Studies On α-Glucosidase Of Thermoanaerobacter Ethanolicus JW200

Isomaltooligosaccharide (IMO) is an α-1,6-linked glucooligosaccharide with degrees ofpolymerization (DP) ranging from2to6in the case of commercial products which containisomaltose, panose, isomaltotriose and tetrasaccharides. Generally, ith increasing publicperception against oligosaccharides, the production processes for oligosaccharides haveattracted strong commercial interest.α-glucosidase (EC3.2.1.20, α-D-glucosidase glucohydrolases) are exoenzymeshydrolyzing terminal glycosidic bonds and releasing α-glucose from the non-reducing end ofthe substrate chain. These enzymes are widely distributed in microorganisms, plants, andanimal tissues. Many α-glucosidases can hydrolyze not only oligosaccharides and syntheticα-glycosidic bonds but also catalyses transglucosidation reaction to synthesis isomerizedoligosaccharides. Therefore, α-glucosidase was used to produce high glucose syrup andiso-maltooligosaccharide with α-amylase in the industrial starch processing.The target fragment containing the2260bp open reading frame of the α-glucosidasegene was amplified using the genomic DNA of Thermoanaerobacter ethanolicus JW200as atemplate. The α-glucosidase nucleotide sequence data has been submitted to the GenBankdatabase under accession number EF635970. There was a putative ribosome-bindingsequence,5’-GGAGG-3’, located7bp upstream of the TTG start codon. The ORF wasterminated with a TAA stop codon. The deduced amino acid sequences of the enzyme from T.ethanolicus JW200shared92%identity with those from T. ethanolicus39E and T.ethanolicus X514. According to the primary structures, it belonged to glycoside hydrolasefamilies31According to the primary structures, it belonged to glycoside hydrolase families31.The new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200was cloned andexpressed in E. coli by pTrc99A and heat-shock vector pHsh. The recombinant α-glucosidaseAGL was purified in three steps, heat treatment, ion exchange chromatography and gelfiltration chromatography, and gave a single band on SDS-PAGE (55kDa), and the maximumhydrolytic activity of the purified enzyme was assayed at pH5.0~5.5and70oC. It was quitestable in the range of pH5-7. With p-Nitrophenyl-α-D-glucoside (pNPG) as a substrate andunder the optimal condition (70oC, pH5.5), Km and Vmax of the enzyme was1.72mM and38.87U/mg, respectively. The enzyme also had strong transglycosylation activity whenmaltose was used as sugar donor. The transglucosylation products towards maltose areisomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme couldconvert400g/l maltose to oligosaccharides with a conversion rate of52%, and83%of theoligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose,panose and isomaltotriose).