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Bacillus circulans chitoanase as well as preparation method and application thereof

A technology of Bacillus circulans and chitosanase, which is applied in the field of chitosanase, can solve the problems of large amount of enzyme used, increased production cost of chitosan oligosaccharide, and low ratio, and achieve huge application potential, improved efficiency, and high efficiency The effect of secretory expression

Active Publication Date: 2018-01-16
INST OF PROCESS ENG CHINESE ACAD OF SCI
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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 circulans chitoanase as well as preparation method and application thereof
  • Bacillus circulans chitoanase as well as preparation method and application thereof
  • Bacillus circulans chitoanase as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0032] Codon optimization and total gene synthesis of embodiment 1 chitosanase gene

[0033] Under the premise of not changing the amino acid sequence, the Bacillus circulans chitosanase (GH46 family) was artificially optimized using the preferred codons of Pichia pastoris (as shown in the sequence SEQ ID NO.1, GenBank accession number: BAA01474. 2) The coding gene, see SEQ ID NO.2 for the specific nucleotide sequence. The optimized nucleotide sequence and the original potential chitosanase coding gene sequence, as shown in the sequence SEQ ID NO.3, GenBank accession number: D10624.2 (569-1474), have the highest homology of 76%. The optimized gene sequence was entrusted to Sangong for full synthesis, and the synthesized gene sequence was named chitosanase gene bccsn.

Embodiment 2

[0034] The expression vector construction of embodiment 2 chitosanase gene bccsn

[0035] First, use restriction endonucleases Xho I and Not I to carry out double enzyme digestion on the cloning vector containing chitosanase gene bccsn to obtain the target gene fragment, and use the same endonuclease to carry out double enzyme digestion on the expression vector pGBG1, and recover large fragments. The two recovered products were connected to obtain a recombinant vector named bccsn-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, the target gene fragment appeared between 750bp and 1000bp, which was consistent with the 813bp fragment of bccsn; after Bgl II digestion, the expected two fragmen...

Embodiment 3

[0036] Example 3 The screening of chitosanase Pichia engineering bacteria and the preparation of chitosanase

[0037] After the obtained recombinant plasmid bccsn-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 1 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 screened out. 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 an equal amount of BMMY was added to induce expression. After 24 hours, methanol was added to a final concentration of...

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Abstract

The invention discloses bacillus circulans chitoanase as well as a preparation method and application thereof. According to the preference of pichia pastoris codons, a chitoanase coding gene sequencein bacillus circulans is obtained by utilizing a whole gene synthesis method; an optimized nucleic acid sequence is shown as SEQ ID NO. 2. Furthermore, efficient secreting expression is carried out onan optimized chitoanase coding gene by utilizing a pichia pastoris expression system to obtain the bacillus circulans chitoanase; an amino acid sequence of the bacillus circulans chitoanase is shownas SEQ ID NO. 1. The chitoanase disclosed by the invention has relatively high hydrolysis activity on a chitosan substrate and a crude enzyme solution produced by shake-flask fermentation has a hydrolysis capability of degrading 5g of chitosan by utilizing 1mL of the crude enzyme solution (with the protein content of about 0.77mg); when the chitosan with the same amount is degraded, about 150mg ofneutral proteinase obtained from bacillus subtilis is needed; the efficiency is theoretically improved by about 200 times; the bacillus circulans chitoanase has a very good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of chitosanase, and in particular relates to a Bacillus circulans 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 nee...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12P19/26C12P19/14C12R1/84
Inventor 杜昱光贾培媛冯翠焦思明任立世程功
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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