Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcusalbus

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• 作者：Shigeaki Ito ; Shigeki Hamada ; Hiroyuki Ito ; Hirokazu Matsui ; Tadahiro Ozawa ; Hidenori Taguchi and Susumu Ito
• 关键词：Cellobiose 2 ; epimerase ; Epilactose ; Homology modeling ; Prebiotics ; Ruminococcus albus ; Site ; directed mutagenesis
• 刊名：Biotechnology Letters
• 出版年：2009
• 出版时间：July, 2009
• 年：2009
• 卷：31
• 期：7
• 页码：1065-1071
• 全文大小：970.4 KB

文摘

The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-β-d-glucopyranosyl-d-mannose and 4-O-β-d-galactopyranosyl-d-mannose (epilactose). Based on the sequence alignment with N-acetyl-d-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (α/α)₆ core barrel structure.