Bioinformatic and biochemical analysis of a novel maltose-forming α-amylase of the GH57 family in the hyperthermophilic archaeon Thermococcus sp CL1

Maltose-forming alpha-amylase is a glycoside hydrolase family 57 (GH57) member that is unique because it displays dual hydrolysis activity toward alpha-1,4- and alpha-1,6-glycosidic linkages and only recognizes maltose. This enzyme was previously identified only in Pyrococcus sp. ST04 (PSMA); however, we recently found two homologs subgroups in Thermococcus species. One subgroup (subgroup A) showed relatively high amino acid sequence similarity to PSMA (>71%), while the other subgroup (subgroup B) showed lower homology with PSMA (<59%). To characterize the subgroup B maltose-forming alpha-amylase from Thermococcus species (TCMA), we cloned the CL1_0868 gene from Thermococcus sp. CL1 and then successfully expressed the gene in Escherichia coli. Although TCMA has a different oligomeric state relative to PSMA, TCMA showed similar substrate specificity. However, TCMA was shown to hydrolyze maltooligosaccharides more easily than PSMA. Also, TCMA displayed different optimum conditions depending on the glycosidic linkage of the substrate. TCMA had the highest activity at 85 degrees C and at pH 5.0 for alpha-1,4-glycosidic linkage hydrolysis whereas it showed its maximal activity to cleave alpha-1,6-glycosidic linkages at 98 degrees C and pH 6.0. (C) 2014 Elsevier Inc. All rights reserved.