Biochemical and molecular characterization of a cellobiohydrolase from <Emphasis Type="Italic">Trametes versicolor</Emphasis> |
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Authors: | Karim Lahjouji Reginald Storms Zhizhuang Xiao Kwang-Bo Joung Yun Zheng Justin Powlowski Adrian Tsang Luc Varin |
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Institution: | (1) Centre for Structural and Functional Genomics, Biology Department, Concordia University, 7141 Sherbrooke street West, Montréal, Quebec, H4B 1R6, Canada |
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Abstract: | A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion
signal but does not possess a linker region nor a carbohydrate-binding domain. Two peaks of activity were obtained after TvCel7a
was purified to apparent homogeneity by gel-filtration followed by anion-exchange chromatography. Mass spectrometry performed
on the purified proteins confirmed that both peaks corresponded to the predicted sequence of the T. versicolor cellulase. The biochemical properties of the purified TvCel7a obtained from both peaks were studied in detail. The pH and
temperature optima were 5.0 and 40°C, respectively. The enzyme was stable over a pH range extending from pH 3.0 to 9.0 and
at temperatures lower than 50°C. The kinetic parameters with the substrate p-nitrophenyl β-d-cellobioside (pNPC) were 0.58 mM and 1.0 μmol/min/mg protein for the purified TvCel7a found in both peaks 1 and 2. TvCel7a
catalyzes the hydrolysis of pNPC, filter paper, β-glucan, and avicel to varying extents, but no detectable hydrolysis was
observed when using the substrates carboxymethylcellulose, laminarin and pNPG. |
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Keywords: | Cellulase Cellobiohydrolase Trametes versicolor Expression Substrate specificity Enzymatic properties |
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