Substrate and product hydrolysis specificity in family 11 glycoside hydrolases: an analysis of Penicillium funiculosum and Penicillium griseofulvum xylanases
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Two genes encoding family 11 endo-(1,4)-β-xylanases from Penicillium griseofulvum (PgXynA) and Penicillium funiculosum (PfXynC) were heterologously expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the recombinant enzymes were purified after affinity chromatography and proteolysis. PgXynA and PfXynC were identical to their native counterparts in terms of molecular mass, pI, N-terminal sequence, optimum pH, and enzymatic activity towards arabinoxylan. Further investigation of the rate and pattern of hydrolysis of PgXynA and PfXynC on wheat soluble arabinoxylan showed the predominant production of xylotriose and xylobiose as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing chain length (n) of oligomer up to n = 6, suggesting that the specificity region of both Penicillium xylanases spans about six xylose units. In contrast to PfXynC, PgXynA was found insensitive to the wheat xylanase inhibitor protein XIP-I.
KeywordsGlycoside hydrolase family 11 Heterologous expression Kinetic parameters Wheat arabinoxylan Xylo-oligosaccharides Xylanase inhibitor
We thank Caroline Furniss for providing PfXynC cDNA, Tariq Tahir for providing XIP-I and Claude Villard for mass spectrometry analysis. This study was financially supported by the European Commission in the Communities Sixth Framework Programme, Project Healthgrain (Food-CT-2005-514008). This publication reflects only author’s views, and the Community is not liable for any use that may be made of the information contained in this publication.
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