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Applied and Environmental Microbiology, November 2009, p. 7026-7036, Vol. 75, No. 22
0099-2240/09/$08.00+0     doi:10.1128/AEM.01781-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Gene Cloning, Protein Characterization, and Alteration of Product Selectivity for the Trehalulose Hydrolase and Trehalulose Synthase from "Pseudomonas mesoacidophila" MX-45{triangledown} ,{dagger}

Hildegard Watzlawick* and Ralf Mattes

Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

Received 27 July 2009/ Accepted 18 September 2009

The naturally occurring structural isomer of sucrose, trehalulose, is produced by sucrose isomerase (SI). Screening of chromosomal DNA from "Pseudomonas mesoacidophila" MX-45 with an SI-specific probe facilitated the cloning of two adjacent gene homologs, mutA and mutB. Both genes were expressed separately in Escherichia coli, and their enzyme products were characterized. MutA hydrolyzed the substrates trehalulose, isomaltulose, and sucrose into glucose and fructose. Due to its highest activity on trehalulose, MutA was referred to as trehalulase. mutB encodes the SI (trehalulose synthase) and catalyzes the isomerization of sucrose to mainly trehalulose. From Northern blot analysis it is apparent that the mutB gene is not transcribed as part of an operon and was transcriptionally upregulated when P. mesoacidophila MX-45 cells were grown in sucrose medium, whereas under investigated conditions no transcript for mutA was detected. Mutants of mutB were created by a random mutagenesis approach in order to alter the product specificity of MutB. Two types of mutants have emerged, one type that prefers the hydrolytic reaction on sucrose and another type that still acts as an SI but with a significant shift in the product from trehalulose to isomaltulose. The hydrolytic character of MutB R311C was demonstrated through its higher catalytic efficiency for glucose production over trehalulose production. MutB D442N favored the transfer reaction, with an isomer preference for isomaltulose.

* Corresponding author. Mailing address: Institut für Industrielle Genetik, Allmandring 31, 70569 Stuttgart, Germany. Phone: 49 711 685 66981. Fax: 49 711 685 66973. E-mail: hildegard.watzlawick{at}iig.uni-stuttgart.de

{triangledown} Published ahead of print on 25 September 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, November 2009, p. 7026-7036, Vol. 75, No. 22
0099-2240/09/$08.00+0     doi:10.1128/AEM.01781-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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