Gene Cloning, Characterization, and Molecular Simulations of a Novel Recombinant Chitinase from Chitinibacter Tainanensis CT01 Appropriate for Chitin Enzymatic Hydrolysis

Yeng-Tseng Wang,Po-Long Wu

doi:10.3390/polym12081648

Abstract

Chitin, a polymer of N-acetyl-d-glucosamine (GlcNAc), can be degraded by chitinase, which is produced by higher plants, vertebrates, and bacteria. Chitinases are characterized by the ability to hydrolyze the beta-1,4-linkages in the chitin chain by either an endolytic or an exolytic mechanism. Chitinase 1198 is a novel endochitinase from the genome sequence of Chitinibacter tainanensis CT01. Herein, we report the findings of molecular simulations and bioassays for chitinase 1198. Our experimental results suggest that chitinase 1198 can recognize the nonreducing end of chitin and cleave the second or third glycosidic linkage from the nonreducing end of chitin oligomers. Furthermore, our simulations results revealed that chitinase 1198 is more likely to bind chitin oligomers with the main hydrogen bonds of the Asp440, the second GlcNAc unit of chitin oligomers, and degrade chitin oligomers to (GlcNAc)2 molecules. Moreover, chitinase 1198 is less likely to bind chitin oligomers with the main hydrogen bonds of the Asp440, the third GlcNAc unit of chitin oligomers, and degrade chitin oligomers to (GlcNAc)3 molecules. Lastly, chitinase 1198 can bind (GlcNAc)3 molecules with the main hydrogen bonds of the Asp440, the second GlcNAc of the (GlcNAc)3 molecules, and degrade chitin oligomers to GlcNAc and (GlcNAc)2 molecules.

Highlights

Chitins have been used as functional materials in the food and health fields because of their biocompatibility and nontoxicity
Our preliminary results indicated that chitinase 1198 (CT01GL001198 in Figure S1) had the highest chitinolytic activity toward the pNP-(GlcNAc)3 substrate
The CT01GL001198 gene was cloned into the pET101/D-TOPO expression vector, which produced active recombinant chitinase 1198

Summary

Introduction

Chitins have been used as functional materials in the food and health fields because of their biocompatibility and nontoxicity. The poor solubility and high molecular weight of chitin polymers limit their potential use; this problem can be overcome by using their derived oligomers and monomers [1]. The production of soluble chitin oligomers and monomers is based on a hydrolysis of acetamide group in marine polysaccharides, and there are chemical and enzymatic methods to produce the soluble chitin oligomers and monomers. Chemical deacetylation has many disadvantages, such as high energy consumption, environmental pollution, and a decrease in Polymers 2020, 12, 1648; doi:10.3390/polym12081648 www.mdpi.com/journal/polymers. Polymers 2020, 12, 1648 chitin’s mechanical properties. The enzymatic hydrolysis has several advantages over the traditional chemical methods, such as avoiding the environmental pollution problem and decrease in the chitin mechanical properties, while producing chitin with suitable molecular weight and the desired degree of deacetylation [2]

Methods

Results

Discussion

Conclusion