α-Amylases from Thermoactinomyces vulgaris: characteristics,primary structure and structure prediction

Two amylolytic active protein fractions (named α-amylase 1 and α-amylase 2) were isolated from the bacterium Thermoactinomyces vulgaris strain 94-2A. α-Amylase 1 had a molecular mass of 51.6 kDa, whereas α-amylase 2 consists of two fragments which have molecular masses of 17.0 and 34.6 kDa, respectively. These two fragments are products from a proteolytic cleavage of a-amylase 1 at amino acid position 303 (tryptophan) by a serine protease (thermitase) which is also produced by T. vulgaris. The purified α-amylase 1 and 2 follow the Michaelis-Menten kinetics in the presence of starch as substrate with K_m values of 1.37 ± 0.07 and 1.29 ± 0.18 mg/mL, respectively. In effect they differ in their stability characteristics. The amino acid sequence of α-amylase from T. vulgaris derived from DNA sequence (1) was compared with those of other α-amylases. It reveals high homologies to α-amylases from other microorganisms (e.g. B. polymyxa, A. oryzae, S. occidentalis and S.fibuligera). A three-dimensional structure model for α-amylase 1 on the basis of the 3 Å X-ray structure of Taka-amylase was constructed.