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

Abstract

ABSTRACT An α-galactosidase gene from Thermus brockianus ITI360 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 of Thermotoga neapolitana and Thermotoga maritima and 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 with para -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. The K m 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.