CN110857443A - Method for producing inositol by complete phosphorization of cellulose - Google Patents

Method for producing inositol by complete phosphorization of cellulose Download PDF

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CN110857443A
CN110857443A CN201810972707.7A CN201810972707A CN110857443A CN 110857443 A CN110857443 A CN 110857443A CN 201810972707 A CN201810972707 A CN 201810972707A CN 110857443 A CN110857443 A CN 110857443A
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游淳
孟冬冬
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention relates to the field of enzyme-catalyzed preparation of inositol, in particular to an enzymatic preparation method for producing inositol by completely phosphorylating cellulose. The preparation method of the inositol disclosed by the invention takes pretreated cellulose (the main component is cellopolysaccharide) as a substrate of enzyme catalytic reaction; the complete phosphorylation conversion of the fibrous polysaccharide into inositol is catalyzed by fibrous polysaccharide phosphorylase, cellobiose phosphorylase, polyphosphoric acid glucokinase, glucose phosphoglucomutase, inositol-1-phosphate synthase and inositol monophosphate enzyme. By optimizing the multienzyme reaction process and controlling the temperature of the reaction process in stages, the conversion efficiency of the substrate and the yield of inositol are obviously improved, and the reaction time is shortened. The technical method realizes the complete phosphorolysis of the cellulose substrate, and can also be used for catalyzing fibrous polysaccharide to produce hydrogen and electricity or produce other bio-based chemicals by other in-vitro multi-enzyme reaction systems.

Description

Method for producing inositol by complete phosphorization of cellulose
Technical Field
The invention relates to a preparation method of inositol, in particular to a method for producing inositol by catalyzing complete phosphorylation of cellopolysaccharide through an in-vitro multi-enzyme system, belonging to the field of enzyme catalysis preparation of inositol.
Background
Inositol belongs to water-soluble vitamin B group, is essential substance for growth of human, animal and microorganism, and is widely applied to industries of food, feed, medicine and the like. The main production method of inositol at present is high-temperature pressurized hydrolysis of phytic acid, and the method has high energy consumption, strict requirements on materials of process equipment and serious environmental pollution. In recent years, the use of in vitro multi-enzyme reaction systems for the catalytic production of inositol from starch (You, C., et al. (2017) An in vitro synthetic biochemical transformation for the induced biological fermentation of myo-inositol from starch, Biotechnology, Bioeng.114(8): 1855) 1864) has become a new method of low pollution and high yield.
In the past decades of research, pretreated cellulose is generally hydrolyzed into glucose by the action of cellulase (endoglucanase, exoglucanase, β -glucanase and the like) and the glucose is fermented into chemicals by microorganisms, cellulose phosphorylation is another cellulose degradation mode, namely, pretreated cellulose is catalyzed by cellulose phosphorylase to generate glucose 1-phosphoric acid on the basis of the existence of inorganic phosphorus until the cellulose is phosphorylated into cellobiose, the cellobiose is catalyzed by the cellulose phosphorylase to generate glucose and glucose 1-phosphoric acid in the presence of inorganic phosphorus, the glucose is catalyzed by polyphosphate to generate glucose 6-phosphate in the presence of polyphosphate, the cellulose phosphorylation can completely phosphorylate the glucan chain into the glucose phosphate, and the energy of the glucan chain in the cellulose phosphorylation is more advantageous than that of the cellulose chain in cellulose hydrolysis, such as β cellulose, cellulose is converted into cellulose with high energy, and the cellulase is capable of converting the energy of the glucan chain into the glucose chain into the cellulose chain.
At present, although the preparation of inositol by using cellulose as a substrate has been reported (Chinese patent application No. 201510184621.4), the conversion rate is far lower than the maximum theoretical conversion rate of 100%, and the catalytic reaction time is too long, usually about 72 hours. The disadvantages of both aspects can cause the production cost of the inositol to be increased, and simultaneously, the low conversion rate can cause the high content of the by-products, and the separation and purification of the inositol are difficult, thereby further increasing the cost and being incapable of meeting the industrialization requirement.
Therefore, it is highly desirable to develop a method for preparing inositol by fully phosphorolysis using cellulose, a non-food biomass resource, as a substrate, at a low cost and with a high conversion rate.
Disclosure of Invention
The invention relates to a method for producing inositol by complete phosphorization of cellulose. The method takes cellulose as a substrate, and fully phosphorylates the cellulose by constructing an in-vitro multi-enzyme catalytic reaction system and optimizing the system so as to synthesize inositol with low cost and high conversion rate. Meanwhile, the method has the advantages of renewable raw materials, rich sources, low price, low pollution, environmental friendliness and the like.
The invention is realized by the following technical scheme:
the invention provides a method for producing inositol by complete phosphorization of cellulose, which comprises the following steps:
cellulose is pretreated to obtain the fibrous polysaccharide.
Using cellopolysaccharide as a substrate, cellopolysaccharide phosphorylase (EC2.4.1.49), phosphoglucomutase (EC 5.4.2.2), inositol-1-phosphate synthase (EC5.5.1.4) and inositol monophosphatase (EC 3.1.3.25) were added to perform a multiple enzyme catalytic reaction.
According to the present invention, pretreatment methods such as acid hydrolysis, enzymatic hydrolysis, physical methods and the like can be used in the present invention. Acid hydrolysis is preferred as the pretreatment method.
According to the present invention, fibrous polysaccharides having a degree of polymerization of less than 100 can be used in the present invention. Preferably, the degree of polymerisation is below 50, more preferably below 20, most preferably between 3 and 7.
According to the present invention, the temperature of the multi-enzyme catalyzed reaction is preferably 10 to 95 ℃, more preferably 20 to 80 ℃, more preferably 30 to 60 ℃, and most preferably 55 ℃.
According to the present invention, the reaction time of the multienzyme catalysis is preferably 0.5 to 150 hours, more preferably 1 to 60 hours, more preferably 6 to 48 hours, most preferably 24 to 36 hours.
According to the present invention, preferably, the concentration of the fibrous polysaccharide in the multi-enzyme catalyzed reaction is 1 to 200g/L, more preferably 5 to 50g/L, still more preferably 8 to 20g/L, and most preferably 10 g/L.
According to the present invention, the amount of the cellopolysaccharide phosphorylase used in the multi-enzyme catalytic reaction is preferably 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, and still more preferably 1 to 5U/mL.
According to the present invention, the phosphoglucomutase is preferably used in an amount of 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, and still more preferably 1 to 5U/mL, in the multi-enzyme catalytic reaction.
According to the present invention, the amount of inositol-1-phosphate synthase used in the multi-enzyme catalytic reaction is preferably 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, and still more preferably 1 to 5U/mL.
According to the present invention, the amount of the phytase to be used in the multi-enzyme catalytic reaction is preferably 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, and still more preferably 1 to 5U/mL.
In a preferred embodiment, the multi-enzyme catalyzed reaction further comprises adding cellobiose phosphorylase (EC 2.4.1.20) for catalyzing cellobiose and phosphate to generate glucose 1-phosphate and glucose.
Preferably, cellobiose phosphorylase is further added after the above-mentioned multi-enzyme catalytic reaction has been carried out for a certain period of time.
Preferably, the cellobiose phosphorylase is used in an amount of 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, and still more preferably 1 to 5U/mL in the multi-enzyme catalytic reaction.
Preferably, the cellobiose phosphorylase is added at 10-95 deg.C for 0.25-10 hr, more preferably at 20-80 deg.C for 0.5-5 hr, even more preferably at 30-60 deg.C for 1-3 hr, and most preferably at 55 deg.C for 2 hr. Preferably, the reaction is continued at 10-95 ℃ for 0.25-50 hours, more preferably at 20-80 ℃ for 5-30 hours, even more preferably at 30-60 ℃ for 10-25 hours, and most preferably at 55 ℃ for 12-24 hours after cellobiose phosphorylase is added in the multi-enzyme catalytic reaction.
In a preferred embodiment, the multi-enzyme catalyzed reaction further comprises the addition of polyphosphate glucokinase (EC 2.7.1.63) and polyphosphate for phosphorylating the by-product glucose to glucose 6-phosphate.
Preferably, the amount of polyphosphate glucokinase used in the multienzyme catalyzed reaction is 0.1-50U/mL, more preferably 0.5-10U/mL, and still more preferably 1-5U/mL.
Preferably, the amount of polyphosphate used in the multi-enzyme catalyzed reaction is between 0.1 and 50mM, more preferably between 1 and 30mM, even more preferably between 5 and 20mM, and most preferably 10 mM.
Preferably, the multienzyme catalyzed reaction is carried out at 10-95 ℃ for 0.25-15 hours, more preferably at 20-80 ℃ for 0.5-10 hours, even more preferably at 30-60 ℃ for 2-8 hours, and most preferably at 55 ℃ for 6 hours, with the addition of polyphosphate glucokinase and polyphosphate.
Preferably, the reaction is continued at 10-95 ℃ for 0.25-50 hours, more preferably at 20-80 ℃ for 5-30 hours, even more preferably at 30-60 ℃ for 10-25 hours, and most preferably at 55 ℃ for 12-24 hours after adding polyphosphate glucokinase and polyphosphate in the multienzyme catalyzed reaction.
According to the present invention, preferably, a buffer, a phosphate, a magnesium salt, and a reducing agent are further added to the multi-enzyme catalytic reaction.
It will be understood by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer such as sodium citrate buffer, etc., preferably, the buffer is HEPES buffer. Preferably, the pH of the buffer is 5.0-8.5, more preferably 6.0-8, and most preferably 7.5. Preferably, the concentration of the buffer in the reaction system is 10 to 500mM, more preferably 20 to 150mM, still more preferably 50 to 120mM, and most preferably 100 mM.
It will be appreciated by those skilled in the art that various phosphates may be used in the present invention, such as potassium phosphate, sodium phosphate, and the like, preferably the phosphate is potassium phosphate. Preferably, the concentration of phosphate in the reaction system is 1 to 50mM, more preferably 2 to 30mM, still more preferably 5 to 25mM, and most preferably 20 mM.
It will be appreciated by those skilled in the art that various magnesium salts may be used in the present invention, such as magnesium chloride, magnesium sulfate, and the like, preferably the magnesium salt is magnesium chloride. Preferably, the concentration of the magnesium salt in the reaction system is 1 to 20mM, more preferably 2 to 15mM, still more preferably 3 to 10mM, and most preferably 5 mM.
Preferably, the reducing agent is dithiothreitol. Preferably, the concentration of dithiothreitol in the reaction catalyzed by the multienzyme is 1-20mM, more preferably 2-15mM, even more preferably 3-10mM, and most preferably 5 mM.
In order to further increase the yield of inositol, in a preferred embodiment, the multi-enzyme catalyzed reaction is supplemented with magnesium ions after a period of time.
Preferably, magnesium ions are additionally added when the concentration of magnesium ions is below 0.1-5mM, more preferably, magnesium ions are additionally added when the concentration of magnesium ions is below 0.2-3mM, even more preferably, magnesium ions are additionally added when the concentration of magnesium ions is below 0.5-1.5mM, and most preferably, magnesium ions are additionally added when the concentration of magnesium ions is below 1 mM.
Preferably, the concentration of the additionally added magnesium ions is 1 to 30mM, more preferably 5 to 20mM, most preferably 15 mM.
In order to shorten the reaction time and increase the yield of inositol, in a preferred embodiment, the reaction temperature of the catalytic reaction is increased after a period of time. Preferably, the reaction temperature of the multiple enzyme catalysis reaction is increased after the reaction is performed for 0.5 to 20 hours, further preferably, the reaction temperature of the multiple enzyme catalysis reaction is increased after the reaction is performed for 1 to 15 hours, more preferably, the reaction temperature of the multiple enzyme catalysis reaction is increased after the reaction is performed for 5 to 10 hours, and most preferably, the reaction temperature of the multiple enzyme catalysis reaction is increased after the reaction is performed for 8 hours.
Preferably, the reaction temperature of the multi-enzyme catalyzed reaction is increased to 60-100 deg.C, more preferably 65-80 deg.C, and most preferably 70 deg.C after a period of time.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. the method adopts the cellopolysaccharide phosphorylase, the cellobiose phosphorylase and the polyphosphoric acid glucose kinase to construct an in-vitro multi-enzyme reaction system, and fully phosphorylates the cellopolysaccharide into glucose phosphate by accurately controlling the adding sequence and the adding time of the enzymes, so that the complete phosphorylation of cellulose is realized;
2. the invention firstly converts the cellulose into the inositol by controlling the reaction temperature of the multi-enzyme catalytic reaction and the content of the magnesium salt, and compared with the constant reaction temperature, the invention shortens the reaction time and improves the conversion rate of the inositol by improving the temperature of a later reaction system. Meanwhile, the substrate of the invention is cellulose, has the advantages of low price, rich source, renewability and the like, and can realize the cheap synthesis and production of inositol;
3. the method has the advantages of high product conversion rate (close to the theoretical value of 100%), short reaction time, low production cost, environmental friendliness and the like, and is favorable for industrial production of inositol.
4. In addition, the method for completely phosphorylating cellulose can also be applied to an in-vitro multi-enzyme reaction system for producing hydrogen, electricity or other bio-based chemicals.
Drawings
FIG. 1 is a schematic diagram of the in vitro multi-enzyme catalytic pathway for complete phosphorylation of cellulose to myo-inositol and other biological energy sources. Wherein, CDP is cellopolysaccharide phosphorylase, CBP is cellobiose phosphorylase, PPGK is polyphosphate glucokinase, PGM is phosphoglucomutase, IPS is inositol-1-phosphate synthase, and IMP is inositol monophosphatase; piIs inorganic phosphorus, (P)i)nAnd (P)i)n-1Is sodium polyphosphate.
FIG. 2 shows the in vitro multi-enzyme system consisting of CDP, PGM, IPS and IMP catalyzing the production of inositol from cellopolysaccharide. FIG. 2A shows SDS-PAGE detecting 4 enzymes, where M is Marker, 1 is CDP, 2 is PGM, 3 is IPS, and 4 is IMP; FIG. 2B is a graph showing the time course of inositol production from 4 enzymes catalyzed by cellopolysaccharide at 55 ℃.
FIG. 3 shows the in vitro multi-enzyme system consisting of CDP, CBP, PGM, IPS and IMP catalyzing the production of inositol from cellopolysaccharide. Wherein, FIG. 3A shows the detection of CBP by SDS-PAGE, and M is Marker; FIG. 3B is a graph showing the time course of inositol production from cellobiose catalyzed by 5 enzymes at 55 ℃. The reaction was run for 2 hours and CBP was added.
FIG. 4 shows the condition of inositol production from cellopolysaccharide catalyzed by multi-enzyme system consisting of CDP, CBP, PPGK, PGM, IPS and IMP in vitro. Wherein, FIG. 4A shows the detection of PPGK by SDS-PAGE, and M is Marker; FIG. 4B is a graph showing the time course of inositol production from cellobiose catalyzed by 6 enzymes at 55 ℃. The reaction was carried out for 6 hours, and PPGK and sodium polyphosphate were added.
FIG. 5 is a time course curve of in vitro multi-enzyme system composed of PPGK, sodium polyphosphate, magnesium chloride, CDP, CBP, PPGK, PGM, IPS and IMP for catalyzing the production of inositol from cellopolysaccharide when the reaction is carried out for 6 hours at 55 ℃.
FIG. 6 is a graph showing the time course of the reaction for 8 hours, the reaction temperature is increased from 55 ℃ to 70 ℃, and the inositol is produced by catalyzing the cellopolysaccharide with the in vitro multi-enzyme system consisting of CDP, CBP, PPGK, PGM, IPS and IMP. In curve a, reacting for 6 hours, and adding PPGK, sodium polyphosphate and magnesium chloride into the reaction system; curve b, reaction for 6 hours, PPGK and sodium polyphosphate were added to the reaction system.
FIG. 7 shows the inositol production from 50g/L cellopolysaccharide or corn stalk hydrolysate catalyzed by an in vitro multi-enzyme system consisting of CDP, CBP, PPGK, PGM, IPS and IMP.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Experimental Material
Microcrystalline cellulose, Avicel PH-105, product of FMC (Philadelphia, PA, USA);
pET20b vector, Novagen, Madison, WI;
coli expression strain BL21(DE3), Invitrogen, Carlsbad, CA;
all enzymes (except for phytase and polyphosphate glucokinase) in the present invention can be purchased from Sigma company, and all enzymes can also be obtained by prokaryotic expression according to a genetic engineering method.
Example 1 production of inositol from cellulose Using a Cellulose Phosphorylase
The catalytic pathways for complete phospholysis of cellulose to inositol or hydrogen and electricity production by in vitro multi-enzyme catalytic systems are shown in figure 1. Among the key enzymes involved in the pathway for the production of inositol from cellopolysaccharides are: (1) a cellopolysaccharide phosphorylase (CDP, ec2.4.1.49) for releasing glucose 1-phosphate from cellopolysaccharide up to cellobiose; (2) cellobiose phosphorylase (CBP, EC 2.4.1.20) for phospho-lysis of cellobiose to glucose 1-phosphate and glucose; (3) polyphosphate glucokinase (PPGK, EC2.7.1.63) for phosphorylating glucose to glucose 6-phosphate; (4) glucose phosphoglucomutase (PGM, EC 5.4.2.2) for isomerising glucose 1-phosphate into glucose 6-phosphate; (5) inositol-1-phosphate synthase (IPS, EC5.5.1.4) for converting glucose 6-phosphate into inositol 1-phosphate; (6) inositol monophosphatase (IMP, ec3.1.3.25) for dephosphorylation of inositol 1-phosphate to inositol.
In the present invention, cellulose is completely phosphorylated to form glucose 6-phosphate, which can enter other In vitro multi-enzyme reaction systems to form hydrogen gas (Kim et al, (2017) Advanced water separation for green hydrogen production through complete oxidation of state by In vitro catalysis In which carbon dioxide is introduced, method. Eng.44: 246. 252), electricity production (Zhu, Z., et al, (2017) In vitro catalysis In which carbon dioxide is introduced, or other chemicals (Wang, W., et al, (2017) ATP-front of glucose-mediated synthesis In which carbon dioxide is introduced, method. Eng.39: 110. 116) or other chemicals (Wang, W., et al, (2017) ATP-front of glucose-mediated synthesis In which carbon dioxide is introduced, method. end. 1. 32. 3542).
In this example, microcrystalline cellulose Avicel PH-105 was subjected to acid hydrolysis (Zhang, Y-HP., et al (2003), cellulose precipitation by mixed-acid hydrolysis and chromatography biochemistry.322(2):225-232), prepared into a cellulose having a degree of polymerization of 3 to 7 (average degree of polymerization of 4.4), and added to a multienzyme reaction system for catalytic reaction.
In this example, the cellopolysaccharide phosphorylase is derived from Clostridium thermocellum (Clostridium thermocellum) with gene number Cth _ 2989; the phosphoglucomutase is derived from thermophilic archaea Thermococcus kodakarensis, and the gene number of the phosphoglucomutase is TK 1108; inositol-1-phosphate synthetase is derived from Archaeoglobus fulgidus, and has a gene number of AF-1794; inositol monophosphatase is derived from Thermotoga maritima (Thermotoga maritima) and has the gene number TM 1415. These genomic DNAs are all available from the ATCC's official website (www.atcc.org). The corresponding expression vectors pET21C-ctcdp, pET20 b-tkmm, pET20 pg 20b-afips and pET20b-tmimp were obtained by PCR from the corresponding genomic DNA using different primers, respectively, and cloned into pET vector (Novagen, Madison, Wis.) by the method of Simple Cloning (You C, Zhang XZ, Zhang Y-HP.2012.Simple Cloning via direct transformation of PCR products (DNA Multimer) to Escherichia coli and Bacillus subtilis). Then, these plasmids were transformed into E.coli BL21(DE3) (Invitrogen, Carlsbad, Calif.) respectively, and protein expression and purification were carried out, and the results of protein purification are shown in FIG. 2A.
The information on the enzymes used in this example is shown in Table 1.
A1.0 mL reaction system contained 10g/L of a fibrous polysaccharide having an average degree of polymerization of 4.4, 100mM of HEPES buffer (pH 7.5), 5mM of magnesium chloride, 20mM of potassium phosphate, 5mM of dithiothreitol, 1U/mL of a fibrous polysaccharide phosphorylase, 1U/mL of a phosphoglucomutase, 2U/mL of an inositol-1-phosphate synthase, and 1.5U/mL of a phytase. The catalytic reaction was carried out at 55 ℃ for 24 hours. The concentrations of inositol and cellobiose produced by the reaction system were determined by high performance liquid chromatography. The conditions of the high performance liquid chromatography are as follows: HPX-87H chromatographic column (Bio-Rad), 5mM sulfuric acid solution as mobile phase, column temperature 60 deg.C, differential refractometer. The concentration of glucose produced in the reaction system was measured by a glucose kit (Beijing prilley Gene technology Co., Ltd., product No. E1010).
As shown in FIG. 2B, the concentration of inositol gradually increased with time to reach 4.64g/L after 12 hours and remained constant; the concentration of cellobiose increased rapidly, reaching 4.61g/L after 2 hours, and remained constant. Thus, the mass conversion of inositol to cellopolysaccharide was 46%.
Example 2 production of inositol from cellulose Using Cellulose Phosphorylase and Cellobiose Phosphorylase
The cellopolysaccharide phosphorylase cannot completely phosphorylate the cellopolysaccharide to generate glucose 1-phosphate, and the cellobiose phosphorylase is added into a reaction system to improve the substrate conversion rate.
In this example, cellobiose phosphorylase is derived from Clostridium thermocellum (Clostridium thermocellum) with gene number Cth _ 0275; the gene is obtained by PCR and cloned into pET20b vector by Simple Cloning method, and the corresponding expression vector pET20b-ctcbp is obtained. Then, this plasmid was transformed into E.coli expression strain BL21(DE3) and protein expression and purification were carried out, and the results of protein purification are shown in FIG. 3A. The basic information of cellobiose phosphorylase used in this example is shown in Table 1.
According to the experimental result of example 1, when the concentration of cellobiose in the reaction system reached the maximum value, i.e., the reaction was carried out for 2 hours, 1U/mL cellobiose phosphorylase was added to the multi-enzyme catalyst system, and the catalytic reaction was carried out at 55 ℃ for 24 hours.
Through high performance liquid chromatography detection, as shown in FIG. 3B, the concentration of cellobiose gradually decreased, and at 6 hours, the concentration was 0.11 g/L; the concentration of inositol gradually increased, at 6 hours and 24 hours, the concentrations were 5.62g/L and 5.99g/L, respectively. At 24 hours, the glucose concentration was 2.34 g/L. Thus, the mass conversion of inositol to cellopolysaccharide was 60%.
Example 3 in vitro Multi-enzyme System complete phosphohydrolysis of cellulose to inositol
In order to further improve the conversion rate of inositol, polyphosphate glucokinase (PPGK, EC2.7.1.63) and sodium polyphosphate are added into a reaction system for phosphorylating glucose to generate glucose 6-phosphate, and then the glucose is converted into inositol.
In this example, polyphosphate glucokinase is derived from Thermobifida fusca (Thermobifida fusca) and has the gene number of Tfu _ 1811; the gene was obtained by PCR and cloned into pET20b vector by Simple Cloning to obtain the corresponding expression vector pET20 b-tfuppggk. The polyphosphate glucokinase used in this example is a mutant 4-1(Zhou W, Huang R, Zhu Z, Zhang Y. -H.P.J., Cooviolation of bath thermal stability and activity of polyphosphate glucokinase from Thermobifida fusca YX.Appl.Environ.Microbiol.2018.doi:10.1128/AEM.01224-18) which is modified in thermal stability, and the results of protein expression and purification are shown in FIG. 4A, and the basic information is shown in Table 1.
According to the experimental result of example 2, when the concentration of glucose in the reaction system reached the maximum value, i.e., the reaction was carried out for 6 hours, 1U/mL of polyphosphoric acid glucokinase and 10mM of sodium polyphosphate were added to the multi-enzyme catalytic system, and the catalytic reaction was carried out at 55 ℃ for 24 hours.
As shown in FIG. 4B, the glucose concentration was close to 0g/L at 8 hours. The inositol concentration is 6.89g/L at 24 hours by high performance liquid chromatography detection. Thus, the mass conversion of inositol to cellopolysaccharide was 69%. The reaction was carried out for 8 hours by using a magnesium ion concentration measuring kit (Beijing Soilebao Tech Co., Ltd., cat. No.: BC2790), and the magnesium ion concentration was 0.38mM, which was much lower than that of the initially charged magnesium ion.
Example 4 optimization of the in vitro Multi-enzyme reaction System to further increase the yield of inositol
Low concentrations of magnesium ions reduce the specific enzyme activity of inositol-1-phosphate synthase and inositol monophosphatase in the pathway leading to inositol. According to the experimental result of example 3, 1U/mL of glucokinase polyphosphate, 10mM of sodium polyphosphate and 15mM of magnesium chloride were added to the reaction system at 6 hours of the reaction. The catalytic reaction was carried out at 55 ℃ for up to 60 hours.
As shown in FIG. 5, the concentration of inositol was 9.81g/L at 60 hours. Thus, the mass conversion of inositol to cellopolysaccharide was 98%.
Example 5 increasing the temperature of the in vitro multienzyme reaction System and shortening the reaction time
Inositol-1-phosphate synthetase has very low specific enzyme activity at a temperature lower than 60 ℃ (Chen, L.J., et al. (2000) inhibitor-1-phosphate synthase from Archaeoglobus fulgidis a class II biochemical, 39(40): 12415) 12423), and can improve the catalytic capability of Inositol-1-phosphate synthetase by increasing the reaction temperature of the reaction system, thereby shortening the reaction time. The specific activities of phosphoglucomutase, inositol-1-phosphate synthase and phytase at 70 ℃ are shown in Table 1.
According to the experimental result of example 4, reaction was carried out for 6 hours by adding 1U/mL of polyphosphoric acid glucokinase, 10mM of sodium polyphosphate and 15mM of magnesium chloride to the reaction system; after 8 hours of reaction, the temperature of the multienzyme system was raised to 70 ℃. As shown in FIG. 6, curve a, the concentration of inositol was 9.9g/L at 24 hours. Thus, the mass conversion of inositol to cellopolysaccharide was 99%.
Reacting for 6 hours, and adding 1U/mL polyphosphoric acid glucokinase and 10mM sodium polyphosphate into a reaction system; after 8 hours of reaction, the temperature of the multienzyme system was raised to 70 ℃. The reaction was carried out for 12 hours, and the concentration of magnesium ions in the reaction system was 4.6mM, which was equivalent to the concentration of magnesium ions initially charged, which was 5 mM. At 24 hours, the concentration of inositol was 9.78g/L, as shown in FIG. 6, Curve b. Thus, the mass conversion of inositol to cellopolysaccharide was 98%.
Example 6 production of inositol Using an in vitro Multi-enzyme reaction System to catalyze high concentrations of cellulosic substrates
In this example, the substrate used was 50g/L of a cellulose polysaccharide (average degree of polymerization of 4.4) or corn stover hydrolysate. The corn straws pretreated by gas explosion (1 MPa for 10 minutes) are crushed and sieved by a 40-60 mesh sieve. Corn stover hydrolysate was prepared according to the literature (Zhang, Y-HP., et al (2003), cellulosic hydrolysis by inorganic and biochemical separation. 322(2): 225-232). Carrying out acidolysis for 4 hours at 22 ℃ by using a mixed solution of sulfuric acid and hydrochloric acid, and dissolving the fibrous polysaccharide into the aqueous solution through the steps of acetone precipitation, vacuum filtration and the like. And regulating the pH value to 7.0 by barium hydroxide to prepare the corn straw hydrolysate.
According to the procedure of example 5, a HEPES buffer solution having a pH of 7.5 was added to a reaction system at a concentration of 100mM, a potassium phosphate concentration of 50mM, a magnesium chloride concentration of 20mM, a dithiothreitol concentration of 5mM, a cellopolysaccharide or corn stover hydrolysate concentration of 50g/L, a cellopolysaccharide phosphorylase amount of 5U/mL, a phosphoglucomutase amount of 5U/mL, an inositol-1-phosphate synthase amount of 10U/mL, and a phytase amount of 7.5U/mL; the reaction system is carried out for 2 hours at 55 ℃, and the dosage of the added cellobiose phosphorylase is 5U/mL; the reaction system is carried out for 6 hours at 55 ℃, the dosage of the polyphosphate glucokinase is 5U/mL, and the dosage of the polyphosphate sodium is 50 mM; the reaction system is carried out for 8 hours at the temperature of 55 ℃, and the temperature of the reaction system is increased to 70 ℃; finally, in the reaction system taking the fiber polysaccharide or the corn stalk hydrolysate as the substrate, the output of inositol is 36.3g/L and 15.3g/L respectively (figure 7).
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
TABLE 1 information on the enzymes used in the present invention
Figure BDA0001776627090000111
aThe measurement of the specific enzyme activity of the cellopolysaccharide phosphorylase was carried out at 55 ℃ using a cellopolysaccharide having an average degree of polymerization of 4.4 as a substrate.

Claims (10)

1. A method for preparing inositol by using in vitro enzyme catalysis reaction is characterized by comprising the following steps: (1) pretreating cellulose to obtain fibrous polysaccharide; (2) with cellopolysaccharide as a substrate, cellopolysaccharide phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2), inositol-1-phosphate synthase (EC 5.5.1.4) and inositol monophosphatase (EC 3.1.3.25) were added to perform a multiple enzyme catalytic reaction.
2. The method of claim 1, wherein the multi-enzyme catalyzed reaction further comprises adding cellobiose phosphorylase (EC 2.4.1.20) for catalyzing cellobiose and phosphate to glucose 1-phosphate and glucose.
Preferably, the cellobiose phosphorylase is added after the multi-enzyme catalytic reaction has been carried out for a period of time.
Preferably, the cellobiose phosphorylase is used in an amount of 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, and still more preferably 1 to 5U/mL.
Preferably, the reaction catalyzed by the multi-enzyme is carried out at 10-95 ℃ for 0.25-10 hours, more preferably at 20-80 ℃ for 0.5-5 hours, even more preferably at 30-60 ℃ for 1-3 hours, and most preferably at 55 ℃ for 2 hours, and cellobiose phosphorylase is added.
Preferably, the reaction is continued at 10-95 ℃ for 0.25-50 hours, more preferably at 20-80 ℃ for 5-30 hours, even more preferably at 30-60 ℃ for 10-25 hours, and most preferably at 55 ℃ for 12-24 hours after adding cellobiose phosphorylase.
3. The method of claim 2, wherein the multi-enzyme catalyzed reaction further comprises the addition of polyphosphate glucokinase (EC 2.7.1.63) and polyphosphate for catalyzing the production of glucose 6-phosphate from glucose and polyphosphate.
Preferably, the multienzyme catalyzed reaction is carried out for a period of time before the addition of polyphosphate glucokinase and polyphosphate.
Preferably, the amount of polyphosphate glucokinase is 0.1-50U/mL, more preferably 0.5-10U/mL, and still more preferably 1-5U/mL.
Preferably, the polyphosphate is used in an amount of 0.1 to 50mM, more preferably 1 to 30mM, more preferably 5 to 20mM, most preferably 10 mM.
Preferably, the multienzyme catalyzed reaction is carried out at 10-95 ℃ for 0.25-15 hours, more preferably at 20-80 ℃ for 0.5-10 hours, even more preferably at 30-60 ℃ for 2-8 hours, and most preferably at 55 ℃ for 6 hours, with the addition of polyphosphate glucokinase and polyphosphate.
Preferably, the reaction is continued at 10-95 ℃ for 0.25-50 hours, more preferably at 20-80 ℃ for 5-30 hours, even more preferably at 30-60 ℃ for 10-25 hours, and most preferably at 55 ℃ for 12-24 hours after adding polyphosphate glucokinase and polyphosphate in the multi-enzyme catalyzed reaction.
4. The method of any one of claims 1 to 3, wherein the multi-enzyme catalytic reaction further comprises a buffer.
Preferably, the buffer is selected from the group consisting of HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer such as sodium citrate buffer;
preferably, the buffer is HEPES buffer.
Preferably, the pH of the buffer is 5.0-8.5, more preferably 6.0-8.0, and most preferably 7.5.
Preferably, the buffer concentration is 10-500mM, more preferably 20-150mM, more preferably 50-120mM, most preferably 100 mM.
Preferably, the multi-enzyme catalytic reaction further comprises phosphate.
Preferably, the phosphate is selected from potassium phosphate, sodium phosphate; preferably, the phosphate is potassium phosphate.
Preferably, the phosphate concentration is 1-50mM, more preferably 2-30mM, more preferably 5-25mM, most preferably 20 mM.
Preferably, the multi-enzyme catalytic reaction further comprises a magnesium salt.
Preferably, the magnesium salt is selected from magnesium chloride, magnesium sulfate; preferably, the magnesium salt is magnesium chloride.
Preferably, the concentration of magnesium salt is 1-20mM, more preferably 2-15mM, more preferably 3-10mM, most preferably 5 mM.
Preferably, the multi-enzyme catalytic reaction further comprises a reducing agent.
Preferably, the reducing agent is dithiothreitol.
Preferably, the concentration of the reducing agent is 1-20mM, more preferably 2-15mM, more preferably 3-10mM, most preferably 5 mM.
5. The method according to any one of claims 1 to 4, wherein the cellulose is pretreated by a method comprising acid hydrolysis, enzymatic hydrolysis, physical method, preferably acid hydrolysis.
6. The method according to any of claims 1 to 5, wherein the temperature of the multi-enzyme catalyzed reaction is 10 to 95 ℃, further preferably 20 to 80 ℃, more preferably 30 to 60 ℃, and most preferably 55 ℃.
Preferably, the time for the multi-enzyme catalytic reaction is 0.5 to 150 hours, more preferably 1 to 60 hours, more preferably 6 to 48 hours, most preferably 24 to 36 hours.
Preferably, the concentration of the fibrous polysaccharide in the multi-enzyme catalyzed reaction is 1-200g/L, more preferably 5-50g/L, more preferably 8-20g/L, and most preferably 10 g/L.
Preferably, the dosage of the cellopolysaccharide phosphorylase in the multi-enzyme catalytic reaction is 0.1-50U/mL, more preferably 0.5-10U/mL, and still more preferably 1-5U/mL.
Preferably, the amount of glucose phosphate converted into enzyme in the multi-enzyme catalytic reaction is 0.1-50U/mL, more preferably 0.5-10U/mL, and still more preferably 1-5U/mL.
Preferably, the amount of inositol-1-phosphate synthase used in the multi-enzyme catalyzed reaction is 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, and still more preferably 1 to 5U/mL.
Preferably, the amount of the phytase used in the multi-enzyme catalytic reaction is 0.1-50U/mL, more preferably 0.5-10U/mL, and still more preferably 1-5U/mL.
7. The method according to any one of claims 4 to 6, wherein magnesium ions are additionally added when the concentration of magnesium ions is reduced in the multi-enzyme catalytic reaction.
Preferably, magnesium ions are additionally added when the concentration of magnesium ions is lower than 0.1-5mM in the multi-enzyme catalytic reaction, more preferably, magnesium ions are additionally added when the concentration of magnesium ions is lower than 0.2-3mM, even more preferably, magnesium ions are additionally added when the concentration of magnesium ions is lower than 0.5-1.5mM, and most preferably, magnesium ions are additionally added when the concentration of magnesium ions is lower than 1 mM.
Preferably, the concentration of the additionally added magnesium ions is 1 to 30mM, more preferably 5 to 20mM, most preferably 15 mM.
8. The method of any one of claims 1 to 7, wherein the temperature of the catalytic reaction is increased after a period of time.
9. The method according to claim 8, wherein the reaction temperature of the multi-enzyme catalysis reaction is increased after the reaction is carried out for 0.5 to 20 hours; further preferably, the reaction temperature of the multi-enzyme catalysis reaction is increased after the reaction is carried out for 1 to 15 hours; more preferably, the temperature of the multi-enzyme catalyzed reaction is increased after the reaction has proceeded for 5 to 10 hours; most preferably, the temperature of the multi-enzyme catalyzed reaction is increased after the reaction has been carried out for 8 hours.
10. A method according to claim 8 or 9, characterized in that the catalytic reaction temperature is increased to 60-100 ℃, further preferably 65-80 ℃, most preferably 70 ℃.
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