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Bacillus subtilis for producing chitosanase and application thereof

A technology of Bacillus subtilis and chitosanase, applied in the direction of glycosylase, enzyme, bacteria, etc., can solve the problems of high optimum temperature, limited application range, high energy consumption, etc., achieve good enzyme activity and save production The effect of high cost and high output

Pending Publication Date: 2018-05-11
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, a report titled "Research Overview of Microbial Chitosanases" pointed out that most of the existing chitosanases are basic proteins, and the optimum pH is generally concentrated between 5 and 8, and occasionally a few The optimal pH of each enzyme is 4, but the corresponding optimal temperature is too high, resulting in high energy consumption and limited scope of application in actual production and application

Method used

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  • Bacillus subtilis for producing chitosanase and application thereof
  • Bacillus subtilis for producing chitosanase and application thereof
  • Bacillus subtilis for producing chitosanase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the screening of producing chitosanase bacterial strain

[0035] 1. Material preparation:

[0036] (1) Soil samples: Soil samples were collected from the asparagus growing area in Mianyang.

[0037] (2) Medium:

[0038] 1) Chitosanase screening medium (g / L): colloidal chitosan 2.0, K 2 HPO 4 0.7, KH 2 PO 4 0.3, (NH 4 ) 2 SO 4 5, MgSO 4 ﹒ 7H 2 O 0.5, FeSO 4 ﹒ 7H 2 0 0.01, agar 12.0, pH=7.0;

[0039] 2) Broth medium (g / L): peptone 10.0, beef extract 3.0, NaCl 5.0, pH=7.0.

[0040] 3) Chitosanase induction medium (g / L): colloidal chitosan 5.0, K 2 HPO 4 0.7, KH 2 PO 4 0.3, (NH 4 ) 2 SO 4 1.0, MgSO 4 ﹒ 7H 2 O 0.5, FeSO 4 ﹒ 7H 2 0 0.01, pH=7.0;

[0041] 4) LB medium (g / L): peptone 10.0, yeast extract 5.0, NaCl 10.0.

[0042] (3) Reagents:

[0043] 1) Colloidal chitosan: Weigh 10 g of high-polymer fine powder chitosan, add appropriate amount of concentrated hydrochloric acid, and stir on a magnetic stirrer for 4-6 hours to full...

Embodiment 2

[0069] Embodiment 2: bacterial strain identification

[0070] 1. 16SrDNA sequencing identification

[0071] Extract the genomic DNA of the strain to be tested, and then clone the 16SrDNA from the genomic DNA with the universal primers of bacterial 16SrDNA.

[0072] The general primers are:

[0073] 27F: AGA GTT TGA TCC TGG CTC AG;

[0074] 1492R:TAC GGT TAC CTT GTT ACG ACT T.

[0075] The PCR results were sent to Sangon Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared with BLAST on the Gene Bank website (http: / / www.nicbi.nlm.nih.gov / ), and the gene sequences of the standard strains were found and downloaded. The sequencing results are shown as SEQ ID Shown in NO 1. Use MEGA7.0 to calculate the genetic distance between different strains, and draw the phylogenetic tree of bacteria, such as Figure 4 shown.

[0076] Comparing the 16SrDNA of the tested strain with the Bacillus type strain, the genetic distances with Bacillus subtilis (Bacillus...

Embodiment 3

[0088] Embodiment 3: construct recombinant chitosanase CsnMY002

[0089] 1. Recombinant expression and purification of chitosanase

[0090] (1) Cloning and sequencing of chitosanase gene

[0091] Through the multiple sequence alignment of the known Bacillus subtilis chitosanase gene in Genebank (https: / / www.ncbi.nlm.nih.gov / genbank / ), the primers were designed as follows:

[0092] F:5'-CGC GGA TCC ATG AAA ATC AGT ATG CAA AAA GC-3';

[0093] R: 5'-A CGC GTC GAC TTA TTT GAT TAC AAA ATT ACC G-3'.

[0094] The entire genome of the strain was extracted using a bacterial whole genome DNA extraction kit, and polymerase chain reaction (PCR) was carried out using this as a template, and the chitosanase gene (CsnMY002) of the strain was successfully cloned, and its gene nucleoside The acid sequence is shown in SEQ ID NO 4, and the protein amino acid sequence is shown in SEQ ID NO 5.

[0095] Sequencing results showed that the full-length chitosanase gene of Bacillus subtilis MY002 w...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a bacillus subtilis for producing chitosanase and application thereof. The chitosanase produced by the bacillus subtilis has high yield and strong enzyme activity, and has a different protein structure from ordinary chitosanase, and provides a new idea for in-depth study of chitosanase molecule mechanism. The ordinarychitosanase has good activity only when the ordinary chitosanase is in weakly alkaline to weakly acidic conditions, and the chitosanase found in the invention has better performance in weak alkaline,neutral, weakly acidic and relatively strong acidic conditions. At the same time, the chitosanase discovered in the invention can not only act on soluble chitosan but also on insoluble high-poly chitosan, omitting pre-treatment of chitosan degradation; final reaction products are dipolyglucosamine and tripolyglucosamine, the bacillus subtilis for producing the chitosanase is simple in composition, is convenient for subsequent further processing applications, and greatly saves production costs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chitosanase-producing bacillus subtilis and an application thereof. Background technique [0002] Glucosamine is an important pharmaceutical precursor and chemical raw material, which has a wide range of uses. Chitosan derived from shrimp and crab shells is a polymer formed by connecting D-glucosamine (or a small amount of N-acetyl-D-glucosamine) through β-1,4-glycosidic bonds. Sugar exceeds 100 billion tons, and a large amount of glucosamine can be produced by hydrolyzing chitosan. [0003] At present, the commonly used chitosan hydrolysis methods are: acid hydrolysis, oxidative degradation, physical degradation and enzymatic hydrolysis. Among them, enzymatic degradation of chitosan has the advantages of low environmental pollution, mild reaction conditions, and no by-products. It is currently the most widely used chitosan degradation method and has been gradually us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12P19/26C12P19/14C12R1/125
CPCC12N9/2402C12P19/14C12P19/26C12Y302/01132C12N1/205C12R2001/125
Inventor 王刚刚苟艳谢天刘忠川
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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