Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.
The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.
Fang
Tsuei-Yun
TY
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan.tyfang@mail.ntou.edu.tw
Tseng
Wen-Chi
WC
Guo
Meng-Shin
MS
Shih
Tong-Yuan
TY
Hung
Xing-Guang
XG
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005959:Glucosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D014199:Trehalose / Q000096:biosynthesis
D011994:Recombinant Proteins; D014199:Trehalose; D005959:Glucosidases; C099156:maltooligosyl trehalose trehalohydrolase