Chitin deacetylase:from molecular structure to practical applications

Chitosan and chitooligosaccharides(COS),as derivatives of chitin through deacetylation reaction,have broad applications due to their good biodegradability,biocompatibility,and solubility.In addition,chitosan and COS are involved in cell wall morphogenesis and host–pathogen interactions in vivo.Chitin deacetylases(CDAs)are enzymes that can catalyze the deN-acetylation of chitin.They are widely distributed in protozoa,algae,bacteria,fungi,and insects with important physiological functions.Compared with the traditional chemical method,enzymatic catalysis by CDAs provides an enzymatic catalysis method to produce chitosan and COS with controllable deacetylation site and environmental friendliness.These characteristics attract researchers to produce CDAs by fungicides or pesticides.However,researches on heterologous expression and directed evolution of CDAs are still lacking.In this review,we summarize the latest knowledge of CDAs,especially for heterologous expression systems and directed evolution strategies,which may contribute to the industrial production and future application of CDAs.

Biocatalytic production of chitosan polymers from shrimp shells,using a recombinant enzyme produced by Pichia pastoris

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 3.5.1.41) catalyzes the hydrolysis of the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, converting it to chitosan and releasing acetate. The entire ORF of the CDA2 gene encoding one of the two isoforms of chitin deacetylase from Saccharomyces cerevisiae was cloned in Pichia pastoris. The Tg (Cda2-6xHis)p was expressed at high levels as a soluble intracellular protein after induction of the recombinant yeast culture with methanol, and purified using nickel-nitrilotriacetic acid chelate affinity chromatography, resulting in a protein preparation with a purity of >98% and an overall yield of 79%. Chitin deacetylase activity was measured by a colorimetric method based on the O-phthalaldehyde reagent, which detects primary amines remaining in chitinous substrate after acetate release. The recombinant enzyme could deacetylate chitin, chitobiose, chitotriose and chitotetraose, with an optimum temperature of 50°C and pH 8.0, determined using oligochitosaccharides as the substrates. The recombinant protein was also able to deacetylate its solid natural substrate, shrimp chitin, to a limited extent, producing chitosan with a degree of acetylation (DA) of 89% as determined by Fourier transform infrared spectroscopy. The degree of deacetylation was increased by pre-hydrolysis of crystalline shrimp chitin by chitinases, which increased the deacetylation ratio triggered by chitin deacetylase, producing chito-oligosaccharides with a degree of acetylation of 33%. The results described here open the possibility to use the rCda2p, combined with chitinases, for biocatalytic conversion of chitin to chitosan with controlled degrees of deacetylation. We show herein that the crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequential enzyme treatment is performed.

Green Synthesis and Physical and Chemical Characterization of Chitosans with a High Degree of Deacetylation, Produced by a Binary Enzyme System

Chitosan is a biopolymer obtained from chitin, where the N-acetylglucosamine monomer is in its deacetylated form; this polymer is useful for a wide variety of industrial applications. The properties and uses of chitosan depend on its physical and chemical characteristics, which result from the treatments used for its production. In this study, we report the preparation and characterization ofchitosan oligosaccharides by a green synthesis from crystalline shrimp chitin, using a sequential enzyme treatment by chitinase and chitin deacetylase. Chitinases were purified from grapes and used to rupture the crystalline shrimp chitin structure, modifying the crystallinity index from 57.6% to 15.9%. The resultant polymers were deacetylated using a recombinant chitin deacetylase from Saccharomyces cerevisiae, which was cloned and expressed in Pichia pastoris. The chitosans produced showed an estimated DA （degree of acetylation） of approximately 20%, and the molecular weights ranged from -7,600 to -3,700 after treatment in pH 3.0 and pH 6.0 for 10 min and 40 min, respectively. Physical and chemical characterization of the products indicated that enzyme fragmentation of chitin probably makes the acetamide groups more accessible to deacetylation, forming homogeneous polymers that are free of hazardous sub-products, have defined low molecular weights, and are highly deacetylated.

Characterization and functional analysis of chitinase family genes involved in nymph-adult transition of Sogatella furcifera

Chitinase degrades chitin in the old epidermis or peritrophic matrix of insects,which ensures normal development and metamorphosis.In our previous work,we comprehensively studied the function of SfCht7 in Sogatella furcifera.However,the number and function of chitinase genes in S.furcifera remain unknown.Here,we identified 12 full-length chitinase transcripts from S.furcifera,which included nine chitinase(Cht),two imaginal disc growth factor(IDGF),and one endo-β-N-acetylglucosaminidase(ENGase)genes.Expression analysis results revealed that the expression levels of eight genes(SfCht3,SfCht5,SfCht6-1,SfCht6-2,SfCht7,SfCht8,SfCht10,and SfIDGF2)with similar transcript levels peaked prior to molting of each nymph and were highly expressed in the integument.Based on RNA interference(RNAi),description of the functions of each chitinase gene indicated that the silencing of SfCht5,SfCht10,and SfIDGF2 led to molting defects and lethality.RNAi inhibited the expressions of SfCht5,SfCht7,SfCht10,and SfIDGF2,which led to downregulated expressions of chitin synthase 1(SfCHS1,SfCHS1a,and SfCHS1b)and four chitin deacetylase genes(SfCDA1,SfCDA2,SfCDA3,and SfCDA4),and caused a change in the expression level of two trehalase genes(TRE1 and TRE2).Furthermore,silencing of SfCht7 induced a significant decrease in the expression levels of three wing development-related genes(SfWG,SfDpp,and SfHh).In conclusion,SfCht5,SfCht7,SfCht10,and SfIDGF2 play vital roles in nymph–adult transition and are involved in the regulation of chitin metabolism,and SfCht7 is also involved in wing development;therefore,these genes are potential targets for control of S.furcifera.

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix （PM）, composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins （insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1） and the chitin biosynthetic or modifying enzymes （chitin synthase-B and chitin deacetylase 1） in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing （by 24 h） for the gene encoding chitin deacetylase 1 and a slower rate of silencing （by 72 h） for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.