To achieve a thermostable beta-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 degrees C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80 degrees C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100 degrees 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.