Biochemical Characterization of a GH46 Chitosanase Provides Insights into the Novel Digestion Specificity.

Endo-chitosanases (EC 3.2.1.132) are generally considered to selectively release functional chito-oligosaccharides (COSs) with degrees of polymerization (DPs) ≥ 2. Although numerous endo-chitosanases have been characterized, the digestion specificity of endo-chitosanases needs to be further explored. In this study, a GH46 endo-chitosanase OUC-CsnPa was cloned, expressed, and characterized from Paenibacillus sp. 1-18. The digestion pattern analysis indicated that OUC-CsnPa could produce monosaccharides from chitotetraose [(GlcN)4], the smallest recognized substrate, in a random endo-acting manner. Especially, the enzyme specificities during chitosan digestion including the regulation of product abundance through a transglycosylation reaction were also evaluated. It was hypothesized that an insertion region in OUC-CsnPa may form a strong force to be involved in stabilizing (GlcN)4 at its negative subsite for efficient hydrolysis. This is the first comprehensive report to reveal the digestion specificity and subsite specificity of monosaccharide production by endo-chitosanases. Overall, OUC-CsnPa described here highlights the previously unknown digestion properties of the endo-acting chitosanases and provides a unique example of possible structure-function relationships.

[1]  G. Aktuganov,et al.  Constitutive chitosanase from Bacillus thuringiensis B-387 and its potential for preparation of antimicrobial chitooligomers , 2022, World Journal of Microbiology and Biotechnology.

[2]  Xianghui Zhao,et al.  Characterization of a New Xylanase Found in the Rumen Metagenome and Its Effects on the Hydrolysis of Wheat. , 2022, Journal of agricultural and food chemistry.

[3]  Xiangzhao Mao,et al.  New Insights into Bifunctional Chitosanases with Hydrolysis Activity toward Chito- and Cello-Substrates. , 2022, Journal of agricultural and food chemistry.

[4]  Xiangzhao Mao,et al.  Heterogenously‐expressed chitosanase combining a green ball milling method for enzymatic degradation , 2022, Food Bioengineering.

[5]  Xiangzhao Mao,et al.  Biochemical characterization and cleavage pattern analysis of a novel chitosanase with cellulase activity , 2022, Applied Microbiology and Biotechnology.

[6]  Q. Yan,et al.  Biochemical characterization of a novel bifunctional chitosanase from Paenibacillus barengoltzii for chitooligosaccharide production , 2021, World Journal of Microbiology and Biotechnology.

[7]  Zhongchuan Liu,et al.  Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose. , 2021, Colloids and surfaces. B, Biointerfaces.

[8]  Xiangzhao Mao,et al.  Engineering a carbohydrate binding module to enhance chitinase catalytic efficiency on insoluble chitinous substrate. , 2021, Food chemistry.

[9]  M. J. Swamy,et al.  New Class of Chitosanase from Bacillus amyloliquefaciens for the Generation of Chitooligosaccharides. , 2021, Journal of agricultural and food chemistry.

[10]  Liming Zhao,et al.  Structure-function analysis of Gynuella sunshinyii chitosanase uncovers the mechanism of substrate binding in GH family 46 members. , 2020, International journal of biological macromolecules.

[11]  C. Xue,et al.  Marine-polysaccharide degrading enzymes: Status and prospects. , 2020, Comprehensive reviews in food science and food safety.

[12]  Jian Tian,et al.  Identification of a chitosanase from the marine metagenome and its molecular improvement based on evolution data , 2020, Applied Microbiology and Biotechnology.

[13]  Xiaobin Yu,et al.  Biocatalysis of Heterogenously-Expressed Chitosanase for the Preparation of Desirable Chitosan Oligosaccharides Applied against Phytopathogenic Fungi , 2020 .

[14]  Lihua Jiang,et al.  High level production of a Bacillus amlyoliquefaciens chitosanase in Pichia pastoris suitable for chitooligosaccharides preparation. , 2020, International journal of biological macromolecules.

[15]  B. Moerschbacher,et al.  The Pattern of Acetylation Defines the Priming Activity of Chitosan Tetramers. , 2019, Journal of the American Chemical Society.

[16]  Yanhua Fan,et al.  Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein. , 2020, Protein expression and purification.

[17]  Yanhe Ma,et al.  Tunnel engineering to accelerate product release for better biomass-degrading abilities in lignocellulolytic enzymes , 2019, Biotechnology for Biofuels.

[18]  Shangyong Li,et al.  Cloning and Characterization of a Cold-adapted Chitosanase from Marine Bacterium Bacillus sp. BY01 , 2019, Molecules.

[19]  Jinbao Liu,et al.  Cloning, expression and characterization of a novel chitosanase from Streptomyces albolongus ATCC 27414. , 2019, Food chemistry.

[20]  Qiming Chen,et al.  Efficient Immobilization of Bacterial GH Family 46 Chitosanase by Carbohydrate-Binding Module Fusion for the Controllable Preparation of Chitooligosaccharides. , 2019, Journal of agricultural and food chemistry.

[21]  M. Sohail,et al.  Chitosan oligosaccharide (COS): An overview. , 2019, International journal of biological macromolecules.

[22]  B. Moerschbacher,et al.  Structural and biochemical insight into mode of action and subsite specificity of a chitosan degrading enzyme from Bacillus spec. MN , 2019, Scientific Reports.

[23]  Laurent David,et al.  Enzymatic Production and Enzymatic-Mass Spectrometric Fingerprinting Analysis of Chitosan Polymers with Different Nonrandom Patterns of Acetylation. , 2019, Journal of the American Chemical Society.

[24]  Laihao Li,et al.  Cloning, purification and characterization of a novel GH46 family chitosanase, Csn-CAP, from Staphylococcus capitis , 2018, Process Biochemistry.

[25]  Lihua Jiang,et al.  Biochemical properties of a novel chitosanase from Bacillus amyloliquefaciens and its use in membrane reactor , 2018, LWT.

[26]  C. Xue,et al.  Cloning, characterization and substrate degradation mode of a novel chitinase from Streptomyces albolongus ATCC 27414. , 2018, Food chemistry.

[27]  Qiming Chen,et al.  Expression and characterization of a novel cold-adapted chitosanase suitable for chitooligosaccharides controllable preparation. , 2018, Food chemistry.

[28]  Xiangzhao Mao,et al.  Discovery and Characterization of a Novel Chitosanase from Paenibacillus dendritiformis by Phylogeny-Based Enzymatic Product Specificity Prediction. , 2018, Journal of agricultural and food chemistry.

[29]  V. Eijsink,et al.  Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bacillus subtilis (BsCsn46A). , 2018, Carbohydrate polymers.

[30]  Fakhra Liaqat,et al.  Chitooligosaccharides and their biological activities: A comprehensive review. , 2018, Carbohydrate polymers.

[31]  B. Moerschbacher,et al.  Chitinosanase: A fungal chitosan hydrolyzing enzyme with a new and unusually specific cleavage pattern. , 2017, Carbohydrate polymers.

[32]  G. Guebitz,et al.  Cellobiohydrolases Produce Different Oligosaccharides from Chitosan. , 2016, Biomacromolecules.

[33]  R. Brzezinski,et al.  Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes , 2015, Marine drugs.

[34]  Weizhi Liu,et al.  Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01. , 2015, Biochimica et biophysica acta.

[35]  P. V. Suresh,et al.  Enzymatic degradation of chitosan and production of d-glucosamine by solid substrate fermentation of exo-β-d-glucosaminidase (exochitosanase) by Penicillium decumbens CFRNT15 , 2015 .

[36]  Koichiro Tamura,et al.  MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. , 2013, Molecular biology and evolution.

[37]  Romy K. Scheerle,et al.  Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost , 2013, Applied Microbiology and Biotechnology.

[38]  R. Prasanna,et al.  Purification and characterization of a novel antifungal endo-type chitosanase from Anabaena fertilissima , 2012, Annals of Microbiology.

[39]  Abdul-Rahman Allouche,et al.  Gabedit—A graphical user interface for computational chemistry softwares , 2011, J. Comput. Chem..

[40]  V. Eijsink,et al.  Degradation of chitosans with a family 46 chitosanase from Streptomyces coelicolor A3(2). , 2010, Biomacromolecules.

[41]  Torsten Schwede,et al.  BIOINFORMATICS Bioinformatics Advance Access published November 12, 2005 The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling , 2022 .

[42]  T. Tanabe,et al.  Novel Chitosanase from Streptomyces griseus HUT 6037 with Transglycosylation Activity , 2003, Bioscience, biotechnology, and biochemistry.

[43]  Y. Higuchi,et al.  Crystal Structure of Chitosanase from Bacillus circulans MH-K1 at 1.6-Å Resolution and Its Substrate Recognition Mechanism* , 1999, The Journal of Biological Chemistry.

[44]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.