Pharmaceutical Research Center, PCSIR Laboratories Complex, Karachi, Pakistan.
AAPS PharmSciTech. 2011 Mar;12(1):255-61. doi: 10.1208/s12249-011-9586-1. Epub 2011 Jan 14.
Purification of extracellular α-amylase from Bacillus subtilis KIBGE HAS was carried out by ultrafiltration, ammonium sulfate precipitation and gel filtration chromatography. The enzyme was purified to homogeneity with 96.3-fold purification with specific activity of 13011 U/mg. The molecular weight of purified α-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular α-amylase showed that the enzyme had a Km and V (max) value of 2.68 mg/ml and 1773 U/ml, respectively. The optimum activity was observed at pH 7.5 in 0.1 M phosphate buffer at 50 °C. The amino acid composition of the enzyme showed that the enzyme is rich in neutral/non polar amino acids and less in acidic/polar and basic amino acids. The N-terminal protein sequence of 10 residues was found to be as Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly (S-S-N-K-L-T-T-S-W-G). Furthermore, the protein was not N-terminally blocked. The sequence of α-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported α-amylases from Bacillus strain.
从枯草芽孢杆菌 KIBGE HAS 中提取胞外α-淀粉酶，采用超滤、硫酸铵沉淀和凝胶过滤层析进行纯化。该酶经过 96.3 倍的纯化，比活为 13011 U/mg，达到均一性。SDS-PAGE 发现纯化的α-淀粉酶的分子量为 56000 Da。胞外α-淀粉酶的特性表明，该酶的 Km 和 V（max）值分别为 2.68 mg/ml 和 1773 U/ml。在 50°C、pH7.5 的 0.1 M 磷酸盐缓冲液中，酶的活性最高。酶的氨基酸组成表明，该酶富含中性/非极性氨基酸，而酸性/极性和碱性氨基酸较少。酶的 N 端 10 个残基的序列为 Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly（S-S-N-K-L-T-T-S-W-G）。此外，该蛋白未被 N 端封闭。枯草芽孢杆菌 KIBGE HAS 的α-淀粉酶序列是一个新序列，与其他报道的枯草芽孢杆菌来源的α-淀粉酶没有同源性。
