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Trichoderma reesei chitinase and preparation method and applications thereof

A technology of chitinase and Trichoderma reesei, which is applied in the field of chitinase, can solve the problems of increased production cost of chitosan oligosaccharides and large amount of enzymes, and achieve efficiency improvement, huge application potential, and high-efficiency secretion expression Effect

Active Publication Date: 2017-10-17
ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the enzymes with chitosan 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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  • Trichoderma reesei chitinase and preparation method and applications thereof
  • Trichoderma reesei chitinase and preparation method and applications thereof
  • Trichoderma reesei chitinase and preparation method and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 Codon optimization and total gene synthesis of chitinase gene

[0020] On the premise of not changing the amino acid sequence, the codons of the chitinase (GH18 family) coding gene derived from Trichoderma reesei were optimized, and all the optimized codons were Pichia pastoris preferred codons. For the specific sequence, see SEQ ID NO.2. Compared with the original sequence (as shown in SEQ ID NO.3, GenBankaccession number: XM_006968075), the optimized nucleotide sequence trchi18 has 246 nucleotides changed, and the nucleotide sequence homology is 80%. At the same time, in order to enable efficient and stable secretion and expression of chitinase in Pichia pastoris, the optimized chitinase gene lacks 22 amino acids encoding the 5' signal peptide sequence. The optimized gene sequence was entrusted to Sangong for full synthesis, and the synthesized gene sequence was named chitinase gene trchi18.

Embodiment 2

[0021] The expression vector construction of embodiment 2 chitinase gene trchi18

[0022] First, the cloning vector containing the chitinase gene trchi18 was double-digested with restriction endonucleases Xho I and Not I to obtain the target gene fragment, and the expression vector pGBG1 was double-digested with the same endonucleases, and recovered large fragments. The two recovered products were connected to obtain a recombinant vector named trchi18-pGBG1. In order to confirm that the target chitinase gene has been constructed into the vector, we used Xho I / Not I and Bgl II to carry out double and single digestion of the recombinant vector respectively, and performed agarose gel electrophoresis on the product. The results are as follows: figure 1 Shown: After double enzyme digestion, the target gene fragment appeared between 1000bp and 1500bp, which was consistent with the fragment 1242bp of trchi18; after Bgl II digestion, the expected two fragments appeared, followed by t...

Embodiment 3

[0023] Example 3 Screening of Chitinase Pichia Pichia Engineering Bacteria and Preparation of Chitinase

[0024] After the obtained recombinant plasmid trchi18-pGBG1 was linearized by the restriction endonuclease BglII, gel electrophoresis was used to separate and excise the nucleotide fragment containing the gene of interest (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 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. Add methanol to the final concentration of 1% after 24 hour...

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Abstract

The invention discloses a trichoderma reesei chitinase and a preparation method and applications thereof. The encoding gene of chitinase in the Trichoderma reesei chitinase is optimized according to the preference of pichia pastoris, and the optimized nucleotide sequence is shown as SEQ ID NO.2. The optimized encoding gene of chitinase is subjected to further high-efficiency secretory expression by utilizing a pichia pastoris expression system to obtain trichoderma reesei chitinase, the amino acid sequence of which is shown as SEQ ID NO.1. The obtained trichoderma reesei chitinase has higher hydrolysis activity to low-deacetylation-degree chitosan substrate, the crude enzyme liquid generated by shaking and fermentation has the hydrolysis capability of degrading 0.5g by weight of chitosan with 1mL (the content of protein is about 0.17mg), degrading of the same amount of chitosan needs about 50mg of non-specific commercial enzyme, so that the efficiency is theoretically increased by 300 times; and the trichoderma reesei chitinase and the preparation method and applications thereof have good industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of chitinase, in particular to a chitinase from Trichoderma reesei and its preparation method and application. Background technique [0002] Chitinase (Chitinase, EC.3.2.1.14) widely exists in archaea, bacteria and eukaryotes, mainly in glycoside hydrolases (Glycoside Hydrolases, GH) families 18 and 19. In industry, due to the lack of economical and efficient specific chitosan glycohydrolase (including chitinase and chitosanase), non-specific commercial enzymes such as protease and cellulase are often used to hydrolyze chitosan to prepare Chitooligosaccharides. Because the enzymes with chitosan hydrolysis activity account for a very low proportion in 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 to develop a series of economical and efficient chitosan hydrolases to...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12P19/26C12P19/14C12R1/84
CPCC12N9/2442C12N15/815C12N2800/22C12P19/14C12P19/26C12Y302/01014
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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