Cloning and Expression of a New Chitinase from Carnivorous Plants Drosera capensis
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ing and Expression of a New Chitinase from Carnivorous Plants Drosera capensis I. G. Sinelnikova, *, I. N. Zorova, c, K. S. Bolotovab, A. P. Sinitsyna, c, and A. M. Rozhkovaa aFederal bFederal
Research Center “Fundamentals of Biotechnology,” Russian Academy of Sciences, Moscow, 119071 Russia State Autonomous Educational Institution of Higher Education, Lomonosov Northern (Arctic) Federal University, Arkhangelsk, 163002 Russia cDepartment of Chemistry, Moscow State University, Moscow, 119991 Russia *e-mail: [email protected] Received April 10, 2020; revised April 12, 2020; accepted April 20, 2020
Abstract—A novel chi19 gene encoding the chitinase, belonging to the glycosyl hydrolase family 19 from the predatory plant Drosera capensis is cloned and expressed in the E. coli bacterial system. The amino acid sequence of the translated enzyme consists of 325 a.a. divided into four functional parts: the N-terminal signal sequence and of the catalytic and chitin-binding domains that were linked by the –S–P- linker. The chi19 gene is expressed in two forms: with and without a chitin-binding domain. Refolding is carried out and homogeneous soluble forms are obtained for both variants of the enzyme. The chitinases obtained exhibit predominantly specific activity towards crystalline chitin, with the optimal pH level ranging from 5.0 to 5.5 and the optimum temperature in the range of 52 to 55°C for both forms of the enzyme. The catalytic activity of the full-sized form of chitinase was 360 U/g with a 64% decrease of the catalytic activity for the form with the chitin-binding domain removed. Keywords: glycoside hydrolases of family 19, chitinase, Drosera capensis, TAIL-PCR DOI: 10.3103/S0027131420050077
INTRODUCTION Chitin is one of the most common polysaccharides in nature (ranked second after cellulose) and is found in the exoskeletons of insects and the cell wall of fungi and yeast. Chitinolytic enzymes, or chitinases, are becoming increasingly important for applied biotechnological processes, in particular for controlling plant pathogens. Chitinases (EC 3.2.1.14) are an extensive class of enzymes capable of breaking down β-1,4-glycosidic bonds in a chitin macromolecule with the release of chitooligosaccharides (n = 2–6) or free N-acetylglucosamine. Chitinases are divided into two main families of glycosyl hydrolases (GH): GH18 (Chit18) and GH19 (Chit19) [1]. GH18 chitinases are present in almost all organisms and have a wide profile of physiological functions. In the 1960s, producers of chitinolytic enzymes began to attract attention as agents for controlling phytopathogens [2]. GH18 chitinase producers, such as Bacillus, Vibrio, Trichoderma, are able to effectively destroy the cell wall of the pathogen; however, homogeneous GH18 chitinases cannot significantly inhibit the growth of phytopathogens [3]. GH19 plant chitinases first appeared in higher plants as immune response proteins secreted during an infection with a phytopathogenic fungi [4], but later
they were found in bacteria of the genus Streptomyces and some act
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