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Protein Engineering vol. 16 no. 7 pp. 505-514, 2003
© 2003 Oxford University Press

{alpha}-Amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity

Manuel Heriberto Rivera, Agustín López-Munguía, Xavier Soberón and Gloria Saab-Rincón1

Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, México

1 To whom correspondence should be addressed. e-mail: gsaab{at}ibt.unam.mx

The {alpha}-amylase from Bacillus licheniformis is the most widely used enzyme in the starch industry owing to its hyperthermostability, converting starch to medium-sized oligosaccharides. Based on sequence alignment of homologous amylases, we found a semi-conserved sequence pattern near the active site between transglycosidic and hydrolytic amylases, which suggested that hydrophobicity may play a role in modifying the transglycosylation/hydrolysis ratio. Based on this analysis, we replaced residue Val286 by Phe and Tyr in Bacillus licheniformis {alpha}-amylase. Surprisingly, the two resultant mutant enzymes, Val286Phe and Val286Tyr, showed two different behaviors. Val286Tyr mutant was 5-fold more active for hydrolysis of starch than the wild-type enzyme. In contrast, the Val286Phe mutant, differing only by one hydroxyl group, was 3-fold less hydrolytic than the wild-type enzyme and apparently had a higher transglycosylation/hydrolysis ratio. These results are discussed in terms of affinity of subsites, hydrophobicity and electrostatic environment in the active site. The engineered enzyme reported here may represent an attractive alternative for the starch transformation industries as it affords direct and substantial material savings and requires no process modifications.

Received April 28, 2003; revised May 29, 2003; accepted June 2, 2003.


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