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Purification, properties and characterization of a high‐molecular‐mass b‐galactosidase isoenzyme from Thermusaquaticus YT‐I
Author(s) -
Berger JeanLuc,
Lee Byong H.,
Lacroix Christophe
Publication year - 1997
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1997.tb00411.x
Subject(s) - chemistry , size exclusion chromatography , molecular mass , chromatography , thermostability , enzyme assay , enzyme , cellobiose , monosaccharide , thermus , biochemistry , cellulase , thermophile
An unusual high‐molecular‐mass b‐galactosidase isoenzyme from Thermus aquaticus YT‐1 was purified to homogeneity by chromatography using gel filtration (Ultrogel AcA 34), anion‐exchange (Mono Q) and gel filtration (Superose‐12). The molecular mass of the purified enzyme was > 700 kDa as estimated by gel filtration, and its subunit molecular mass was 59 ± 1 kDa as determined by SDS/PAGE. A single protein band of pI 4.9 was obtained by isoelectric‐focusing gel electrophoresis. The optimum tempera‐ture and pH for enzyme activity were 80 ° C and pH 5.5 respectively. The enzyme was stable over a wide pH range (pH 3‐12), and the thermostability of the enzyme was enhanced by CaCl 2 . The enzyme was significantly activated by alkali and alkaline‐earth‐metal salts. Whereas reducing agents enhanced b‐galactosidase activity, thiol‐binding agents drastically decreased the enzyme activity. The enzyme was strongly inhibited by the end products of the lactose hydrolysis reaction. The b‐galactosidase was specific for b‐D anomeric linkages, and the identity of the aglycone moiety also influenced enzyme activity dramatically. Oligosaccharide (OS) formation rates were directly related to lactose concentration. At a concentration of 19.4% (w/v) lactose, OS accounted for approx. 20′ of the total carbohydrates, and only two types of OS were observed. Moreover, once formed, OS were not rehydrolysed to monosaccharides, even during long residence times. OS synthesis was also observed with cellobiose and lactulose. Fructose, xylose, lyxose, rhamnose and arabinose were active as galactosyl acceptors, in contrast with glucose, ribose, fucose, tagatose and N ‐acetyl‐glucosamine.