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Applied and Environmental Microbiology, August 2008, p. 5168-5177, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00121-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Enhancement of the Alcoholytic Activity of {alpha}-Amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by Site-Directed Mutagenesis{triangledown}

Juanita Yazmin Damián-Almazo, Alina Moreno, Agustin López-Munguía, Xavier Soberón, Fernando González-Muñoz, and Gloria Saab-Rincón*

Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62271, México

Received 15 January 2008/ Accepted 7 June 2008

AmyA, an {alpha}-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal {alpha}-1,4-glycosidic bonds in various {alpha}-glucans at 85°C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some {alpha}-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside.

* Corresponding author. Mailing address: Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México. Phone: (52) (777) 329-1640. Fax: (52) (777) 317-2388. E-mail: gsaab{at}ibt.unam.mx

{triangledown} Published ahead of print on 13 June 2008.

Applied and Environmental Microbiology, August 2008, p. 5168-5177, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00121-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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