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Streptomyces thermoviolaceus chitinase, as well as preparation method and application of streptomyces thermoviolaceus chitinase

A technology of chitinase and Streptomyces violaceum, applied in the field of chitinase, can solve the problems of increased production cost of chitosan oligosaccharides and large amount of enzymes, and achieve the effect of improved efficiency and efficient secretion and expression

Active Publication Date: 2018-01-19
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

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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  • Streptomyces thermoviolaceus chitinase, as well as preparation method and application of streptomyces thermoviolaceus chitinase
  • Streptomyces thermoviolaceus chitinase, as well as preparation method and application of streptomyces thermoviolaceus chitinase
  • Streptomyces thermoviolaceus chitinase, as well as preparation method and application of streptomyces thermoviolaceus chitinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Codon optimization and total gene synthesis of chitinase gene

[0038] On the premise of not changing the amino acid sequence, using the preferred codon of Pichia pastoris, the chitinase (GH19 family) of Streptomyces thermoviolets was artificially designed (as shown in the sequence SEQ ID NO.1, GenBank accession number: BAA88833 ) coding gene, see SEQ ID NO.2 for the specific nucleotide sequence. The optimized nucleotide sequence has the highest homology of 78% with the original coding gene sequence (as shown in sequence SEQ ID NO.3, GenBank accession number: AB016842.1). The optimized gene sequence was entrusted to Sankon for full synthesis, and the synthesized gene sequence was named chitinase gene stchi19.

Embodiment 2

[0039] The expression vector construction of embodiment 2 chitinase gene stchi19

[0040] First, use restriction endonucleases Xho I and Not I to double-enzyme digest the cloning vector containing chitinase gene stchi19 to obtain the target gene fragment, and use the same endonuclease to double-enzyme digest the expression vector pGBG1, recover large fragments. The two recovered products were connected to obtain a recombinant vector named stchi19-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 500bp-750bp, which was consistent with the 690bp fragment of stchi19; after Bgl II digestion, the expected two fragments appeared, followed by the large fra...

Embodiment 3

[0041] Example 3 Screening of Chitinase Pichia Pichia Engineering Bacteria and Preparation of Chitinase

[0042] After the obtained recombinant plasmid stchi19-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. After 24 hours, add methanol to a final concentration of 1%...

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Abstract

The invention discloses streptomyces thermoviolaceus chitinase (GH19 family), as well as a preparation method and application of streptomyces thermoviolaceus chitinase. According to the method, a chitinase gene sequence in streptomyces thermoviolaceus can be obtained by utilizing a gene synthesis method according to the codon preference of pichia pastoris, and the optimized nucleotide sequence isas shown in SEQ ID NO.2. The optimized chitinase coding gene is efficiently secreted and expressed by further utilizing a pichia pastoris expression system to obtain the streptomyces thermoviolaceus chitinase (GH19 family) having an amino acid sequence as shown in SEQ ID NO.1. The streptomyces thermoviolaceus chitinase (GH19 family) has relatively high hydrolytic activity for chitosan primer withlow degree of deacetylation, 1mL of crude enzyme (the content of protein is about 0.30mg) generated by fermentation of a shake flask has the hydrolysis ability for degrading 0.5g of chitosan, and theefficiency is improved by over 300 times theoretically since about 100mg of commercial cellulose is required for degrading the same amount of chitosan. The streptomyces thermoviolaceus chitinase has excellent industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of chitinase, and in particular relates to a Streptomycesthermoviolaceus chitinase (GH19 family) and a preparation method and application thereof. 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 hydrolytic enzymes (including chitinase and chitosanase), non-specific commercial enzymes such as protease and cellulase are often used to hydrolyze chitosan to prepare chitosan. Oligosaccharides. 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 ec...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12P19/26C12P19/14
Inventor 杜昱光冯翠任立世贾培媛焦思明程功
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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