Cloning of the Gene Encoding a Novel Thermostable α-Galactosidase from Thermus brockianus ITI360

Abstract

An α-galactosidase gene from Thermus brockianusITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified. The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53,810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the α-galactosidases ofThermotoga neapolitana and Thermotoga maritimaand a low-level sequence similarity to α-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93°C withpara-nitrophenyl-α-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80°C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. TheKm s for melibiose and raffinose at 80°C were determined as 4.1 and 11.0 mM, respectively. The α-galactosidase gene in T. brockianus ITI360 was inactivated by integrational mutagenesis. Consequently, no α-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.

Cloning and characterization of a novel α-galactosidase fromBifidobacterium breve203 capable of synthesizing Gal-α-1,4 linkage

“…Figure 1 shows a multiple sequence alignment of the deduced amino acid sequences of Aga2 and several other microbial a-galactosidases. The characteristic consensus motif of a-galactosidase ( (Fridjonsson et al, 1999) was found to exist in this protein (…”

Production of soya milk containing low flatulence‐causing oligosaccharides in a packed bed reactor using immobilised α‐galactosidase

“…Stachyose (C 24 H 42 O 21 , galactose a-1,6-raffinose) is a tetrasaccharide consisting of fructose, glucose and two molecules of galactose both in an a-(1-6) glycosidic linkage to each other and to glucose. The a-1,6 glycosidic bond that joins the residue of galactose to the glucose present in raffinose and stachyose can be easily hydrolysed by the enzyme a-galactosidase (Fridjonsson et al, 1999). Because humans lack this enzyme, these oligosaccharides remain unhydrolysed in the upper intestine.…”

“…Most studies on purification and characterisation of a-galactosidases indicate that the enzyme functions as a tetramer (Brumer et al, 1999;Fridjonsson et al, 1999;Baik et al, 2000;Fujimoto et al, 2003). However, Ishiguro et al (2001) purified a recombinant a-galactosidase and is shown to be a hexamer.…”

“…For a large number of enzymes, the presence of metal ions is crucial for its activity. But it has been shown that the activity of a-galactosidase is not influenced by metal ions such as Na + , K + , Li + , Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ (Garro et al, 1996;Liebl et al, 1998;Fridjonsson et al, 1999) or, in other words, the enzyme does not have any metal cofactor requirement. The nonrequirement of metal ions as cofactors for enzyme activity gives some advantages to the use of a-galactosidase in the immobilised form by avoiding the necessity of coimmobilising the enzyme and the metal ion.…”

Galactomannan hydrolysis and mannose metabolism inCellvibrio mixtus

“…Ultimately, the substrate specificity of these enzymes is not restricted to the debranching activity of hemicelluloses, as a-galactosidases have been shown to be crucial in other biological systems. For example, removal of galactose from short chain galactooligosaccharides, such as melibiose, raffinose and stachyose that are carbohydrate reserves in leguminous plants, is catalysed by plant and microbial a-galactosidases usually located in families 4 and 36 (Fridjonsson et al, 1999). In eukaryotes, lysosomal a-galactosidases are involved in the catabolism of large macromolecules, such as glycoproteins and glycolipids, and these enzymes belong to family 27 (Garman & Garboczi, 2004).…”