Bacillus sp. MB809에서의 알카리성 α-amylase의 말단부위 서열조사 및 알카리 특성에 관한 연구

Abstract

Alkaline α-amylase expressed in the transformant Bacillus subtilis containing alkaline amylase gene from the alkalophilic Bacillus sp.MB809 was purified through several separation processes. The molecular weight of the α-amylase is approximately 59,000 daltons on SDS-PAGE (polyacrylamide gel electrophoresis) and Sephadex G-100 gel filtration. Amino acid sequencing of terminal portion was done with eluted amylase fraction from the SDS-PAGE gel. NH_(2)-Terminal portion amino acid sequence of α-amyalse determined by the Edman degradation method and HPLC isocratic solvent elution resulted in NH_(2)-Ser-Thr- Ala-Pro-Ser-(Ile)-Lys-Ala-Gly-Thr-(Ile)-Leu-. COOH- Terminal portion amino acid sequence was determined by digestion of the α-amylase with gradient solvent elution resulted in -Thr-Trp-Pro-Lys-COOH. The chemical modification study on the α-amylase revealed that Histidine, Tyrosine and Tryptophane are active site component amino acids. In connection with these results, proteolytic modification of α-amylase COOH-Terminal portion has been practiced. The COOH-terminal portion deleted α-amylase exhibited a maximum stability and activity at pH 7 environment, which indicate that the COOH-terminal portion has very important roles in alkalophilicity mechanism of alkaline α-amylase from Bacillus sp MB809.