Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts

• Published in: International journal of biological macromolecules 2024-06, Vol.269 (Pt 2), p.131924-131924, Article 131924
• Main Authors: Tue, Nguyen Hoang, Phuc, Nguyen Hoang, Hoa, Phung Thi Bich, Tien, Nguyen Quang Duc, Loc, Nguyen Hoang
• Format: Article
• Language: English
• Subjects: 42 kDa chitinase; Agroinfiltration; antifungal properties; Aqueous two-phase system; Athelia rolfsii; Chi42 gene; chitinase; Chitinases - chemistry; Chitinases - isolation & purification; Chitinases - metabolism; enzyme activity; fruits; hydrolysis; Nicotiana - enzymology; Nicotiana benthamiana; Phosphates - chemistry; polyacrylamide gel electrophoresis; polyethylene glycol; Polyethylene Glycols - chemistry; potassium phosphates; Recombinant Proteins - isolation & purification; Salts - chemistry; Salts - pharmacology; sodium phosphate; Trichoderma - enzymology; Trichoderma asperellum; vegetables; Water - chemistry
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.131924

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm–3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation. •N. benthamiana expressed the Chi42 gene from T. asperellum to make 42 kDa chitinase.•42 kDa chitinase was then successfully purified by the aqueous two phase system.•The activity of pure chitinase (834 unit/mg) is 3.32 times higher than the control.•This enzyme shows notable efficacy against the hazardous fungus Sclerotium rolfsii.•High antifungal activity points to possible uses for chitinase in plant protection.