Chitosanase ouc-t613 and its application
A technology of OUC-T613 and chitosanase, which is applied in the field of functional enzyme technology and genetic engineering, can solve problems such as complex phenotypes
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Embodiment 1
[0027] The preparation of embodiment 1 recombinant chitosanase
[0028] (1) Amplification of chitosanase parent
[0029]First, amplify the three chitosanase proteins that the applicant has obtained by PCR method, respectively Paenibacillus sp.1-18 (OUC-CsnPa), Paenibacillus tyrfis (OUC-CsnPT) and Chromobacterium sp.ATCC53434 (OUC-CsnCA), The size is about 1000bp, wherein the amino acid sequence of OUC-CsnPa is shown in SEQ ID NO.3, the sequence of the coding gene is shown in SEQ ID NO.4; the amino acid sequence of OUC-CsnPT is shown in SEQ ID NO.5, The sequence of the coding gene is shown in SEQ ID NO.6; the amino acid sequence of OUC-CsnCA is shown in SEQ ID NO.7, and the sequence of the coding gene is shown in SEQ ID NO.8. Then the PCR product was recovered, and the concentration of the three fragments of the parent was measured with a micro-nucleic acid analyzer. The three parental fragments were digested with the digestive enzyme DNsae I in an optimized digestion system,...
Embodiment 2
[0050] The screening of embodiment 2 highly active recombinant chitosanase mutants
[0051] Compared with the transparent circle on the solid plate in embodiment 1, the clone with the bigger transparent circle than the parent is inoculated into the 5mL5052 medium test tube and cultivated for 48h, after centrifugation and buffer solution cleaning the thalline, carry out full-blown with colloidal chitosan Cell catalytic screening of highly active mutants. Sequencing was performed to screen out highly active positive strains relative to the OUC-CsnPa mutation. The highly active positive strains screened were amplified and fermented in high-density self-inducing medium ZYP-5052, and the supernatant crude enzyme was obtained through ultrasonic crushing, and the hydrolysis activity of chitosanase was used as a screening standard, and the enzyme activity was compared ( figure 2 ), the enzyme activity of the recombinase OUC-T613 was increased by nearly 104%, which indicated that a c...
Embodiment 3
[0052] Embodiment 3 Highly active chitosanase mutant OUC-T613 property determination
[0053] The thermal stability of parental and mutant OUC-T613 is mainly determined by the parameter T 50 value is evaluated. T 50 Defined as the temperature at which 50% of the enzyme is inactivated within 10 minutes. In detail, the purified enzyme (0.01 mg / mL) was incubated at various temperatures (30-70° C.) for 10 minutes, and then the remaining activity was measured. Determine T 50 Values were obtained by fitting a shifted sigmoid function to the thermal inactivation curve. from the result( image 3 ) It can be seen that the mutant combines the excellent thermal stability of the parental OUC-CsnPT and OUC-CsnCA, and has enhanced thermal stability compared to the wild OUC-CsnPa.
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