Method for efficiently synthesizing epiinositol through double-enzyme cascade catalysis of myo-inositol

Abstract

The invention discloses a method for efficiently synthesizing epiinositol by catalyzing muscle inositol through double-enzyme cascade, and belongs to the field of biological catalysis engineering. According to the invention, scyllo-inositol dehydrogenase is mutated to obtain a beneficial mutant C261R, and the beneficial mutant C261R is coupled with heat-resistant myo-inositol dehydrogenase to efficiently catalyze and synthesize the epiinositol, and belongs to the field of biological catalysis engineering. The new way for synthesizing the epiinositol is completely different from reported synthesis methods of scyllo-inositol and D-chiro-inositol, and comprises the following steps of: firstly dehydrogenating the C4 position of the muscle inositol to form an intermediate product ketone, and then hydrogenating to realize chiral inversion of hydrogen at the C4 position, so as to obtain the final product epiinositol. The invention provides a brand new way for biocatalytically synthesizing the epiinositol, the yield of the obtained epiinositol is 1.39 g / L, the conversion rate is 38.7%, the yield and the conversion rate are the highest level which is publically reported at present, the biosynthesis process is simple to operate, low in cost, green and environment-friendly, and a solid research foundation is laid for industrial production of the epiinositol.

Description

Technical Field

[0001] The invention relates to a method for efficiently synthesizing epi-inositol from myo-inositol by double-enzyme cascade catalysis, and belongs to the field of biocatalysis 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 is as effective as 100nM insulin in promoting GLUT4-dependent glucose absorption in rat L6 myotubes.

[0003] The synthesis of epi-inositol is still at the stage of chemical synthesis, mainly by reducing epi-inositol with sodium borohydride under alkaline conditions. However, this reaction has the disadvantages of dangerous reagents, high pollution and high cost. The defects of the chemical production method make the price of epi-inositol extremely high, with a market price of about 14,000 yuan per gram.

[0004] In recent years, with the development of bioengineering technology, biotransformation synthesis of inositol isomers has become a new development trend. 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, and 1.8g / L of scyllo-inositol can be produced from 20g / L of glucose within 72h. Ji et al. obtained a scyllo-inositol production strain DCI-7 by co-expressing inositol isomerase IolI2 and inositol dehydrogenase IolG in Corynebacterium glutamicum. The strain can produce 6.96g / L of 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 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 substrate and product structures 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 and approach 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] Since the structures of the substrate myo-inositol and the product epi-inositol are extremely symmetrical, it is often difficult for enzymes to specifically recognize them during the catalytic process. Therefore, the synthesis of different chiral compounds of inositol has always been an important problem in the field of biocatalysis.

[0007] In view of the problems existing in the existing method for preparing epi-inositol, the present invention provides a method for producing epi-inositol by catalyzing myo-inositol in a two-step whole-cell method using scyllo-inositol dehydrogenase (sIDH) that can catalyze the dehydrogenation of the hydroxyl group at C4 and a heat-resistant myo-inositol dehydrogenase (IDH) that can catalyze the hydrogenation of the carbonyl group at C4. The scyllo-inositol dehydrogenase is subjected to structural analysis and virtual mutation screening based on sequence conservation, and a mutant with higher catalytic efficiency is obtained by directed evolution. 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.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] 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; and adding heat-resistant myo-inositol dehydrogenase or a genetically engineered bacterium expressing heat-resistant myo-inositol dehydrogenase to catalyze the 1L-epi-2-inositol to synthesize epi-inositol.

[0010] In one embodiment of the present invention, the method is:

[0011] (1) engineered bacteria and / or mutants expressing scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase respectively;

[0012] (2) fermenting the engineered bacteria and / or mutants constructed in step (1) to obtain whole cells;

[0013] (3) subjecting the heat-resistant myo-inositol dehydrogenase obtained in step (2) to cell membrane permeability treatment to obtain permeable whole cells;

[0014] (4) using the whole cell expressing scyllo-inositol dehydrogenase obtained in step (2) to catalyze myo-inositol to synthesize 1L-epi-2-inosose;

[0015] (5) Using the permeabilized whole cells expressing the thermostable myo-inositol dehydrogenase obtained in step (3) to catalyze the synthesis of epi-inositol (epi-inositol) from 1L-epi-2-inosose (1L-epi-2-inosose).

[0016] 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 shown.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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 amino acid of the scyllo-inositol dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1.

[0023] Most preferably, 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 or lysine.

[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 conditions for whole-cell catalysis of 1L-epi-2-inosose are: subjecting the reaction system to heat treatment before conducting the reaction; 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 20 minutes.

[0028] In one embodiment of the present invention, the added cell concentration of the genetically engineered bacteria is 10-100 g / L; further preferably, the cell concentration is 20-80 g / L; more preferably, the cell concentration is 30-60 g / L; most preferably, the cell concentration is 50 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, 20-50°C for 2-24 hours; preferably, reaction at pH 8.0-9.5, 25-40°C for 6-14 hours; most preferably, reaction at pH 9.0, 25-30°C for 10-12 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, Zn 2+ 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 PCR product was 1 mM, and the addition amount of whole cells was 50 g / 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 50-55°C for 14-16 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 50-55°C for 14-16 hours.

[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 to arginine or lysine, and is named C261R or C261K.

[0039] The parent enzyme sequence of the scyllo-inositol dehydrogenase mutant (SEQ ID NO.1) is:

[0040] MTATQKSLGVALIGTGFMGKCHAMAWRNLATAFGGMPPRLEVLADTPAERAAELASAFGFARGTGDWRAAVTDPAVDVVSITTPNGMHREMAALAAGKHVWLEKPMALTVEDAEAMETAARAAGRVTILGYNYTRSPAFRAAADLVAQGAIGRPIHFRGVYDEDYMADPELPWSWRLTRAEGGL GALGDLGCHLVSVMLALMGPVARVQAQMDTVVTERPHQGGTATVENEDQAQALIRFASGASGEFSCSRVARGYRCRLAWEVQGTEGTLRFDQERMNELWLYQPGQAETDGFRRILTGPAQPGFAAFCPAAGHNFGFNEQKVVEAEMLRQAIAGGIPAWPDFTAGLTIERIIHGMARSARTGQPVLF

[0041] The base sequence encoding the scyllo-inositol dehydrogenase mutant parent enzyme (SEQ ID NO.3) is:

[0042]

[0043] In one embodiment of the present invention, the catalytic efficiency of the mutant is based on myo-inositol as substrate and NAD + The enzyme activity is measured by measuring the change in absorbance at 340 nm using a microplate reader. One unit of enzyme activity (U) is defined as the amount of enzyme activity that catalyzes the oxidation of 1 μmol of NADH to NAD per minute under the measurement conditions. + The amount of enzyme.

[0044] 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.

[0045] 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(+).

[0046] 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).

[0047] 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.

[0048] 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.

[0049] The present invention provides a gene encoding the scyllo-inositol dehydrogenase and its mutant or the heat-resistant myo-inositol dehydrogenase.

[0050] The heat-resistant myo-inositol dehydrogenase sequence (SEQ ID NO.2) is:

[0051] MTRVKVGILGAGGIAKVHTSILKKDERVQIIGVADIVEERAASLAKEAGEAKAVKSLEELFDLGVDAVYVTTPNTMHVEPVLKCLENNVHVFSEKPMATSLEGAEQIRQAAERSKAIYNLGMNRRYASVYKRVKELIDSGEVTPYLAHVKMNRGELLNPPWTANPKV TGGFLYETPFHLMDLCRYLFGEVQTVYCEAKQNISTEELDTFAIMMTFVSGTIVNFVTYAHAGWSFPFESLEVYGKYSTVATQELEKVMYAPGLQQAAQIYDFYQLSIEEKWGYKEEDRLFIDAIIHGAKPPVTAEDGFRSIQLLEAIYESAKTGKMIDFRQTALSK

[0052] The base sequence encoding the heat-resistant muscle inositol dehydrogenase (SEQ ID NO.4) is:

[0053]

[0054] The invention also provides a recombinant vector carrying the gene.

[0055] The present invention also provides a recombinant cell expressing the mutant, or carrying the gene, or carrying the recombinant vector.

[0056] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as host cells.

[0057] The present invention provides a vector for expressing scyllo-inositol dehydrogenase and a vector for expressing heat-resistant myo-inositol dehydrogenase.

[0058] The invention provides the above-mentioned engineering bacteria and mutants expressing scyllo-inositol dehydrogenase and engineering bacteria expressing heat-resistant myo-inositol dehydrogenase.

[0059] According to the present invention, the engineered bacteria and mutants expressing scyllo-inositol dehydrogenase include a vector expressing scyllo-inositol dehydrogenase; the engineered bacteria expressing heat-resistant myo-inositol dehydrogenase include a vector expressing heat-resistant myo-inositol dehydrogenase.

[0060] The present invention provides a method for improving substrate affinity and catalytic efficiency of scyllo-inositol dehydrogenase, the method comprising 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;

[0061] The present invention provides a recombinant enzyme catalyst containing the above-mentioned scyllo-inositol dehydrogenase mutant, which is any one of the following forms:

[0062] (1) culturing a recombinant expression transformant containing the scyllo-inositol dehydrogenase mutant, and isolating a transformant cell containing the recombinant scyllo-inositol dehydrogenase mutant enzyme;

[0063] (2) culturing a recombinant expression transformant containing the scyllo-inositol dehydrogenase mutant, isolating transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme, and disrupting the transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme to obtain a cell disrupted liquid;

[0064] (3) culturing a recombinant expression transformant containing the scyllo-inositol dehydrogenase mutant, isolating transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme, disrupting the transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant scyllo-inositol dehydrogenase mutant enzyme to obtain a lyophilized enzyme powder.

[0065] 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.

[0066] 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 muscular inositol dehydrogenase, or the above-mentioned gene, or the above-mentioned recombinant vector, or 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.

[0067] Beneficial Effects

[0068] (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, low cost, and green environmental protection.

[0069] (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.

[0070] (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.

[0071] (4) The present invention provides a mutant of scyllo-inositol dehydrogenase, the catalytic efficiency (k cat / K m ) was 2.2 times that of the wild type, and the 1L-epi-2-inosose yield was increased by 42.1% compared with the wild type.

[0072] (5) The present invention optimizes the reaction conditions of the mutant of scyllo-inositol dehydrogenase and the thermostable myo-inositol dehydrogenase, and explores the optimal reaction temperature, pH, cell amount, substrate amount, reaction time and other conditions. In the optimized dual-enzyme cascade two-step method for catalyzing myo-inositol to prepare epi-inositol, the total yield of epi-inositol is 38.7%.

[0073] (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

[0074] Figure 1: Chemical structures of myo-inositol, 1L-epi-2-inositol, and epi-inositol.

[0075] Figure 2 : Schematic diagram of the whole cell catalyzing myo-inositol to produce epi-inositol of the present invention. myo-inositol: myo-inositol; epi-inositol: scyllo-inositol; 1L-epi-2-inosose: 1L-epi-2-inosose; sIDH: scyllo-inositol dehydrogenase; IDH: myo-inositol dehydrogenase.

[0076] 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.

[0077] Figure 4 : Molecular docking map of substrate and enzyme; A is the binding pocket of myo-inositol in PksIDH A shows the amino acid residues in the PksIDH binding pocket; B shows the binding of myo-inositol in the PksIDH binding pocket.

[0078] Figure 5 : PksIDH binding pocket in myo-inositol The changes in binding energy when virtual saturation mutations are performed on the semi-conservative amino acids in the

[0079] 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.

[0080] Figure 7 : SDS-PAGE analysis of recombinant engineering bacteria E. coli BL21 / PksIDH-C261R and E. coli BL21 / GtIDH; M, protein marker; 1, E. coli BL21GtIDH; 2, E. coli BL21 / PksIDH-C261R.

[0081] Figure 8 : The yield of the intermediate product 1L-epi-2-inosose under different catalytic 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.

[0082] Fig. 9 : The yield of the final product epi-inositol catalyzed by 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

[0083] 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.

[0084] Technical terms

[0085] 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.

[0086] 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.

[0087] 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.

[0088] The culture medium involved in the following examples is as follows:

[0089] LB liquid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, natural pH.

[0090] The detection methods involved in the following embodiments are as follows:

[0091] Detection of scyllo-inositol dehydrogenase activity:

[0092] 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 changes at 340 nm using the Bio-Tek Cytation 5 cell imaging multifunctional detection system.

[0093] 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.

[0094] The calculation formula of enzyme activity is: enzyme activity (U) = EW × V × 10 3 / (6220×0.625);

[0095] The calculation formula of specific activity is: specific activity (U / mg) = enzyme activity (U) / protein amount (mg);

[0096] 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).

[0097] Determination of kinetic parameters:

[0098] According to the Michaelis equation v = V max ×[S] / K m +[S], calculate the K of the enzyme for different substrates m value.

[0099] 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 .

[0100] When the substrate concentration is saturated, k cat =V max / Et, calculate k cat value.

[0101] Among them, V max : maximum reaction rate; Et: enzyme concentration.

[0102] Reaction system for determination of kinetic parameters:

[0103] 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 Km Value, obtained by data conversion cat Value and k cat / K m Values ​​(Table 2).

[0104] 1L-Table-2-Detection of inositol production:

[0105] 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 6 As shown in A.

[0106] Detection of Epi-inositol Production:

[0107] 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 6 As shown in B.

[0108] Preparation of whole cells of recombinant engineering bacteria involved in the following examples

[0109] (1) Preparation of whole cells of recombinant engineered bacteria for scyllo-inositol dehydrogenase

[0110] Pick the recombinant engineered bacteria E.coli BL21 / PksIDH or its mutants and inoculate them in 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 an inoculum of 1%, 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 8000×g for 10 minutes, discard the supernatant, and obtain whole cells expressing scyllo-inositol dehydrogenase.

[0111] (2) Preparation of whole cells of thermotolerant myo-inositol dehydrogenase recombinant bacteria

[0112] 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 cultured bacterial liquid in the 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, add IPTG for induction, and the final concentration is 0.5mM. 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.

[0113] Example 1: Mining of scyllo-inositol dehydrogenase and thermostable myo-inositol dehydrogenase and construction of recombinant engineering bacteria

[0114] The specific steps are as follows:

[0115] 1. Discovery of scyllo-inositol dehydrogenase and construction of recombinant engineering bacteria

[0116] (1) Construction of genetically engineered bacteria

[0117] 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;

[0118] 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.).

[0119] (2) Enzyme screening

[0120] 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;

[0121] 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.

[0122] 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.

[0123] 2. Discovery of heat-resistant muscle inositol dehydrogenase and construction of recombinant engineering bacteria

[0124] (1) Construction of genetically engineered bacteria

[0125] 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;

[0126] 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.).

[0127] (2) Enzyme screening

[0128] 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.

[0129] 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.

[0130] 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.

[0131] Example 2: Screening of scyllo-inositol dehydrogenase mutants

[0132] The specific steps are as follows:

[0133] 1. Structural analysis of scyllo-inositol dehydrogenase

[0134] By combining PksIDH with myo-inositol and cofactor NAD + Molecular docking was performed and the results were as follows Figure 5 As shown, 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.

[0135] 2. Virtual screening of scyllo-inositol dehydrogenase mutation sites

[0136] 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 site C261 showed the most significant change in binding energy with the protein molecule, indicating that this site is a key residue for binding to the substrate myo-inositol, and this site was selected as the mutation site for directed evolution.

[0137] (1) Directed evolution of scyllo-inositol dehydrogenase mutants

[0138] The mutation hotspot C261 in step 2 was selected as the mutation starting point, 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:

[0139] Table 1: Primer design for scyllo-inositol dehydrogenase mutant enzyme gene

[0140]

[0141] 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.

[0142] (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.).

[0143] 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.

[0144] 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 and E. coli BL21 / pET21a-PksIDH-C261K.

[0145] (3) Kinetic parameters of scyllo-inositol dehydrogenase mutants

[0146] 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.

[0147] Table 2: Kinetic parameters and specific enzyme activities of scyllo-inositol dehydrogenase and its mutants

[0148]

[0149] The results showed that the catalytic efficiency of mutant E. coli BL21 / pET21a-PksIDH-C261R for myo-inositol was cat / K m It is 2.2 times that of the wild type.

[0150] Example 3: Preparation of 1L-epi-2-inositol by whole cells containing the scyllo-inositol dehydrogenase mutant PksIDH-C261R

[0151] 1. Reactor system:

[0152] In 1 mL reaction system, add 20 mM myo-inositol, 20 mM NAD + , 30 g / L E. coli BL21 / pET21a-PksIDH-C261R whole cells, reaction buffer 50 mM Tris-HCl (pH 7.5), 30°C, 200 rpm for 12 h, and samples were taken for high performance liquid chromatography (HPLC) analysis.

[0153] 2. Optimization of reaction conditions

[0154] (1) Optimum pH

[0155] The optimal reaction pH of the recombinant engineered bacterium E. coli BL21 / PksIDH-C261R 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).

[0156] like Figure 8 As shown in A, when the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R reacted in a pH 9.0 system, the yield of 1L-epi-2-inosose was the highest, at 33.8%.

[0157] (2) Optimum temperature

[0158] The optimum temperature of the recombinant engineered bacterium E. coli BL21 / PksIDH-C261R is determined by the yield of the product 1L-epi-2-inosose in a reaction system with whole cell catalysis at pH 9.0 and within the range of 20 to 50°C (other reaction conditions are the same as in step 1).

[0159] like Figure 8 As shown in B, when the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R reacted at 28°C, the yield of 1L-epi-2-inosose was the highest, at 36.9%.

[0160] (3) Optimal cell mass

[0161] The optimal cell mass of the recombinant engineered bacterium E. coli BL21 / pET21a-PksIDH-C261R is determined by the yield of the product 1L-epi-2-inosose in a reaction system with a cell mass range of 10 to 100 g / L under the conditions of pH 9.0 and 28°C (other reaction conditions are the same as in step 1).

[0162] like Figure 8 As shown in C, when the whole cell addition amount of the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R was 80 g / L, the yield of 1L-epi-2-inosose was the highest, which was 55.8%.

[0163] (4) Optimal substrate amount

[0164] The optimal amount of substrate for the recombinant engineered bacterium E. coli BL21 / pET21a-PksIDH-C261R was determined by the yield of the product 1L-epi-2-inositol in a reaction system with 1-100 mM myo-inositol in a whole-cell catalysis at pH 9.0, 28°C, and 80 g / L cells. + The final concentration is consistent with the final concentration of the substrate myo-inositol.

[0165] like Figure 8 As shown in D, when the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R was reacted under the condition of adding 20mM myo-inositol, the yield of 1L-epi-2-inosose was the highest, which was 57.3%.

[0166] (5) Optimal response time

[0167] The optimal reaction time of the recombinant engineered bacterium E. coli BL21 / pET21a-PksIDH-C261R was determined by the yield of the product 1L-epi-2-inosose under the conditions of pH 9.0, 28°C, 80 g / L cells, and 20 mM myo-inositol for 2 to 24 hours of whole-cell catalysis.

[0168] 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 was the highest at 62.3% when the reaction was carried out for 12 hours.

[0169] Example 4: Preparation of epi-inositol by catalyzing 1L-epi-2-inositol with whole cells containing thermostable myo-inositol dehydrogenase GtIDH

[0170] 1. Reaction system:

[0171] In a 1 mL reaction system, add 1 L-epi-2-inositose with a final concentration of 20 mM, 20 mM NADH, 0.5 mM Zn 2+ , 30 g / L E. coli BL21 / pET28a-GtIDH whole cells, reaction buffer 50 mM Tris-HCl (pH 7.5). The above system was first heat treated at 60°C for 20 min, then continued to react at 45°C and 200 rpm for 12 h, and samples were taken for high performance liquid chromatography (HPLC) analysis.

[0172] 2. Optimization of reaction conditions

[0173] (1) Optimal heat treatment conditions

[0174] The optimal heat treatment conditions for the recombinant engineered bacteria E. coli BL21 / pET28a-GtIDH are determined by the yield of the product epi-inositol after whole-cell catalysis at 50-70°C for 10-30 min (other reaction conditions are the same as step 1).

[0175] like Fig. 9As shown in A, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH was heat-treated at 65°C for 20 minutes, the yield of epi-inositol was the highest, which was 18.6%.

[0176] (2) Optimum pH

[0177] The optimal reaction pH of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH 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 20 min, and other reaction conditions are the same as step 1).

[0178] like Fig. 9 As shown in B, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH reacts in a system of pH 7.5, the yield of epi-inositol is the highest, which is 20.7%.

[0179] (3) Optimum temperature

[0180] The optimum temperature of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH is determined by the yield of the product epi-inositol in a reaction system ranging from 35 to 70°C after heat treatment at 65°C for 20 min at pH 7.5 (other reaction conditions are the same as in step 1).

[0181] like Fig. 9 As shown in C, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH reacts at 50°C, the yield of epi-inositol is the highest, which is 25.1%.

[0182] (4) Optimal cell mass

[0183] The optimal cell weight of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH 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 20 min (other reaction conditions are the same as in step 1).

[0184] like Fig. 9 As shown in D, when the whole cell addition amount of the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH was 50 g / L, the yield of epi-inositol was the highest, which was 32.7%.

[0185] (5) Optimal substrate amount

[0186] The optimal substrate amount of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH 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 50 g / L cells after heat treatment at pH 7.5 and 65°C for 20 min. The final concentration of NADH was consistent with the final concentration of the substrate 1L-epi-2-inosose.

[0187] like Fig. 9 As shown in FIG. 8E , when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH was reacted under the condition of adding 40 mM 1L-epi-2-inositol, the yield of epi-inositol was the highest, which was 36.7%.

[0188] (6) Optimal Zn 2+ Addition amount

[0189] The optimal substrate amount for the recombinant engineered bacteria E. coli BL21 / pET28a-GtIDH was 0.1-5.0 mM Zn at 50°C, 50 g / L cells, 40 mM 1L-ep-2-inositose, and 20 min of heat treatment at pH 7.5 and 65°C. 2+ The range of reactions performed in whole-cell systems was determined by the yield of the product epi-inositol.

[0190] like Fig. 9 As shown in F, the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH 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 40.9%.

[0191] (7) Optimal response time

[0192] The optimal substrate amount for the recombinant engineered bacteria E. coli BL21 / pET28a-GtIDH was 50°C, 50 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.

[0193] like Fig. 9 As shown in G, the yield of 1L-epi-2-inosose in the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH was the highest at 49.3% when the reaction lasted for 14 hours.

[0194] Example 5: Two-step whole-cell catalysis of myo-inositol to produce epi-inositol

[0195] The specific steps are as follows:

[0196] 1. Preparation of epi-inositol by wild-type enzyme dual enzyme

[0197] (1) To a 10 mL reaction system containing 50 mM Tris-HCl (pH 9.0) buffer, add 20 mM myo-inositol and 20 mM NAD + , 80 g / L of E. coli BL21 / pET21a-PksIDH whole cells, and reacted at 28°C and 200 rpm for 12 h.

[0198] (2) Add Zn to the system after the reaction in step (1) 2+ and E. coli BL21 / pET28a-GtIDH whole cells, Zn 2+ The final concentration was 1 mM, and the final cell concentration was 50 g / L. The reaction system was heat-shocked at 65°C for 20 min, and then reacted at 50°C, 200 rpm for 14 h, and samples were taken for high performance liquid chromatography (HPLC) analysis.

[0199] 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 15.3% and the output is 0.55 g / L.

[0200] 2. Preparation of epi-inositol by scyllo-inositol dehydrogenase mutant and thermostable myo-inositol dehydrogenase

[0201] (1) To a 10 mL reaction system containing 50 mM Tris-HCl (pH 9.0) buffer, add 20 mM myo-inositol and 20 mM NAD + , 80 g / L of E. coli BL21 / pET21a-PksIDH-C261R whole cells, and reacted at 28°C and 200 rpm for 12 h.

[0202] (2) Add Zn to the reaction system obtained in step (1) 2+ and E. coli BL21 / pET28a-GtIDH whole cells, Zn 2+ The final concentration was 1 mM, and the final cell concentration was 50 g / L. The reaction system was heat-shocked at 65°C for 20 min, and then reacted at 50°C, 200 rpm for 14 h, and samples were taken for high performance liquid chromatography (HPLC) analysis.

[0203] 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 38.7% and the output was 1.39 g / L.

[0204] 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 scyllo-inositol dehydrogenase mutant, characterized in that: 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 or lysine.

2. A gene encoding the scyllo-inositol dehydrogenase mutant according to claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant according to claim 1 or carrying the gene or the recombinant vector according to claim 2, preferably, the recombinant cell uses bacteria or fungi as host cells.

4. A recombinant enzyme catalyst comprising the scyllo-inositol dehydrogenase mutant according to claim 1, characterized in that: Is any of the following: (1) culturing a recombinant expression transformant containing the scyllo-inositol dehydrogenase mutant, and isolating a transformant cell containing the recombinant scyllo-inositol dehydrogenase mutant enzyme; (2) culturing a recombinant expression transformant containing the scyllo-inositol dehydrogenase mutant, isolating transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme, and disrupting the transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme to obtain a cell disrupted liquid; (3) culturing a recombinant expression transformant containing the scyllo-inositol dehydrogenase mutant, isolating transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme, disrupting the transformant cells containing the recombinant scyllo-inositol dehydrogenase mutant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant scyllo-inositol dehydrogenase mutant enzyme to obtain a lyophilized enzyme powder.

5. A method for improving the substrate affinity and catalytic efficiency of scyllo-inositol dehydrogenase, characterized in that: The method comprises the following steps: mutating the cysteine ​​at position 261 of the scyllo-inositol dehydrogenase of the amino acid sequence as shown in SEQ ID NO. 1 into arginine or lysine.

6. Use of the scyllo-inositol dehydrogenase mutant and / or thermostable myo-inositol dehydrogenase according to claim 1 in the preparation of epi-inositol, characterized in that: The thermostable myo-inositol dehydrogenase is derived from Laceyella putida, Geobacillus thermoleovorans, Parageobacillus thermoglucosidasius or Marini thermofilumabyssi; the scyllo-inositol dehydrogenase mutant catalyzes the synthesis of 1L-epi-2-inositose from myo-inositol as a substrate; the thermostable myo-inositol dehydrogenase catalyzes the carbonyl group at the C4 position of 1L-epi-2-inositol to prepare epi-inositol.

7. A method for preparing epi-inositol, characterized in that: The method comprises the following steps: using scyllo-inositol dehydrogenase from Paracoccuskondratievae or Roseibium suaedae, or the scyllo-inositol dehydrogenase mutant according to claim 1, or the recombinant cell according to claim 3 or 4, to catalyze the synthesis of 1L-epi-2-inositol from myo-inositol; and adding heat-resistant myo-inositol dehydrogenase or a genetically engineered bacterium expressing heat-resistant myo-inositol dehydrogenase to catalyze the synthesis of epi-inositol from 1L-epi-2-inositol. The thermostable myo-inositol dehydrogenase is derived from Laceyella putida, Geobacillus thermoleovorans, Parageobacillus thermoglucosidasius or Marinithermofilum abyssi.

8. The preparation method according to claim 7, characterized in that: The reaction system for catalyzing myo-inositol is as follows: the concentration of the substrate myo-inositol is 1-100 mM, NAD + The concentration is 1 to 100 mM, and the amount of the recombinant cells added is 10 to 100 g / L; Preferably, the concentration of the substrate myo-inositol is 10-80 mM, and the amount of the recombinant cells added is 30-90 g / L; Preferably, the concentration of the substrate myo-inositol is 15-60 mM, NAD + The concentration is 15-60 mM, and the amount of the recombinant cells added is 40-80 g / L; Preferably, the concentration of the substrate myo-inositol is 20 mM, NAD + The concentration of is 20mM, the amount of the recombinant cells added is 80g / L; the reaction conditions for catalyzing myo-inositol are to react at pH 6.0-10.0 and 20-50°C for 2-24h; Preferably, the reaction is carried out at pH 8.0 to 9.5 and 25 to 40° C. for 6 to 14 hours; Preferably, the reaction is carried out at pH 9.0 and 25-30° C. for 10-12 hours.

9. The preparation method according to claim 7 or 8, wherein the reaction system of the catalytic substrate 1L-epi-2-inosose is that the concentration of the substrate 1L-epi-2-inosose is 1 to 100 mM, Zn 2+ The addition amount of the genetically engineered bacteria is 0.1-5 mM, and the addition amount of the genetically engineered bacteria is 10-100 g / L; Preferably, the concentration of the substrate 1L-epi-2-inosose is 10-80 mM, Zn 2+ The amount of the added product is 0.5-4 mM, and the amount of the added product of the genetically engineered bacteria is 20-80 g / L; Preferably, the concentration of the substrate 1L-epi-2-inosose is 20-60 mM, Zn 2+ The addition amount of the genetically engineered bacteria is 1-3 mM, and the addition amount of the genetically engineered bacteria is 30-60 g / L; Preferably, the concentration of the substrate 1L-epi-2-inosose is 40 mM, Zn 2+ The addition amount of is 1mM, and the addition amount of the genetically engineered bacteria is 50g / L; Preferably, the reaction system of the catalytic substrate 1L-epi-2-inosose is subjected to heat treatment and then continues to react, and the heat treatment temperature is 50-70°C; further, the heat treatment temperature is 55-70°C; further, the heat treatment temperature is 60-65°C; further, the heat treatment temperature is 65°C; Preferably, the heat treatment time is 10 to 30 minutes; further, the heat treatment time is 20 to 30 minutes; further, the heat treatment time is 20 minutes; The reaction conditions of the heat-treated reaction system are: reacting at pH 6.0-10.0 and 35-70°C for 2-24 hours; further, reacting at pH 6.5-8.0 and 40-60°C for 8-16 hours; further, reacting at pH 7.5 and 50-55°C for 14-16 hours.

10. Use of the mutant according to claim 1, or the gene or recombinant vector according to claim 2, or the recombinant cell according to claim 3, or the recombinant enzyme catalyst according to claim 4, or the method according to claim 5, or the use according to claim 6, or the preparation method according to any one of claims 7 to 9 in the preparation of epi-inositol or a product containing epi-inositol.

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