Labbé Geneviève, Bezaire Jeremy, de Groot Sarah, How Christine, Rasmusson Tim, Yaeck Jason, Jervis Eric, Dmitrienko Gary I, Guillemette J Guy
Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, Ont., Canada N2L 3G1.
Protein Expr Purif. 2007 Jan;51(1):110-9. doi: 10.1016/j.pep.2006.06.020. Epub 2006 Jun 28.
The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes.
来自稻瘟病病原体稻瘟病菌的II类果糖1,6 - 二磷酸醛缩酶被亚克隆到大肠杆菌载体pT7 - 7中。该酶在小型台式反应器中通过补料分批发酵进行过量表达。从1.5升培养物中总共获得了275克细胞和1.3克比活性为70 U/mg的高度纯化的酶。纯化后的酶是由39.6 kDa亚基组成的同型二聚体，活性位点含有一个锌离子。动力学表征表明，该酶催化果糖1,6 - 二磷酸裂解时，K(m)为51 microM，k(cat)为46 s(-1)，最适pH为7.8。所报道的发酵系统程序例证了使用实验室规模生物反应器大规模生产重组酶的潜力。
