1,3-1,4-beta-glucanase mutant

A technology of dextranase and mutants, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of not being able to adapt to industrial applications, thermal stability cannot be adapted to industrial applications, etc., and achieve the effect of improving specific activity

Active Publication Date: 2015-08-26
SHANGHAI BAILANG BIOTECHOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the thermal stability of the modified enzyme BglTM obtained so far is still not suitable for industrial applications.
[0005] The optimal temperature of the transformed 1,3-1,4-β-glucanase (BglTM) derived from Bacillus tequila CGX 5-1 used in the present invention is 60°C, and the specific activity is 3936.4U / mg, does not meet the requirements of industrial applications

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Mutation site analysis

[0025] Submit the deduced amino acid sequence of β-glucanase to the SWISSMODEL online server for homology modeling, and input the modeled PDB file into Disulfide by Design software for calculation, so as to predict that when the mutation is cysteine, it can Potential amino acid pairs to form disulfide bonds. It can be seen from Table 1 that a total of 29 pairs of amino acids are predicted to form disulfide bonds.

[0026] It can be seen from Table 1 that a total of 29 pairs of amino acids were predicted to form disulfide bonds, and then these amino acid pairs were screened. Firstly, the amino acid pair (C32-C61) and the conflicting amino acid pair C32-F59C in β-glucanase were excluded. After that, in order to ensure the integrity of the active center, 9 pairs located in the active center were excluded. Amino acid pairs in the range. According to literature reports, amino acid pairs forming disulfide bonds differ in sequence by le...

Embodiment 2

[0033] Example 2 Preparation and expression of β-glucanase mutants

[0034] (1) Site-directed mutation

[0035] Using the plasmid pET28a(+)-BglTM (Niu C, Zhu L, Zhu P, LiQ.2015.Lysine-Based Site-Directed Mutagenesis Increased Rigidβ-Sheet Structure and Thermostability of Mesophilic 1,3–1,4-β-Glucanase. Journal of Agricultural and Food Chemistry 63:5249-5256) was used as a template, and the G3C- Q68C, N31C-T187C, E63C-N182C, K83C-A141C, and P102C-N125C genes.

[0036] The site-directed mutagenesis primers (sequences shown in SEQ ID NO.7 and SEQ ID NO.8) that introduce G3C codons are:

[0037] Forward primer: 5'-cggctcaaacaTGTggatcgttttttga-3', capital letters are mutant bases,

[0038] Reverse primer: 5'-tcaaaaaacgatccACAtgtttgagccg-3', capital letters are mutant bases;

[0039] The site-directed mutagenesis primers (sequences shown in SEQ ID NO.9 and SEQ ID NO.10) for introducing the Q68C codon are:

[0040] Forward primer: 5'-aaaccgttctgttTGTacatatggcta-3', capital lette...

Embodiment 3

[0077] Embodiment 3: Enzyme activity and protein concentration analysis

[0078] (1) Enzyme activity assay method:

[0079]3,5-Dinitrosalicylic acid (DNS) method combined with improved AZO assay method for the determination of β-glucanase activity:

[0080] Enzyme activity definition: 1 mL of enzyme solution under the conditions of 40°C and pH value of 6.5, the amount of hydrolyzing β-glucan per minute to produce glucose reducing substances equivalent to 1 μmol is 1 enzyme activity unit, expressed in U / mL.

[0081] Determination of the enzyme activity of the fermentation broth: after the fermentation broth is centrifuged, the supernatant is diluted to an appropriate multiple to measure its enzyme activity.

[0082] Drawing of glucose standard curve: draw 1% glucose standard solution 2.0, 3.0, 4.0, 5.0, 6.0mL respectively into 50mL volumetric flask, dilute to the mark with distilled water, and make each milliliter contain glucose 200, 400, 600, 800 , 1000, 1200μg dilute stand...

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Abstract

The invention discloses a 1,3-1,4-beta-glucanase mutant, belonging to the fields of genetic engineering and enzyme engineering. According to the invention, for the 1,3-1,4-beta-glucanase mutant sourced from modified bacillus terquilensis CGX5-1, the 31st asparagine, 187th threonine, 102nd proline and 125th asparaginate are subjected to an overlap extension PCR method to become cysteine to respectively obtain single mutants N31C-T187C and P102C-N125C. Two mutation sites are integrated and mutated to obtain N31C-T187C / P102C-N125C double disulfide bond mutant enzymes. Three mutant enzymes show good thermal stability. Compared with wild enzymes, the mutant enzymes is beneficial to industrial application.

Description

technical field [0001] The invention relates to a 1,3-1,4-beta-glucanase mutant and belongs to the fields of genetic engineering and enzyme engineering. Background technique [0002] β-glucan is a kind of non-starch polysaccharide existing in the cell wall of gramineous plants, and its content is very high in economical grains such as barley, wheat, and rice. It is composed of thousands of β-D-glucose residues arranged linearly through β-1,3 or β-1,4 glycosidic bonds, and has a very high molecular weight. It can be dissolved in water, and the resulting solution has a high viscosity, which brings many disadvantages to the beer industry and the feed industry. Malt, the main raw material of the beer industry, contains a large amount of β-glucan. The presence of undegraded β-glucan in the wort will cause the viscosity of the wort to be too high, resulting in difficulty in filtration, prolonging the filtration time of wort mash, and reducing the extract. Content, for the finish...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21
CPCC12N9/2405C12Y302/01073
Inventor 朱林江钮成拓李崎李永仙王金晶刘春凤郑飞云
Owner SHANGHAI BAILANG BIOTECHOLOGY
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