PEDS Advance Access originally published online on June 8, 2007
Protein Engineering Design and Selection 2007 20(7):319-325; doi:10.1093/protein/gzm023
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Concurrent mutations in six amino acids in ß-glucuronidase improve its thermostability
1 Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, China 2Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA 3Department of Plant Science, University of Connecticut, Storrs, CT 06269, USA 4 College of Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China 5 College of Bioscience and Biotechnology, Yangzhou University, 88 S. Daxue Road, Yangzhou 225009, China
6 To whom correspondence should be addressed. E-mail: yaoquanhongcn{at}yahoo.com.cn
To achieve a thermostable ß-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70°C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80°C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100°C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.
Keywords: ß-glucuronidase/directed evolution/enzyme properties/structure-function analysis/thermostability
Received January 14, 2007; revised April 29, 2007; accepted April 29, 2007.