ENZYMATIC-PROPERTIES OF A NOVEL LIQUEFYING ALPHA-AMYLASE FROM AN ALKALIPHILIC BACILLUS ISOLATE AND ENTIRE NUCLEOTIDE AND AMINO-ACID-SEQUENCES

A novel liquefying a-amylase (LAMY) was found in cultures of an alkali philic Bacillus isolate, KSM-1378, The specific activity of purified L AMY was approximately 5,000 U mg of protein(-1), a value two- to fivef old greater between pH 5 and 10 than that of an industrial, thermostab le Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 t o 8.5 and displayed maximum activity at 55 degrees C. The molecular ma ss deduced from sodium dodecyl sulfate-polyacrylamide gel electrophore sis was approximately 53 kDa, and the apparent isoelectric point was a round pH 9, This enzyme efficiently hydrolyzed various carbohydrates t o yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction, Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzym e. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a s ignal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da, LAMY exhibited relatively l ow amino acid identity to other liquefying amylases, such as the enzym es from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, d esignated I, II, III, and IV, and the putative catalytic triad were fo und in the deduced amino acid sequence of LAMY, Essentially, the seque nce of LAMY was consistent with the tertiary structures of reported am ylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (alpha/beta)(8) barrel motif in domain A.