2024-03-28T17:20:43Zhttps://eprints.lib.hokudai.ac.jp/dspace-oai/requestoai:eprints.lib.hokudai.ac.jp:2115/387542022-11-17T02:08:08Zhdl_2115_20074hdl_2115_161Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albusIto, ShigeakiHamada, ShigekiIto, HiroyukiMatsui, HirokazuOzawa, TadahiroTaguchi, HidenoriIto, SusumuCellobiose 2-epimeraseRuminococcus albusHomology modelingSite-directed mutagenesisEpilactosePrebiotics464Cellobiose 2-epimerase (EC 5.1.3.11) from Ruminococcus albus (RaCE) catalyzes the reversible 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 structure-based amino acid sequence alignment with N-acetyl-D-glucosamine 2-epimerases (EC 5.1.3.8) from porcine kidney and Anabaena sp. CH1 and on the computer-aided model building of the tertiary structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. It was found that R52, H243, E246, W249, M251, W304, E308, H374, and M378 were absolutely required for the activity of RaCE. F114 and W303 (and possibly R377) also contributed to catalysis. These possible catalytic protruded into or near the active-site cleft surrounded by the inner α-helices in the predicted (α/α)6 core barrel structure of RaCE.Springer NetherlandsJournal Articleapplication/pdfhttp://hdl.handle.net/2115/38754https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/38754/1/31-7_p1065-1071.pdf0141-5492Biotechnology Letters317106510712009-07enginfo:pmid/19330485info:doi/10.1007/s10529-009-9979-3The original publication is available at
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