Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate

Abstract

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 ␤-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both ␤-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-␤-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ ␤-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 ␤-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.Glycosyl hydrolases (EC 3.2.1 to 3.2.3) cleave the glycosidic bond between two or more carbohydrates or between a carbohydrate and another moiety. Traditionally, glycosyl hydrolases were classified based on functional similarity. More recently, however, Henrissat and his coworkers have organized these enzymes into 90 glycosyl hydrolase families characterized by hydrophobicity plots, amino acid sequence similarities, and reaction mechanisms (10-12). This system also identifies possible common structural domains, thereby defining evolutionary connections and suggesting reaction mechanisms for the glycosyl hydrolases. Based on these criteria, the once-unified group of enzymes that exhibit ␤-galactosidase activity (EC 3.2.1.23) are now subdivided into four different families: 1, 2, 35, and 42.Of these, the best studied is family 2, which includes the well-characterized ␤-galactosidase from Escherichia coli that is encoded by the lacZ gene. Although there is considerable information about the regulation (1), biochemistry (18,23,35,47), reaction mechanism (17, 45), and structure (16) of this LacZ ␤-galactosidase, few other ␤-galactosidases within this family have been characterized biochemically (4, 7, 13-15, 26, 27, 43), while most examples exist only as a published sequence. Because of the emphasis on the E. coli LacZ enzyme, opportunities to lea...

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

“…The lacZ gene was also placed into p⌬␣18 (pLacZ). The lacZ gene was from a pET28a(ϩ) construct (6), and an XbaI site was created in a noncoding 5Ј region using the QuikChange site-directed mutagenesis kit (Stratagene) to obtain constructs without a six-His tag (Table 1).…”

Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7

“…We have been investigating psychrophilic microorganisms and their cold-active glycoside hydrolases (7,9,27,37) with particular interest in their physiological roles. As part of this work, we enriched for and isolated numerous psychrophilic spore-forming bacteria to obtain phylogenetically related organisms with ␤-galactosidases active on the chromogen 5-bromo-4-chloro-3-indoyl-␤-D-galactopyranoside (X-Gal) at low temperatures.…”

Isolation and identification of a strain producing cold-adapted β galactosidase, and purification and characterisation of the enzyme

“…The β-galactosidase gene from E.coli has been so well studied and exploited as a reporter gene, which is coded for by the lacZ gene. However, a number of other important genes coding for cold active β-galactosidase have been detected in arthrobacter (Coker et al 2003;Nakagawa et al 2003), Pseudoalteromons (Hoyoux et al 2001;Fernandes et al 2002), Rahnella aquatilis (Park et al 2006), and even in yeast (Nakagawa et al 2006a, b). Cold-adapted β-galactosidase with high activity levels at low temperatures might prove to be useful for removing lactose from refrigerated milk enabling it to be consumed by lactose-intolerant individuals, and for converting lactose in whey into glucose and galactose.…”

“…Studies on the isolation of cold-adapted β-galactosidase producing strains have been performed from 1993 to now (Loveland et al 1993(Loveland et al ,1994Trimbur et al 1994;Hoyoux et al 2001;Fernandes et al 2002;Coker et al 2003Coker et al , 2006Nakagawa et al 2003Nakagawa et al , 2006aNakagawa et al , b, 2007Turkiewica et al 2003;Hubert et al 2005;Park et al 2006)..…”