Purification and Characterization of Chitosanase from Bacillus sp. Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides

ABSTRACT For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.

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A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40Tand Its Transglycosylase Activity

Applied and Environmental Microbiology ◽

10.1128/aem.00635-16 ◽

2016 ◽

Vol 82 (14) ◽

pp. 4340-4349 ◽

Cited By ~ 9

Author(s):

Damao Wang ◽

Do Hyoung Kim ◽

Nari Seo ◽

Eun Ju Yun ◽

Hyun Joo An ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Marine Bacterium ◽

Enzymatic Reaction ◽

Glycoside Hydrolase Family ◽

Reaction Products ◽

Brown Macroalgae ◽

Fungal Cell ◽

Content Type ◽

Saccharophagus Degradans ◽

Hydrolase Family

ABSTRACTIn this study, we characterized Gly5M, originating from a marine bacterium, as a novel β-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. Thegly5Mgene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) inSaccharophagus degradans2-40T. Thegly5Mgene was cloned and overexpressed inEscherichia coli. Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry, Gly5M was identified as a novel β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75) in GH5. The β-1,3-endoglucanase and β-1,6-endoglucanase activities were detected by using laminarin (a β-1,3-glucan with β-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a β-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes.IMPORTANCEIn this study, we have discovered a novel β-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium,Saccharophagus degradans2-40T. Gly5M was identified as the newly found β-1,3-endoglucanase and bacterial β-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucans and β-1,6-glucans. Gly5M also possesses a transglycosylase activity toward β-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides.

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Xylanases of glycoside hydrolase family 30 – An overview

Biotechnology Advances ◽

10.1016/j.biotechadv.2021.107704 ◽

2021 ◽

Vol 47 ◽

pp. 107704

Author(s):

Vladimír Puchart ◽

Katarína Šuchová ◽

Peter Biely

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Hydrolase Family ◽

Glycoside Hydrolase Family 30

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Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

Applied Sciences ◽

10.3390/app11094048 ◽

2021 ◽

Vol 11 (9) ◽

pp. 4048

Author(s):

Javier A. Linares-Pastén ◽

Lilja Björk Jonsdottir ◽

Gudmundur O. Hreggvidsson ◽

Olafur H. Fridjonsson ◽

Hildegard Watzlawick ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Ligand Docking ◽

Glycoside Hydrolase Family ◽

Ligand Interactions ◽

Tim Barrel ◽

Branching Point ◽

The Difference ◽

Dynamics Simulations ◽

And Function ◽

Hydrolase Family

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

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Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)84038-3 ◽

2006 ◽

Vol 281 (42) ◽

pp. 31254-31267

Author(s):

Claire Moulis ◽

Gilles Joucla ◽

David Harrison ◽

Emeline Fabre ◽

Gabrielle Potocki-Veronese ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Polymerization Mechanism ◽

Hydrolase Family

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Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005134 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18296-18308 ◽

Cited By ~ 10

Author(s):

Chelsea Vickers ◽

Feng Liu ◽

Kento Abe ◽

Orly Salama-Alber ◽

Meredith Jenkins ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Biological Activities ◽

Bacterial Species ◽

Structural Data ◽

Side Chain ◽

Glycoside Hydrolase Family ◽

X Ray Crystallography ◽

Catalytic Mechanisms ◽

Thickening Agents ◽

Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

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Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity

FEBS Journal ◽

10.1111/febs.12424 ◽

2013 ◽

Vol 280 (18) ◽

pp. 4560-4571 ◽

Cited By ~ 3

Author(s):

Takatsugu Miyazaki ◽

Megumi Ichikawa ◽

Gaku Yokoi ◽

Motomitsu Kitaoka ◽

Haruhide Mori ◽

...

Keyword(s):

Substrate Specificity ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Hydrolase Family

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Substrate Specificity in Glycoside Hydrolase Family 10

Journal of Biological Chemistry ◽

10.1074/jbc.275.30.23020 ◽

2000 ◽

Vol 275 (30) ◽

pp. 23020-23026 ◽

Cited By ~ 51

Author(s):

Valérie Ducros ◽

Simon J. Charnock ◽

Urszula Derewenda ◽

Zygmunt S. Derewenda ◽

Zbigniew Dauter ◽

...

Keyword(s):

Substrate Specificity ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Glycoside Hydrolase Family 10 ◽

Hydrolase Family

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The α-glucuronidase Agu1 from Schizophyllum commune is a member of a novel glycoside hydrolase family (GH115)

Applied Microbiology and Biotechnology ◽

10.1007/s00253-011-3157-y ◽

2011 ◽

Vol 90 (4) ◽

pp. 1323-1332 ◽

Cited By ~ 31

Author(s):

Sun-Li Chong ◽

Evy Battaglia ◽

Pedro M. Coutinho ◽

Bernard Henrissat ◽

Maija Tenkanen ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Schizophyllum Commune ◽

Glycoside Hydrolase Family ◽

Hydrolase Family

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Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

Biochemical Journal ◽

10.1042/bj1970629 ◽

1981 ◽

Vol 197 (3) ◽

pp. 629-636 ◽

Cited By ~ 19

Author(s):

J L McKenzie ◽

A K Allen ◽

J W Fabre

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Human Brain ◽

Affinity Chromatography ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Antibody Affinity ◽

Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

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