Method for efficiently synthesizing epiinositol by using heat-resistant myo-inositol dehydrogenase mutant cascade system

Abstract

The invention discloses a method for efficiently synthesizing epiinositol by using a heat-resistant myo-inositol dehydrogenase mutant cascade system, and belongs to the field of biocatalysis engineering. Whole cells for expressing a scyllo-inositol dehydrogenase mutant and a heat-resistant scyllo-inositol dehydrogenase mutant are utilized to catalyze the scyllo-inositol to synthesize the epiinositol in a two-step cascade manner, and different from reported scyllo-inositol and D-chiral inositol synthesis methods, in the process of synthesizing the epiinositol by taking the scyllo-inositol as a substrate, the C4 position of the scyllo-inositol is dehydrogenated firstly, then the C4 position of the scyllo-inositol is dehydrogenated, and the C4 position of the D-chiral inositol According to the present invention, the structure of the substrate and the structure of the product are symmetric, and the enzyme is difficult to specifically recognize during the catalysis process, such that the synthesis of the different chiral compounds of the inositol is the important problem in the biological catalysis field. The invention provides a brand-new way for biocatalytically synthesizing the epiinositol, the yield of the epiinositol is increased to 1.89 g / L through enzyme modification, the conversion rate is 52.6%, and the yield and the conversion rate are the highest level reported publicly at present.

Description

Technical Field

[0001] The invention relates to a method for efficiently synthesizing epi-inositol by a heat-resistant myo-inositol dehydrogenase mutant cascade system, and belongs to the field of biocatalytic engineering. Background Art

[0002] Myo-inositol is an important nutrient that has many benefits for the human body as a dietary supplement. Inositol has 8 stereoisomers, including epi-inositol, allo-inositol, muco-inositol, neo-inositol, L-chiro-inositol, D-chiro-inositol, and scyllo-inositol, which exist in nature, as well as cis-inositol, which has not been observed in nature. Epi-inositol, as a stereoisomer of inositol, also has certain value in food health and medicine. Studies have shown that daily intraperitoneal injection of epi-inositol in rats can reduce anxiety levels and is more effective than inositol. 100μM epi-inositol and 100nM insulin are equivalent in promoting GLUT4-dependent glucose absorption in rat L6 myotubes.

[0003] The synthesis technology of epi-inositol is still limited to the stage of chemical synthesis, which is mainly synthesized by using sodium borohydride to catalyze the reduction of epi-inositol under alkaline conditions. However, the chemical synthesis has disadvantages such as dangerous and unstable reaction reagents, large environmental pollution, and high preparation costs, resulting in the extremely expensive sales price of epi-inositol in the market, which is about 14,000 yuan / gram.

[0004] In recent years, with the development of synthetic biology technology, biotransformation synthesis of inositol isomers has become a new research hotspot. Fujisawa et al. used maltodextrin phosphorylase (MalP) and phosphoglucomutase (PGM), inositol-3-phosphate synthase (MIPS) and inositol monophosphatase (IMPase) to construct an in vitro enzyme system for producing inositol using starch as raw material. After 7 hours of reaction, 2.9g of inositol can be produced from 2.9g of starch, and the molar conversion rate is about 96%. Paul et al. achieved the conversion from glucose to scyllo-inositol by transforming Corynebacterium glutamicum, catalyzing 20g / L glucose substrate within 72h to synthesize 1.8g / L scyllo-inositol. Ji et al. co-expressed inositol isomerase IolI2 and inositol dehydrogenase IolG in Corynebacterium glutamicum, and obtained a scyllo-inositol production strain DCI-7, which can produce 6.96g / L D-chiro-inositol in the presence of 40g / L inositol.

[0005] Myo-inositol dehydrogenase (myo-inositol 2-dehydrogenase, EC 1.1.1.18, referred to as IDH) reported so far is almost used to catalyze the C2 position dehydrogenation of myo-inositol to obtain scyllo-inositol, or common scyllo-inositol dehydrogenase (scyllo-inositol 2-dehydrogenase, EC 1.1.1.370, referred to as sIDH) is used to catalyze scyllo-inositol to obtain synthetic scyllo-inositol. Different from the common scyllo-inositol and D-chiro-inositol synthesis methods, in the process of synthesizing epi-inositol with myo-inositol as substrate, the C4 position needs to be dehydrogenated first, and then hydrogenated to achieve chiral inversion, and because the structures of substrates and products are very symmetrical structures, enzymes are often difficult to specifically identify during the catalytic process, so the synthesis of different chiral compounds of inositol has always been an important problem in the field of biocatalysis. So far, there is no report on the biosynthesis method of epi-inositol. Therefore, it is urgent to develop a biosynthesis method of epi-inositol with simple production process, less pollution and low cost. Summary of the invention

[0006] In view of the difficulties in the biosynthesis of epi-inositol, the present invention provides a method for synthesizing epi-inositol by catalyzing myo-inositol through a whole-cell two-step method: the first step is to use scyllo-inositol dehydrogenase (sIDH for short) to catalyze the dehydrogenation of the C4 hydroxyl group to produce the intermediate product 1L-epi-2-inosose, and the second step is to use heat-resistant myo-inositol dehydrogenase (IDH for short) to catalyze the hydrogenation of the C4 carbonyl group to produce epi-inositol. Based on the structural analysis of sequence conservation and virtual mutation screening, the two enzymes are directed to evolve, and mutants with higher catalytic efficiency are obtained. The present invention has the advantages of simple production process, low production cost, green environmental protection, etc., and lays a solid foundation for the industrial preparation of epi-inositol.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The invention provides a method for preparing epi-inositol. The method comprises the following steps: using scyllo-inositol dehydrogenase or a genetically engineered bacterium expressing scyllo-inositol dehydrogenase to catalyze myo-inositol to synthesize 1L-epi-2-inositose with whole cells; and adding heat-resistant myo-inositol dehydrogenase or a genetically engineered bacterium expressing heat-resistant myo-inositol dehydrogenase to catalyze 1L-epi-2-inositol with whole cells.

[0009] In one embodiment of the present invention, the method is:

[0010] (1) engineered bacteria and / or mutants expressing scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase, respectively;

[0011] (2) fermenting the engineered bacteria and / or mutants constructed in step (1) to obtain whole cells;

[0012] (3) subjecting the heat-resistant myo-inositol dehydrogenase obtained in step (2) to cell membrane permeability treatment to obtain permeable whole cells;

[0013] (4) using the whole cell expressing scyllo-inositol dehydrogenase obtained in step (2) to catalyze myo-inositol to synthesize 1L-epi-2-inosose;

[0014] The permeabilized whole cells expressing the thermostable myo-inositol dehydrogenase obtained in step (3) or the whole cell lysate expressing the thermostable myo-inositol dehydrogenase are used to catalyze 1L-epi-2-inosose to synthesize epi-inositol (epi-inositol).

[0015] In one embodiment of the present invention, according to the present invention, the catalytic pathway comprises: scyllo-inositol dehydrogenase in NAD + In the presence of NADH, the substrate myo-inositol is oxidized to the intermediate 1L-epi-2-inosose; the intermediate 1L-epi-2-inosose is reduced to the product epi-inositol by a thermostable myo-inositol dehydrogenase in the presence of NADH, such as Figure 2 In the catalytic pathway of the present invention, the required NADH comes from scyllo-inositol dehydrogenase in NAD + The process of oxidizing the substrate myo-inositol to the intermediate 1L-epi-2-inosose in the presence of NADH, that is, the method of the present invention does not include the step of additionally adding NADH.

[0016] In one embodiment of the present invention, according to the present invention, in step (1), the engineered bacteria comprises a vector expressing scyllo-inositol dehydrogenase, or comprises a vector expressing thermostable myo-inositol dehydrogenase.

[0017] Those skilled in the art will appreciate that the vectors and engineered bacteria involved in the present invention can be prepared by conventional methods known in the art.

[0018] In one embodiment of the present invention, according to the present invention, in step (1), the scyllo-inositol dehydrogenase is an enzyme having the function of oxidizing myo-inositol to 1L-epi-2-inosose and / or reducing 1L-epi-2-inosose to myo-inositol; further preferably, the scyllo-inositol dehydrogenase is derived from a Gfo / Idh / MocA family protein, such as a scyllo-inositol dehydrogenase derived from Paracoccus cavernae, Paracoccus kondratievae or Roseibium suaedae; more preferably, the scyllo-inositol dehydrogenase is derived from Paracoccus kondratievae; or the amino acid sequence of the scyllo-inositol dehydrogenase is derived from Paracoccus kondratievae. The scyllo-inositol dehydrogenase of kondratievae has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity.

[0019] In one embodiment of the present invention, according to the present invention, in step (1), the thermostable myo-inositol dehydrogenase refers to an enzyme having the function of reducing 1L-epi-2-inosose to epi-inositol and / or oxidizing epi-inositol to 1L-epi-2-inosose at above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, or above 80°C; further preferably, the thermostable myo-inositol dehydrogenase is derived from a Gfo / Idh / MocA family protein, such as from Laceyella putida, Geobacillus thermoleovorans, Paraageobacillus thermoleovorans, thermoglucosidasius); more preferably, the thermostable muscle inositol dehydrogenase is derived from Geobacillus thermoleovorans; or the amino acid sequence of the thermostable muscle inositol dehydrogenase is at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identical to the thermostable muscle inositol dehydrogenase derived from Geobacillus thermoleovorans.

[0020] In one embodiment of the present invention, according to the present invention, in step (1), the scyllo-inositol dehydrogenase mutant is obtained by mutating the 261st and / or 322nd amino acids of the scyllo-inositol dehydrogenase whose amino acid sequence is shown in SEQ ID NO.1; more preferably, the scyllo-inositol dehydrogenase mutant is obtained by mutating the 261st and 322nd amino acids.

[0021] Most preferably, the scyllo-inositol dehydrogenase mutant is obtained by mutating the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 to arginine or lysine; or the scyllo-inositol dehydrogenase mutant is obtained by mutating the phenylalanine at position 322 of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 to threonine; or the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to arginine, and the phenylalanine at position 322 is mutated to threonine.

[0022] In one embodiment of the present invention, according to the present invention, in step (1), the heat-resistant myo-inositol dehydrogenase mutant is obtained by mutating the amino acid at position 177 of the heat-resistant myo-inositol dehydrogenase as shown in SEQ ID NO.2.

[0023] Most preferably, the thermostable myo-inositol dehydrogenase mutant is obtained by mutating the phenylalanine at position 177 of the thermostable myo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.2 to lysine or glutamic acid.

[0024] In one embodiment of the present invention, in step (2), the preparation of the whole cells is performed using methods known in the art. The fermentation can use any culture medium suitable for the expression of foreign proteins, including but not limited to LB culture medium, TB culture medium, etc.

[0025] In one embodiment of the present invention, in step (3), the cell membrane permeability treatment includes but is not limited to heat treatment. Preferably, the cell membrane permeability treatment is heat treatment. The purpose of permeability treatment of the cell membrane is to allow extracellular myo-inositol to enter the cell through the cell membrane.

[0026] In one embodiment of the present invention, the reaction condition of the whole cell catalyzing the 1L-epi-2-inosose is that the whole cell is subjected to heat treatment and then the reaction is carried out; the heat treatment conditions are: the heat treatment temperature is 50-70°C; further preferably, the heat treatment temperature is 55-70°C; more preferably, the heat treatment temperature is 60-65°C; most preferably, the heat treatment temperature is 65°C.

[0027] The heat treatment time is 10 to 30 minutes; more preferably, the heat treatment time is 20 to 30 minutes; most preferably, the heat treatment time is 25 minutes.

[0028] In one embodiment of the present invention, the heat-treated cell concentration is 10-100 g / L; further preferably, the heat-treated cell concentration is 20-80 g / L; more preferably, the heat-treated cell concentration is 30-60 g / L; most preferably, the heat-treated cell concentration is 40 g / L.

[0029] In one embodiment of the present invention, according to the present invention, the heat treatment can be carried out in a buffer-free system or a buffer system; preferably, the heat treatment is carried out in a buffer system, and the buffer can be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, etc.

[0030] In one embodiment of the present invention, according to the present invention, in step (4), the reaction system in which myo-inositol is catalyzed by scyllo-inositol dehydrogenase to prepare 1L-epi-2-inosose is:

[0031] The concentration of the substrate myo-inositol is 1-100 mM, NAD + The concentration of the substrate myo-inositol is 1-100 mM, and the amount added to the whole cell is 10-100 g / L; preferably, the concentration of the substrate myo-inositol is 10-80 mM, and the amount added to the whole cell is 30-90 g / L; more preferably, the concentration of the substrate myo-inositol is 15-60 mM, NAD + The concentration of NAD is 15-60 mM, and the amount added to the whole cell is 40-80 g / L; most preferably, the concentration of the substrate myo-inositol is 20 mM, + The concentration was 20 mM and the amount of whole cells added was 80 g / L.

[0032] The reaction conditions for catalyzing myo-inositol to prepare 1L-epi-2-inosose are: reaction at pH 6.0-10.0 and 20-50°C for 2-24 hours; preferably, reaction at pH 8.0-9.5 and 25-40°C for 6-14 hours; most preferably, reaction at pH 9.5 and 28°C for 12 hours.

[0033] In one embodiment of the present invention, according to the present invention, in step (4), the reaction system in which 1L-epi-2-inositol is prepared by catalyzing 1L-epi-2-inositol with heat-resistant myo-inositol dehydrogenase is:

[0034] The concentration of substrate 1L-epi-2-inosose is 1-100 mM, Zn 2+ The addition amount of Zn is 0.1-5mM, and the addition amount of whole cells is 10-100g / L; preferably, the concentration of substrate 1L-epi-2-inosose is 10-80mM, Zn2+ The addition amount of Zn is 0.5-4 mM, and the addition amount of whole cells is 20-80 g / L; more preferably, the concentration of substrate 1L-epi-2-inosose is 20-60 mM, Zn 2+ The addition amount of Zn is 1-3mM, and the addition amount of whole cells is 30-60g / L; most preferably, the concentration of substrate 1L-epi-2-inosose is 40mM, Zn 2+ The addition amount of 1mM is for 100 μg / L and the addition amount of whole cells is 40g / L.

[0035] The reaction conditions for catalyzing 1L-epi-2-inosose to prepare epi-inositol are: reaction at pH 6.0-10.0 and 35-70°C for 2-24 hours; preferably, reaction at pH 6.5-8.0 and 40-60°C for 8-16 hours; most preferably, reaction at pH 7.5 and 50°C for 14 hours.

[0036] In one embodiment of the present invention, the catalytic reaction can be carried out in a buffer-free system or a buffer system; preferably, it is carried out in a buffer system, and the buffer can be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, etc. Among them, the phosphate buffer is, for example, sodium phosphate buffer, potassium phosphate buffer, etc.

[0037] In one embodiment of the present invention, according to the present invention, in step (4), the reaction conditions for preparing epi-inositol by catalyzing myo-inositol in two steps are as follows: after the reaction of preparing 1L-epi-2-inositol from myo-inositol is completed, the pH of the reaction system is adjusted to 7.5, and then the whole cells of thermostable myo-inositol dehydrogenase and Zn are directly added to the reaction system. 2+ After mixing, the cells were heat treated and reacted at pH 7.5 and 50°C for 14 h.

[0038] The present invention provides a scyllo-inositol dehydrogenase mutant. The scyllo-inositol dehydrogenase mutant is obtained by mutating the amino acid at position 261 shown in SEQ ID NO.1 into arginine, and is named C261R.

[0039] Or the scyllo-inositol dehydrogenase mutant is obtained by mutating the phenylalanine at position 322 of the scyllo-inositol dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 to threonine, and is named F322T.

[0040] Or the scyllo-inositol dehydrogenase mutant is obtained by mutating the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 to arginine, and mutating the phenylalanine at position 322 to threonine, and is named C261R / F322T;

[0041] The parent enzyme sequence of the scyllo-inositol dehydrogenase mutant (SEQ ID NO.1) is:

[0042] MTATQKSLGVALIGTGFMGKCHAMAWRNLATAFGGMPPRLEVLADTPAERAAELASAFGFARGTGDWRAAVTDPAVDVVSITTPNGMHREMAEAALAAGKHVWLEKPMALTVEDAEAMETAARAAGRVTILGYNYTRSPAFRAAADLVAQGAIGRPIHFRGVYDEDYMADPELPWSWRLTRAEGGL GALGDLGCHLVSVMLALMGPVARVQAQMDTVVTERPHQGGTATVENEDQAQALIRFASGASGEFSCSRVARGYRCRLAWEVQGTEGTLRFDQERMNELWLYQPGQAETDGFRRILTGPAQPGFAAFCPAAGHNFGFNEQKVVEAEMLRQAIAGGIPAWPDFTAGLTIERIIHGMARSARTGQPVLF

[0043] The base sequence encoding the scyllo-inositol dehydrogenase mutant parent enzyme (SEQ ID NO.3) is:

[0044]

[0045] The base sequence encoding the scyllo-inositol dehydrogenase mutant C261R / F322T (SEQ ID NO.5):

[0046]

[0047] The present invention provides a heat-resistant myo-inositol dehydrogenase mutant. The heat-resistant myo-inositol dehydrogenase mutant is obtained by mutating the amino acid at position 177 shown in SEQ ID NO.2 to lysine or glutamic acid, and is named F177K or F177E.

[0048] The heat-resistant myo-inositol dehydrogenase mutant parent enzyme sequence (SEQ ID NO.2) is:

[0049] MTRVKVGILGAGGIAKVHTSILKKDERVQIIGVADIVEERAASLAKEAGEAKAVKSLEELFDLGVDAVYVTTPNTMHVEPVLKCLENNVHVFSEKPMATSLEGAEQIRQAAERSKAIYNLGMNRRYASVYKRVKELIDSGEVTPYLAHVKMNRGELLNPPWTANPKV TGGFLYETPFHLMDLCRYLFGEVQTVYCEAKQNISTEELDTFAIMMTFVSGTIVNFVTYAHAGWSFPFESLEVYGKYSTVATQELEKVMYAPGLQQAAQIYDFYQLSIEEKWGYKEEDRLFIDAIIHGAKPPVTAEDGFRSIQLLEAIYESAKTGKMIDFRQTALSK

[0050] The base sequence encoding the thermostable myo-inositol dehydrogenase mutant parent enzyme (SEQ ID NO.4) is:

[0051]

[0052] The base sequence encoding the heat-resistant myo-inositol dehydrogenase mutant F177K (SEQ ID NO.6) is:

[0053]

[0054] In one embodiment of the present invention, the catalytic efficiency of the mutant is based on myo-inositol or 1L-epi-2-inosose as substrate and NAD + Or NADH as a cofactor to react, measured by the change in absorbance at 340nm detected by a microplate reader. One unit of enzyme activity (U) is defined as the catalytic oxidation of 1μmol NADH to NAD per minute under the measurement conditions. + The amount of enzyme.

[0055] In one embodiment of the present invention, according to the present invention, the vector expressing scyllo-inositol dehydrogenase comprises a promoter, a scyllo-inositol dehydrogenase gene, and a terminator; the vector expressing heat-resistant muscle inositol dehydrogenase comprises a promoter, a heat-resistant muscle inositol dehydrogenase gene, and a terminator.

[0056] Those skilled in the art will appreciate that various expression vectors known in the art can be used in the present invention, including but not limited to pET series, pGEX series, pACYCDuet-1, pRSFDuet-1, etc. Preferably, the vector is pET-21a(+), pET-28a(+).

[0057] Those skilled in the art will appreciate that various host cells known in the art can be used in the present invention, including but not limited to Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, etc. Preferably, the host bacteria is Escherichia coli BL21 (DE3).

[0058] Those skilled in the art will appreciate that various promoters known in the art can be used as the promoter of the present invention, including but not limited to T7 promoter, lac promoter, tac promoter, trc promoter, PR promoter, etc. Preferably, the promoter is T7 promoter.

[0059] Those skilled in the art will appreciate that various terminators known in the art can be used as the terminator of the present invention, including but not limited to T7 terminator, rrnB T1 terminator, rrnB T2 terminator, etc. Preferably, the terminator is T7 terminator.

[0060] The present invention provides a gene encoding the scyllo-inositol dehydrogenase mutant or the heat-resistant myo-inositol dehydrogenase mutant.

[0061] The invention also provides a recombinant vector carrying the gene.

[0062] The present invention also provides a recombinant cell expressing the mutant, or carrying the gene, or carrying the recombinant vector.

[0063] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as host cells.

[0064] The present invention relates to the above-mentioned vector for expressing scyllo-inositol dehydrogenase and the vector for expressing heat-resistant myo-inositol dehydrogenase.

[0065] The present invention relates to the above-mentioned engineering bacteria and mutants expressing scyllo-inositol dehydrogenase and engineering bacteria and mutants expressing heat-resistant myo-inositol dehydrogenase.

[0066] According to the present invention, the engineered bacteria and mutants expressing scyllo-inositol dehydrogenase include a vector expressing scyllo-inositol dehydrogenase; the engineered bacteria and mutants expressing heat-resistant myo-inositol dehydrogenase include a vector expressing heat-resistant myo-inositol dehydrogenase.

[0067] The present invention provides a method for improving substrate affinity and catalytic efficiency of scyllo-inositol dehydrogenase, wherein the method comprises mutating the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase of the amino acid sequence as shown in SEQ ID NO.1 to arginine;

[0068] or mutating the phenylalanine at position 322 of the scyllo-inositol dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 to threonine;

[0069] Or the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to arginine, and the phenylalanine at position 322 is mutated to threonine.

[0070] The present invention provides a method for improving the substrate affinity and catalytic efficiency of heat-resistant myo-inositol dehydrogenase, the method comprising:

[0071] The phenylalanine at position 177 of the heat-resistant myo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.2 is mutated to lysine or glutamic acid.

[0072] The present invention also provides the use of the above-mentioned scyllo-inositol dehydrogenase mutant, or a gene encoding the above-mentioned scyllo-inositol dehydrogenase mutant, or a recombinant vector carrying the above-mentioned scyllo-inositol dehydrogenase mutant, or a recombinant cell expressing the above-mentioned scyllo-inositol dehydrogenase mutant, or the above-mentioned method for improving the substrate affinity and catalytic efficiency of scyllo-inositol dehydrogenase in the preparation of 1L-epi-2-inositose or a product containing 1L-epi-2-inositose.

[0073] The present invention also provides the above-mentioned heat-resistant myo-inositol dehydrogenase mutant, or a gene encoding the above-mentioned heat-resistant myo-inositol dehydrogenase mutant, or a recombinant vector carrying the above-mentioned heat-resistant myo-inositol dehydrogenase mutant, or a recombinant cell expressing the above-mentioned heat-resistant myo-inositol dehydrogenase mutant, or the use of the above-mentioned method for improving the substrate affinity and catalytic efficiency of heat-resistant myo-inositol dehydrogenase in the preparation of epi-inositol or a product containing epi-inositol.

[0074] The present invention also provides a method for preparing the above-mentioned epi-inositol, or the use of the above-mentioned scyllo-inositol dehydrogenase mutant, the heat-resistant myo-inositol dehydrogenase mutant, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant cell, or the above-mentioned method for improving enzyme substrate affinity and catalytic efficiency in the preparation of epi-inositol or a product containing epi-inositol.

[0075] Beneficial Effects

[0076] (1) The present invention is the first to utilize a biosynthetic method to prepare epi-inositol using myo-inositol as a substrate. Compared with the chemical method, this method has the advantages of simple operation, mild reaction conditions, high efficiency, low cost, and environmental protection.

[0077] (2) Different from the enzymes commonly used to synthesize scyllo-inositol and D-chiro-inositol, the present invention provides a scyllo-inositol dehydrogenase that can catalyze the dehydrogenation of the C4 position of myo-inositol, thereby solving the problem of enzyme regioselectivity in the process of epi-inositol biosynthesis.

[0078] (3) The present invention is the first to use a two-step method of whole cells expressing scyllo-inositol dehydrogenase and whole cells expressing thermostable myo-inositol dehydrogenase to synthesize epi-inositol from myo-inositol, thus developing a new method for preparing epi-inositol that is simple and easy to scale up.

[0079] (4) The present invention provides a mutant of scyllo-inositol dehydrogenase and a mutant of thermostable myo-inositol dehydrogenase, respectively. The catalytic efficiency (k cat / K m ) was 3.2 times that of the wild type, and the yield of 1L-epi-2-inosose was 4.4 times that of the wild type; the catalytic efficiency (k cat / K m ) was 1.7 times that of the wild type, and the yield of epi-inositol was 5.2 times that of the wild type.

[0080] (5) The present invention optimizes the reaction conditions of the mutants of scyllo-inositol dehydrogenase and the mutants of thermostable myo-inositol dehydrogenase, and explores the optimal reaction temperature, pH, cell amount, substrate amount, reaction time and other conditions. In the two-step process of preparing epi-inositol from myo-inositol catalyzed by the optimized dual enzyme mutant, the total yield of epi-inositol was 52.6%.

[0081] (6) The present invention does not require the addition of NADH, greatly reduces production costs, and has important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 : Chemical structures of myo-inositol, 1L-epi-2-inositol, and epi-inositol.

[0083] Figure 2 : Schematic diagram of the whole cell catalysis of myo-inositol production of epi-inositol. myo-inositol: myo-inositol; epi-inositol: scyllo-inositol; 1L-epi-2-inosose: 1L-epi-2-inosose; sIDH: scyllo-inositol dehydrogenase; IDH: myo-inositol dehydrogenase.

[0084] Figure 3 : The initial yield of the enzyme reaction; wherein A is the initial yield of myo-inositol converted into 1L-table-2-inosose catalyzed by scyllo-inositol dehydrogenase from different sources; B is the initial yield of 1L-table-2-inositol converted into table-inositol catalyzed by thermostable myo-inositol dehydrogenase from different sources.

[0085] Figure 4 : Molecular docking map of substrate and enzyme; A is the position of myo-inositol in the PksIDH binding pocket The amino acid residues in the binding pocket of PksIDH; B is the binding of myo-inositol in the binding pocket of PksIDH; C is the binding of 1L-epi-2-inosose in the binding pocket of GtIDH The amino acid residues in the GtIDH binding pocket are shown in Figure 2; D is the binding status of 1L-epi-2-inosose in the GtIDH binding pocket.

[0086] Figure 5 :Virtual screening of mutation sites; A is the myo-inositol in the PksIDH binding pocket The binding energy changes when the semi-conservative amino acids in the binding pocket of GkIDH are subjected to virtual saturation mutation; B is the binding energy changes when the semi-conservative amino acids in the binding pocket of GkIDH are subjected to virtual saturation mutation; The changes in binding energy when virtual saturation mutations are performed on the semi-conservative amino acids in the

[0087] Figure 6 : HPLC spectrum of the product; wherein A is the HPLC spectrum of 1L-table-2-inosose, and B is the HPLC spectrum of epi-inositol.

[0088] Figure 7 : SDS-PAGE analysis of recombinant engineering bacteria E. coli BL21 / PksIDH-C261R / F322T and E. coli BL21 / GtIDH-F177K; M, protein marker; 1, E. coli BL21 / PksIDH-C261R / F322T; 2, E. coli BL21 / GtIDH-F177K.

[0089] Figure 8: The yield of 1L-epi-2-inosose synthesized by catalysis under different conditions; wherein, AE are the changes in the yield of the intermediate 1L-epi-2-inosose under different pH, reaction temperature, cell amount, substrate amount and reaction time, respectively.

[0090] Fig. 9 : The yield of epi-inositol synthesized under different conditions; AG represents different heat treatment conditions, reaction temperature, pH, cell amount, substrate amount, Zn 2+ The yield of the product inositol changes under the addition amount and reaction time. DETAILED DESCRIPTION

[0091] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below by specific examples. However, it should be understood that the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0092] Technical terms

[0093] Expression: The term "expression" includes any step involved in the production of scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0094] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding the scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase of the present invention and operably linked to control sequences that provide for the expression thereof.

[0095] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The host cell can be any cell useful in the production of scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase, such as a prokaryotic cell or a eukaryotic cell. The prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanic Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Mycobacterium, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. Host cells can also be eukaryotic organisms, such as mammals, insects, plants, or fungal cells.

[0096] The culture medium involved in the following examples is as follows:

[0097] LB liquid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, natural pH.

[0098] The detection methods involved in the following embodiments are as follows:

[0099] Detection of scyllo-inositol dehydrogenase activity:

[0100] The enzyme activity of scyllo-inositol dehydrogenase in oxidizing myo-inositol was measured at 30°C by measuring the absorbance change rate of NADH at 340 nm. Enzyme activity assay system: The reaction volume was 200 μL, and 5 mM NAD + and 50 mM myo-inositol, incubated in a 30°C metal bath for 3 min, added 20 μL of pure enzyme at an appropriate concentration, and scanned the absorbance change at 340 nm using the Bio-Tek Cytation 5 Cell Imaging Multifunctional Detection System.

[0101] Enzyme activity is defined as: under the above conditions, catalyzing 1 μmol NAD per minute. + The amount of enzyme reduced to NADH is defined as one unit U.

[0102] The calculation formula of enzyme activity is: enzyme activity (U) = EW × V × 10 3 / (6220×0.625);

[0103] The calculation formula of specific activity is: specific activity (U / mg) = enzyme activity (U) / protein amount (mg);

[0104] Wherein, EW: change in absorbance at 340 nm within 1 min; V: volume of reaction solution (mL); 6220: molar extinction coefficient (L / mol / cm); 0.625: optical path distance (cm).

[0105] Determination of kinetic parameters:

[0106] According to the Michaelis equation v = V max ×[S] / K m +[S], calculate the K of the enzyme for different substrates m value.

[0107] Where, v: reaction rate (U / mg); V max : maximum reaction rate (U / mg); [S]: substrate concentration (mM); K m :The reaction speed v reaches half 1 / 2V max The substrate concentration at .

[0108] When the substrate concentration is saturated, k cat =V max / Et, calculate k cat value.

[0109] Among them, V max : maximum reaction rate; Et: enzyme concentration.

[0110] Kinetic parameter determination:

[0111] The total reaction volume was 200 μL, and 20 mM Tris-HCl buffer (pH 7.5) and 5 mM NAD + , keep warm at 30℃ for 3min, add 20μL of pure enzyme at appropriate concentration, and measure the specific enzyme activity under different concentrations (1~100mM) of substrate myo-inositol. The enzyme kinetic curve was fitted by GraphPad software, with substrate concentration and specific enzyme activity as horizontal and vertical coordinates, respectively, and the Michaelis-Menten equation model was selected for nonlinear fitting to obtain V max Value and K mValue, obtained by data conversion cat Value and k cat / K m Values ​​(Table 2).

[0112] Detection of heat-resistant muscle inositol dehydrogenase activity:

[0113] The enzyme activity of thermostable myo-inositol dehydrogenase reducing 1L-epi-2-inosose was measured at 45°C by measuring the absorbance change rate of NADH at 340nm. Enzyme activity assay system: 200μL reaction volume, 5mM NADH and 50mM 1L-epi-2-inosose were added to 20mM Tris-HCl buffer (pH7.5), incubated in a 45°C metal bath for 3min, 20μL of pure enzyme of appropriate concentration was added, and the absorbance change at 340nm was scanned using the Bio-Tek Cytation 5 Cell Imaging Multifunctional Detection System.

[0114] Enzyme activity is defined as: under the above conditions, catalyzing the oxidation of 1 μmol NADH to NAD per minute. + The amount of enzyme is defined as one unit U.

[0115] The calculation formula of enzyme activity is: enzyme activity (U) = EW × V × 10 3 / (6220×0.625);

[0116] The calculation formula of specific activity is: specific activity (U / mg) = enzyme activity (U) / protein amount (mg);

[0117] Wherein, EW: change in absorbance at 340 nm within 1 min; V: volume of reaction solution (mL); 6220: molar extinction coefficient (L / mol / cm); 0.625: optical path distance (cm).

[0118] Determination of kinetic parameters:

[0119] According to the Michaelis equation v = V max ×[S] / K m +[S], calculate the K of the enzyme for different substrates m value.

[0120] Where, v: reaction rate (U / mg); V max : maximum reaction rate (U / mg); [S]: substrate concentration (mM); K m :The reaction speed v reaches half 1 / 2V max The substrate concentration at .

[0121] When the substrate concentration is saturated, k cat =V max / Et, calculate kcat value.

[0122] Among them, V max : maximum reaction rate; Et: enzyme concentration.

[0123] Kinetic parameter determination:

[0124] The total reaction volume was 200 μL. 20 mM Tris-HCl buffer (pH 7.5) and 5 mM NADH were added. The temperature was kept at 45 °C for 3 min. 20 μL of pure enzyme of appropriate concentration was added. The specific enzyme activity under different concentrations (1-100 mM) of substrate 1L-epi-2-inosose was measured. The enzyme kinetic curve was fitted by GraphPad software. The substrate concentration and specific enzyme activity were used as the horizontal and vertical coordinates, respectively. The Michaelis-Menten equation model was selected for nonlinear fitting to obtain V max Value and K m Value, obtained by data conversion cat Value and k cat / K m Values ​​(Table 4).

[0125] 1L-Table-2-Detection of inositol production:

[0126] The mixture was reacted at 30°C and 200 rpm for 12 hours, and samples were taken for high performance liquid chromatography (HPLC) analysis. The HPLC detection conditions are as follows: the chromatographic column is Sepax HP-Amino Column; the mobile phase is acetonitrile / water (v:v, 80:20); the flow rate is 1 mL / min; the column temperature is 40°C; the detector is an ultraviolet detector with a detection wavelength of 210 nm; and the injection volume is 10 μL. The liquid chromatogram of 1L-epi-2-inosose is shown in FIG. Figure 7 As shown in A.

[0127] Detection of Epi-inositol Production:

[0128] After heat treatment at 60℃ for 20min, the mixture was reacted at 45℃ and 200rpm for 12h, and then sampled for high performance liquid chromatography (HPLC) analysis. The HPLC detection conditions are as follows: the chromatographic column is Bio-Rad Aminex HPX-87H Column; the mobile phase is 5mMH2SO4; the flow rate is 0.5mL / min; the column temperature is 60℃; the detector is a differential detector; and the injection volume is 10μL. The liquid chromatogram of epi-inositol is shown in Figure 7 As shown in B.

[0129] Preparation of whole cells of recombinant engineering bacteria involved in the following examples

[0130] (1) Preparation of whole cells of recombinant engineered bacteria for scyllo-inositol dehydrogenase

[0131] Pick the recombinant engineered bacteria E.coli BL21 / PksIDH or its mutants and inoculate them into LB liquid test tube culture medium containing ampicillin, and shake culture at 37°C for 8 hours. Inoculate the cultured bacterial liquid in the LB liquid test tube into the LB liquid shake flask culture medium containing ampicillin antibiotics at a 1% inoculation rate, and continue to culture at 37°C for 2 hours. When the OD value is 0.4-0.6, start cooling to 25°C, add IPTG for induction, and the final concentration is 0.3mM. Collect the cells by low-temperature refrigerated centrifuge 14 hours after induction, centrifuge at 8000rpm for 10 minutes, discard the supernatant, and obtain whole cells expressing scyllo-inositol dehydrogenase.

[0132] (2) Preparation of whole cells of thermotolerant myo-inositol dehydrogenase recombinant bacteria

[0133] Pick the recombinant engineered bacteria E.coli BL21 / GtIDH or its mutants and inoculate them in LB liquid test tube culture medium containing kanamycin, and shake culture at 37°C for 8 hours. Inoculate the bacterial solution in the cultured LB liquid test tube into the LB liquid shake flask culture medium containing kanamycin at a 1% inoculation rate, and continue to culture at 37°C for 2 hours. When the OD value is 0.4-0.6, start cooling to 28°C, and add IPTG with a final concentration of 0.5mM to induce protein expression. Collect the cells by low-temperature refrigerated centrifuge 12 hours after induction, centrifuge at 8000×g for 10 minutes, discard the supernatant, and obtain whole cells expressing heat-resistant myo-inositol dehydrogenase.

[0134] Example 1: Mining of scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase and construction of recombinant engineering bacteria

[0135] The specific steps are as follows:

[0136] 1. Discovery of scyllo-inositol dehydrogenase and construction of recombinant engineering bacteria

[0137] (1) Construction of genetically engineered bacteria

[0138] The scyllo-inositol dehydrogenase from Paracoccus cavernae (NCBI No. WP_377687665.1), the scyllo-inositol dehydrogenase from Paracoccus kondratievae (NCBI No. WP_152368211.1), the scyllo-inositol dehydrogenase from Roseibium suaedae (NCBI No. WP_073014975.1), the scyllo-inositol dehydrogenase from Celeribacter baekdonensis (NCBI No. WP_339692564.1), the scyllo-inositol dehydrogenase from Sinirhodobacter populi (NCBI No. WP_128233564.1), and the scyllo-inositol dehydrogenase from Donghicola eburneus scyllo-inositol dehydrogenase (NCBI No.: WP_092465240.1), Jiellapacifica scyllo-inositol dehydrogenase (NCBI No.: WP_163463107.1), Thalassospira lohafexi scyllo-inositol dehydrogenase (NCBI No.: WP_101299495.1), Pannonibacter indicus scyllo-inositol dehydrogenase (NCBI No.: WP_055457068.1), Devosia marina scyllo-inositol dehydrogenase (NCBI No.: WP_157290965.1) were connected to the pET21a vector to prepare recombinant vectors respectively;

[0139] The prepared recombinant vectors were introduced into E. coli BL21 (DE3) to prepare recombinant strains (the gene synthesis and the construction of recombinant engineering bacteria were completed by Sangon Biotech (Shanghai) Co., Ltd.).

[0140] (2) Enzyme screening Figure 3-4 )

[0141] The above recombinant strains were inoculated into LB liquid test tube culture medium containing ampicillin, and cultured at 37°C for 8 hours. The bacterial liquid in the cultured LB liquid test tube was inoculated into LB liquid shake flask culture medium containing ampicillin antibiotics at an inoculum of 1% (v / v), and cultured at 37°C for 2 hours; when the OD value was 0.4-0.6, the temperature was lowered to 25°C, and IPTG was added for induction at a final concentration of 0.3mM; after 14 hours of induction, the cells were collected by low-temperature refrigerated centrifuge, centrifuged at 8000×g for 10 minutes, and the supernatant was discarded to obtain whole cells expressing scyllo-inositol dehydrogenase;

[0142] Reaction system: Add 1 mL of reaction mixture (pH 7.5) to the whole cells at a concentration of 30 g / L and resuspend. The reaction mixture includes 20 mM NAD + and 20 mM myo-inositol (dissolved in 50 mM Tris-NaCl buffer solution) and reacted at 30°C and 200 rpm for 12 hours. After the reaction, the reaction mixture was heat-inactivated to terminate the enzyme activity.

[0143] The initial conversion rate at 20 mM myo-inositol was determined. Figure 3 As shown in A, scyllo-inositol dehydrogenase (PksIDH) from Paracoccus kondratievae has the highest initial conversion rate (15.9%) among the screened enzymes, and this enzyme was selected as the research object; the results show that not all sources of scyllo-inositol dehydrogenase can produce 1L-epi-2-inositose.

[0144] 2. Discovery of heat-resistant muscle inositol dehydrogenase and construction of recombinant engineering bacteria

[0145] (1) Construction of genetically engineered bacteria

[0146] The heat-resistant muscle inositol dehydrogenase from Laceyella putida (NCBI No.: WP_379863543.1), the heat-resistant muscle inositol dehydrogenase from Geobacillus thermoleovorans (NCBI No.: WP_367164351.1), the heat-resistant muscle inositol dehydrogenase from Parageobacillus thermoglucosidasius (NCBI No.: WP_064550463.1), the heat-resistant muscle inositol dehydrogenase from Anoxybacillus tepidamans (NCBI No.: WP_183254894.1), the heat-resistant muscle inositol dehydrogenase from Marinithermofilum abyssi (NCBI No.: WP_229751859.1), and the heat-resistant muscle inositol dehydrogenase from Parageobacillus thermoglucosidasius (NCBI No.: WP_064550463.1) were respectively toebii (NCBI No.: WP_255265174.1), Caldalkalibacillus mannanilyticus (NCBI No.: WP_025027206.1), Ectobacillus funiculus (NCBI No.: MFC6605139.1), Salifodinibacter halophilus (NCBI No.: NNC22636.1), and Acidobacteriota bacterium (NCBI No.: MBI1748953.1) were connected to the pET28a vector to prepare recombinant vectors respectively;

[0147] The prepared recombinant vectors were introduced into E. coli BL21 (DE3) respectively; and recombinant strains were prepared respectively (the gene synthesis and the construction of the recombinant engineering bacteria were completed by Sangon Biotech (Shanghai) Co., Ltd.).

[0148] (2) Enzyme screening

[0149] The recombinant strain was selected and inoculated into LB liquid test tube medium containing kanamycin, and cultured at 37°C for 8 hours. The bacterial liquid in the cultured LB liquid test tube was inoculated into LB liquid shake flask medium containing kanamycin at an inoculum of 1% (v / v), and cultured at 37°C for 2 hours; when the OD value was 0.4-0.6, the temperature was lowered to 28°C, IPTG was added for induction, and the final concentration was 0.5mM. After 12 hours of induction, the cells were collected by low-temperature refrigerated centrifuge, centrifuged at 8000×g for 10 minutes, and the supernatant was discarded to obtain whole cells expressing heat-resistant myo-inositol dehydrogenase.

[0150] Reaction system: Add 1 mL of reaction mixture (pH 7.5) to the whole cells at a concentration of 20 g / L for resuspending. The reaction mixture includes 10 mM NADH and 10 mM 1L-epi-2-inosose (dissolved in 50 mM Tris-NaCl buffer solution), heat-treated at 60°C for 20 min, and reacted at 45°C and 200 rpm for 12 hours. After the reaction is completed, the reaction mixture is heat-inactivated to terminate the enzyme activity.

[0151] The initial conversion rate was determined at 20 mM 1L-epi-2-inosose. Figure 3 As shown in B, the thermostable muscle inositol dehydrogenase (GtIDH) from Geobacillus thermoleovorans showed the highest initial conversion rate (10.2%) among the screened enzymes, and this enzyme was selected as the research object.

[0152] Example 2: Screening of scyllo-inositol dehydrogenase mutants

[0153] The specific steps are as follows:

[0154] 1. Structural analysis of scyllo-inositol dehydrogenase

[0155] By combining PksIDH with myo-inositol and cofactor NAD + Molecular docking was performed and the results were as follows Figure 5 As shown in A in FIG, several semi-conserved sites such as Y163, Y167, L192, R260, C261 and F322 present in the substrate binding region were identified, and these semi-conserved sites were selected for virtual mutation.

[0156] 2. Virtual screening of scyllo-inositol dehydrogenase mutation sites

[0157] By performing virtual saturation mutation on the amino acid residues obtained by screening in step 1, the results are as follows Figure 6As shown in A, the mutation sites C261 and F322 showed significant changes in binding energy to the protein molecule, proving that these three sites are key residues for binding to the substrate myo-inositol, and these sites were selected as mutation sites for directed evolution.

[0158] (1) Directed evolution of scyllo-inositol dehydrogenase mutants

[0159] The mutation hotspots C261 and F322 in step 2 were selected as the mutation starting points, and then random mutations were introduced at the corresponding sites by PCR using degenerate primers (NNK-MNN); the sequence of the degenerate primers (NNK-MNN) is shown in Table 1; wherein N represents a random one of the four nucleotides ATCG, K represents a random one of the two nucleotides GT, and M represents a random one of the two nucleotides AC:

[0160] Table 1: Primer design for scyllo-inositol dehydrogenase mutant enzyme gene

[0161]

[0162] The PCR reaction mixture system was as follows: upstream primer 1 μL, downstream primer 1 μL, template plasmid 1 μL, 2×Primstar 5 μL, ddH2O 2 μL. Pre-denaturation temperature 98°C, 30 s, denaturation temperature 98°C, 15 s, annealing temperature 55°C, 30 s, extension temperature 72°C, 80 s.

[0163] (2) The wild-type recombinant expression vector and the recombinant expression vector with random mutations were transformed into E. coli BL21 (DE3) competent cells to prepare recombinant expression strains (the gene synthesis and the construction of recombinant engineering bacteria were completed by Sangon Biotech (Shanghai) Co., Ltd.).

[0164] First, the monoclonal recombinant strains introduced with random mutations were cultured and induced for expression in 96-well plates, and the wild type was used as a control (the culture conditions were the same as the preparation of whole cells in Example 1). After centrifugation and removal of the supernatant, 1 mL of the reaction mixture (pH 7.5) was added in situ for resuspending. The reaction mixture included 10 mM NADH and 10 mM myo-inositol (dissolved in 50 mM Tris-NaCl buffer solution) and reacted at 30°C and 200 rpm for 12 hours. After the reaction was completed, the reaction mixture was heat-inactivated to terminate the activity of the enzyme.

[0165] Subsequently, the absorbance was measured at 340 nm using a BioTek-Cytation-5 cell imaging multimode reader to determine the NADH concentration in the reaction mixture. Samples with significantly higher NADH content than the control group were screened out, rescreened by HPLC, and mutants with significantly increased 1L-epi-2-inosose production were sequenced, and finally recombinant strains containing scyllo-inositol dehydrogenase mutants with significant effects were obtained: E. coli BL21 / pET21a-PksIDH-C261R, E. coli BL21 / pET21a-PksIDH-F322T, and E. coli BL21 / pET21a-PksIDH-C261R / F322T.

[0166] (3) Kinetic parameters of scyllo-inositol dehydrogenase mutants

[0167] Enzyme activity and kinetic data were obtained using a BioTek-Cytation-5 Cell Imaging Multimode Reader at 30°C and pH 7.5. The results are shown in Table 2.

[0168] Table 2: Kinetic parameters and specific enzyme activities of scyllo-inositol dehydrogenase and its mutants

[0169]

[0170] The results showed that the catalytic efficiency of mutant E. coli BL21 / pET21a-PksIDH-C261R / F322T for myo-inositol was cat / K m It is 3.2 times that of the wild type.

[0171] Example 3: Preparation of 1L-epi-2-inositol by whole-cell catalysis of myo-inositol using the scyllo-inositol dehydrogenase mutant E. coli BL21 / pET21a-PksIDH-C261R / F322T

[0172] 1. Reaction system:

[0173] In 1 mL reaction system, add 20 mM myo-inositol, 20 mM NAD + , 30 g / L E. coli BL21 / pET21a-PksIDH-C261R / F322T whole cells, reaction buffer 50 mM Tris-HCl (pH 7.5), reaction at 30°C, 200 rpm for 12 h, sampling for high performance liquid chromatography (HPLC) analysis.

[0174] 2. Optimization of reaction conditions

[0175] (1) Optimum pH

[0176] The optimal reaction pH of the recombinant engineered bacteria E. coli BL21 / pET21a-PksIDH-C261R / F322T is determined by the yield of the product 1L-epi-2-inosose in a reaction system with whole cell catalysis in the pH range of 6.0 to 10.0 (other reaction conditions are the same as in step 1).

[0177] like Figure 8 As shown in A, when the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R / F322T reacts in a pH 9.5 system, the yield of 1L-epi-2-inosose is the highest, which is 43.4%.

[0178] (2) Optimum temperature

[0179] The optimum temperature of the recombinant engineered bacterium E. coli BL21 / pET21a-PksIDH-C261R / F322T is determined by the yield of the product 1L-epi-2-inosose in a reaction system at pH 9.5 and 20 to 50°C (other reaction conditions are the same as in step 1).

[0180] like Figure 8 As shown in B, when the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R / F322T reacts at 28°C, the yield of 1L-epi-2-inosose is the highest, which is 47.2%.

[0181] (3) Optimal cell mass

[0182] The optimal cell weight of the recombinant engineered bacterium E. coli BL21 / pET21a-PksIDH-C261R / F322T is determined by the yield of the product 1L-epi-2-inosose by whole-cell catalysis in a reaction system with a cell weight range of 10 to 100 g / L under the conditions of pH 9.5 and 28°C (other reaction conditions are the same as in step 1).

[0183] like Figure 8 As shown in C, when the whole cell addition amount of the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R / F322T was 80 g / L, the yield of 1L-epi-2-inosose was the highest, which was 64.4%.

[0184] (4) Optimal substrate amount

[0185] The optimal amount of substrate for the recombinant engineered bacterium E. coli BL21 / pET21a-PksIDH-C261R / F322T was determined by the yield of the product 1L-epi-2-inositol in a reaction system with 1-100 mM myo-inositol under the conditions of pH 9.5, 28°C, 80 g / L cells, and NAD + The final concentration is consistent with that of the substrate myo-inositol (other reaction conditions are the same as step 1).

[0186] like Figure 8 As shown in D, when the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R / F322T was reacted under the condition of adding 20mM myo-inositol, the yield of 1L-epi-2-inosose was the highest, which was 67.1%.

[0187] (5) Optimal response time

[0188] The optimal reaction time of the recombinant engineered bacteria E. coli BL21 / pET21a-PksIDH-C261R / F322T is determined by the yield of the product 1L-epi-2-inosose after 2 to 24 hours of whole-cell catalysis at pH 9.5, 28°C, 80 g / L cells, and 20 mM myo-inositol (other reaction conditions are the same as step 1).

[0189] like Figure 8 As shown in Figure E, the yield of 1L-epi-2-inosose in the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R / F322T was the highest at 70.5% when the reaction was carried out for 12 hours.

[0190] Example 4: Screening of heat-resistant myo-inositol dehydrogenase mutants

[0191] The specific steps are as follows:

[0192] 1. Structural analysis of thermostable muscle inositol dehydrogenase

[0193] Molecular docking of GtIDH with 1L-epi-2-inosose and cofactor NADH revealed the following results: Figure 5 As shown in C, several semi-conserved sites such as M122, R124, Y173 and F177 present in the substrate binding region were identified, and these semi-conserved sites were selected for virtual mutation.

[0194] 2. Virtual screening of heat-resistant muscle inositol dehydrogenase mutation sites

[0195] By performing virtual saturation mutation on the amino acid residues obtained by screening in step 1, the results are as follows Figure 6As shown in B, the binding energy of mutation sites R124 and F177 to the protein molecule showed significant changes, proving that this site is the key residue for binding to the substrate 1L-epi-2-inosose, and this site was selected as the mutation site for directed evolution.

[0196] 3. Directed evolution of heat-resistant myo-inositol dehydrogenase mutants

[0197] The mutation hot spots R124 and F177 in step 2 were selected as the starting points for mutation, and then random mutations were introduced at the corresponding sites by PCR using degenerate primers (NNK-MNN); the sequence of the degenerate primers (NNK-MNN) is as follows:

[0198] Table 3: Primer design for heat-resistant myo-inositol dehydrogenase mutant enzyme gene

[0199]

[0200] The PCR reaction mixture system was as follows: upstream primer 1 μL, downstream primer 1 μL, template plasmid 1 μL, 2×Primstar 5 μL, ddH2O 2 μL. Pre-denaturation temperature 98°C, 30 s, denaturation temperature 98°C, 15 s, annealing temperature 55°C, 30 s, extension temperature 72°C, 80 s.

[0201] (2) The wild-type recombinant expression vector and the recombinant expression vector with random mutations introduced were transformed into E. coli BL21 (DE3) competent cells to prepare recombinant expression strains (the gene synthesis and the construction of recombinant engineering bacteria were completed by Sangon Biotech (Shanghai) Co., Ltd.).

[0202] First, the monoclonal recombinant strains introduced with random mutations were cultured and induced for expression in 96-well plates, and the wild type was used as a control (the culture conditions were the same as the preparation of whole cells in Example 1). After centrifugation and removal of the supernatant, 1 mL of the reaction mixture (pH 7.5) was added in situ for resuspending. The reaction mixture included 10 mM NADH and 10 mM 1L-epi-2-inosose (dissolved in 50 mM Tris-NaCl buffer solution), and after heat treatment at 60°C for 20 min, the reaction was carried out at 45°C and 200 rpm for 12 hours. After the reaction was completed, the reaction mixture was heat-inactivated to terminate the activity of the enzyme.

[0203] Subsequently, the absorbance was measured at 340 nm using a BioTek-Cytation-5 Cell Imaging Multimode Reader to determine the NADH concentration in the reaction mixture. Samples with significantly lower NADH content than that of the control group were screened out, rescreened by HPLC, and mutants with significantly increased epi-inositol production were sequenced. Finally, recombinant strains containing heat-resistant myo-inositol dehydrogenase mutants were obtained: E. coli BL21 / pET28a-GtIDH-F177K, E. coli BL21 / pET28a-GtIDH-F177E, E. coli BL21 / pET28a-GtIDH-F177R, E. coli BL21 / pET28a-GtIDH-F177S, E. coli BL21 / pET28a-GtIDH-R124W, and E. coli BL21 / pET28a-GtIDH-R124C.

[0204] (3) Kinetic parameters of thermostable myo-inositol dehydrogenase mutants

[0205] Kinetic data were obtained using a BioTek-Cytation-5 Cell Imaging Multimode Reader at 45°C and pH 7.5. The results are shown in Table 4.

[0206] Table 4: Kinetic parameters and specific enzyme activities of thermostable myo-inositol dehydrogenase and its mutants

[0207]

[0208] The results showed that the catalytic efficiency of mutant E. coli BL21 / pET28a-GtIDH-F177K on 1L-epi-2-inosose was cat / K m It is 1.7 times that of the wild type.

[0209] Example 5: Preparation of epi-inositol by catalyzing 1L-epi-2-inositol with whole cells containing a thermostable myo-inositol dehydrogenase mutant E. coli BL21 / pET28a-GtIDH-F177K

[0210] 1. Reaction system:

[0211] In a 1 mL reaction system, add 1 L-epi-2-inositose, 20 mM NADH, 0.5 mM Zn 2+, 30 g / L E. coli BL21 / pET28a-GtIDH-F177K whole cells, reaction buffer 50 mM Tris-HCl (pH 7.5). After heat treatment at 60°C for 20 min, reaction was carried out at 45°C and 200 rpm for 12 h, and samples were taken for high performance liquid chromatography (HPLC) analysis of substrate and product concentrations.

[0212] 2. Optimization of reaction conditions

[0213] (1) Optimal heat treatment conditions

[0214] The optimal heat treatment condition for the recombinant engineered bacteria E. coli BL21 / pET28a-GtIDH-F177K is to heat shock at 50-70°C for 10-30 min (specific conditions are as follows Fig. 9 The whole-cell catalysis (as shown in A) was carried out and the yield of the product epi-inositol was determined (other reaction conditions were the same as step 1).

[0215] like Fig. 9 As shown in A, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH-F177K was heat-treated at 65°C for 30 minutes, the yield of epi-inositol was the highest, which was 24.38%.

[0216] (2) Optimum pH

[0217] The optimal reaction pH of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH-F177K is determined by the yield of the product epi-inositol in a reaction system with whole cell catalysis in the pH range of 6.0 to 10.0 (heat treated at 65° C. for 30 min, and other reaction conditions are the same as step 1).

[0218] like Fig. 9 As shown in B, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH-F177K reacts in a system of pH 7.5, the yield of epi-inositol is the highest, which is 25.16%.

[0219] (3) Optimum temperature

[0220] The optimum temperature of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH-F177K is determined by the yield of the product epi-inositol by whole-cell catalysis in a reaction system within the range of 35-70°C after heat treatment at 65°C for 30 min at pH 7.5 (other reaction conditions are the same as in step 1).

[0221] like Fig. 9As shown in C, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH-F177K reacts at 50°C, the yield of epi-inositol is the highest, which is 29.33%.

[0222] (4) Optimal cell mass

[0223] The optimal cell weight of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH-F177K is determined by the yield of the product epi-inositol by whole-cell catalysis in a reaction system with a cell weight range of 10 to 100 g / L at 50°C after heat treatment at pH 7.5 and 65°C for 30 min (other reaction conditions are the same as in step 1).

[0224] like Fig. 9 As shown in D, when the whole cell addition amount of the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH-F177K was 40 g / L, the yield of epi-inositol was the highest, which was 36.74%.

[0225] (5) Optimal substrate amount

[0226] The optimal substrate amount of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH-F177K was determined by the yield of the product epi-inositol in a reaction system with a concentration range of 1 to 100 mM 1L-epi-2-inosose at 50°C and 40 g / L cells after heat treatment at pH 7.5 and 65°C for 30 min. The final concentration of NADH was consistent with the final concentration of the substrate 1L-epi-2-inosose.

[0227] like Fig. 9 As shown in FIG. 8E , when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH-F177K was reacted under the condition of 40 mM 1L-epi-2-inositol addition, the yield of epi-inositol was the highest, which was 39.71%.

[0228] (6) Optimal Zn 2+ Addition amount

[0229] The optimal substrate amount for the recombinant engineered bacteria E. coli BL21 / pET28a-GtIDH-F177K was 0.1-5 mM Zn at 50°C, 40 g / L cells, 40 mM 1L-ep-2-inositose, and 30 min of heat treatment at pH 7.5 and 65°C. 2+ Within the range of reaction systems, whole-cell catalysis was determined by the yield of the product epi-inositol.

[0230] like Fig. 9As shown in F, the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH-F177K was cultured in 1 mM Zn 2+ When the reaction was carried out under the addition amount conditions, the yield of epi-inositol was the highest, which was 44.25%.

[0231] (7) Optimal response time

[0232] The optimal substrate amount for the recombinant engineered bacteria E. coli BL21 / pET28a-GtIDH-F177K was 50°C, 40 g / L cells, 40 mM 1L-episode-2-inositose, 1 mM Zn 2+ The yield of the product epi-inositol was determined by whole-cell catalysis for 2 to 24 hours under the same conditions.

[0233] like Fig. 9 As shown in G, the yield of epi-inositol of the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH-F177K was the highest at 52.96% when the reaction was carried out for 14 hours.

[0234] Example 6: Two-step whole-cell catalysis of myo-inositol to produce epi-inositol

[0235] The specific steps are as follows:

[0236] 1. Preparation of epi-inositol by wild-type enzyme dual enzyme

[0237] (1) Add 20 mM myo-inositol and 20 mM NAD to a reaction system containing 10 mL of 50 mM Tris-HCl (pH 9.5) buffer. + , 80 g / L of E. coli BL21 / pET21a-PksIDH whole cells (the construction method is the same as in Example 1), and reacted at 28° C. and 200 rpm for 12 h.

[0238] (2) Add Zn to the system after the reaction in step (1) 2+ and E. coli BL21 / pET28a-GtIDH whole cells (construction method is the same as in Example 1), Zn 2+ The final concentration was 1 mM, and the final cell concentration was 40 g / L. The reaction system was heat-shocked at 65°C for 30 min, and then reacted at 50°C, 200 rpm for 14 h. Samples were taken for high performance liquid chromatography (HPLC) analysis of the concentrations of substrates and products.

[0239] The experimental results show that in the process of preparing epi-inositol from myo-inositol by a two-step whole-cell method using scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase, the total yield of epi-inositol is 14.8% and the output is 0.53 g / L.

[0240] 2. Preparation of epi-inositol by scyllo-inositol dehydrogenase mutant and thermostable myo-inositol dehydrogenase

[0241] (1) Add 20 mM myo-inositol and 20 mM NAD to a reaction system containing 10 mL of 50 mM Tris-HCl (pH 9.5) buffer. + , 80 g / L of E. coli BL21 / pET21a-PksIDH-C261R / F322T whole cells (the construction method is the same as in Example 2), and reacted at 28° C. and 200 rpm for 12 h.

[0242] (2) Add Zn to the system after the reaction in step (1) 2+ and E. coli BL21 / pET28a-GtIDH whole cells (construction method is the same as in Example 1), Zn 2+ The final concentration was 1 mM, and the final cell concentration was 40 g / L. The reaction system was heat-shocked at 65°C for 30 min, and then reacted at 50°C, 200 rpm for 14 h, and samples were taken for high performance liquid chromatography (HPLC) analysis.

[0243] The experimental results showed that in the process of preparing epi-inositol from myo-inositol using a two-step whole-cell method catalyzed by scyllo-inositol dehydrogenase mutant and thermostable myo-inositol dehydrogenase, the total yield of epi-inositol was 45.2% and the output was 1.63 g / L.

[0244] 3. Preparation of epi-inositol by scyllo-inositol dehydrogenase mutants and thermostable myo-inositol dehydrogenase mutants

[0245] (1) Add 20 mM myo-inositol and 20 mM NAD to a reaction system containing 10 mL of 50 mM Tris-HCl (pH 9.5) buffer. + , 80 g / L of E. coli BL21 / pET21a-PksIDH-C261R / F322T whole cells (the construction method is the same as in Example 2), and reacted at 28° C. and 200 rpm for 12 h.

[0246] (2) Add Zn to the reaction system obtained in step (1) 2+ and E. coli BL21 / pET28a-GtIDH-F177K whole cells (construction method is the same as in Example 4), Zn 2+ The final concentration was 1 mM, and the final cell concentration was 40 g / L. The reaction system was heat-shocked at 65°C for 30 min, and then reacted at 50°C, 200 rpm for 14 h, and samples were taken for high performance liquid chromatography (HPLC) analysis.

[0247] The experimental results showed that in the two-step whole-cell method of using scyllo-inositol dehydrogenase mutants and thermostable myo-inositol dehydrogenase mutants to catalyze myo-inositol to prepare epi-inositol, the total yield of epi-inositol was 52.6%, and the output was 1.89 g / L.

[0248] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing epi-inositol, characterized in that: The method comprises the following steps: using a scyllo-inositol dehydrogenase mutant or a genetically engineered bacterium expressing a scyllo-inositol dehydrogenase mutant to catalyze myo-inositol to synthesize 1L-epi-2-inositose; and adding a heat-resistant myo-inositol dehydrogenase or a mutant thereof or a genetically engineered bacterium expressing a heat-resistant myo-inositol dehydrogenase or a mutant thereof to catalyze the 1L-epi-2-inositol to synthesize epi-inositol. The scyllo-inositol dehydrogenase mutant is obtained by mutating the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 to arginine, and mutating the phenylalanine at position 322 to threonine; or by mutating the phenylalanine at position 322 of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 to threonine; The thermostable myo-inositol dehydrogenase is derived from Laceyella putida, Geobacillus thermoleovorans, Parageobacillus thermoglucosidasius or Marinithermofilum abyssi; the thermostable myo-inositol dehydrogenase mutant is obtained by mutating the phenylalanine at position 177 of the thermostable myo-inositol dehydrogenase with a amino acid sequence as shown in SEQ ID NO.2 to lysine or glutamic acid.

2. The preparation method according to claim 1, characterized in that: The reaction system for catalyzing myo-inositol is as follows: the concentration of substrate myo-inositol is 1-100 mM, NAD + The concentration is 1-100 mM, and the amount of genetically engineered bacteria added is 10-100 g / L; Preferably, the concentration of the substrate myo-inositol is 10-80 mM, and the amount of genetically engineered bacteria added is 30-90 g / L; Preferably, the concentration of the substrate myo-inositol is 15-60 mM, NAD + The concentration is 15-60mM, and the amount of genetically engineered bacteria added is 40-80g / L; The reaction conditions for the whole cell catalysis of myo-inositol are: pH 6.0-10.0, 20-50° C., for 2-24 hours; Preferably, the whole cell catalyzes the reaction of myo-inositol at a pH of 8.0 to 9.5 and at 25 to 40° C. for 6 to 14 hours.

3. The preparation method according to claim 1 or 2, wherein the reaction system for catalyzing the 1L-epi-2-inosose is: the concentration of the substrate 1L-epi-2-inosose is 1 to 100 mM, and the concentration of Zn 2+ The addition amount of is 0.1~5mM, and the addition amount of genetically engineered bacteria is 10~100g / L; Preferably, the concentration of the substrate 1L-epi-2-inosose is 10-80 mM, Zn 2+ The addition amount of is 0.5~4mM, and the addition amount of genetically engineered bacteria is 20~80g / L; Preferably, the concentration of the substrate 1L-epi-2-inosose is 20-60 mM, Zn 2+ The addition amount of is 1~3mM, and the addition amount of genetically engineered bacteria is 30~60g / L; The reaction conditions for catalyzing the 1L-epi-2-inosose are: heat-treating the whole cells before reacting; the heat-treating conditions are: the heat-treating temperature is 50-70° C.; Preferably, the heat treatment temperature is 55 to 70°C; preferably, the heat treatment temperature is 60 to 65°C; Preferably, the heat treatment time is 10 to 30 minutes; preferably, the heat treatment time is 20 to 30 minutes; The conditions for reacting the whole cells after heat treatment are: The reaction is carried out at pH 6.0-10.0 and 35-70°C for 2-24 hours; preferably, the reaction is carried out at pH 6.5-8.0 and 40-60°C for 8-16 hours; most preferably, the reaction is carried out at pH 7.5 and 50-55°C for 14-16 hours.

4. A scyllo-inositol dehydrogenase mutant, characterized in that: The scyllo-inositol dehydrogenase mutant is obtained by mutating the phenylalanine at position 322 of the scyllo-inositol dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 to threonine; Or the amino acid sequence of scyllo-inositol dehydrogenase as shown in SEQ ID NO.1 is obtained by mutating the cysteine ​​at position 261 to arginine and mutating the phenylalanine at position 322 to threonine.

5. A heat-resistant myo-inositol dehydrogenase mutant, characterized in that: The thermostable myo-inositol dehydrogenase mutant is obtained by mutating the phenylalanine at position 177 of the thermostable myo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.2 to lysine or glutamic acid.

6. A gene encoding the scyllo-inositol dehydrogenase mutant according to claim 4 or the heat-resistant myo-inositol dehydrogenase mutant according to claim 5.

7. A recombinant vector carrying the gene according to claim 6.

8. A recombinant cell expressing the mutant according to claim 4 or 5, or carrying the gene according to claim 6, or carrying the recombinant vector according to claim 7, preferably, the recombinant cell uses bacteria or fungi as host cells.

9. A method for improving enzyme substrate affinity and catalytic efficiency, characterized in that: The method comprises mutating the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 to arginine, and mutating the phenylalanine at position 322 to threonine; or mutating the phenylalanine at position 322 of the scyllo-inositol dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 to threonine; Or the phenylalanine at position 177 of the heat-resistant myo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.2 is mutated to lysine or glutamic acid.

10. Use of the preparation method according to any one of claims 1 to 3, or the mutant according to claim 4 or 5, or the gene according to claim 6, or the recombinant vector according to claim 7, or the recombinant cell according to claim 8, or the method according to claim 9 in the preparation of epi-inositol or a product containing epi-inositol.

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