Improving the Thermostability of Raw-Starch-Digesting Amylase from a Cytophaga sp. by Site-Directed Mutagenesis

Abstract

A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca 2؉ -independent mechanism.Raw starch is the most abundant source of glucose in the world. Industrially, starch granules are gelatinized in water to improve the conversion of starch molecules into maltodextrins and simple sugars by ␣-amylase and/or other amylolytic enzymes (13). This gelatinization of starch granules requires heating and a long reaction time. Therefore, finding enzymes capable of digesting raw starch directly would provide a possible replacement for such an expensive and energy-consuming process. Many organisms, including fungi, yeast, and bacteria, are known to produce raw-starch-digesting amylases (RSDAs) (1,2,3,22). Some of these RSDAs have been produced and applied to the beverage, food, paper, and textile industries as well as to dishwashing and laundry detergent production (11). However, the relatively low reaction temperature optimal for RSDA on granular starch results in not only a low reaction rate but also a high risk of bacterial contamination. Therefore, increasing the thermostability of RSDAs for industrial utilization is an important goal of protein engineering.Studies have shown that a loop area within domain B of ␣-amylase plays a crucial role in improving the thermostability of this protein (17). Deletion of an Arg-Gly dipeptide from both the liquefying ␣-amylase (LAMY) of Bacillus strain KSM-1378 and Bacillus amyloliquefaciens amylase (BAA) increased their thermostability (5, 18). In previous reports, an RSDA from a Cytophaga sp. has been shown to exhibit an excellent starch digestion activity on raw cornstarch (7,8). By comparing the amino acid sequences of this RSDA and other amylases (5,12,19,23), a loop structure within domain B in Cytophaga sp. RSDA similar to those in other amylases was also found. In this study, we demonstrated that removing Arg and Gly residues from the corresponding loop area in this RSDA improved the stability of the enzyme both at high temperatures and in stringent environments. To the best of our knowledge, this is the first thermostable RSDA that has been reported to date.The mutant RSDA, with residues 178 and 179 deleted, was generated by site-directed mutagenesis by the method of splicing by overlap extension based on PCR (4). A recombinant plasmid (unpublished data), pHR, was constructed by ligation of pHY300PLK (TaKaRa, Shing, Japan) and a DNA fragment containing the rsda gene. To generate the deletion mutant, the nucleotide sequence of the rsda gene was PCR amplified on pHR by using primer 1 (5Ј-CGGACCGTACGATTTGTACG ATCTAGG-3Ј, where the BsiWI recognition sequence is underlined) and primer 2 (5Ј-CCCATGCTTTGCCGG...

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