Yao Qiang, Sun Tingting, Chen Guanjun, Liu Weifeng
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, 250100, P.R. China.
Biotechnol Lett. 2007 Aug;29(8):1243-7. doi: 10.1007/s10529-007-9379-5. Epub 2007 May 23.
The endoglucanase CelA from Clostridium thermocellum was strongly expressed in Bacillus subtilis. The enzyme was purified by Ni(2+)-affinity chromatography. Site-directed substitution of D278 with an asparagine or an alanine residue surprisingly did not decrease the apparent k(cat) value. Further substitutions of two other potentially critical residues, Y215 and D152, resulted in a 2-fold decrease in apparent k(cat) value for Y215P and complete loss of activity for D152N.
来自嗜热栖热放线菌的内切葡聚糖酶CelA在枯草芽孢杆菌中得到了高效表达。该酶通过镍离子亲和层析进行纯化。将D278定点替换为天冬酰胺或丙氨酸残基，令人惊讶的是并未降低表观催化常数（k(cat)）值。另外两个潜在关键残基Y215和D152的进一步替换，导致Y215P的表观催化常数降低了2倍，而D152N则完全丧失了活性。
