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Applied and Environmental Microbiology, September 2007, p. 5607-5612, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00499-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Changes in the Catalytic Properties of Pyrococcus furiosus Thermostable Amylase by Mutagenesis of the Substrate Binding Sites{triangledown}

Sung-Jae Yang,1 Byoung-Chul Min,2 Young-Wan Kim,1 Sang-Mok Jang,1 Byong-Hoon Lee,3 and Kwan-Hwa Park1*

Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University, Seoul 151-921,1 Starch and Sweetener Research and Development Center, BNS Division, Daesang Corporation, Ichon 467-813, Korea,2 Department of Food Science and Agricultural Chemistry, McGill University, Quebec H9X 3V9, Canada3

Received 5 March 2007/ Accepted 30 June 2007

Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced {alpha}-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over ß-CD, calculated by comparing kcat/Km ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes.

* Corresponding author. Mailing address: Department of Food Science and Biotechnology, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151-921, Korea. Phone: 82-2-8804852. Fax: 82-2-8735095. E-mail: parkkh{at}plaza.snu.ac.kr

{triangledown} Published ahead of print on 13 July 2007.

Applied and Environmental Microbiology, September 2007, p. 5607-5612, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00499-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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