Suen Wen-Chen, Zhang Ningyan, Xiao Li, Madison Vincent, Zaks Aleksey
Biotransformations Group, Schering-Plough Research Institute, Union, NJ 07083, USA.
Protein Eng Des Sel. 2004 Feb;17(2):133-40. doi: 10.1093/protein/gzh017. Epub 2004 Jan 12.
DNA family shuffling was used to create chimeric lipase B proteins with improved activity toward the hydrolysis of diethyl 3-(3',4'-dichlorophenyl)glutarate (DDG). Three homologous lipases from Candida antarctica ATCC 32657, Hyphozyma sp. CBS 648.91 and Crytococcus tsukubaensis ATCC 24555 were cloned and shuffled to generate a diverse gene library. A high-throughput screening assay was developed and used successfully to identify chimeric lipase B proteins having a 20-fold higher activity toward DDG than lipase B from C.antarctica ATCC 32657 and a 13-fold higher activity than the most active parent derived from C.tsukubaensis ATCC 24555. In addition, the stability characteristics of several highly active chimeric proteins were also improved as a result of family shuffling. For example, the half-life at 45 degrees C and melting point (T(m)) of one chimera exceeded those of lipase B from C.antarctica ATCC 32657 by 11-fold and 6.4 degrees C, respectively, which closely approached the stability characteristics of the most thermostable parent derived from Hyphozyma sp. CBS 648.91.
利用DNA家族改组技术构建了对3-(3',4'-二氯苯基)戊二酸二乙酯(DDG)水解活性更高的嵌合脂肪酶B蛋白。克隆并改组了来自南极假丝酵母ATCC 32657、Hyphozyma sp. CBS 648.91和筑波隐球菌ATCC 24555的三种同源脂肪酶，以生成一个多样化的基因文库。开发了一种高通量筛选测定法，并成功用于鉴定对DDG的活性比来自南极假丝酵母ATCC 32657的脂肪酶B高20倍、比来自筑波隐球菌ATCC 24555的活性最高的亲本高13倍的嵌合脂肪酶B蛋白。此外，由于家族改组，几种高活性嵌合蛋白的稳定性特征也得到了改善。例如，一种嵌合体在45℃的半衰期和熔点(Tm)分别比来自南极假丝酵母ATCC 32657的脂肪酶B长11倍和高6.4℃，这与来自Hyphozyma sp. CBS 648.91的最耐热亲本的稳定性特征非常接近。
