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Preparation method and application of immobilized cells for mannose production

A technology for immobilizing cells and mannose, which is applied in the field of bioengineering and mannose production and preparation, can solve the problems of high mannose production cost, low enzyme recovery utilization rate, cumbersome production steps, etc., and achieve simple immobilization process, effective It is beneficial to separation and purification, and the effect of simplifying the production and preparation process

Active Publication Date: 2021-08-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problems existing in the existing multi-enzyme catalyzed method for preparing mannose, such as cumbersome production steps of the enzyme, complicated separation and purification, low enzyme recovery utilization rate, difficulty in recycling and high production cost of mannose, the purpose of the present invention It aims to provide a method for immobilized cells to produce mannose, simplify the production steps of enzymes in the mannose production process, simplify the separation and purification of products and enzymes in the mannose preparation process, and realize the synthesis of multiple enzymes in the new multi-enzyme synthesis route of mannose Recycling, reducing the production cost of mannose, and realizing the industrial production of mannose

Method used

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  • Preparation method and application of immobilized cells for mannose production
  • Preparation method and application of immobilized cells for mannose production
  • Preparation method and application of immobilized cells for mannose production

Examples

Experimental program
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Embodiment 1

[0039] Embodiment 1: the preparation of the Bacillus subtilis fermented liquid expressing enzyme

[0040] (1) Construction of pMA5-Pylb-aGP

[0041] In this example, the agp sequence (NCBI-ProteinID: BAD85595), the gene encoding thermostable a-glucan phosphorylase, was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and spliced ​​into a common plasmid. The thermostable α-glucan phosphorylase gene agp gene was obtained by PCR using a pair of primers (1-IF and 1-IR). The linear backbone of pMA5‐Pylb was obtained by PCR using a pair of primers (1-VF and 1-VR). The thermostable α-glucan phosphorylase gene fragment and the pMA5-Pylb vector backbone were then assembled using POE-PCR. The ligation product was transferred into competent SCK6 by the calcium chloride method, and the transformant was selected for colony PCR, double enzyme digestion identification and sequencing verification to obtain an expression vector, which was named pMA5-Pylb-aGP.

[0042] 1-IF: AGAAACAAC...

Embodiment 2

[0072] Embodiment 2: Production of mannose by immobilized Bacillus subtilis

[0073] According to the OD600 ratio of 1:1:1:1:1, the Bacillus subtilis fermentation broth expressing thermostable α-glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented subtilis fermentation broth expressing thermostable glucose phosphoisomerase, Bacillus subtilis fermentation broth expressing thermostable mannose 6-phosphate isomerase, fermentation broth of Bacillus subtilis expressing thermostable mannose 6-phosphate phosphatase The solution was mixed so that OD 600 = 100, and 2% w / v montmorillonite was added to the bacterial suspension, and stirred evenly. Subsequently, 1% w / v polyethyleneimine aqueous solution with a molecular weight of 600 was added to flocculate at room temperature. Then, 0.5% v / v glutaraldehyde aqueous solution was added to crosslink for 2 h at room temperature. The filter cake is obtained after va...

Embodiment 3

[0075] Embodiment 3: Production of mannose by immobilized Bacillus subtilis

[0076] According to the OD600 ratio of 1:1:1:1:1, the Bacillus subtilis fermentation broth expressing thermostable α-glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented subtilis fermentation broth expressing thermostable glucose phosphoisomerase, Bacillus subtilis fermentation broth expressing thermostable mannose 6-phosphate isomerase, fermentation broth of Bacillus subtilis expressing thermostable mannose 6-phosphate phosphatase The solution was mixed so that OD 600 = 100, and 5% w / v diatomaceous earth was added to the bacterial suspension, and stirred evenly. Subsequently, 0.5% w / v polyethyleneimine aqueous solution with a molecular weight of 70000 was added to flocculate at room temperature. Then, 0.5% v / v glutaraldehyde aqueous solution was added to crosslink for 2 h at room temperature. The filter cake was obtained ...

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Abstract

The invention discloses a preparation method of immobilized cells for mannose production and a method for producing mannose by using the immobilized cells. When immobilized bacillus subtilis is subjected to continuous catalytic reaction, the yield of an initial product can reach 65% at most, and after continuous catalysis is carried out for 25 batches, the yield of the product can still be maintained at 45%. When immobilized Escherichia coli is used for continuous catalytic reaction, the yield of an initial product can be up to 65% at most, and after continuous catalysis is carried out for 25 batches, the yield of the product can still be maintained at 44%. According to the method, enzyme separation and purification steps are simplified, the enzyme recycling rate is increased, and enzyme recycling is achieved. The method has the advantages of easiness in product separation, simple production process, low cost and the like, and provides reference for industrial application.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the field of production and preparation of mannose. Background technique [0002] Mannose is a six-carbon sugar that is an isomer of glucose. Mannose has the functions of regulating immunity, resisting bacterial and viral infection, promoting glycoprotein synthesis in the body, and anticancer. Therefore, mannose is widely used in the fields of biology and medicine. Mannose can not only be used as a raw material for the synthesis of some pharmaceutical chemical raw materials and important sugar drug precursors, but also can be used as a sweetener for food and beverages. In recent years, the demand for mannose in the fields of functional food, feed additive and medicine has increased significantly, and it has huge economic development value. [0003] The main methods of industrial production of mannose are extraction and isomerization. The extraction method mainly uses c...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/10C12N11/089C12N11/087C12N11/082C12P19/02C12R1/19C12R1/125
CPCC12N11/14C12N11/10C12N11/089C12N11/087C12N11/082C12P19/02C12Y204/01001C12Y504/02002C12Y503/01009C12Y503/01008C12N11/06
Inventor 马延和石婷韩平平
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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