Department of PG Studies and Research in Biochemistry, Jnana Sahyadri, Kuvempu University, Shankaraghatta, Karnataka, India.
J Basic Microbiol. 2014 May;54(5):386-96. doi: 10.1002/jobm.201200656. Epub 2013 Jun 17.
The extracellular thermoalkaline lipase from Geobacillus sp. Iso5 was purified to homogeneity by ultrafiltration, 6% cross-linked agarose and Phenyl spehrose HIC column chromatography. The final purified lipase resulted in 8.7-fold with 6.2% yield. The relative molecular weight of the enzyme was determined to be a monomer of 47 kDa by SDS-PAGE and MALDI-TOF MS/MS spectroscopy. The purified enzyme exhibit optimum activity at 70 °C and pH 8.0. The enzyme retained above 90% activity at temperatures of 70 °C and about 35% activity at 85 °C for 2 h. However, the stability of the enzyme decreased at the temperature over 90 °C. The enzyme activity was promoted in the presence of Ca(2+) and Mg(2+) and strongly inhibited by HgCl2 , PMSF, DTT, K(+) , Co(2+) , and Zn (2+) . EDTA did not affect the enzyme activity. The secondary structure of purified lipase contains 36% α-helix and 64% β-sheet which was determined by Circular dichromism, FTIR, and Raman Spectroscopy.
从地芽孢杆菌 Iso5 中提取的胞外嗜热碱性脂肪酶经超滤、6%交联琼脂糖和苯基琼脂糖 HIC 柱层析纯化至均一性。最终纯化的脂肪酶的产率为 6.2%，比活提高了 8.7 倍。SDS-PAGE 和 MALDI-TOF MS/MS 光谱法测定该酶的相对分子质量为单体 47 kDa。纯化酶在 70°C 和 pH 8.0 时表现出最佳活性。该酶在 70°C 下保持超过 90%的活性，在 85°C 下保持约 35%的活性 2 小时。然而，该酶的稳定性在超过 90°C 的温度下下降。该酶在 Ca(2+)和 Mg(2+)的存在下活性增强，HgCl2 、PMSF、DTT、K(+)、Co(2+)和 Zn(2+)强烈抑制其活性。EDTA 不影响酶活性。通过圆二色性、傅里叶变换红外光谱和拉曼光谱测定，纯化脂肪酶的二级结构含有 36%的α-螺旋和 64%的β-折叠。
