Bacillus subtilis chitosanase as well as preparation method and application thereof

A technology of Bacillus subtilis and chitosanase, which is applied in the field of chitosanase, can solve the problems of low ratio, large amount of enzyme used, and increased production cost of chitosan oligosaccharides, and achieve the effect of efficient secretion and expression

Active Publication Date: 2017-10-10
ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the enzymes with chitosanase hydrolysis activity account for a very low proportion of these commercial enzymes, the amount of enzymes used is relatively large, and the production cost of chitosan oligosaccharides also increases accordingly.

Method used

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  • Bacillus subtilis chitosanase as well as preparation method and application thereof
  • Bacillus subtilis chitosanase as well as preparation method and application thereof
  • Bacillus subtilis chitosanase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Codon optimization and total gene synthesis of embodiment 1 chitosanase gene

[0022] On the premise of not changing the amino acid sequence, the codons of the chitosanase coding gene derived from Bacillus subtilis were optimized, and all the optimized codons were Pichia pastoris preferred codons. The specific sequence is shown in SEQ ID NO.2. Compared with the original sequence (GenBank accession number: AL009126, shown as SEQ ID NO.3), the optimized nucleotide sequence bscsn has 195 nucleotides changed, and the nucleotide sequence homology is 74%. At the same time, in order to enable efficient and stable secretion and expression of chitosanase in Pichia pastoris, the optimized chitosanase gene lacks 35 amino acids encoding the 5' signal peptide sequence. See SEQ ID NO.1 for the specific sequence . The optimized gene sequence was entrusted to Sangong for full synthesis, and the synthesized gene sequence was named chitosanase gene bscsn.

Embodiment 2

[0023] The expression vector construction of embodiment 2 chitosanase gene bscsn

[0024] First, use restriction endonucleases XhoI and NotI to double-enzyme-digest the cloning vector containing chitosanase gene bscsn to obtain the target gene fragment, and use the same endonuclease to double-enzyme-digest the expression vector pGBG1 to recover the large fragment . The two recovered products were connected to obtain a recombinant vector named bscsn-pGBG1. In order to confirm that the target chitosanase gene has been constructed into the vector, we respectively use Xho I / Not I and Bgl II to carry out double digestion and single digestion of the recombinant vector, and perform agarose gel electrophoresis on the product, the results are as follows figure 1 Shown: After double digestion, a fragment slightly larger than 750bp appeared, which was consistent with the 762bp fragment of bscsn; after digestion with Bgl II, two expected fragments appeared, which were the large fragment ...

Embodiment 3

[0025] Example 3 The screening of chitosanase Pichia engineering bacteria and the preparation of chitosanase

[0026] After the obtained recombinant plasmid bscsn-pGBG1 was linearized by the restriction endonuclease BglII, gel electrophoresis was used to separate and excise the nucleotide fragment containing the target gene (such as figure 2 shown in the larger fragment), electroporation introduced into Pichia pastoris GS115, and the recombinant obtained by screening on the histidine auxotrophic MD plate was spread on the BMMY agar plate containing colloidal chitosan (0.5%) for cultivation , from which the monoclonal strain with the largest hydrolytic circle was further screened. A single colony of the screened monoclonal strain was inoculated in 200 mL of BMGY medium, cultured at 30°C and 250 rpm for 48 hours, the supernatant was discarded by centrifugation, and 200 mL of BMMY medium was added to induce expression. After 24 hours, methanol was added to a final concentration...

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Abstract

The invention discloses a bacillus subtilis chitosanase as well as a preparation method and application thereof. The invention optimizes an encoding gene of bacillus subtilis chitosanase according to the preference of pichia pastoris codon. The optimized nucleotide sequence is shown as SEQ ID NO.2. A pichia pastoris expression system is further utilized to perform efficient secretory expression on the optimized chitosanase encoding gene, so as to obtain the bacillus subtilis chitosanase with the amino acid sequence as SEQ ID NO.1. The bacillus subtilis chitosanase obtained according to the invention has higher hydrolytic activity to chitosan substrates at different degrees of deacetylation; the crude enzyme generated through shake-flask fermentation has the hydrolysis capacity of degrading 5g of chitosan by 1mL crude enzyme (0.3mg of protein), about 150mg of non-specific commercial enzyme is required for degrading the same amount of chitosan, and the efficiency is theoretically increased by 500 times; and the bacillus subtilis chitosanase has excellent industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of chitosanase, and in particular relates to a bacillus subtilis chitosanase and a preparation method and application thereof. Background technique [0002] Chitosanases (Chitosanases, EC.3.2.1.132) widely exist in archaea, bacteria and eukaryotes, in glycoside hydrolases (Glycoside Hydrolases, GH) family 3, 5, 7, 8, 46, 75 and 80 All of them are distributed, among them, families 46, 75 and 80 only contain chitosanase. In industry, due to the lack of economical and efficient specific chitosanase, non-specific commercial enzymes such as protease and cellulase are often used to hydrolyze chitosan to prepare chitooligosaccharides. Because the enzymes with chitosanase hydrolysis activity account for a very low proportion of these commercial enzymes, the amount of enzymes used is relatively large, and the production cost of chitosan oligosaccharides also increases accordingly. Therefore, there is an urgent need...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12P19/26C12P19/14C12R1/84
Inventor 杜昱光程功任立世焦思明孙明冯翠
Owner ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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