Two exo-β-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases

Abstract

A GlcNase (exo-beta-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with K(m) and kcat values of 0.16 mg/ml and 2832 min(-1). On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative beta-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable beta-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with beta-galactosidase, beta-glucuronidase or beta-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.

Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

“…The catalytic module of P. barcinonensis Xyn30D shows homology to the biochemically characterized GH30 xylanases XynC from Bacillus subtilis (38), Xyn5B from Bacillus sp. BP-7 (18), and XynA from Erwinia chrysanthemi (22, 47) (81, 79, and 41% identity, respectively), while the carbohydrate-binding module of P. barcinonensis Xyn30D shows 41% identity to CBM35 from exo-␤-D-glucosaminidase Csxa from Amycolatopsis orientalis (14), 38% identity to rhamnogalacturonan acetyl esterase Rgae12A from Clostridium thermocellum (Che_3141), and 35% identity to CBM35 from Xyn10B from Cellvibrio japonicus (24).…”

Microbial enzyme systems for biomass conversion: emerging paradigms

“…Free enzyme systems of aerobic microorganisms are characterized by enzymes, which are usually secreted directly into the extracellular milieu in relatively large quantities and with essentially no residence time on Exo-b-d-glucosaminidase CBM35 Amycolatopsis orientalis [125] Cel48A Cellobiohydrolase CBM37 Ruminococcus albus [97] Cel9B Endoglucanase CBM37 R. albus [97] Cel9C Endoglucanase CBM37 R. albus [98] Cel5G Endoglucanase CBM37 R. albus [98] Xyn11C Xylanase CBM37 R. albus [98] CBM35, CBM37, CBMs from families 35 and 37, respectively. CBM: Cellulose binding module; SLH: S-layer homology module; Ssm: Sortase-signal motif (LPXTG).…”

Chitosan-Soyprotein Interaction as Determined by Thermal Unfolding Experiments

“…In recent years, we have been working with chitosan-degrading enzymes, such as endochitosanase and exo--glucosaminidase. [10][11][12] In the course of these studies, we have developed several strategies for examining chitosan-protein interaction. [13][14][15] Among these strategies, thermal unfolding experiments are most convenient for simple evaluation of the ability of chitosan interaction with the enzyme protein.…”