Expression and Refolding of the Plant Chitinase From Drosera capensis for Applications as a Sustainable and Integrated Pest Management.

Igor G Sinelnikov,Arkady P Sinitsyn,Larisa A Shcherbakova,Ivan N Zorov,Andrey M Chulkin,Aleksandra M Rozhkova,Ulrich Schwaneberg,Mehdi D Davari,Olga A Sinitsyna,Niklas E Siedhoff

doi:10.3389/fbioe.2021.728501

Abstract

Recently, the study of chitinases has become an important target of numerous research projects due to their potential for applications, such as biocontrol pest agents. Plant chitinases from carnivorous plants of the genus Drosera are most aggressive against a wide range of phytopathogens. However, low solubility or insolubility of the target protein hampered application of chitinases as biofungicides. To obtain plant chitinase from carnivorous plants of the genus Drosera in soluble form in E.coli expression strains, three different approaches including dialysis, rapid dilution, and refolding on Ni-NTA agarose to renaturation were tested. The developed « Rapid dilution » protocol with renaturation buffer supplemented by 10% glycerol and 2M arginine in combination with the redox pair of reduced/oxidized glutathione, increased the yield of active soluble protein to 9.5 mg per 1 g of wet biomass. A structure-based removal of free cysteines in the core domain based on homology modeling of the structure was carried out in order to improve the soluble of chitinase. One improved chitinase variant (C191A/C231S/C286T) was identified which shows improved expression and solubility in E. coli expression systems compared to wild type. Computational analyzes of the wild-type and the improved variant revealed overall higher fluctuations of the structure while maintaining a global protein stability. It was shown that free cysteines on the surface of the protein globule which are not involved in the formation of inner disulfide bonds contribute to the insolubility of chitinase from Drosera capensis. The functional characteristics showed that chitinase exhibits high activity against colloidal chitin (360 units/g) and high fungicidal properties of recombinant chitinases against Parastagonospora nodorum. Latter highlights the application of chitinase from D. capensis as a promising enzyme for the control of fungal pathogens in agriculture.

Highlights

Chitin is the second most common natural polymer, predominantly consisting of b-1,4 linked molecules of N-acetyl-D-glucosamine, which forms the basis of the cell walls of fungi, the exoskeleton of insects and shells of crustaceans
We demonstrated that the optimum action of the chitinase from Drosera capensis is 55°C and pH 5.0 (Sinelnikov et al, 2020)
We have chosen the optimal composition of the refolding buffer and the optimal concentrations of arginine and glycerol to obtain the maximum yield of soluble Shit19

Summary

Introduction

Chitin is the second most common natural polymer, predominantly consisting of b-1,4 linked molecules of N-acetyl-D-glucosamine, which forms the basis of the cell walls of fungi, the exoskeleton of insects and shells of crustaceans. Chitinases (EC 3.2.1.14) divided into two groups: exochitinases and endochitinases. Exochitinases, in turn, are further divided into two groups: chitobiosidases and 1,4-β-glucosaminidases. The endochitinases of GH18 are characteristic enzymes for a wide range of organisms: bacteria, fungi, insects, arthropods, and mammals. These enzymes play a key role in the development of the cell wall of organisms using chitin as a structural component, as well as these are the main enzymes involved in the degradation of chitin in nature (Javed et al, 2013)

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