Inositol preparation method

Abstract

An inositol preparation method by enzymatic catalysis uses starch and cellulose or substrates thereof as substrates. Raw materials are converted to inositol by in vitro multi-enzyme reaction system in one pot. The yield from the substrate to inositol is significantly improved by process optimization and adding new enzymes. The new enzymes can promote the phosphorolysis of starch or cellulose and utilization of glucose, which is the final production after the phosphorolysis of starch and cellulose. The inositol preparation method described herein has great potentials in industrial production of inositol because of high inositol yield, easy scale-up, low production cost, and lower impact to environment.

Claims

1. A method for preparing inositol, comprising: (1) using starch or starch derivative as substrate, adding -glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase to establish an in vitro multi-enzyme reaction system and perform an enzyme-catalyzed reaction; and (2) separating and purifying reaction product to obtain inositol, wherein the starch derivative of step (1) is selected from the group consisting of partially hydrolyzed starch, starch dextrin, maltodextrin, malto-oligosaccharide, and maltose, wherein the enzyme-catalyzed reaction is performed at 60-80 C., wherein the multi-enzyme reaction system further comprises starch debranching enzyme, -amylase, and one or both of maltose phosphorylase and glucanotransferase, wherein the starch debranching enzyme is one or both of isoamylase and pullulanase, wherein the multi-enzyme reaction system further comprises polyphosphate glucokinase and polyphosphate; wherein the polyphosphate is sodium polyphosphate.

2. The preparation method according to claim 1, wherein the multi-enzyme reaction system further comprises the following components: buffer, inorganic phosphate anion, divalent magnesium ion, and either zinc ion or manganese ion.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic showing the multi-enzymatic catalytic pathway of converting starch to inositol in vitro. Wherein: IA, isoamylase; PA, pullulanase; GP, -glucan phosphorylase; PGM, phosphoglucomutase; IPS, inositol 3-phophate synthase; IMP, inositol monophosphatase; MP, maltose phosphorylase (which can be replaced by glucanotransferase); PPGK, polyphosphate glucokinase.

(2) FIG. 2 shows the detection of four key enzymes by SDS-PAGE. Wherein: lane 1, -glucan phosphorylase; lane 2, phosphoglucomutase; lane 3, inositol 3-phophate synthase; lane 4, inositol monophosphatase.

(3) FIGS. 3a and 3b show analysis of inositol by HPLC. FIG. 3a shows the identification of inositol, glucose, glucose-1-phosphate and glucose-6-phosphate by HPLC; FIG. 3b shows the inositol concentration by HPLC quantitative analysis, which is calculated from the height of inositol peak.

(4) FIG. 4 shows the product detected by HPLC after enzyme-catalyzed reaction in which soluble starch is used as substrate. The arrow indicates the characteristic peak of inositol.

(5) FIG. 5 shows the product detected by HPLC after enzyme-catalyzed reaction in which maltodextrin is used as substrate. The arrow indicates the characteristic peak of the inositol.

(6) FIG. 6 is a schematic showing the multi-enzymatic catalytic pathway of converting cellulose to inositol in vitro. Wherein: cellulase, cellulase; CDP, cellodextrin phosphorylase; CBP, cellobiose phosphorylase; PGM, phosphoglucomutase; IPS, inositol 3-phosphate synthase; IMP, inositol monophosphatase; PPGK, polyphosphate glucokinase.

(7) FIG. 7 shows the detection of two key enzymes in the conversion of cellulose to inositol by SDS-PAGE. Wherein: lane 1, cellobiose phosphorylase; lane 2, cellodextrin phosphorylase.

(8) FIG. 8 shows the product detected by HPLC after enzyme-catalyzed reaction in which microcrystalline cellulose (Avicel) is used as substrate. The arrow indicates the characteristic peak of the inositol.

DETAILED DESCRIPTION

(9) The present disclosure is further described in connection with specific examples and the advantages and features of the present disclosure will be more apparent from the description. It is to be understood that the examples are exemplary only and are not intended to limit the scope of the present disclosure. It will be understood by those of ordinary skill in the art that the details and form of the invention may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications or replacements fall within the scope of the present disclosure.

(10) Experimental Materials

(11) Soluble starch, ACROS, Cat No. 424490020

(12) Maltodextrin, ALDRICH, Cat No. 419672

(13) pET20b vector, Novagen (Madison, Wis., USA)

(14) Escherichia coli expressing bacteria BL21 (DE3), Invitrogen (Carlsbad, Calif., USA)

(15) Most of the enzymes in the present disclosure (except inositol monophosphatase, polyphosphate glucokinase and glucanotransferase) are commercially available from Sigma. They can all be obtained by prokaryotic expression through genetic engineering methods.

(16) Cellulase, purchased from Sigma, Cat No. C2730

(17) Maltose phosphorylase, purchased from Sigma, Cat No. M8284

(18) -amylase, purchased from Sigma, Cat No. 10065

(19) Avicel, microcrystalline cellulose, purchased from Sigma, Cat No. 11365

EXAMPLES

Example 1. Conversion of Starch to Inositol Through In Vitro Multi-Enzyme Catalysis

(20) Starch is converted to inositol through an in vitro multi-enzyme catalyzed system (FIG. 1). The key enzymes include: (1) -glucan phosphorylase (GP, EC 2.4.1.1), releasing glucose 1-phosphate from starch; (2) phosphoglucomutase (PGM, EC 5.4.2.2), catalyzing glucose 1-phosphate to glucose-6-phosphate; (3) inositol 3-phophate synthase (IPS, EC 5.5.1.4), catalyzing glucose 6-phosphate to inositol 3-phosphate; (4) inositol monophosphatase (IMP, EC 3.1.3.25), catalyzing the dephosphorylation of inositol 3-phosphate to inositol. Since the last two enzymatic reactions are irreversible, the enzyme-catalyzed system has a very high yield.

(21) In the present disclosure, -glucan phosphorylase was from Thermotoga maritime and the gene number on KEGG is TM1168; phosphoglucomutase was also from T. maritime and the gene number on KEGG was TM0769; inositol 3-phophate synthase is from Archaeoglobus fulgidus and the gene number on KEGG is AF1794; inositol monophosphatase is also from T. maritime and the gene number on KEGG is TM1415. All these genomic DNAs are available from ATCC official website (www.atcc.org). The four genes were obtained from the corresponding genomic DNA by PCR using different primers and cloned into the pET20b vector (Novagen, Madison, Wis.) by Simple Cloning (You, C., et al. (2012), Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. 78(5): 1593-1595.), to obtain corresponding expression vectors: pET20b-TmGP, pET20b-AfIPS, pET20b-TmPGM and pET20b-TmIMP. These four plasmids were transformed into E. coli BL21 (DE3) (Invitrogen, Carlsbad, Calif.), and protein expression and purification were carried out. The results of protein purification were shown in FIG. 2.

(22) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 0.05 U/mL -glucan phosphorylase, 1 U/mL phosphoglucomutase, 0.05 U/mL inositol-3-phosphate synthase, 2 U/mL inositol monophosphate and 10 g/L of soluble starch, was subjected to catalytic reaction at 60 C. for 40 hours.

(23) Depending on the retention time, HPLC can be used to distinguish inositol, glucose, glucose 1-phosphate or glucose 6-phosphate in the reaction solution (FIG. 3a); the inositol can be quantified by HPLC, as shown in FIG. 3b, the concentration of inositol is proportional to the height of the inositol characteristic peak in HPLC. The mobile phase of HPLC was 5 mM dilute sulfuric acid.

(24) After the completion of reaction, the final concentration of the final inositol (FIG. 4) was 1.6 g/L and the yield was 16%.

Example 2. Conversion of Starch to Inositol Through In Vitro Multi-Enzyme Catalysis

(25) The preparations of -glucan phosphorylase, phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1.

(26) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 0.05 U/mL -glucan phosphorylase, 1 U/mL phosphoglucomutase, 0.05 U/mL inositol-3-phosphate synthase, 2 U/mL inositol monophosphate and 10 g/L of soluble starch, was subjected to catalytic reaction at 40 C. for 40 hours.

(27) After the completion of reaction, the final concentration of the final inositol was 0.9 g/L and the yield was 9%.

Example 3. Conversion of Starch to Inositol Through In Vitro Multi-Enzyme Catalysis

(28) The preparations of -glucan phosphorylase, phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1.

(29) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 0.05 U/mL -glucan phosphorylase, 1 U/mL phosphoglucomutase, 0.05 U/mL inositol-3-phosphate synthase, 2 U/mL inositol monophosphate and 10 g/L of soluble starch, was subjected to catalytic reaction at 80 C. for 40 hours.

(30) After the completion of reaction, the final concentration of the final inositol was 3.6 g/L and the yield was 36%.

Example 4. Conversion of Starch to Inositol by In Vitro Multi-Enzyme Catalysis Through Process Optimization and Addition of Enzyme that Promotes Starch Hydrolysis

(31) The use of glucan phosphorylase alone cannot completely hydrolyze starch, since starch has branch chain which is linked to main chain with -1,6 glycosidic bond, and -glucan phosphorylase only acts on -1,4 glycosidic bond. This requires the addition of isoamylase (EC 3.2.1.68) to hydrolyze the -1,6 glycosidic bond. Finally, the final products of starch hydrolysis by these two enzymes are maltose and glucose. In order to convert the final products to inositol, further adding of maltose phosphorylase (EC 2.4.1.8) and polyphosphate glucokinase (EC 2.7.1.63) is needed.

(32) In the present disclosure, isoamylase is from Sulfolobus tokodaii and the gene number on KEGG is ST0928; the genomic DNA of this strain is kindly provided by Professor Georg Fuchs in Albert-Ludwigs-Universitt Freiburg (Germany). Polyphosphate glucokinase is from Thermobifida fusca and the gene number on KEGG is Tfu1811; the genomic DNA of this strain is kindly provided by Professor David Wilson in Cornell University (USA). Glucanotransferase is from Thermococcus litoralis and the gene number on KEGG is OCC_10078; the genomic DNA of this strain is available from ATCC official website (www.atcc.org). These three genes are obtained from the corresponding genomic DNAs by PCR using different primers and cloned into the pET20b vector by Simple Cloning (You, C., et al. (2012), Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. 78(5): 1593-1595.), to obtain corresponding expression vectors: pET20b-StIA, pET20b-TfuPPGK and pET20b-T14GT. These three plasmids were transformed into E. coli BL21 (DE3) (Invitrogen, Carlsbad, Calif.), and protein expression and purification were carried out.

(33) The preparation of -glucan phosphorylase, phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1. Maltose phosphorylase was purchased from Sigma, Cat No. M8284.

(34) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL -glucan phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol-3-phosphate synthase, 2 U/mL inositol monophosphate, 1 U/mL isoamylase, 1 U/mL maltose phosphorylase, 1 U/mL polyphosphate glucokinase, 10 mM sodium polyphosphate and 10 g/L of soluble starch, was subjected to catalytic reaction at 80 C. for 40 hours. The final concentration of the final inositol (FIG. 4) was 7.2 g/L and the yield reached 72%.

Example 5. Conversion of Starch to Inositol by In Vitro Multi-Enzyme Catalysis Through Process Optimization and Addition of Enzyme that Promotes Starch Hydrolysis

(35) The preparation of -glucan phosphorylase, phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1. The preparation of polyphosphate glucokinase is the same as in Example 4. Pullulanase (EC 3.2.1.41) was purchased from Sigma, Cat No. P1067; maltose phosphorylase was purchased from Sigma, Cat No. M8284.

(36) Since the pullulanase purchased from sigma does not react at high temperatures (80 C.), soluble starch was treated with pullulanase at 37 C. first, followed by adding other enzymes. The reaction was carried out at 80 C.

(37) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 1 U/mL pullulanase, 10 mM sodium polyphosphate and 10 g/L of soluble starch, was subjected to catalytic reaction at 37 C. After 10 hours, 5 U/mL -glucan phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol 3-phosphate synthase, 2 U/mL inositol monophosphate, 1 U/mL maltose phosphorylase and 1 U/mL polyphosphate glucokinase were added and the catalytic reaction was carried out at 80 C. for 40 hours. The final concentration of the final inositol (FIG. 4) was 7.3 g/L and the yield reached 73%.

(38) Subsequently, a small amount of -amylase was added to the reaction system to promote the hydrolysis of the residual starch and increase the production of inositol. The amount of -amylase was 0.1 U/ml and the reaction was performed at 37 C. for 6 hours then at 80 C. for 24 hours. The final concentration of the final inositol was 8.8 g/L and the yield reached 88%.

Example 6. Conversion of Maltodextrin to Inositol Through In Vitro Multi-Enzyme Catalysis

(39) The preparation of -glucan phosphorylase, phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1. The preparation of isoamylase and polyphosphate glucokinase are the same as in Example 4. Maltose phosphorylase was purchased from Sigma, Cat No. M8284.

(40) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL -glucan phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol 3-phosphate synthase, 2 U/mL inositol monophosphate, 1 U/mL isoamylase, 1 U/mL maltose phosphorylase, 1 U/mL polyphosphate glucokinase, 10 mM sodium polyphosphate and 10 g/L of maltodextrin (ALDRICH, Cat No. 419672), was subjected to catalytic reaction at 80 C. for 40 hours. The final concentration of the final inositol (FIG. 5) was 7.8 g/L and the yield reached 78%.

Example 7. Conversion of Cellulose to Inositol Through In Vitro Multi-Enzyme Catalysis

(41) Schematic of conversion of cellulose to inositol through multi-Enzyme catalysis in vitro is shown in FIG. 6.

(42) Cellulase is from Sigma, Cat No. C2730; the preparation of phosphoglucomutase, inositol-3-phophate synthase and inositol monophosphatase are the same as in Example 1.

(43) Cellodextrin phosphorylase (Cthe_2989) and cellobiose phosphorylase (Cthe_0275) are both from Clostridium thermocellum. These two genes are obtained from the corresponding genomic DNAs (genomic DNA is available from ATCC official website (www.atcc.org)) by PCR using different primers and cloned into the pET20b vector by Simple Cloning (You, C., et al. (2012)) to obtain corresponding expression vectors: pET20b-CthCDP and pET20b-CthCBP. The two plasmids were both transformed into E. coli BL21 (DE3) and protein expression and purification were carried out. The results of protein purification were shown in FIG. 7.

(44) In this experiment, microcrystalline cellulose (Avicel) was used as substrate. First, commercial cellulase (5 U/ml) and cellulose (10 g/L) were mixed in an ice-water bath and stood for 5 minutes; the mixture was centrifuged at 4 C. and supernatant was discarded. The pellet was a mixture of cellulose and cellulase which can bind to cellulose. This treatment can remove almost all glucosidase from commercial cellulase, avoiding the generation of large amount of glucose from hydrolyzation of cellobiose by glucosidase, so that the major hydrolysates are cellobiose and cellodextrin.

(45) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiose phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol 3-phosphate synthase, 2 U/mL inositol monophosphate and 10 g/L of the mixture of cellulose and cellulase, was subjected to catalytic reaction at 50 C. for 72 hours. The final concentration of the final inositol was 1.4 g/L and the yield reached 14%, as shown in FIG. 8.

Example 8. Conversion of Cellulose to Inositol Through In Vitro Multi-Enzyme Catalysis

(46) Cellulase is from Sigma, Cat No. C2730; the preparation of phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1; the preparation of cellodextrin phosphorylase and cellobiose phosphorylase are the same as in Example 7.

(47) In this experiment, regenerated amorphous cellulose (RAC) was used as substrate, which is the product of Avicel after concentrated phosphoric acid treatment (Zhang, Y. H. P., et al. (2006), A Transition from Cellulose Swelling to Cellulose Dissolution by o-Phosphoric Acid: Evidence from Enzymatic Hydrolysis and Supramolecular Structure. Biomacromolecules 7(2): 644-648.). First, commercial cellulase (5 U/ml) and cellulose (10 g/L) were mixed in an ice-water bath and stood in the ice-water bath for 5 minutes; the mixture was centrifuged at 4 C. and supernatant was discarded. The pellet was a mixture of cellulose and cellulase which can bind to cellulose.

(48) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiose phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol-3-phosphate synthase, 2 U/mL inositol monophosphate and 10 g/L of the mixture of cellulose and cellulase, was subjected to catalytic reaction at 50 C. for 72 hours. The final concentration of the final inositol was 4.8 g/L and the yield reached 48%.

Example 9. Conversion of Cellulose to Inositol Through In Vitro Multi-Enzyme Catalysis

(49) Cellulase is from Sigma, Cat No. C2730. The preparation of phosphoglucomutase, inositol-3-phophate synthase and inositol monophosphatase are the same as in Example 1; the preparations of cellodextrin phosphorylase and cellobiose phosphorylase are the same as in Example 7.

(50) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiose phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol 3-phosphate synthase, 2 U/mL inositol monophosphate and 10 g/L of the mixture of cellulose and cellulose in Example 8, was subjected to catalytic reaction at 40 C. for 72 hours. The final concentration of the final inositol was 2.3 g/L and the yield reached 23%.

Example 10. Conversion of Cellulose to Inositol Through In Vitro Multi-Enzyme Catalysis

(51) Cellulase is from Sigma, Cat No. C2730; the preparation of phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1; the preparation of cellodextrin phosphorylase and cellobiose phosphorylase are the same as in Example 7.

(52) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiose phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol 3-phosphate synthase, 2 U/mL inositol monophosphate and 10 g/L of the mixture of cellulose and cellulose in Example 8, was subjected to catalytic reaction at 80 C. for 72 hours. The final concentration of the final inositol was 1.9 g/L and the yield reached 19%.

Example 11. Conversion of Cellulose to Inositol Through Multi-Enzyme Catalysis In Vitro

(53) Since the final product after cellulose hydrolysis is glucose, it is necessary to add polyphosphate glucokinase and polyphosphoric acid to convert it to inositol.

(54) Cellulase is from Sigma, Cat No. C2730; the preparation of phosphoglucomutase, inositol 3-phophate synthase and inositol monophosphatase are the same as in Example 1; the preparation of polyphosphate glucokinase is the same as in Example 4; and the preparation of cellodextrin phosphorylase and cellobiose phosphorylase are the same as in Example 7.

(55) A 0.75 ml reaction system comprising 100 mM HEPES buffer (pH 7.2), 10 mM inorganic phosphate, 5 mM divalent magnesium ion, 0.5 mM zinc ion, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiose phosphorylase, 1 U/mL phosphoglucomutase, 5 U/mL inositol 3-phosphate synthase, 2 U/mL inositol monophosphate, 10 g/L of the mixture of cellulose and cellulose in Example 8, 5 U/mL polyphosphate glucokinase and 10 mM sodium polyphosphate, was subjected to catalytic reaction at 50 C. for 72 hours. The final concentration of the final inositol (FIG. 8) was 6.5 g/L and the yield reached 65%.