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A method for producing inositol by complete phosphorylation of cellulose

A technology for inositol and polyphosphoric acid, applied in the field of enzymatic catalysis preparation of inositol, can solve problems such as inability to meet industrialization requirements, increase in inositol production cost, difficulty in separation and purification of inositol, and achieve cheap synthesis and production with low production cost , a rich source of effects

Active Publication Date: 2022-04-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The defects in these two aspects will lead to an increase in the production cost of myo-inositol. At the same time, the low conversion rate will also lead to high content of by-products, and the separation and purification of myo-inositol will be difficult, thereby further increasing the cost and failing to meet the requirements of industrialization.

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  • A method for producing inositol by complete phosphorylation of cellulose
  • A method for producing inositol by complete phosphorylation of cellulose
  • A method for producing inositol by complete phosphorylation of cellulose

Examples

Experimental program
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Embodiment 1

[0057] Example 1 Production of inositol from cellulose catalyzed by cellopolysaccharide phosphorylase

[0058] The catalytic pathways for the complete phosphorylation of cellulose into inositol or the production of hydrogen and electricity through an in vitro multi-enzyme catalytic system are shown in figure 1 . The key enzymes involved in catalyzing the inositol-producing pathway of cellopolysaccharide include: (1) cellopolysaccharide phosphorylase (CDP, EC2.4.1.49), which is used to release glucose 1-phosphate from cellopolysaccharide until cellobiose; ( 2) Cellobiose phosphorylase (CBP, EC 2.4.1.20), used to phosphorylate cellobiose into glucose 1-phosphate and glucose; (3) Polyphosphoglucokinase (PPGK, EC 2.7.1.63) used to Phosphorylation of glucose to glucose 6-phosphate; (4) phosphoglucomutase (PGM, EC 5.4.2.2), for the isomerization of glucose 1-phosphate to glucose 6-phosphate; (5) inositol-1 - phosphate synthase (IPS, EC 5.5.1.4), for the conversion of glucose 6-pho...

Embodiment 2

[0065] Example 2 Utilizing cellopolysaccharide phosphorylase and cellobiose phosphorylase to catalyze the production of inositol from cellulose

[0066] Cellobiose phosphorylase cannot completely phosphorylate cellopolysaccharide to generate glucose 1-phosphate, and adding cellobiose phosphorylase to the reaction system can increase the substrate conversion rate.

[0067] In this example, cellobiose phosphorylase is derived from Clostridium thermocellum (Clostridium thermocellum), and its gene number is Cthe_0275; the gene is obtained by PCR, and cloned into pET20b vector by Simple Cloning method to obtain corresponding expression Vector pET20b-ctcbp. Then, this plasmid was transformed into Escherichia coli expression strain BL21 (DE3), and protein expression and purification were carried out. The results of protein purification were as follows: image 3 As shown in A. The basic information of the cellobiose phosphorylase used in this example is shown in Table 1.

[0068] A...

Embodiment 3

[0070] Example 3 In vitro multi-enzyme system complete phosphorylation of cellulose to produce inositol

[0071] In order to further increase the conversion rate of inositol, polyphosphoglucokinase (PPGK, EC2.7.1.63) and sodium polyphosphate were added to the reaction system to phosphorylate glucose to generate glucose 6-phosphate, which was then converted into inositol.

[0072] In this example, polyphosphoglucokinase is derived from Thermobifida fusca, and its gene number is Tfu_1811; the gene is obtained by PCR, and cloned into pET20b vector by Simple Cloning method to obtain corresponding expression Vector pET20b-tfuppgk. The polyphosphate glucokinase used in this example is the mutant 4-1 (Zhou W, Huang R, Zhu Z, Zhang Y.-H.P.J., Coevolution of both thermostability and activity of polyphosphate glucokinase from Thermobifida fusca) after thermostability modification YX.Appl.Environ.Microbiol.2018.doi:10.1128 / AEM.01224-18), protein expression and purification results are a...

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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 complete phosphorylation of cellulose. The preparation method of myo-inositol disclosed by the present invention takes pretreated cellulose (the main component is cellopolysaccharide) as the substrate of the enzyme-catalyzed reaction; adopts cellopolysaccharide phosphorylase, cellobiose phosphorylase, polyphosphate glucose Kinases, phosphoglutaminase, inositol‑1‑phosphate synthase, and inositol monophosphatase catalyze the complete phosphorolysis of celloglycans to inositol. By optimizing the multi-enzyme reaction process and controlling the temperature of the reaction process in stages, the conversion efficiency of the substrate and the yield of inositol are significantly improved, and the reaction time is shortened. This technical method realizes the complete phosphorylation of cellulose substrates, and can also be used in other in vitro multi-enzyme reaction systems to catalyze cellopolysaccharides to produce hydrogen, generate electricity, or produce other bio-based chemicals.

Description

technical field [0001] The invention relates to a preparation method of inositol, in particular to a method for producing inositol by catalyzing the complete phosphorylation of cellopolysaccharide by an in vitro multi-enzyme system, and belongs to the field of enzyme-catalyzed preparation of inositol. Background technique [0002] Inositol belongs to the water-soluble vitamin B group and is an essential substance for the growth of humans, animals and microorganisms. It is widely used in food, feed, medicine and other industries. At present, the main production method of inositol is high-temperature pressurized hydrolysis of phytic acid, which has high energy consumption, strict requirements on process equipment materials, and serious environmental pollution. In recent years, an in vitro multi-enzyme reaction system was used to catalyze the production of myo-inositol from starch (You, C., et al. (2017). An in vitro synthetic biology platform for the industrial biomanufacturin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/02C12P19/14C12P19/02
CPCC12P7/02C12P19/14C12P19/02C12P2203/00C12P2201/00
Inventor 游淳孟冬冬
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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