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Purification and characterization of galacto‐oligosaccharide‐producing β‐galactosidase from Sirobasidium magnum
Author(s) -
Onishi N.,
Tanaka T.
Publication year - 1997
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1046/j.1472-765x.1997.00351.x
Subject(s) - isoelectric point , chemistry , oligosaccharide , chromatography , lactose , agarose , size exclusion chromatography , enzyme , concanavalin a , molecular mass , isoelectric focusing , gel electrophoresis , agarose gel electrophoresis , biochemistry , in vitro , gene
Galacto‐oligosaccharide‐producing β‐galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p ‐aminobenzyl 1‐thio‐β‐ d ‐galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β‐galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β‐galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The K m values for o ‐nitrophenyl‐β‐ d ‐galactopyranoside and lactose were 14·3 and 5·5 mmol l −1 , respectively, and the V max values for these substrates were 33·4 and 94·5 μmol min −1 mg of protein −1 , respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml −1 galacto‐oligosaccharide was produced from 200 mg ml −1 lactose.