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Journal of Bacteriology, June 2002, p. 3385-3391, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3385-3391.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Thermoadaptation of {alpha}-Galactosidase AgaB1 in Thermus thermophilus

Olafur Fridjonsson,* Hildegard Watzlawick, and Ralf Mattes

Institut für Industrielle Genetik, Universität Stuttgart, 70569 Stuttgart, Germany

Received 17 December 2001/ Accepted 15 March 2002

The evolutionary potential of a thermostable {alpha}-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid {alpha}-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD ({Delta}agaT) by transformation. This selector strain is unable to metabolize melibiose ({alpha}-galactoside) without recombinant {alpha}-galactosidases, because the native {alpha}-galactosidase gene, agaT, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene. With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67°C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant {alpha}-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.

* Corresponding author. Present address: Prokaria Ltd., Gylfaflöt 5, 112 Reykjavik, Iceland. Phone: (354) 570 7914. Fax: (354) 570 7901. E-mail: olafur{at}prokaria.com.

Journal of Bacteriology, June 2002, p. 3385-3391, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3385-3391.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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