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Highly active mannanase obtained through genetic engineering and its mutation site

A technology of mannanase and transgenic engineering, applied in the field of microbial technology engineering, can solve problems such as unfavorable screening and identification, enzyme inactivation, and low mutation frequency

Active Publication Date: 2019-05-07
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key to this method is to find a suitable mutation frequency. If the mutation frequency is too high, the enzyme will be completely inactivated and the library capacity will be reduced. If the mutation frequency is too low, the wild type will occupy a large proportion, which is not conducive to the later screening and identification.

Method used

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  • Highly active mannanase obtained through genetic engineering and its mutation site
  • Highly active mannanase obtained through genetic engineering and its mutation site
  • Highly active mannanase obtained through genetic engineering and its mutation site

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Embodiment Construction

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

[0026] 1 material

[0027] Superrich medium (1L): 20g yeast powder, 25g peptone, 3g dipotassium hydrogen phosphate, 30g glucose.

[0028] Bacillus subtilis WB800 Transformation Reagent:

[0029] SP-A salt solution (1L): (NH 4 ) 2 SO 4 4g, K 2 HPO 4 ·3H 2 O 28g, KH 2 PO 4 12g, sodium citrate 2g, sterilized at 121°C for 20min.

[0030] SP-B salt solution (1L): MgSO 4 ·7H 2 O 0.4g, sterilized at 121°C for 20min.

[0031] 100×CAYE solution (100ml): 2g of casein hydrolyzate, 10g of yeast powder, sterilized at 121°C for 20min.

[0032] SPI Medium (10mL): 4.9mL SP-A salt solution, 4.9mL SP-B salt solution, 100...

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Abstract

The invention discloses high-activity mannanase obtained through genetic engineering modification and a mutation site thereof. An amino acid sequence of the mannanase is obtained by replacing and modifying at least one of 221th, 301th and 122th amino acids of an amino acid sequence as shown in SEQ ID NO.1, wherein the 221th amino acid is replaced with and modified by leucine to form isoleucine, the 301th amino acid is replaced with and modified by lysine to form glutamic acid, and the 122th amino acid is replaced with and modified by glutamine to form arginine. After the genetic engineering modification, the enzyme activity of the mannanase is greatly improved.

Description

technical field [0001] The invention belongs to the field of microbial technology engineering, and relates to a highly active mannanase obtained through genetic engineering transformation and a mutation site thereof. technical background [0002] Plants are the main renewable organic resources in nature, mainly composed of cellulose, hemicellulose and lignin. Hemicellulose accounts for 25%-35% of the dry weight of plants, and its content in nature is second only to cellulose. Compared with cellulose, the structure and composition of hemicellulose are more complex, including xylan, mannan, dextran and arabinan and other components. [0003] β-mannan is a linear polysaccharide linked by 1,4-β-D-mannopyranosidic bonds. If some residues in the main chain are replaced by glucose, or galactose is bonded to mannose through 1,6-α glycosidic bonds The sugar residues are connected to form branches, which become different mannose, mainly including glucomannan, galactomannan and galac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N5/10A23L29/00A23K20/189A23K10/14D21C9/10
CPCC12N9/2494C12Y302/01078D21C9/10
Inventor 张瑞福张稳沈其荣邵佳慧
Owner NANJING AGRICULTURAL UNIVERSITY