Increasing the thermal stability of an oligomeric protein, beta-glucuronidase

被引:54
作者
Flores, H [1 ]
Ellington, AD [1 ]
机构
[1] Univ Texas, Dept Chem & Biochem, Inst Mol & Cellular Biol, Austin, TX 78712 USA
关键词
beta-glucuronidase; directed evolution; thermostability; DNA shuffling; random mutagenesis;
D O I
10.1006/jmbi.2001.5223
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70degreesC, whereas the wild-type enzyme lost function at 65degreesC. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80degreesC), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary, structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads,to enzymes that have kinetic parameters that are optimized for high temperatures. (C) 2002 Academic Press.
引用
收藏
页码:325 / 337
页数:13
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