Gene expression and molecular characterization of a thermostable trehalose phosphorylase from Thermoanaerobacter tengcongensis.
A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli. The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70 degrees C (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual activity after incubation at 50 degrees C for 7 h. Under the optimum reaction conditions, 50 microg crude enzyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L alpha-Glc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L trehalose is degraded within the same time period. Dot blotting revealed that the treP gene in T. tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of the T. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for industrial production of trehalose.
Ren
Yuanyuan
Y
State Key Laboratory of Microbial Resources and Center for Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Dai
Xiuyu
X
Zhou
Jian
J
Liu
Jingfang
J
Pei
Huadong
H
Xiang
Hua
H
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology / Q000235:genetics*; D014199:Trehalose / Q000378:metabolism
D005958:Glucosephosphates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014199:Trehalose; C031590:glucose-1-phosphate; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase; D005947:Glucose