ZCOMPARISON OF PRIMARY STRUCTURES AND SUBSTRATE SPECIFICITIES OF 2 PULLULAN-HYDROLYZING ALPHA-AMYLASES, TVA-I AND TVA-II, FROM THERMOACTINOMYCES-VULGARIS, R-47

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme (1). Both e nzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclod extrins. We cloned and sequenced the TVA I gene. The TVA I gene consis ted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence c onsisting of 29 amino-acid residues. The similarity between the amino- acid sequence of mature TVA I with those of other pullulan/cyclodextri n-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulan ase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas k(cat)/K-m values of TVA I for pullul an were larger than that of TVA II, and TVA II could not hydrolyze sta rch completely. TVA II was inhibited by maltose, the hydrolysate of st arch, which seems to be the reason for inefficient hydrolysis of starc h. These kinetic properties indicate that TVA I and TVA II have differ ential physiological roles in sugar metabolism extracellularly and int racellularly, respectively.