Recombinant expression and characterization of Thermoanaerobacter tengcongensis thermostable alpha-glucosidase with regioselectivity for high yield isomaltooligosaccharides synthesis.
A novel thermostable alpha-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in E. coli and characterized. The TtgluA gene contained 2253 bp, which encodes 750 amino acids. The native TtGluA was a trimer with monomer molecular mass of 89 kDa shown by SDS-PAGE. The purified recombinant enzyme showed hydrolytic activity on maltooligosaccharides, pnitrophenyl-alpha-D-glucopyranide, and dextrin with an exo-type cleavage manner. TtGluA showed preference for short-chain maltooligosaccharides and the highest specific activity for maltose of 3.26 units/mg. Maximal activity was observed at 60 degrees C and pH 5.5. The half-life was 2 h at 60 degrees C. The enzyme showed good tolerance to urea and SDS but was inhibited by Tris. When maltose with the concentration over 50 mM was used as substrate, TtGluA was also capable of catalyzing transglycosylation to produce alpha-1,4-linked maltotriose and alpha-1,6-linked isomaltooligosaccharides. More importantly, TtGluA showed exclusive regiospecificity with high yield to produce alpha-1,6-linked isomaltooligosaccharides when the reaction time extended to more than 10 h.
Zhou
Cheng
C
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Xue
Yanfen
Y
Zhang
Yueling
Y
Zeng
Yan
Y
Ma
Yanhe
Y
D000595:Amino Acid Sequence; D003912:Dextrins / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009578:Nitrobenzenes / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics; D013997:Time Factors; D014312:Trisaccharides / Q000096:biosynthesis; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D003912:Dextrins; D005960:Glucosides; D009578:Nitrobenzenes; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides; C021705:maltooligosaccharides; C008317:maltotriose; D000520:alpha-Glucosidases