Purification, biochemical characterization, and gene cloning of a new extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming alpha-amylase from an endophytic ascomycete Fusicoccum sp. BCC4124.
An endophytic fungus, Fusicoccum sp. BCC4124, showed strong amylolytic activity when cultivated on multi-enzyme induction enriched medium and agro-industry substrates. alpha-Amylase and alpha-glucosidase activities were highly induced in the presence of maltose and starch. The purified target alpha-amylase, Amy-FC1, showed strong hydrolytic activity on soluble starch (kcat/Km=6.47 x 10(3) min(-1)(ml/mg)) and selective activity on gamma- and beta-cyclodextrins, but not on alpha-cyclodextrin. The enzyme worked optimally at 70 degrees C in a neutral pH range with t(1/2) of 240 min in the presence of Ca(2+) and starch. Maltose, matotriose, and maltotetraose were the major products from starch hydrolysis but prolonged reaction led to the production of glucose, maltose, and maltotriose from starch, cyclodextrins, and maltooligosaccharides (G3-G7). The amylase showed remarkable glucose tolerance up to 1 M, but was more sensitive to inhibition by maltose. The deduced protein primary structure from the putative gene revealed that the enzyme shared moderate homology between alpha-amylases from Aspergilli and Lipomyces sp. This thermotolerant, glucose tolerant maltooligosaccharide-forming alpha-amylase is potent for biotechnological application.
Champreda
Verawat
V
Molecular and Enzyme Screening Laboratory, BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, Klong Luang, Patumthani, Thailand. verawat@biotec.or.th
Kanokratana
Pattanop
P
Sriprang
Rutchadaporn
R
Tanapongpipat
Sutipa
S
Eurwilaichitr
Lily
L
D001203:Ascomycota / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D003505:Cyclodextrins / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009844:Oligosaccharides / Q000096:biosynthesis / Q000378:metabolism*; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D003505:Cyclodextrins; D009844:Oligosaccharides; C021705:maltooligosaccharides; D013213:Starch; D000516:alpha-Amylases; D005947:Glucose; D002118:Calcium