Cloning, expression, purification, and characterization of a glycoside hydrolase family 44 cellulase from Clostridium acetobutylicum in Escherichia coli

ABSTRACT A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a β-1,6-galactanase that preferentially debranches β-1,6-galactobiose from the substrate.

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Substrate-Induced Production and Secretion of Cellulases by Clostridium acetobutylicum

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5238-5243.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5238-5243 ◽

Cited By ~ 38

Author(s):

Ana M. López-Contreras ◽

Krisztina Gabor ◽

Aernout A. Martens ◽

Bernadet A. M. Renckens ◽

Pieternel A. M. Claassen ◽

...

Keyword(s):

Catabolite Repression ◽

Clostridium Acetobutylicum ◽

Glycoside Hydrolase ◽

Protein A ◽

Cellulase Activity ◽

Glycoside Hydrolase Family ◽

Extracellular Production ◽

Encoding Genes ◽

Glycoside Hydrolase Family 48 ◽

Hydrolase Family

ABSTRACT Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of β-1,3- and β-1,4-linked β-d-glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli, and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum. Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.

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Characterization of the endoglucanase and glucomannanase activities of a glycoside hydrolase family 45 protein from Penicillium decumbens 114-2

The Journal of General and Applied Microbiology ◽

10.2323/jgam.56.223 ◽

2010 ◽

Vol 56 (3) ◽

pp. 223-229 ◽

Cited By ~ 24

Author(s):

Guodong Liu ◽

Xiaomin Wei ◽

Yuqi Qin ◽

Yinbo Qu

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Penicillium Decumbens ◽

Hydrolase Family

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Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

10.1101/2021.10.27.466152 ◽

2021 ◽

Author(s):

Gregory S Bulmer ◽

Fang Wei Yuen ◽

Naimah Begum ◽

Bethan S Jones ◽

Sabine S Flitsch ◽

...

Keyword(s):

Aspergillus Niger ◽

Glycoside Hydrolase ◽

Biochemical Characterization ◽

Maximum Activity ◽

Glycoside Hydrolase Family ◽

Enzyme Specificity ◽

Fungal Enzymes ◽

Glycoside Hydrolase Family 43 ◽

Hydrolase Family

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

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