Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.
The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the alpha-1,4-glucosidic linkage at the reducing end to an alpha-1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75 degrees C. The enzyme was stable in a pH range of 4.5-11, and the activity remained unchanged after a 2-h incubation at 80 degrees C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2-14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.
Fang
Tsuei-Yun
TY
Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Rd., 202, Keelung, Taiwan. tyfang@mail.ntou.edu.tw
Hung
Xing-Guang
XG
Shih
Tong-Yuan
TY
Tseng
Wen-Chi
WC
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D017124:Conserved Sequence; D017931:DNA Primers; D003912:Dextrins / Q000096:biosynthesis* / Q000378:metabolism*; D004798:Enzymes / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics; D014199:Trehalose / Q000235:genetics / Q000378:metabolism*; D014462:Ultrafiltration; D000516:alpha-Amylases / Q000378:metabolism*
D019843:Archaeal Proteins; D017931:DNA Primers; D003912:Dextrins; D004798:Enzymes; D014199:Trehalose; C499532:trehalosyl dextrin-forming enzyme, Sulfolobus solfataricus; D000516:alpha-Amylases