Chitinase low temperature resistant mutant and application thereof
A technology of chitinase and chitin, which is applied in the direction of application, enzyme, and introduction of foreign genetic material by using vectors, etc., can solve the problem of not many low-temperature chitinases, etc.
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[0023] Example 1 Gene cloning of wild-type chitinase
[0024] The inventors discovered that the prior art discloses a chitinase screened from Pseudoalteromonas sp. whose amino acid sequence is as shown in SEQ ID NO: 1, and its coding nucleotide is as SEQ. ID NO: 2, expressed in E. coli. In short, Shanghai Jierui Biotechnology Co., Ltd. was commissioned to artificially synthesize the gene, and then ligated into the pET-28(+) vector (New England Biolabs) between the EcoR I and HindIII restriction sites, and transformed into the large intestine Bacillus DH5α was screened on an LB plate containing 50 mg / L of ampicillin. The expression vector contained in the positive strain was named pET-chi. The plasmid was extracted and identified by sequencing.
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[0025] Example 2 Mutation Screening
[0026] In order to further improve the low temperature resistance of the above chitinase (amino acid sequence is SEQ ID NO: 1), the applicant used the recombinant plasmid pET-chi constructed in Example 1 as a template to design random mutation primers, respectively, to perform error-prone PCR carried out multiple random mutations on the target gene, and it was found that some mutations had no effect on its low temperature resistance, some mutations even made low temperature resistance worse, and some mutations, although they can improve heat resistance, but after mutation The application range of pH is narrowed and none of them meets the requirements. In the end, the applicant obtained a combination of mutation sites that can significantly improve low-temperature properties and expand the scope of application under acidic conditions: R501L, Y555W, F635P, A673S, E689T; specifically, the Arg mutation at position 501 is Leu, The Tyr mutation at...
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[0029] Example 3 Expression and purification
[0030] The competent cells of Escherichia coli BL21 (DE3) were prepared by methods known in the art, and the wild-type expression vector and mutant expression vector constructed in Example 1 and Example 2 were transformed into competent cells respectively by heat shock method, and the positive results were screened. After cloning PCR and sequencing verification for use.
[0031] Pick up the positive bacteria from the inoculation needle and inoculate it in 5mL LB medium, culture at 30℃, 200r / min for 12h, and then inoculate 400mL LB containing 50ug / mL kanamycin at 2% (V / V) inoculum. In the base, incubate at 30°C and 200r / min for 8h. When the bacterial density OD600 reaches 0.7, add 0.5mM IPTG to induce expression; shake culture overnight at 16°C at 170rpm.
[0032] The cells were collected by centrifugation, washed with PBS, sonicated in ice, and collected by centrifugation. The supernatant was collected by centrifugation. The AKTA affin...
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