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

Autor: Tue NH; Institute of Bioactive Compounds and Department of Biotechnology, University of Sciences, Hue University, 77 Nguyen Hue St., Hue 49000, Viet Nam., Phuc NH; Institute of Bioactive Compounds and Department of Biotechnology, University of Sciences, Hue University, 77 Nguyen Hue St., Hue 49000, Viet Nam., Hoa PTB; Department of Biology, University of Education, Hue University, 34 Le Loi St., Hue 49000, Viet Nam., Tien NQD; Institute of Bioactive Compounds and Department of Biotechnology, University of Sciences, Hue University, 77 Nguyen Hue St., Hue 49000, Viet Nam., Loc NH; Institute of Bioactive Compounds and Department of Biotechnology, University of Sciences, Hue University, 77 Nguyen Hue St., Hue 49000, Viet Nam. Electronic address: nhloc@hueuni.edu.vn.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Jun; Vol. 269 (Pt 2), pp. 131924. Date of Electronic Publication: 2024 Apr 28.
DOI: 10.1016/j.ijbiomac.2024.131924
Abstrakt: 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 10 4 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.
Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest exists.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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