A kind of papaya chitinase and its preparation method and application
A technology of chitinase and chitinase gene, which is applied in the field of chitinase, can solve the problems of increased production cost of oligochitosan and large amount of enzyme, and achieves improved efficiency, great application potential, and efficient secretion expression Effect
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Embodiment 1
[0026] Example 1 Codon optimization and total gene synthesis of chitinase gene
[0027] On the premise of not changing the amino acid sequence, the chitinase (GH19 family) of papaya (as shown in sequence SEQ ID NO.1, GenBank accession number: 3CQL_A) was artificially designed using the preferred codon of Pichia pastoris For the coding gene, see SEQ ID NO.2 for the specific nucleotide sequence. The optimized nucleotide sequence has the highest homology of 72% with the potential chitinase coding gene sequence derived from radish (Raphanus sativus) (as shown in the sequence SEQ ID NO.3, GenBankaccession number: XM_018582027.1) . The optimized gene sequence was entrusted to Sangong to carry out the total synthesis, and the synthesized gene sequence was named as the chitinase gene cpchi19.
Embodiment 2
[0028] The expression vector construction of embodiment 2 chitinase gene cpchi19
[0029] First, the cloning vector containing the chitinase gene cpchi19 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 cpchi19-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 750bp and 1000bp, which was consistent with the 765bp fragment of cpchi19; after Bgl II digestion, the expected two fragments appeared, followed by the...
Embodiment 3
[0030] Example 3 Screening of Chitinase Pichia Pichia Engineering Bacteria and Preparation of Chitinase
[0031] After the obtained recombinant plasmid cpchi19-pGBG1 was linearized by the restriction endonuclease BglII, it was separated by gel electrophoresis and the nucleotide fragment containing the target gene (the larger fragment as shown in Figure 1) was excised and electroporated into Pichia In yeast GS115, the recombinant obtained by screening on the histidine auxotrophic MD plate was plated on the BMMY agar plate containing colloidal chitosan (0.5%) and cultured, and 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%, and then add it once every 2...
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