Improving the thermostability of 42 kDa chitinase from Trichoderma asperellum SH16 expressed in Nicotiana benthamiana via site-directed mutagenesis.

Autor: Tien, Nguyen Quang Duc, Van Thanh, Dang, Ha, Huynh Thi Thu, Tue, Nguyen Hoang, Phuc, Nguyen Hoang, Hoa, Phung Thi Bich, Thanh, Le Thi Ha, Loc, Nguyen Hoang
Zdroj: Plant Cell, Tissue & Organ Culture; Oct2024, Vol. 159 Issue 1, p1-14, 14p
Abstrakt: This study aimed to improve the thermal stability of 42 kDa chitinase from T. asperellum SH16 produced in N. benthamiana through site-directed mutagenesis for application in post-harvest fruit and vegetable preservation. In this work, thermostable mutants were predicted using bioinformatics tools, gene expression was analyzed by SDS-PAGE and Western blot, chitinase activity was determined spectrophotometrically, and the antifungal activity of chitinase was evaluated through inhibition of mycelial growth. The results showed that six selected mutants were successfully expressed in N. benthamiana and had in vitro antifungal activity against Colletotrichum siamense AD1.2. Among them, mutant I168L has a higher optimal temperature, thermal stability, and enzymatic activity than native chitinase and other mutants. Further analysis showed that, after 6 h of treatment at 40 °C, the relative activity of I168L remained about 50%, but that of native chitinase was only more than 15%. Besides, the I168L mutant could maintain a relative activity of about 78% and 50% after 30 min at 60ºC and 70ºC, while the native chitinase had only 38% and 12%, respectively. The Km and Vmax of the I168L mutant were both lower than those of native chitinase: 6.99 mg/mL and 254 units/mg vs. 11.24 mg/mL and 512 units/mg, respectively. Mutant I168L showed the ability to prevent and treat chili anthracnose disease caused by C. siamense CH26.1, which was many times stronger than the native chitinase at 35ºC. This finding suggests that the thermostable chitinase, I168L, has potential for applications in post-harvest fruit and vegetable preservation.Key message: This study screened insilico a mutant chitinase named I168L, derived from Trichoderma asperellum. The I168L exhibited significantly greater thermostability and Collectotrichum antifungal activity than the native chitinase. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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