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| Expression of Trichothecium roseum Chitinase Gene Trchi2 in Pichia pastoris and Its Enzyme Characterization |
| YU Song-Ni1*, JIA Peng-Fei1*, LI Ya-Hua1,2, XIAN Hong-Quan1,2** |
1 College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China;
2 Shandong Provincial Key Laboratory of Applied Mycology, Qingdao 266109, China |
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Abstract Trichothecium roseum is a kind of mycoparasites, which can inhibit the growth of Sclerotinia sclerotiorum and other pathogenic bacteria. Chitin is the major component of the most fungal cell wall, the chitinase of T. roseum plays an important role in the parasitism and bacteriostasis. In order to achieve the expression of the chitinase Trchi2 gene from T. roseum and clarify its enzymological properties, T. roseum chitinase gene Trchi2 was cloned by reverse transcriptase PCR (RT-PCR), the recombinant expression vector was constructed and transferred into Pichia pastoris GS115. The yeast transformants were screened and identified by PCR, and the engineered yeast was cultured with methanol to obtain the protein Trchi2 and studied its enzymatic properties. The results showed that the recombinant plasmid pPIC9K-Trchi2 was successfully constructed, and the engineered yeast GS115-Trchi2-2-8 that expressed chitinase Trchi2 were obtained. The enzyme production was the highest after induction for 7 d, the enzyme activity reached 3.96 U/ mL, the apparent molecular weight of chitinase Trchi2 was 50 kD. The main product of chitin decomposition by chitinase Trchi2 was N, N-diacetyl‐chitobiose. The optimum temperature and pH of enzyme reaction were 45 ℃ and 6.0, respectively. Under 30 to 55 ℃, the enzyme reaction was relatively stable. The enzyme solution treated at 60 ℃ for 1 h still had 25% relative activity. While pH between 4 to 7.5, the relative activity was stable at more than 50%. When there were Ag+ , Hg2+ , Cu2+ and Fe2+ at a concentration of 50 mmol/L in catalytic system, activity was strongly inhibited, and the relative activity was less than 5%. Na+ , K+ and Ca2+ had no significant effect on chitinase Trchi2. The activity range of the expressed chitinase was similar to that of most fungal chitinases, with a wide range of temperature adaptability. This study lays a foundation for the further study of chitinase Trchi2 and also promotes the production and application of chitinase.
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Received: 29 November 2021
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Corresponding Authors:
**hqxian0517@163.com
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