Labbé Geneviève, de Groot Sarah, Rasmusson Timothy, Milojevic Gorica, Dmitrienko Gary I, Guillemette J Guy
Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, ON N2L3G1, Canada.
Protein Expr Purif. 2011 Dec;80(2):224-33. doi: 10.1016/j.pep.2011.06.020. Epub 2011 Jul 7.
Fructose 1,6-bisphosphate (FBP) aldolase has been used as biocatalyst in the synthesis of several pharmaceutical compounds such as monosaccharides and analogs. Is has been suggested that microbial metal-dependant Class II aldolases could be better industrial catalysts than mammalian Class I enzyme because of their greater stability. The Class II aldolases from four microbes were subcloned into the Escherichia coli vector pT7-7, expressed and purified to near homogeneity. The kinetic parameters, temperature stability, pH profile, and tolerance to organic solvents of the Class II enzymes were determined, and compared with the properties of the Class I aldolase from rabbit muscle. Contrary to results obtained previously with the E. coli Class II aldolase, which was reported to be more stable than the mammalian enzyme, other recombinant Class II aldolases were found to be generally less stable than the Class I enzyme, especially in the presence of organic solvents. Class II aldolase from Bacillus cereus showed higher temperature stability than the other enzymes tested, but only the Mycobacterium tuberculosis Class II aldolase had a stability comparable to the Class I mammalian enzyme under assay conditions. The turnover number of the recombinant M. tuberculosis and Magnaporthe grisea Class II type A aldolases was comparable or higher than that of the Class I enzyme. The recombinant B. cereus and Pseudomonas aeruginosa Class II type B aldolases had very low turnover numbers and low metal content, indicating that the E. coli overexpression system may not be suitable for the Class II type B aldolases from these microorganisms.
1,6 - 二磷酸果糖（FBP）醛缩酶已被用作生物催化剂，用于合成多种药物化合物，如单糖及其类似物。有人认为，微生物金属依赖性II类醛缩酶可能比哺乳动物I类酶更适合作为工业催化剂，因为它们具有更高的稳定性。将来自四种微生物的II类醛缩酶亚克隆到大肠杆菌载体pT7 - 7中，进行表达并纯化至接近均一。测定了II类酶的动力学参数、温度稳定性、pH曲线以及对有机溶剂的耐受性，并与兔肌肉I类醛缩酶的性质进行了比较。与之前报道的大肠杆菌II类醛缩酶比哺乳动物酶更稳定的结果相反，发现其他重组II类醛缩酶通常比I类酶更不稳定，尤其是在有机溶剂存在的情况下。蜡样芽孢杆菌的II类醛缩酶比其他测试酶表现出更高的温度稳定性，但在测定条件下，只有结核分枝杆菌的II类醛缩酶具有与I类哺乳动物酶相当的稳定性。重组结核分枝杆菌和稻瘟病菌II类A型醛缩酶的转换数与I类酶相当或更高。重组蜡样芽孢杆菌和铜绿假单胞菌II类B型醛缩酶的转换数非常低且金属含量低，这表明大肠杆菌过表达系统可能不适用于这些微生物的II类B型醛缩酶。
