Dataset Information

A Novel Potent Crystalline Chitin Decomposer: Chitin Deacetylase from Acinetobacter schindleri MCDA01.

ABSTRACT: Chitosan is a functional ingredient that is widely used in food chemistry as an emulsifier, flocculant, antioxidant, or preservative. Chitin deacetylases (CDAs) can catalyze the hydrolysis of acetyl groups, making them useful in the clean production of chitosan. However, the high inactivity of crystalline chitin catalyzed by CDAs has been regarded as the technical bottleneck of crystalline chitin deacetylation. Here, we mined the AsCDA gene from the genome of Acinetobacter schindleri MCDA01 and identified a member of the uraD_N-term-dom superfamily, which was a novel chitin deacetylase with the highest deacetylation activity. The AsCDA gene was expressed in Escherichia coli BL21 by IPTG induction, whose activity to colloidal chitin, α-chitin, and β-chitin reached 478.96 U/mg, 397.07 U/mg, and 133.27 U/mg, respectively. In 12 h, the enzymatic hydrolysis of AsCDA removed 63.05% of the acetyl groups from α-chitin to prepare industrial chitosan with a degree of deacetylation higher than 85%. AsCDA, as a potent chitin decomposer in the production of chitosan, plays a positive role in the upgrading of the chitosan industry and the value-added utilization of chitin biological resources.

SUBMITTER: Yang G

PROVIDER: S-EPMC9416191 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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A Novel Potent Crystalline Chitin Decomposer: Chitin Deacetylase from Acinetobacter schindleri MCDA01.

Yang Guang G Wang Yuhan Y Fang Yaowei Y An Jia J Hou Xiaoyue X Lu Jing J Zhu Rongjun R Liu Shu S

Molecules (Basel, Switzerland) 20220822 16

Chitosan is a functional ingredient that is widely used in food chemistry as an emulsifier, flocculant, antioxidant, or preservative. Chitin deacetylases (CDAs) can catalyze the hydrolysis of acetyl groups, making them useful in the clean production of chitosan. However, the high inactivity of crystalline chitin catalyzed by CDAs has been regarded as the technical bottleneck of crystalline chitin deacetylation. Here, we mined the AsCDA gene from the genome of Acinetobacter schindleri</ ...[more]

PMID: 36014581

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Project description:Vibrio parahaemolyticus RIMD2210633 secretes both chitinase and chitin oligosaccharide deacetylase and produces β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) from chitin. Previously, we reported that GlcNAc-GlcN induces chitinase production by several strains of Vibrio harboring chitin oligosaccharide deacetylase genes (T. Hirano, K. Kadokura, T. Ikegami, Y. Shigeta, et al., Glycobiology 19:1046-1053, 2009). The metabolism of chitin by Vibrio was speculated on the basis of the findings of previous studies, and the role of chitin oligosaccharide produced from chitin has been well studied. However, the role of GlcNAc-GlcN in the Vibrio chitin degradation system, with the exception of the above-mentioned function as an inducer of chitinase production, remains unclear. N,N'-Diacetylchitobiose, a homodisaccharide produced from chitin, is known to induce the expression of genes encoding several proteins involved in chitin metabolism in Vibrio strains (K. L. Meibom, X. B. Li, A. Nielsen, C. Wu, et al., Proc Natl Acad Sci U S A 101:2524-2529, 2004). We therefore hypothesized that GlcNAc-GlcN also affects the expression of enzymes involved in chitin metabolism in the same manner. In this study, we examined the induction of protein expression by several sugars released from chitin using peptide mass fingerprinting and confirmed the expression of genes encoding enzymes involved in chitin metabolism using real-time quantitative PCR analysis. We then confirmed that GlcNAc-GlcN induces the expression of genes encoding many soluble enzymes involved in chitin degradation in Vibrio parahaemolyticus Here, we demonstrate that GlcNAc-GlcN enhances the chitin-metabolizing ability of V. parahaemolyticusIMPORTANCE We demonstrate that β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) enhances the chitin-metabolizing ability of V. parahaemolyticus Members of the genus Vibrio are chitin-degrading bacteria, and some species of this genus are associated with diseases affecting fish and animals, including humans (F. L. Thompson, T. Iida, and J. Swings, Microbiol Mol Biol Rev 68:403-431, 2004; M. Y. Ina-Salwany, N. Al-Saari, A. Mohamad, F.-A. Mursidi, et al., J Aquat Anim Health 31:3-22, 2019). Studies on Vibrio are considered important, as they may facilitate the development of solutions related to health, food, and aquaculture problems attributed to this genus. This report enhances the current understanding of chitin degradation by Vibrio bacteria.

Project description:Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungus Puccinia graminis f. sp. tritici in Escherichia coli The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein from P. graminis (PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed that PgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating that PgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property of PgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications.ImportanceWe successfully achieved heterologous expression of a fungal chitin deacetylase gene from the basidiomycete Puccinia graminis f. sp. tritici in the periplasm of E. coli as a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences.

Dataset Information

A Novel Potent Crystalline Chitin Decomposer: Chitin Deacetylase from Acinetobacter schindleri MCDA01.

Publications

A Novel Potent Crystalline Chitin Decomposer: Chitin Deacetylase from <i>Acinetobacter schindleri</i> MCDA01.

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