A method for efficiently synthesizing epipolyhydroxymate by a double-enzyme cascade catalysis of muscle inositol

Abstract

The application discloses a method for efficiently synthesizing epipolyhydroxymatrine by double-enzyme cascade catalysis of muscle inositol, and belongs to the field of biological catalysis engineering. The application mutates shark inositol dehydrogenase to obtain a beneficial mutant C261R, and couples the mutant with heat-resistant muscle inositol dehydrogenase to efficiently catalyze the synthesis of epipolyhydroxymatrine, and belongs to the field of biological catalysis engineering. The new synthesis method of epipolyhydroxymatrine is completely different from the synthesis methods of shark inositol and D-chiral inositol reported in the prior art. The method is to first dehydrogenate muscle inositol at a C4 position to form an intermediate product ketone, then hydrogenate the intermediate product ketone, realize hydrogen chirality inversion at the C4 position, and obtain the end product epipolyhydroxymatrine. The application provides a brand-new biological catalysis synthesis method of epipolyhydroxymatrine, and the yield of epipolyhydroxymatrine is 1.39 g / L, and the conversion rate is 38.7%, which are the highest levels reported at present. The biological synthesis process is simple in operation, low in cost and green in environmental protection, and lays a solid research foundation for the industrial production of epipolyhydroxymatrine.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for efficiently synthesizing epi-inositol by a two-enzyme cascade catalysis of myo-inositol, belonging to the field of biological catalysis engineering. BACKGROUND

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

[0003] The synthesis of epi-inositol is currently still in the stage of chemical synthesis, mainly by reducing epi-inositol sugar with sodium borohydride under alkaline conditions to produce epi-inositol. 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 14000 yuan per gram.

[0004] In recent years, with the development of bioengineering technology, the biosynthesis of inositol isomers has become a new trend. Fujisawa et al. used malt dextrin phosphorylase (MalP) and phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and myo-inositol monophosphatase (IMPase) to construct an in vitro enzyme system for producing inositol from starch. In 7 h, 2.9 g of inositol was produced from 2.9 g of starch, with a molar conversion rate of about 96%. Paul et al. achieved the conversion from glucose to scyllite by modifying Corynebacterium glutamicum, and 1.8 g / L of scyllite was produced from 20 g / L of glucose in 72 h. Ji et al. obtained a scyllite-producing strain DCI-7 by co-expressing myo-inositol isomerase IolI2 and myo-inositol dehydrogenase IolG in Corynebacterium glutamicum. The strain can produce 6.96 g / L of D-chiro-inositol in the presence of 40 g / L of myo-inositol.

[0005] Most of the reported myo-inositol 2-dehydrogenases (EC 1.1.1.18, IDH for short) are used to catalyze the dehydrogenation of C2 position of myo-inositol to obtain dextro-inosose, or the common scyllo-inositol 2-dehydrogenase (EC 1.1.1.370, sIDH for short) is used to catalyze the dehydrogenation of scyllo-inositol to obtain dextro-inosose. Different from the common synthesis method of scyllo-inositol and D-chiro-inositol, in the process of synthesizing epi-inositol from myo-inositol, the dehydrogenation of C4 position is needed first, and then hydrogenation is needed to realize the chiral inversion. Because the substrate and product structures are very symmetrical structures, the enzyme is often difficult to specifically recognize in the catalytic process, so the synthesis of different chiral compounds of myo-inositol has been an important problem in the field of biological catalysis. So far, there is no report on the method and pathway of the biosynthesis 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

[0006] Because the substrate myo-inositol and the product epi-inositol are extremely symmetrical structures, the enzyme is often difficult to specifically recognize in the catalytic process, so the synthesis of different chiral compounds of myo-inositol has been an important problem in the field of biological catalysis.

[0007] In view of the problems existing in the method for preparing epi-inositol, the present application provides a two-step method for producing epi-inositol from myo-inositol by using whole cells of scyllo-inositol dehydrogenase (sIDH for short) capable of catalyzing the dehydrogenation of C4 position and myo-inositol dehydrogenase (IDH for short) capable of catalyzing the hydrogenation of C4 position. The structure analysis and virtual mutation screening of scyllo-inositol dehydrogenase based on sequence conservation are carried out, and the mutant with higher catalytic efficiency is obtained through directed evolution. The present application has the advantages of simple production process, low production cost, green environmental protection and the like, and lays a solid foundation for the industrial preparation of epi-inositol.

[0008] To solve the above technical problems, the present application adopts the following technical scheme:

[0009] The present application provides a preparation method of epi-inositol, which comprises the following steps: using scyllo-inositol dehydrogenase or genetically engineered bacteria expressing scyllo-inositol dehydrogenase to catalyze the synthesis of 1L-epi-2-inosose from myo-inositol; and adding myo-inositol dehydrogenase or genetically engineered bacteria expressing myo-inositol dehydrogenase to catalyze the synthesis of epi-inositol from the 1L-epi-2-inosose.

[0010] In an embodiment of the present application, the method is as follows:

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

[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 the synthesis of 1L-epi-2-inosose from myo-inositol;

[0015] (5) Using the permeabilized whole cells expressing the heat-resistant myo-inositol dehydrogenase obtained in step (3) to catalyze the synthesis of epi-inositol from 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 heat-resistant 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 a vector expressing heat-resistant 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 less than that of the scyllo-inositol dehydrogenase derived from Paracoccus. 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 heat-resistant 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 a temperature 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 heat-resistant myo-inositol dehydrogenase is derived from a Gfo / Idh / MocA family protein, such as from Laceyella putida, Geobacillus thermoleovorans, Paraglucosidase 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 amino acid at position 261 of the scyllo-inositol dehydrogenase with the 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 whole cells are prepared using methods known in the art. 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: the reaction system is heat-treated before 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 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 produce 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 9.0 and 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 from epi-inositol by catalyzing the use of heat-resistant myo-inositol dehydrogenase:

[0034] The concentration of substrate 1L-epi-2-inosose is 1-100 mM, Zn 2+ The addition amount of is 0.1-5mM, 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 0.5-4mM, and the addition amount of whole cells is 20-80g / L; more preferably, the concentration of substrate 1L-epi-2-inosose is 20-60mM, 2+ The addition amount of is 1-3mM, 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 and the addition amount of whole cells is 50g / 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 h; preferably, reaction at pH 6.5-8.0 and 40-60° C. for 8-16 h; most preferably, reaction at pH 7.5 and 50-55° C. for 14-16 h.

[0036] In an embodiment of the present application, the catalytic reaction can be carried out in a buffer-free system or a buffer system; preferably in a buffer system, which 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 an embodiment of the present application, according to the present application, in step (4), the reaction conditions for preparing epi-cyclohexane-1,2,3,5,6-pentaol from cyclohexane-1,2,3,5,6-pentaol by two-step catalysis are as follows: after the reaction for preparing cyclohexane-1,2,3,5,6-pentaol is completed, the pH of the reaction system is adjusted to 7.5, and then the whole cell of heat-resistant cyclohexane-1,2,3,5,6-pentaol dehydrogenase and Zn 2+ After mixing, the cells are subjected to heat treatment, and the reaction is carried out at pH 7.5, 50-55℃ for 14-16h.

[0038] The present application provides a squalene dehydrogenase mutant, which is obtained by mutating the 261th amino acid in the amino acid sequence shown in SEQ ID NO. 1 to arginine or lysine, and is named C261R or C261K.

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

[0040] MTATQKSLGVALIGTGFMGKCHAMAWRNLATAFGGMPPRLEVLADTPAERAAELASAFGFARGTGDWRAAVTDPAVDVVSITTPNGMHREMAEAALAAGKHVWLEKPMALTVEDAEAMETAARAAGRVTILGYNYTRSPAFRAAADLVAQGAIGRPIHFRGVYDEDYMADPELPWSWRLTRAEGGLGALGDLGCHLVSVMLALMGPVARVQAQMDTVVTERPHQGGTATVENEDQAQALIRFASGASGEFSCSRVARGYRCRLAWEVQGTEGTLRFDQERMNELWLYQPGQAETDGFRRILTGPAQPGFAAFCPAAGHNFGFNEQKVVEAEMLRQAIAGGIPAWPDFTAGLTIERIIHGMARSARTGQPVLF

[0041] The base sequence (SEQ ID NO. 3) encoding the parent enzyme of the squalene dehydrogenase mutant 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 includes a promoter, a scyllo-inositol dehydrogenase gene, and a terminator; the vector expressing heat-resistant muscle inositol dehydrogenase includes 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 the pET series, pGEX series, pACYCDuet-1, pRSFDuet-1, etc. Preferably, the vector is pET-21a(+) or 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 mutants or the heat-resistant myo-inositol dehydrogenase.

[0050] The heat-resistant muscle 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 present application also provides a recombinant vector carrying the gene.

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

[0056] In one embodiment of the present application, the recombinant cell is a bacterial or fungal host cell.

[0057] The present application provides a vector for expressing the scyllo-inositol dehydrogenase and a vector for expressing the thermostable pinitol dehydrogenase.

[0058] The present application provides an engineered bacterium for expressing the scyllo-inositol dehydrogenase and a mutant, and an engineered bacterium for expressing the thermostable pinitol dehydrogenase.

[0059] According to the present application, the engineered bacterium for expressing the scyllo-inositol dehydrogenase and the mutant comprises a vector for expressing the scyllo-inositol dehydrogenase; the engineered bacterium for expressing the thermostable pinitol dehydrogenase comprises a vector for expressing the thermostable pinitol dehydrogenase.

[0060] The present application provides a method for improving the substrate affinity and catalytic efficiency of the scyllo-inositol dehydrogenase, which comprises mutating the cysteine at position 261 of the scyllo-inositol dehydrogenase with the amino acid sequence shown in SEQ ID NO. 1 into arginine or lysine.

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

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

[0063] (2) culturing the recombinant expression transformant containing the mutant of the scyllo-inositol dehydrogenase, and isolating the transformant cell containing the recombinant mutant of the scyllo-inositol dehydrogenase enzyme, crushing the transformant cell containing the recombinant mutant of the scyllo-inositol dehydrogenase enzyme to obtain a cell crushing solution;

[0064] (3) culturing the recombinant expression transformant containing the mutant of the scyllo-inositol dehydrogenase, and isolating the transformant cell containing the recombinant mutant of the scyllo-inositol dehydrogenase enzyme, crushing the transformant cell containing the recombinant mutant of the scyllo-inositol dehydrogenase enzyme to obtain a cell crushing solution, and freeze-drying the cell crushing solution of the recombinant mutant of the scyllo-inositol dehydrogenase enzyme to obtain a freeze-dried 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-inositol or a product containing 1L-epi-2-inositol.

[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, heat-resistant myo-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 products containing epi-inositol.

[0067] Beneficial effects

[0068] (1) The present invention is the first to use 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, solving the problem of enzyme regioselectivity in the biosynthesis of epi-inositol.

[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 heat-resistant myo-inositol dehydrogenase to synthesize epi-inositol from myo-inositol, and develop 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 optimized the reaction conditions for both the mutant scyllo-inositol dehydrogenase and the thermostable myo-inositol dehydrogenase, exploring their optimal reaction temperature, pH, cell number, substrate amount, and reaction time. The optimized dual-enzyme cascade two-step process catalyzed the production of epi-inositol from myo-inositol, achieving a total yield of 38.7% for epi-inositol.

[0073] (6) The present invention does not require the addition of NADH, which greatly reduces production costs and has important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0075] Figure 2 Schematic diagram of whole-cell catalysis of myo-inositol to produce epi-inositol by the present application. myo-inositol: myo-inositol; epi-inositol: epi-inositol; 1L-epi-2-inosose: 1L-epi-2-inosose; sIDH: shark muscle inositol dehydrogenase; IDH: myo-inositol dehydrogenase.

[0076] Figure 3 Initial yield of enzyme reaction; wherein, A is the initial yield of myo-inositol conversion to 1L-epi-2-inosose catalyzed by shark muscle inositol dehydrogenase from different sources; B is the initial yield of 1L-epi-2-inosose conversion to epi-inositol catalyzed by heat-resistant myo-inositol dehydrogenase from different sources.

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

[0078] Figure 5 Binding energy changes when semi-conserved amino acids in myo-inositol in the binding pocket of PksIDH are subjected to virtual saturation mutation.

[0079] Figure 6 HPLC diagram of product; wherein, A is the HPLC diagram of 1L-epi-2-inosose, B is the HPLC diagram 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 BL21 GtIDH; 2, E. coli BL21 / PksIDH-C261R.

[0081] Figure 8 Yield of intermediate 1L-epi-2-inosose catalyzed under different conditions; wherein, A-E are the yield changes of intermediate 1L-epi-2-inosose under different pH, reaction temperature, cell amount, substrate amount and reaction time.

[0082] Figure 9 Yield of end product epi-inositol catalyzed under different conditions; wherein, A-G are the yield changes of end product epi-inositol under different heat treatment conditions, reaction temperature, pH, cell amount, substrate amount, Zn 2+ ​​The production of the product, phytosphingosine, was varied with the amount added and the reaction time. DETAILED DESCRIPTION

[0083] To further clarify the technical means adopted by the present application and its effects, the technical solutions of the present application are further described below through specific examples. However, it should be understood that the examples are exemplary only, and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications or replacements all fall within the protection scope of the present application.

[0084] Technical terms

[0085] Expression: The term "expression" includes any step involving the production of scyllo-inositol dehydrogenase and thermotolerant tachynositol 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 thermotolerant tachynositol dehydrogenase of the present application and operably linked to control sequences that provide for its expression.

[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. Prokaryotic host cells 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, Helicobacterium, 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. The enzyme activity assay system was as follows: the reaction volume was 200 μL, 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 a Bio-Tek Cytation 5 cell imaging multifunctional detection system.

[0093] The enzyme activity is defined as: 1 μmol NADH reduced per minute under the above conditions. + The amount of enzyme that reduces NAD to NADH is defined as one unit (U).

[0094] The formula for calculating the enzyme activity is: enzyme activity (U) = EW x V x 10 3 / (6220 x 0.625).

[0095] The formula for calculating the specific activity is: specific activity (U / mg) = enzyme activity (U) / protein amount (mg).

[0096] Wherein, EW: change in absorbance at 340 nm in 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], the K m value of the enzyme for different substrates is calculated.

[0099] Wherein, v: reaction rate (U / mg); V max : maximum reaction rate (U / mg); [S]: substrate concentration (mM); K m : substrate concentration when the reaction rate v reaches half of 1 / 2 V max .

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

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

[0102] Reaction system for determination of kinetic parameters:

[0103] The total reaction volume is 200 μL, 20 mM Tris-HCl buffer (pH 7.5) and 5 mM NAD + are added, incubated at 30°C for 3 min, 20 μL of pure enzyme of appropriate concentration is added, and the specific enzyme activity under different concentrations (1-100 mM) of substrate myo-inositol is determined. The fitting of enzyme kinetic curve is realized by GraphPad software, with substrate concentration and specific enzyme activity as horizontal and vertical coordinates, respectively, and the Michaelis equation model is selected for non-linear fitting, to obtain V max value and Km Values, k is obtained by data conversion cat Values and k cat / K m Values (Table 2).

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

[0105] 30℃, 200rpm reaction for 12h, sampling for high performance liquid chromatography (HPLC) analysis. 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 1mL / min; the column temperature is 40℃; the detector is a UV detector, and the detection wavelength is 210nm; the injection volume is 10μL. The liquid chromatogram of 1L-Table-2-inositol is shown in Figure A. Figure 6

[0106] Table Inositol production detection:

[0107] 60℃ heat treatment for 20min, 45℃, 200rpm reaction for 12h, sampling for high performance liquid chromatography (HPLC) analysis. HPLC detection conditions are as follows: the chromatographic column is Bio-Rad Aminex HPX-87H Column; the mobile phase is 5mM H2SO4; the flow rate is 0.5mL / min; the column temperature is 60℃; the detector is a differential detector; the injection volume is 10μL. The liquid chromatogram of Table inositol is shown in Figure B. Figure 6

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

[0109] (1) Preparation of whole cells of recombinant engineering bacteria of shark inositol dehydrogenase

[0110] The recombinant engineering bacteria E. coli BL21 / PksIDH or its mutants were picked and inoculated in LB liquid test tube medium containing ampicillin, and cultured at 37℃ for 8h. The bacterial liquid in the cultured LB liquid test tube was inoculated into LB liquid flask medium containing ampicillin at an inoculation amount of 1%, and then cultured at 37℃ for 2h. When the OD value was 0.4-0.6, the temperature was reduced to 25℃, IPTG was added for induction, and the final concentration was 0.3mM. The cells were collected by low-temperature refrigerated centrifuge 14h after induction, centrifuged at 8000xg for 10min, and the supernatant was discarded to obtain whole cells expressing shark inositol dehydrogenase.

[0111] (2) Preparation of whole cells of recombinant engineering bacteria of thermostable inositol dehydrogenase

[0112] ​​The recombinant engineering bacteria E. coli BL21 / GtIDH or its mutant was inoculated in LB liquid test tube medium containing kanamycin and cultured at 37°C for 8 h. The bacteria liquid in the cultured LB liquid test tube was inoculated in LB liquid flask culture medium containing kanamycin at an inoculation amount of 1%, and then cultured at 37°C for 2 h. When the OD value was 0.4-0.6, the temperature was reduced to 28°C, IPTG was added for induction, and the final concentration was 0.5 mM. The cells were collected by low-temperature refrigerated centrifuge 12 h after induction, centrifuged at 8000 x g for 10 min, and the supernatant was discarded to obtain the whole cell expressing heat-resistant muscle inositol dehydrogenase.

[0113] Example 1: Mining of squaline dehydrogenase and heat-resistant muscle inositol dehydrogenase and construction of recombinant engineering bacteria

[0114] The specific steps are as follows:

[0115] 1. Mining of squaline dehydrogenase and construction of recombinant engineering bacteria

[0116] (1) Construction of genetically engineered bacteria

[0117] The squaline dehydrogenase from Paracoccus cavernae (NCBI number: WP_377687665.1), the squaline dehydrogenase from Paracoccus kondratievae (NCBI number: WP_152368211.1), the squaline dehydrogenase from Roseibium suaedae (NCBI number: WP_073014975.1), the squaline dehydrogenase from Celeribacter baekdonensis (NCBI number: WP_339692564.1), the squaline dehydrogenase from Sinirhodobacter populi (NCBI number: WP_128233564.1), the squaline dehydrogenase from Donghicola eburneus (NCBI number: WP_092465240.1), the squaline dehydrogenase from Jiellapacifica (NCBI number: WP_163463107.1), the squaline dehydrogenase from Thalassospira lohafexi (NCBI number: WP_101299495.1), the squaline dehydrogenase from Pannonibacter indicus (NCBI number: WP_055457068.1), and the squaline dehydrogenase from Devosia marina (NCBI number: WP_157290965.1) were connected to the pET21a vector to prepare the recombinant vectors, respectively.

[0118] The prepared recombinant vectors were introduced into E. coli BL21 (DE3) to prepare recombinant strains (gene synthesis and construction of recombinant engineering bacteria were completed by Sangon Biotech (Shanghai) Co., Ltd.).

[0119] (2) Enzyme screening

[0120] The recombinant strains were inoculated into LB liquid test tube culture medium containing ampicillin and cultured at 37°C with shaking for 8 hours. The bacterial solution from the cultured LB liquid test tube was inoculated into LB liquid shake flask culture medium containing ampicillin at a 1% (v / v) inoculum and cultured at 37°C for 2 hours. When the OD value reached 0.4-0.6, the temperature was lowered to 25°C and IPTG was added for induction at a final concentration of 0.3 mM. After 14 hours of induction, the cells were collected using a low-temperature refrigerated centrifuge and centrifuged at 8000×g for 10 minutes. 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 30 g / L whole cells 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 Figure A, scyllo-inositol dehydrogenase (PksIDH) from Paracoccus kondratievae had the highest initial conversion rate (15.9%) among the screened enzymes and was selected as the research object; the results show that not all scyllo-inositol dehydrogenases can produce 1L-epi-2-inosose.

[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 ligated into the pET28a vector to prepare recombinant vectors.

[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 culture medium containing kanamycin and cultured with shaking 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 kanamycin at a 1% (v / v) inoculum and cultured at 37°C for 2 hours. When the OD value reached 0.4-0.6, the temperature was lowered to 28°C and IPTG was added for induction at a final concentration of 0.5mM. After 12 hours of induction, the cells were collected using a low-temperature refrigerated centrifuge and centrifuged at 8000×g for 10 minutes. The supernatant was discarded to obtain whole cells expressing heat-resistant myo-inositol dehydrogenase.

[0129] Reaction system: Whole cells at a concentration of 20 g / L were resuspended in 1 mL of a reaction mixture (pH 7.5). The reaction mixture consisted of 10 mM NADH and 10 mM 1L-epi-2-inosose (dissolved in 50 mM Tris-NaCl buffer). After heat treatment at 60°C for 20 min, the reaction was incubated at 45°C and 200 rpm for 12 hours. Following completion of the reaction, the reaction mixture was heat-inactivated to terminate enzyme activity.

[0130] The initial conversion rate of 1L-epi-2-inosose was determined at 20 mM. Figure 3 As shown in Figure B, the heat-resistant 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 shows the most significant change in the binding energy of the protein molecule, indicating that the site is a key residue for binding with the substrate myo-inositol, and the site is selected as the mutation site for directed evolution.

[0137] (1) Directed evolution of shikimic acid dehydrogenase mutants

[0138] The mutation hot spot C261 in step 2 is selected as the mutation starting point, and then a degenerate primer (NNK-MNN) is used to introduce random mutations at the corresponding site by PCR; the sequence of the degenerate primer (NNK-MNN) is as follows:

[0139] Table 1: Primer design for shikimic acid dehydrogenase mutant enzyme gene

[0140]

[0141] The PCR reaction mixture is as follows: 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of template plasmid, 5 μL of 2×Primstar, and 2 μL of ddH2O. The pre-denaturation temperature is 98℃, 30 s, the denaturation temperature is 98℃, 15 s, the annealing temperature is 55℃, 30 s, and the extension temperature is 72℃, 80 s.

[0142] (2) The wild-type recombinant expression vector and the recombinant expression vector with introduced random mutations are respectively transformed into E. coli BL21 (DE3) competent cells to prepare recombinant expression strains (the synthesis of genes and the construction of recombinant engineering bacteria are completed by Shengong Bioengineering (Shanghai) Co., Ltd.).

[0143] First, the monoclonal recombinant strains with introduced random mutations are respectively cultured and induced for expression in 96-well plates, with the wild type as a control (the culture conditions are the same as those for the preparation of whole cells in Example 1). After centrifugation and removal of the supernatant, 1 mL of reaction mixture (pH 7.5) is added in situ for resuspension. The reaction mixture includes 10 mM NADH and 10 mM myo-inositol (dissolved in 50 mM Tris-NaCl buffer solution), and the reaction is carried out at 30℃, 200 rpm for 12 hours. After the reaction is completed, the reaction mixture is heat-inactivated to terminate the activity of the enzyme.

[0144] Subsequently, 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 levels than the control were screened and rescreened by HPLC. Mutants with significantly increased 1L-epi-2-inosose production were sequenced, ultimately yielding recombinant strains containing the scyllo-inositol dehydrogenase mutants with significant effects: 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 acquired 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, 50 mM Tris-HCl (pH 7.5) reaction buffer, 30°C, 200 rpm for 12 h, and samples were collected for high performance liquid chromatography (HPLC) analysis.

[0153] 2. Optimization of reaction conditions

[0154] (1) Optimum pH

[0155] The optimum reaction pH of the recombinant engineering bacteria E. coli BL21 / PksIDH-C261R was determined by the yield of product 1L-epi-2-inositol in the reaction system in the pH range of 6.0-10.0 (other reaction conditions were the same as step 1).

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

[0157] (2) Optimum temperature

[0158] The optimum temperature of the recombinant engineering bacteria E. coli BL21 / PksIDH-C261R was determined by the yield of product 1L-epi-2-inositol in the reaction system in the temperature range of 20-50°C at pH 9.0 (other reaction conditions were the same as step 1).

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

[0160] (3) Optimal cell amount

[0161] The optimal cell amount of the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R was determined by the yield of product 1L-epi-2-inositol in the reaction system in the cell amount range of 10-100 g / L at pH 9.0 and 28°C (other reaction conditions were the same as step 1).

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

[0163] (4) Optimal substrate amount

[0164] The optimal substrate amount of the recombinant engineering bacteria E. coli BL21 / pET21a-PksIDH-C261R was determined by the yield of product 1L-epi-2-inositol in the reaction system in the substrate amount range of 1-100 mM inositol at pH 9.0, 28°C and 80 g / L of cells, and the final concentration of NAD + The final concentration of NAD was consistent with the final concentration of substrate inositol.

[0165] As shown in D of Figure 8 , the recombinant engineered bacteria E. coli BL21 / pET21a-PksIDH-C261R had the highest yield of 1L-epi-2-cymarose of 57.3% under the condition of 20 mM myo-inositol addition when the reaction was carried out.

[0166] (5) Optimal reaction time

[0167] The optimal reaction time of the recombinant engineered bacteria E. coli BL21 / pET21a-PksIDH-C261R was determined by the yield of the product 1L-epi-2-cymarose when the whole-cell catalysis was carried out for 2-24 h under the condition of pH 9.0, 28℃, 80 g / L cells, and 20 mM myo-inositol.

[0168] As shown in E of Figure 8 , the recombinant engineered bacteria E. coli BL21 / pET21a-PksIDH-C261R had the highest yield of 1L-epi-2-cymarose of 62.3% when the reaction was carried out for 12 h.

[0169] Example 4: Preparation of epi-inositol from 1L-epi-2-cymarose using whole-cell catalysis containing heat-resistant myo-inositol dehydrogenase GtIDH

[0170] 1. Reaction system:

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

[0172] 2. Optimization of reaction conditions

[0173] (1) Optimal heat treatment condition

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

[0175] As shown in D of Figure 9As shown in Figure 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, at 18.6%.

[0176] (2) Optimum pH

[0177] The optimal reaction pH 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 whole-cell catalysis in the pH range of 6.0 to 10.0 (heat treated at 65°C for 20 minutes, and other reaction conditions were the same as in step 1).

[0178] like Figure 9 As shown in B, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH reacts in a pH 7.5 system, 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 was determined by the yield of the product, epi-inositol, in a reaction system catalyzed by whole-cell reaction at 35-70°C after heat treatment at pH 7.5 at 65°C for 20 minutes (other reaction conditions were the same as in step 1).

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

[0182] (4) Optimal cell mass

[0183] The optimal cell size of the recombinant engineered bacterium E. coli BL21 / pET28a-GtIDH was determined by heat treatment at pH 7.5 and 65°C for 20 minutes, followed by whole-cell catalysis at 50°C in a reaction system with a cell size range of 10 to 100 g / L (other reaction conditions were the same as in step 1).

[0184] like Figure 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 for the recombinant engineered E. coli BL21 / pET28a-GtIDH was determined by whole-cell catalysis at 50°C, 50 g / L cells, and the yield of the product, epi-inositol, after heat treatment at pH 7.5 and 65°C for 20 minutes in a reaction system with a concentration range of 1-100 mM 1L-epi-2-inosose. The final NADH concentration was consistent with the final concentration of the substrate 1L-epi-2-inosose.

[0187] like Figure 9 As shown in Figure E, when the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH was reacted under the condition of adding 40mM 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 is 0.1-5.0 mM Zn at 50°C, 50 g / L cells, 40 mM 1L-epi-2-inositose, and 20 min after heat treatment at pH 7.5 and 65°C. 2+ The yield of the product epi-inositol was determined by whole-cell catalysis in the reaction system.

[0190] like Figure 9 As shown in F, the recombinant engineering bacteria E. coli BL21 / pET28a-GtIDH was cultured in 1mM 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 reaction time

[0192] The optimal substrate amount for the recombinant engineered bacteria E. coli BL21 / pET28a-GtIDH was 50 g / L cells, 40 mM 1 L-epi-2-inositose, 1 mM Zn 2+ The yield of the product epi-inositol was determined by whole-cell catalysis under the same conditions for 2 to 24 hours.

[0193] like Figure 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% after 14 hours of reaction.

[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, 20 mM NAD + , 80 g / L 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. Samples were taken for high performance liquid chromatography (HPLC) analysis.

[0199] 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 and thermostable myo-inositol dehydrogenase, the total yield of epi-inositol was 15.3% and the output was 0.55 g / L.

[0200] 2. Preparation of Epi-inositol by Scyllo-inositol Dehydrogenase Mutants 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, 20 mM NAD + , 80 g / L 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. Samples were taken for high performance liquid chromatography (HPLC) analysis.

[0203] The experimental results showed that in the two-step whole-cell method of using scyllo-inositol dehydrogenase mutant and thermostable myo-inositol dehydrogenase to catalyze the preparation of epi-inositol from myo-inositol, the total yield of epi-inositol was 38.7%, and the output was 1.39 g / L.

[0204] While the application has been described by way of example and in terms of the preferred embodiment, it is to be understood that certain modifications can be made to the disclosed apparatus without departing from the scope of the application, and the scope of the application should be determined not by the embodiment but by the appended 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 shown in SEQ ID NO. 1 to arginine or lysine.

2. A gene encoding the scyllo-inositol dehydrogenase mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant cell expressing the mutant according to claim 1, carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3.

5. An 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 transformant cells containing the scyllo-inositol dehydrogenase mutant enzyme; (2) culturing a recombinant expression transformant containing the scyllo-inositol dehydrogenase mutant, isolating transformant cells containing the scyllo-inositol dehydrogenase mutant enzyme, and disrupting the transformant cells containing the 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 scyllo-inositol dehydrogenase mutant enzyme, disrupting the transformant cells containing the scyllo-inositol dehydrogenase mutant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the scyllo-inositol dehydrogenase mutant enzyme to obtain a freeze-dried enzyme powder.

6. A method for improving the affinity and catalytic efficiency of scyllo-inositol dehydrogenase for the substrate myo-inositol, characterized in that: The method comprises the following steps: 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.

7. Use of the scyllo-inositol dehydrogenase mutant and the heat-resistant myo-inositol dehydrogenase according to claim 1 in the preparation of epi-inositol, characterized in that: The amino acid sequence of the heat-resistant myo-inositol dehydrogenase is shown in SEQ ID NO.2; The scyllo-inositol dehydrogenase mutant catalyzes the synthesis of 1L-epi-2-inositol from myo-inositol as a substrate; the heat-resistant myo-inositol dehydrogenase catalyzes the carbonyl group at the C4 position of 1L-epi-2-inositol to prepare epi-inositol.

8. A method for preparing epi-inositol, characterized in that: The method comprises the following steps: using the scyllo-inositol dehydrogenase mutant of claim 1 or the recombinant cell of claim 4 to catalyze the conversion of the substrate myo-inositol into 1L-epi-2-inosose; and adding a heat-resistant myo-inositol dehydrogenase or a genetically engineered bacterium expressing a heat-resistant myo-inositol dehydrogenase to catalyze the conversion of the 1L-epi-2-inositol into epi-inositol. The amino acid sequence of the heat-resistant muscle inositol dehydrogenase is shown in SEQ ID NO.

2.

9. The preparation method according to claim 8, 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 of the recombinant cells is 1-100 mM, and the amount of the recombinant cells added is 10-100 g / L.

10. The preparation method according to claim 9, characterized in that The concentration of the substrate myo-inositol is 10-80 mM, and the amount of the recombinant cells added is 30-90 g / L.

11. The preparation method according to claim 9, characterized in that The concentration of the substrate myo-inositol is 15-60 mM, NAD + The concentration of the recombinant cells is 15-60 mM, and the amount of the recombinant cells added is 40-80 g / L.

12. The preparation method according to claim 9, characterized in that The concentration of the substrate myo-inositol was 20 mM, NAD + The concentration of the recombinant cells is 20 mM, and the amount of the recombinant cells added is 80 g / L. The reaction conditions for catalyzing myo-inositol are as follows: pH 6.0-10.0, 20-50° C., and 2-24 h.

13. The preparation method according to claim 12, characterized in that The reaction conditions for catalyzing myo-inositol are as follows: pH 8.0-9.5, 25-40° C., and 6-14 h.

14. The preparation method according to claim 12, characterized in that The reaction conditions for catalyzing myo-inositol are as follows: pH 9.0, 25-30° C., and 10-12 h.

15. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4, or the enzyme catalyst according to claim 5, or the method according to claim 6, or the preparation method according to any one of claims 8 to 14 in the preparation of epi-inositol or a product containing epi-inositol.

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