Lindenmuth Benjamin E, McDonald Karen A
Department of Chemical Engineering & Materials Science, University of California, Davis, CA 95616, USA.
Protein Expr Purif. 2011 Jun;77(2):153-8. doi: 10.1016/j.pep.2011.01.006. Epub 2011 Jan 22.
The endoglucanase (E1) from Acidothermus cellulolyticus has been used extensively in cellulase research. The goal of this work was to produce high levels of this enzyme in a system that facilitates purification. A codon-optimized synthetic gene for A. cellulolyticus E1 with a C-terminal histidine tag was cloned into the genome of Pichia pastoris. Strain KM71H expressed the most enzyme, with a yield of 550mg/L culture supernatant. The temperature optimum (80°C) and pH optimum (5.1) of the purified enzyme agree with previously determined values for the enzyme produced in other systems. Michaelis-Menten kinetic parameters were determined, using a fluorescent substrate (methylumbelliferyl-β-d-cellobioside) at various temperatures. This thermostable enzyme can be used in future cellulosic biofuels-related research.
嗜热栖热放线菌的内切葡聚糖酶（E1）已在纤维素酶研究中得到广泛应用。这项工作的目标是在一个便于纯化的系统中大量生产这种酶。将带有C端组氨酸标签的嗜热栖热放线菌E1的密码子优化合成基因克隆到毕赤酵母基因组中。KM71H菌株表达的酶最多，培养上清液中的产量为550mg/L。纯化酶的最适温度（80°C）和最适pH（5.1）与先前在其他系统中产生的该酶的测定值一致。使用荧光底物（甲基伞形酮基-β-D-纤维二糖苷）在不同温度下测定了米氏动力学参数。这种热稳定酶可用于未来与纤维素生物燃料相关的研究。
