[0001] The invention relates to an ectoine hydroxylase mutant and application thereof in the preparation of 5-hydroxyectoine, belonging to the field of synthetic biotechnology. Background Art

[0002] 5-Hydroxytetrahydropyrimidine (1, 4, 5, 6-tetrahydro-2-methyl-5-hydroxy-4-pyrimidinecarboxylic acid) is a derivative of tetrahydropyrimidine. It also has the osmotic regulation function of tetrahydropyrimidine and has the function of protecting and stabilizing nucleic acids, cell membranes, proteins and other substances in adverse environments such as high temperature, dryness, hypertonicity, and freezing. Compared with tetrahydropyrimidine, 5-hydroxytetrahydropyrimidine has a better protective effect on nucleic acids, cell membranes, proteins, etc. In the field of cosmetics, based on the osmotic protection function of 5-hydroxytetrahydropyrimidine, it can be added to cosmetics as a moisturizer to prevent skin dryness and aging, and reduce the damage of ultraviolet rays to the skin; in the field of medicine, 5-hydroxytetrahydropyrimidine can not only be added to drugs for the treatment of neurological diseases, but also as a protective agent for healthy cells during chemotherapy; in the application of genetic engineering technology, 5-hydroxytetrahydropyrimidine can increase the melting temperature of double-stranded DNA, so it can be used as an enhancer for polymerase chain reaction.

[0003] In microorganisms, tetrahydropyrimidine hydroxylase uses Fe2 + As a cofactor, α-ketoglutarate is a cosubstrate, catalyzing the conversion of ectoine to 5-hydroxyectoine, and is a key enzyme in the process of microbial fermentation to produce 5-hydroxyectoine. The activity of ectoine hydroxylase is crucial to the biosynthesis of hydroxypyrimidine and is extremely important for the production and preparation of 5-hydroxyectoine. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide an ectoine hydroxylase mutant and its application in the preparation of 5-hydroxyectoine, and to improve the synthesis efficiency of 5-hydroxyectoine by preparing the ectoine hydroxylase mutant, constructing a recombinant expression strain and preparing 5-hydroxyectoine by fermentation.

[0005] In order to solve the above technical problems, the technical solutions provided by the present invention are as follows:

[0006] The present invention provides a tetrahydropyrimidine hydroxylase mutant, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleic acid sequence of which is shown in SEQ ID NO.2.

[0007] Furthermore, the ectoine hydroxylase mutant is obtained by mutation of the ectoine hydroxylase gene ectD.

[0008] Furthermore, the ectohydropyrimidine hydroxylase mutant is obtained by simulation mutation using bioinformatics software based on the tertiary predicted structure of the ectohydropyrimidine hydroxylase gene ectD and the chemical structures of the substrate ectohydropyrimidine and the cosubstrate α-ketoglutarate.

[0009] Furthermore, the amino acid sequence of the tetrahydropyrimidine hydroxylase gene ectD is shown as SEQ ID NO.3, and the nucleic acid sequence is shown as SEQ ID NO.4.

[0010] Furthermore, the tetrahydropyrimidine hydroxylase gene is derived from Halobacillus sp. FL-2423, which has the characteristic of tetrahydropyrimidine secretion. The strain was deposited in the General Microbiology Center of the China Culture Collection Administration on November 1, 2019, with the deposit number CGMCCNO.18783, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] The present invention also provides a construction of a recombinant expression strain containing an ectohydropyrimidine hydroxylase mutant, wherein the recombinant expression strain is BL21 (DE3) / pET32a (+) -ectD mut -6×His, the recombinant expression vector used was pET32a(+)-ectD mut -6×His.

[0012] Furthermore, the construction of a recombinant expression strain containing a tetrahydropyrimidine hydroxylase mutant comprises the following steps:

[0013] (1) Design of primer ectD mut -F / R, the sequences are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, using high-fidelity polymerase Phanta HS and ectD mut As a template, the nucleic acid sequence is as shown in SEQ ID NO.2, and ectD is obtained by amplification. mut -6×His gene fragment;

[0014] (2) Design primers pET32a(+)-F / R, the sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. Use high-fidelity polymerase Phanta HS and pET32a(+) plasmid as a template to amplify and obtain the pET32a(+) linearized fragment;

[0015] (3) Use homologous recombination enzyme ClonExpress II One Step Cloning Kit to connect ectD mut-6×His gene fragment and pET32a (+) linearized fragment were transformed into DH5α and screened for ampicillin resistance to obtain the recombinant expression vector pET32a (+)-ectD mut -6×His;

[0016] (4) Insert the recombinant expression vector pET32a(+)-ectD mut -6×His was transformed into E. coli BL21 (DE3) to construct the recombinant expression strain BL21 (DE3) / pET32a (+)-ectD mut -6×His.

[0017] The present invention also provides an application of a recombinant expression strain containing an ectoine hydroxylase mutant in the preparation of 5-hydroxyectoine, comprising the following steps:

[0018] (1) Seed plate activation: Streak the recombinant expression strain on an eggplant-shaped bottle containing solid LB medium containing resistance in an ultra-clean workbench and culture at 37°C for 12 hours to obtain activated seeds;

[0019] (2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium, at a temperature of 37°C, dissolved oxygen of 20-30%, and pH 7.0 adjusted with ammonia water. The culture was continued until the OD value reached 10 to obtain seed liquid.

[0020] (3) Fermentation of expression strain: The seed liquid was inoculated into a fermentation tank containing 8 L of fermentation medium, the temperature was 37 ° C, the dissolved oxygen was 20-30%, and the pH was adjusted to 7.0 with ammonia water. After the substrate sugar was consumed, IPTG was added to the fermentation medium at an IPTG concentration of 0.1 mM / L. A glycerol solution with a concentration of 500 g / L was fed, and the growth rate of OD was adjusted to 2-4 / h. The fermentation was terminated when OD grew to 60, and the fermented bacteria were obtained;

[0021] (4) Collecting bacteria by centrifugation: circulating chilled water was introduced into the jacket to cool the fermented bacteria to 4°C, and the mixture was centrifuged at 10,000 rpm and 4°C for 5 min. The supernatant was discarded and the bacteria were retained. The mixture was then resuspended in 4°C precooled PBS buffer with a pH of 7.0 and 0.01 M. The wet bacteria were collected by centrifugation at 10,000 rpm and 4°C for 5 min, and the wet bacteria were stored at 4°C.

[0022] (5) Pretreatment of bacteria: resuspend the wet bacteria in 4°C precooled pH 7.0, 0.02M PBS to prepare a suspension with a wet bacterial concentration of 500 g / L, add hydroxytyrosol to obtain a mixed solution, heat it to 42°C and maintain it for 10 min, then cool it to 35°C to obtain a pretreated bacterial suspension;

[0023] (6) Catalytic preparation of 5-hydroxytetrahydropyrimidine: The pretreated bacterial suspension is added to the reaction solution to obtain a mixed solution, the pH of which is adjusted to 7.0 with phosphoric acid and liquid alkali, and the mixture is fermented at a temperature of 35°C and a dissolved oxygen content of 10-20%. After additional treatment, the fermentation is terminated to obtain a reaction solution containing 5-hydroxytetrahydropyrimidine.

[0024] Note: IPTG is isopropyl-β-D-thiogalactoside.

[0025] Furthermore, in step (2), the seed culture medium components are: 20.0 g / L glucose, 1.5 g / L peptone, 6.8 g / L yeast powder, 0.68 g / L citric acid monohydrate, 1.6 g / L potassium dihydrogen phosphate, 0.68 g / L magnesium sulfate heptahydrate, and 0.33 g / L GPE defoamer.

[0026] Furthermore, in step (3), the seed liquid accounts for 10% of the fermentation medium by volume.

[0027] Furthermore, in step (3), the fermentation medium components are: 20.0 g / L glucose, 10.5 g / L peptone, 3.8 g / L yeast powder, 4.6 g / L corn steep liquor powder, 2.5 g / L citric acid monohydrate, 3.8 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, and 0.85 g / L GPE defoamer.

[0028] Furthermore, in step (5), the concentration of hydroxytyrosol in the mixed solution is 100-200 ppm.

[0029] Furthermore, in step (6), the mixed solution comprises: 30-80 g / L ectoine, 0.01 M PBS buffer with a pH of 7.0, 30-100 g / L α-ketoglutaric acid, 0.1-0.2 g / L ferrous sulfate, and 50-200 g / L wet bacteria.

[0030] Furthermore, in step (6), the specific operation of the supplementation treatment is: sampling and determining the content of 5-hydroxytetrahydropyrimidine and α-ketoglutaric acid in the mixed solution every 0.5 h; when the concentration of α-ketoglutaric acid in the solution is less than 5 g / L, adding α-ketoglutaric acid until the concentration of α-ketoglutaric acid in the solution is 5 g / L; when the concentration growth rate of 5-hydroxytetrahydropyrimidine is less than 2 (g / L) / h, raising the temperature to 42°C for fermentation for 2-4 h, and ending the fermentation.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention provides a novel ectoine hydroxylase mutant, which has enhanced activity compared with the original hydroxylase, and the efficiency of catalyzing the conversion of ectoine to 5-hydroxyectoine is increased by 45.8%.

[0033] 2. The present invention applies an ectoine hydroxylase mutant to construct a recombinant expression strain and ferment it to synthesize 5-hydroxyectoine, thereby improving the synthesis efficiency of 5-hydroxyectoine, reducing the amount of residual ectoine in the reaction system, making 5-hydroxyectoine easy to separate and extract, and reducing the preparation cost of 5-hydroxyectoine, which is beneficial to the popularization and application of 5-hydroxyectoine.

[0034] 3. The application method of the ectoine hydroxylase mutant of the present invention has a large conversion amount per unit volume, reduces production costs, and is conducive to the large-scale production of 5-hydroxyectoine. DETAILED DESCRIPTION

[0035] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. At the same time, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials, and instruments are all prior art and can be obtained from commercial channels unless otherwise specified.

[0036] Example 1 Construction of a recombinant expression strain containing a tetrahydropyrimidine hydroxylase mutant

[0037] (1) Based on the predicted tertiary structure of ectoine hydroxylase ectD (amino acid sequence as shown in SEQ ID NO.3, nucleic acid sequence as shown in SEQ ID NO.4) and the chemical structures of substrate ectoine and co-substrate α-ketoglutarate, a mutant ectoine hydroxylase with improved binding ability to substrate ectoine and co-substrate and good stability was obtained after simulated mutation using bioinformatics software. mut The amino acid sequence is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2.

[0038] (2) Design of primer ectD mut -F / R, the sequences are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, using high-fidelity polymerase Phanta HS and ectD mut As a template, the nucleic acid sequence is as shown in SEQ ID NO.2, and ectD is obtained by amplification. mut -6×His gene fragment;

[0039] (3) Design primers pET32a(+)-F / R, the sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and use high-fidelity polymerase Phanta HS to amplify the pET32a(+) linearized fragment using the pET32a(+) plasmid as a template;

[0040] (4) Use homologous recombination enzyme ClonExpress II One Step Cloning Kit to connect ectD mut -6×His gene fragment and pET32a (+) linearized fragment were transformed into DH5α and screened for ampicillin resistance to obtain the recombinant expression vector pET32a (+)-ectD mut -6×His;

[0041] (5) Insert the recombinant expression vector pET32a(+)-ectD mut -6×His was transformed into E. coli BL21 (DE3) to construct the recombinant expression strain BL21 (DE3) / pET32a (+)-ectD mut -6×His.

[0042] SEQ ID NO.1

[0043] MSVQTSSTRPLEQATQFIVVDSPDPQSRVRSAPLPGEDRYPTRLSEPLDLPWLSRREPVVKGHDAELPLSAAQLSAFERQGFLFEPGFISGDELAELRRELNALLERDDFRGRDFAITEPQVNEIRSLFAVHFLSRVFGRLAHDPRLTGRARQILSGEPYVHQSRI NDKPGVEGQGFNWHSDFETWHAEDGMPAMHAVSASLVLTDNHTFNGPLMLVPGSHGVFVPCLGETPDGHHRQSLKRQALGVPGHEALAALAERHGIEAPTGAAGGLLLFDCNTLHGANANMSPDPRSNAFFVYNRRDNRCVEPYAAGKRRPRFLAHDPDERWTPEA*

[0044] SEQ ID NO.2

[0045] atgtcagtgcagacatcgtccacccgaccgctggaacaggcgacccagttcatcgtcgtggattctcccgatccccagagccgggtgcgcagcgcgccgcttcccggggaggatcgctaccccacccggctgtccgagcccctcgacctgccctggctgagccgccgggagccggtggtgaagggtcatgacgccgagctcccgctctccgcggcgcagctctccgcgttcgagcgccaggggttcctcttcgagcccggtttcatcagcggcgacgagctcgccgagctgcgccgggagcttaacgcgctgctggagcgcgatgacttccgggggcgcgacttcgccatcaccgagcctcaggtcaacgagatccgctcgctgttcgccgtgcatttcctctcgcgggtcttcggccgcctggcccatgatccgcgcctgaccggtcgcgcccggcagatcctcagcggcgagccctacgtgcatcagtcgcgcatcaacgacaagccgggcgtcgaggggcagggcttcaactggcactccgacttcgagacctggcacgccgaggacggcatgccggccatgcacgcggtgagcgcctccctcgtgctgaccgacaaccacaccttcaatggcccgctgatgctggtgcccggctcccacggcgtcttcgtgccctgcctgggcgagacaccggatggccatcatcgccagtcgctgaagcggcaggcgctcggcgtgccgggccacgaggcgcttgccgcgctggccgagcgtcacggcatcgaggcccccaccggggcggccggcggcttgctgctgttcgactgcaacaccctgcacggcgccaacgccaacatgtcgccggacccgcgcagcaatgccttcttcgtctacaaccgccgcgacaatcgctgcgtcgagccctacgccgccggcaagcgccgcccgcgcttcctggctcacgatcccgacgagcggtggacgccggaggcctga

[0046] SEQ ID NO.3

[0047] MSVQTSSNRPLEQATQFTVVDSPDPQSRVRSAPLPGEDPYPTRLSEPLDLPWLSRREPVVKGHDGEGPLSAAQLDAFERQGFLFEPGFISGDELAELRRELAALLERDDFRGRDFAITEPQGNEIRSLFAVHFLSRVFGRLTHDPRLTGRARQILGGEPYVHQSRINYKPGFEGQGFNWHSDFETWHAEDGMPAMHAVSASIVLTDNHTFNGPLMLVPGSHRVFVPCLGETPDGNHRQSLKRQALGVPGHEALAALAERHGIEAPTGAAGGLLLFDCNTLHGSNANMSPDPRSNAFFVYNRRDNRCVEPYAAGRRRPRFLAHDPDERWTPEA*

[0048] SEQ ID NO.4

[0049] atgtcagtgcagacatcgtccaaccgaccgctggaacaggcgacccagttcaccgtcgtggattctcccgatccccagagccgggtgcgcagcgcgccgcttcccggggaggatccctaccccacccggctgtccgagcccctcgacctgccctggctgagccgccgggagccggtggtgaagggtcatgacggcgagggcccgctctccgcggcgcagctcgacgcgttcgagcgccaggggttcctcttcgagcccggtttcatcagcggcgacgagctcgccgagctgcgccgggagcttgccgcgctgctggagcgcgatgacttccgggggcgcgacttcgccatcaccgagcctcagggcaacgagatccgctcgctgttcgccgtgcatttcctctcgcgggtcttcggccgcctgacccatgatccgcgcctgaccggtcgcgcccggcagatcctcggcggcgagccctacgtgcatcagtcgcgcatcaactacaagccgggcttcgaggggcagggcttcaactggcactccgacttcgagacctggcacgccgaggacggcatgccggccatgcacgcggtgagcgcctccatcgtgctgaccgacaaccacaccttcaatggcccgctgatgctggtgcccggctcccaccgggtcttcgtgccctgcctgggcgagacaccggatggcaaccatcgccagtcgctgaagcggcaggcgctcggcgtgccgggccacgaggcgcttgccgccctggccgagcgtcacggcatcgaggcccccaccggggcggccggcggcttgctgctgttcgactgcaacaccctgcacggctccaacgccaacatgtcgccggacccgcgcagcaatgccttcttcgtctacaaccgccgcgacaatcgctgcgtcgagccctacgccgccggcaggcgccgcccgcgcttcctggctcacgatcccgacgagcggtggacgccggaggcctga

[0050] Example 2 Application of a recombinant expression strain containing an ectoine hydroxylase mutant in the preparation of 5-hydroxyectoine

[0051] (1) Seed plate activation: In a clean bench, the recombinant expression strain BL21 (DE3) / pET32a (+)-ectD mut -6×His was streaked on an eggplant-shaped bottle containing solid LB medium containing resistance, and cultured at 37°C for 12 hours to obtain activated seeds;

[0052] (2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at 37 °C, dissolved oxygen 25%, pH 7.0 regulated with ammonia water, and cultured until the OD reached 10 to obtain seed liquid;

[0053] (3) Fermentation of expression strains: The seed liquid was inoculated into a fermentation tank containing 8 L of fermentation medium at a transfer rate of 10%, the temperature was 37°C, the dissolved oxygen was 25%, and the pH was adjusted to 7.0 with ammonia water. After the substrate sugar was consumed, IPTG was added to a concentration of 0.1 mM / L, and a glycerol solution with a concentration of 500 g / L was fed. The growth rate of OD was adjusted to 3 / h. The fermentation was terminated when OD grew to 60, and the fermented bacteria were obtained;

[0054] (4) Collecting bacteria by centrifugation: circulating chilled water was introduced into the jacket to cool the fermented bacteria to 4°C, and the mixture was centrifuged at 10,000 rpm and 4°C for 5 min. The supernatant was discarded and the bacteria were retained. The mixture was then resuspended in 4°C precooled PBS buffer with a pH of 7.0 and 0.01 M. The wet bacteria were collected by centrifugation at 10,000 rpm and 4°C for 5 min, and the wet bacteria were stored at 4°C.

[0055] (5) Pretreatment of bacteria: resuspend the wet bacteria in 4°C precooled pH 7.0, 0.02M PBS to prepare a suspension with a wet bacteria concentration of 500 g / L, add hydroxytyrosol until the concentration of hydroxytyrosol in the mixed solution is 100 ppm, and obtain a mixed solution. Raise the temperature to 42°C and maintain for 10 min, then cool to 35°C to obtain a pretreated bacterial suspension.

[0056] (6) Catalytic preparation of 5-hydroxytetrahydropyrimidine: The pretreated bacterial suspension is added to the reaction solution to obtain a mixed solution, the pH of which is adjusted to 7.0 with phosphoric acid and liquid alkali, and fermented at a temperature of 35°C and a dissolved oxygen content of 15%. Samples are taken every 0.5 h to determine the content of 5-hydroxytetrahydropyrimidine and α-ketoglutaric acid in the mixed solution. When the concentration of α-ketoglutaric acid in the solution is less than 5 g / L, α-ketoglutaric acid is added until the concentration of α-ketoglutaric acid in the solution is 5 g / L. When the concentration growth rate of 5-hydroxytetrahydropyrimidine is less than 2 (g / L) / h, the temperature is raised to 42°C for fermentation for 2 h, and the fermentation is terminated to obtain a reaction solution containing 5-hydroxytetrahydropyrimidine.

[0057] The mixed solution comprises: 50 g / L ectoine, 0.01 M PBS buffer with a pH of 7.0, 55 g / L α-ketoglutaric acid, 0.18 g / L ferrous sulfate, and 160 g / L wet bacteria.

[0058] Example 3

[0059] Steps (1) to (4) are the same as steps (1) to (4) in Example 2.

[0060] (5) Bacterial pretreatment: The wet bacteria were resuspended in 4°C precooled pH 7.0, 0.02 M PBS to prepare a wet bacterial suspension with a concentration of 500 g / L. Hydroxytyrosol was added to the mixed solution until the concentration of hydroxytyrosol in the mixed solution was 200 ppm. The mixed solution was heated to 42°C and maintained for 10 min. The mixture was cooled to 35°C to obtain a pretreated bacterial suspension.

[0061] Step (6) is the same as step (6) in Example 2.

[0062] Example 4

[0063] Steps (1) to (5) are the same as steps (1) to (5) in Example 2.

[0064] (6) Catalytic preparation of 5-hydroxytetrahydropyrimidine: The pretreated bacterial suspension was added to the reaction solution, and the pH was adjusted to 7.0 with phosphoric acid and liquid alkali, the temperature was 35°C, and the dissolved oxygen was 15% to obtain a mixed solution, which was then fermented. Samples were taken every 0.5 h to determine the content of 5-hydroxytetrahydropyrimidine and α-ketoglutaric acid in the mixed solution. When the concentration of α-ketoglutaric acid in the solution was less than 5 g / L, α-ketoglutaric acid was added until the concentration of α-ketoglutaric acid in the solution was 5 g / L. When the concentration growth rate of 5-hydroxytetrahydropyrimidine was less than 2 (g / L) / h, the temperature was raised to 42°C for fermentation for 4 h, and the fermentation was terminated to obtain a reaction solution containing 5-hydroxytetrahydropyrimidine.

[0065] The reaction solution comprises: 50 g / L ectoine, 0.01 M PBS buffer with a pH of 7.0, 55 g / L α-ketoglutaric acid, 0.18 g / L ferrous sulfate, and 160 g / L wet bacteria.

[0066] Example 5

[0067] Steps (1) to (4) are the same as steps (1) to (4) in Example 2.

[0068] (5) Bacterial pretreatment: The wet bacteria were resuspended in 4°C precooled pH 7.0, 0.02 M PBS to prepare a wet bacterial suspension with a concentration of 500 g / L. Hydroxytyrosol was added to the mixed solution until the concentration of hydroxytyrosol in the mixed solution was 150 ppm. The mixed solution was heated to 42°C and maintained for 10 min. The mixture was cooled to 35°C to obtain a pretreated bacterial suspension.

[0069] (6) Catalytic preparation of 5-hydroxytetrahydropyrimidine: The pretreated bacterial suspension is added to the reaction solution to obtain a mixed solution, the pH of which is adjusted to 7.0 with phosphoric acid and liquid alkali, and fermented at a temperature of 35°C and a dissolved oxygen content of 15%. Samples are taken every 0.5 h to determine the content of 5-hydroxytetrahydropyrimidine and α-ketoglutaric acid in the mixed solution. When the concentration of α-ketoglutaric acid in the solution is less than 5 g / L, α-ketoglutaric acid is added until the concentration of α-ketoglutaric acid in the solution is 5 g / L. When the concentration growth rate of 5-hydroxytetrahydropyrimidine is less than 2 (g / L) / h, the temperature is raised to 42°C and fermented for 4 h. The fermentation is terminated to obtain a reaction solution containing 5-hydroxytetrahydropyrimidine.

[0070] The mixed solution comprises: 80 g / L ectoine, 0.01 M PBS buffer with a pH of 7.0, 100 g / L α-ketoglutaric acid, 0.2 g / L ferrous sulfate, and 200 g / L wet bacteria.

[0071] Comparative Example 1

[0072] (1) Design primers ectD-F / R, the sequences of which are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively, and use high-fidelity polymerase Phanta HS to amplify the gene fragment of ectD-6×His with SEQ ID NO.4;

[0073] (2) Design primers pET32a(+)-F / R, use high-fidelity polymerase Phanta HS, and use pET32a(+) plasmid as a template to amplify and obtain the pET32a(+) linearized fragment;

[0074] (3) Using the homologous recombination enzyme ClonExpress II One Step Cloning Kit, the gene fragment of ectD-6×His and the linearized fragment of pET32a(+) were connected, transformed into DH5α, and screened for ampicillin resistance to obtain the recombinant expression vector pET32a(+)-ectD-6×His;

[0075] (4) The recombinant expression vector pET32a(+)-ectD-6×His was transformed into Escherichia coli BL21(DE3) to construct the recombinant expression strain BL21(DE3) / pET32a(+)-ectD-6×His.

[0076] The above primer sequences are shown in Table 1.

[0077] Table 1. Primer sequences

[0078]

[0079] Comparative Example 2

[0080] (1) Seed plate activation: Streak the strain BL21 (DE3) / pET32a (+) -ectD-6 × His on an eggplant-shaped bottle containing solid LB medium containing resistance in an ultra-clean workbench, and culture at 37°C for 12 h to obtain activated seeds;

[0081] (2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at 37 °C, dissolved oxygen 25%, pH 7.0 regulated with ammonia water, and cultured until the OD reached 10 to obtain seed liquid;

[0082] (3) Fermentation of expression strains: The seed liquid was inoculated into a fermentation tank containing 8 L of fermentation medium at a transfer rate of 10%, the temperature was 37°C, the dissolved oxygen was 25%, and the pH was adjusted to 7.0 with ammonia water. After the substrate sugar was consumed, IPTG was added to a concentration of 0.1 mM / L, and a glycerol solution with a concentration of 500 g / L was fed. The growth rate of OD was adjusted to 3 / h. The fermentation was terminated when OD grew to 60, and the fermented bacteria were obtained;

[0083] (4) Collecting bacteria by centrifugation: circulating chilled water was introduced into the jacket to cool the temperature to 4°C, and the fermented bacteria were centrifuged at a speed of 10,000 rpm and a temperature of 4°C for 5 min. The supernatant was discarded, and the bacteria were retained. The bacteria were then resuspended in a 4°C precooled pH 7.0, 0.01 M PBS buffer, and the wet bacteria were collected by centrifugation at a speed of 10,000 rpm and a temperature of 4°C for 5 min, and the wet bacteria were stored at 4°C.

[0084] (5) Pretreatment of bacteria: The wet bacteria were resuspended in 4°C precooled pH 7.0, 0.02M PBS to prepare a suspension with a wet bacteria concentration of 500 g / L, and hydroxytyrosol was added until the concentration of hydroxytyrosol in the suspension was 100 ppm to obtain a mixed solution, which was heated to 42°C and maintained for 10 min, and then cooled to 35°C to obtain a pretreated bacterial suspension;

[0085] (6) Catalytic preparation of 5-hydroxytetrahydropyrimidine: The pretreated bacterial suspension is added to the reaction solution to obtain a mixed solution, the pH of which is adjusted to 7.0 with phosphoric acid and liquid alkali, and fermented at a temperature of 35°C and a dissolved oxygen content of 15%. Samples are taken every 0.5 h to determine the content of 5-hydroxytetrahydropyrimidine and α-ketoglutaric acid in the mixed solution. When the concentration of α-ketoglutaric acid in the solution is less than 5 g / L, α-ketoglutaric acid is added to a concentration of 5 g / L. When the concentration growth rate of 5-hydroxytetrahydropyrimidine is less than 2 (g / L) / h, the temperature is raised to 42°C and fermented for 2 h. The fermentation is terminated to obtain a reaction solution containing 5-hydroxytetrahydropyrimidine.

[0086] The mixed solution comprises: 50 g / L ectoine, 0.01 M PBS buffer with a pH of 7.0, 55 g / L α-ketoglutaric acid, 0.18 g / L ferrous sulfate, and 160 g / L wet bacteria.

[0087] Comparative Example 3

[0088] Step (5) of bacterial cell pretreatment was not performed, and the remaining steps were the same as those in Example 2.

[0089] Comparative Example 4

[0090] Steps (1) to (4) are the same as steps (1) to (4) in Example 2.

[0091] (5) Bacterial pretreatment: The wet bacteria were resuspended in 4°C precooled pH 7.0, 0.02 M PBS to prepare a wet bacterial suspension with a concentration of 500 g / L. The mixture was heated to 42°C and maintained for 10 min. The mixture was then cooled to 35°C to obtain a pretreated bacterial suspension.

[0092] Step (6) is the same as step (6) in Example 2.

[0093] Comparative Example 5

[0094] Steps (1) to (4) are the same as steps (1) to (4) in Example 2.

[0095] (5) Bacterial pretreatment: The wet bacteria were resuspended in 4°C precooled pH 7.0, 0.02 M PBS to prepare a wet bacterial suspension with a concentration of 500 g / L. Hydroxytyrosol was added to the mixed solution until the concentration of hydroxytyrosol in the mixed solution was 100 ppm. The mixed solution was heated to 35°C to obtain a pretreated bacterial suspension.

[0096] Step (6) is the same as step (6) in Example 2.

[0097] Comparative Example 6

[0098] Steps (1) to (5) are the same as steps (1) to (5) in Example 2.

[0099] (6) Catalytic preparation of 5-hydroxytetrahydropyrimidine: The pretreated bacterial suspension is added to the reaction solution to obtain a mixed solution, the pH of which is adjusted to 7.0 with phosphoric acid and liquid alkali, and fermented at a temperature of 35°C and a dissolved oxygen content of 15%. Samples are taken every 0.5 h to determine the content of 5-hydroxytetrahydropyrimidine and α-ketoglutaric acid in the mixed solution. When the concentration of α-ketoglutaric acid in the solution is less than 5 g / L, α-ketoglutaric acid is added to a concentration of 5 g / L. When the concentration growth rate of 5-hydroxytetrahydropyrimidine is less than 2 (g / L) / h, the temperature is raised to 36°C and fermented for 2 h. The fermentation is terminated to obtain a reaction solution containing 5-hydroxytetrahydropyrimidine.

[0100] The reaction solution comprises: 50 g / L ectoine, 0.01 M PBS buffer with a pH of 7.0, 55 g / L α-ketoglutaric acid, 0.18 g / L ferrous sulfate, and 160 g / L wet bacteria.

[0101] Experimental Example 1 Enzyme Activity Assay

[0102] The wet cells collected in step (4) of Example 2 and Comparative Example 2 were tested for specific enzyme activity according to the following method.

[0103] Method for determining enzyme activity: first prepare the reaction system: 10 g / L ectoine, 0.01 M PBS buffer with a pH of 7.0, 10 g / L α-ketoglutaric acid, 0.1 g / L ferrous sulfate, 50 g / L wet bacteria, and mix well. Pour the mixture into a triangular flask at a volume of 50 ml / 500 ml, place it in a shaking table at 30 ° C and 250 rpm for 30 min, and heat it to 90 ° C for 5 min to stop the reaction. The reaction system without humidified bacteria was used as a blank control, and the amount of 5-hydroxyectoine generated was detected by high performance liquid chromatography (HPLC).

[0104] Tetrahydropyrimidine hydroxylase specific activity (U / g) = the amount of 5-hydroxytetrahydropyrimidine generated / (wet cell volume × reaction time)

[0105] U represents the amount of wet bacteria that catalyzes the production of 1 mg of 5-hydroxytetrahydropyrimidine per minute.

[0106] The measured data are listed in Table 2.

[0107] Table 2. Specific enzyme activity data

[0108]

[0109] From the data in Table 2, we can see that the tetrahydropyrimidine hydroxylase mutant ectD mut The specific enzyme activity of the mutant ectD was 1.72 U / g, and the specific enzyme activity of the original ectoine hydroxylase ectD was 1.18 U / g. mut The specific enzyme activity was increased by 45.8%, indicating that the efficiency of the mutant enzyme of the tetrahydropyrimidine hydroxylase provided by the present invention in catalyzing the conversion of tetrahydropyrimidine into 5-hydroxytetrahydropyrimidine was increased by 45.8% compared with the original tetrahydropyrimidine hydroxylase, and the enzyme activity was greatly improved.

[0110] Experimental Example 2 Conversion Rate Calculation

[0111] After the catalytic reaction of Example 2-5 and Comparative Example 2-6 was completed, the concentration of 5-hydroxytetrahydropyrimidine in the reaction solution was determined and the conversion rate was calculated according to the following method, and the determination was performed three times respectively, and the average value was taken. The results are listed in Table 3.

[0112] The determination method is as follows: the reaction solution is centrifuged at 12000rpm for 5 minutes, the supernatant is taken, and after dilution with deionized water, the high performance liquid chromatography is used for determination, and a ZORBAX SB-C18 column is selected, the mobile phase is a 2% acetonitrile solution, the column temperature is 35°C, the flow rate is 1mL / min, the injection volume is 10μL, the ultraviolet detection wavelength is 210nm, the retention time is 10min, the peak area is measured, and the concentration of 5-hydroxytetrahydropyrimidine in the reaction solution is obtained. The conversion rate is calculated according to the following formula:

[0113] Conversion rate = c (5-hydroxytetrahydropyrimidine) / c (initial tetrahydropyrimidine) * 100%,

[0114] c (ectoine) = ectoine concentration (g / L) / 142.16 (g / mol),

[0115] c(5-hydroxytetrahydropyrimidine) = 5-hydroxytetrahydropyrimidine concentration (g / L) / 158.16 (g / mol).

[0116] Where c is the molar concentration in mol / L.

[0117] Table 3. Data comparison of conversion rate

[0118]

[0119] From the data in Table 3, it can be seen that the conversion time of Example 2 is 10.5h, the 5-hydroxytetrahydropyrimidine concentration is 51.4 g / L, and the conversion rate is 92.45%. The conversion time of Comparative Example 2 is 15.5h, the 5-hydroxytetrahydropyrimidine concentration is 41.9 g / L, and the conversion rate is 75.36%. It can be seen that the conversion time of Example 2 is shorter, the 5-hydroxytetrahydropyrimidine concentration is higher, and the conversion rate is higher, indicating that the efficiency of the tetrahydropyrimidine hydroxylase mutant provided by the present invention in catalyzing the conversion of tetrahydropyrimidine into hydroxytetrahydropyrimidine is significantly improved compared with the efficiency and effect of the original tetrahydropyrimidine hydroxylase, which is beneficial to the preparation of 5-hydroxytetrahydropyrimidine.

[0120] In Example 2, the conversion time was 10.5 h, the 5-hydroxytetrahydropyrimidine concentration was 51.4 g / L, and the conversion rate was 92.45%. In Example 3, the conversion time was 8.5 h, the 5-hydroxytetrahydropyrimidine concentration was 50.6 g / L, and the conversion rate was 91.01%. In Example 4, the conversion time was 12.5 h, the 5-hydroxytetrahydropyrimidine concentration was 53.7 g / L, and the conversion rate was 96.58%. All of them are significantly better than the comparative example 3, which has a conversion time of 20h, a 5-hydroxytetrahydropyrimidine concentration of 45.3 g / L, and a conversion rate of 81.47%, the comparative example 4 has a conversion time of 14h, a 5-hydroxytetrahydropyrimidine concentration of 48.5 g / L, and a conversion rate of 87.23%, the comparative example 5 has a conversion time of 14.5h, a 5-hydroxytetrahydropyrimidine concentration of 47.2g / L, and a conversion rate of 84.89%, and the comparative example 6 has a conversion time of 10.5h, a 5-hydroxytetrahydropyrimidine concentration of 43.8 g / L, and a conversion rate of 78.78%. It is shown that the application method of the tetrahydropyrimidine hydroxylase mutant provided by the present invention can effectively improve the efficiency of the mutant in catalyzing the conversion of tetrahydropyrimidine into 5-hydroxytetrahydropyrimidine, the conversion time is short, the conversion rate is above 90.0%, and the conversion rate is high.

[0121] In Example 5, when the substrate tetrahydropyrimidine was 80 g / L, the conversion rate was 93.27%, and the content of 5-hydroxytetrahydropyrimidine reached 83.0 g / L, indicating that the preparation method of the present invention has a large conversion rate per unit volume and a low production cost, which is conducive to the large-scale production of 5-hydroxytetrahydropyrimidine.

[0122] In summary, the ectoine hydroxylase mutant and application provided by the present invention solve the problem of 5-hydroxyectoine production, are conducive to the large-scale production and preparation of 5-hydroxyectoine, can effectively reduce the production cost of 5-hydroxyectoine, and are of great significance to the promotion and application of hydroxyectoine.

Claims

1. A tetrahydropyrimidine hydroxylase mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.

2.

2. The tetrahydropyrimidine hydroxylase mutant according to claim 1, characterized in that: The ectoine hydroxylase mutant is obtained by mutation of the ectoine hydroxylase gene ectD.

3. The tetrahydropyrimidine hydroxylase mutant according to claim 2, characterized in that: The amino acid sequence of the tetrahydropyrimidine hydroxylase gene ectD is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.

4.

4. The tetrahydropyrimidine hydroxylase mutant according to claim 2 or 3, characterized in that: The tetrahydropyrimidine hydroxylase gene is derived from a halophilic Bacillus having the property of secreting tetrahydropyrimidine. Halobacillus sp.) FL-2423, which was deposited in the General Microbiology Center of China Microorganism Culture Collection on November 1, 2019, with the deposit number CGMCC No.18783, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

5. A construction of a recombinant expression strain based on the tetrahydropyrimidine hydroxylase mutant according to claim 1, characterized in that: The recombinant expression strain is BL21 (DE3) / pET32a (+)-ectD mut -6×His, the recombinant expression vector used was pET32a(+)-ectD mut -6×His.

6. The construction of a recombinant expression strain containing an ectohydropyrimidine hydroxylase mutant according to claim 5, characterized in that: The following steps are involved: (1) Design of primer ectD mut -F / R, the sequences are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, using high-fidelity polymerase Phanta HS and ectD mut As a template, the nucleotide sequence is as shown in SEQ ID NO.2, and ectD is obtained by amplification. mut -6×His gene fragment; (2) Design primers pET32a(+)-F / R, the sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. Use high-fidelity polymerase Phanta HS and pET32a(+) plasmid as a template to amplify and obtain the pET32a(+) linearized fragment; (3) Use homologous recombination enzyme ClonExpress II One Step Cloning Kit to connect ectD mut -6×His gene fragment and pET32a (+) linearized fragment were transformed into DH5α and screened for ampicillin resistance to obtain the recombinant expression vector pET32a (+)-ectD mut -6×His; (4) Insert the recombinant expression vector pET32a(+)-ectD mut -6×His was transformed into E. coli BL21 (DE3) to construct the recombinant expression strain BL21 (DE3) / pET32a (+)-ectD mut -6×His.

7. Use of the recombinant expression strain of the ectoine hydroxylase mutant according to claim 5 in the preparation of 5-hydroxyectoine, characterized in that: The following steps are involved: (1) Seed plate activation: Streak the recombinant expression strain on an eggplant-shaped bottle containing solid LB medium containing resistance in an ultra-clean workbench and culture at 37°C for 12 hours to obtain activated seeds; (2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium, at a temperature of 37°C, dissolved oxygen of 20-30%, and pH 7.0 adjusted with ammonia water. The culture was continued until the OD value reached 10 to obtain seed liquid. (3) Fermentation of expression strain: The seed liquid was inoculated into a fermentation tank containing 8 L of fermentation medium, the temperature was 37 ° C, the dissolved oxygen was 20-30%, and the pH was adjusted to 7.0 with ammonia water. After the substrate sugar was consumed, IPTG was added to the fermentation medium at an IPTG concentration of 0.1 mM / L. A glycerol solution with a concentration of 500 g / L was fed, and the growth rate of OD was adjusted to 2-4 / h. The fermentation was terminated when OD grew to 60, and the fermented bacteria were obtained; (4) Collecting bacteria by centrifugation: circulating chilled water was introduced into the jacket to cool the fermented bacteria to 4°C, and the mixture was centrifuged at 10,000 rpm and 4°C for 5 min. The supernatant was discarded and the bacteria were retained. The mixture was then resuspended in 4°C precooled PBS with a pH of 7.0 and 0.01 M. The wet bacteria were collected by centrifugation at 10,000 rpm and 4°C for 5 min, and the wet bacteria were stored at 4°C. (5) Pretreatment of bacteria: resuspend the wet bacteria in 4°C precooled pH 7.0, 0.02M PBS buffer to prepare a suspension with a wet bacterial concentration of 500 g / L, add hydroxytyrosol to obtain a mixed solution, heat it to 42°C and maintain it for 10 min, then cool it to 35°C to obtain a pretreated bacterial suspension; (6) Catalytic preparation of 5-hydroxytetrahydropyrimidine: The pretreated bacterial suspension is added to the reaction solution to obtain a mixed solution, the pH of which is adjusted to 7.0 with phosphoric acid and liquid alkali, and the mixture is fermented at a temperature of 35°C and a dissolved oxygen content of 10-20%. After additional treatment, the fermentation is terminated to obtain a reaction solution containing 5-hydroxytetrahydropyrimidine.

8. Use of the recombinant expression strain of the ectoine hydroxylase mutant according to claim 7 in the preparation of 5-hydroxyectoine, characterized in that: In step (5), the concentration of hydroxytyrosol in the mixed solution is 100-200 ppm.

9. Use of the recombinant expression strain of the ectoine hydroxylase mutant according to claim 7 or 8 in the preparation of 5-hydroxyectoine, characterized in that: In step (6), the mixed solution comprises: 30-80 g / L of ectoine, 0.01 M of PBS buffer with a pH of 7.0, 30-100 g / L of α-ketoglutaric acid, 0.1-0.2 g / L of ferrous sulfate, and 50-200 g / L of wet bacteria.

10. Use of the recombinant expression strain of the ectoine hydroxylase mutant according to claim 9 in the preparation of 5-hydroxyectoine, characterized in that: In step (6), the specific operation of the supplementation treatment is: sampling and determining the content of hydroxyectoine and α-ketoglutaric acid in the mixed solution every 0.5 h; when the concentration of α-ketoglutaric acid in the solution is less than 5 g / L, adding α-ketoglutaric acid until the concentration of α-ketoglutaric acid in the solution is 5 g / L; when the concentration growth rate of hydroxyectoine is less than 2 (g / L) / h, raising the temperature to 42°C for fermentation for 2-4 h, and ending the fermentation.

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