Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

Applied and Environmental Microbiology
C Y LeeJ G Zeikus

Abstract

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and p...Continue Reading

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Citations

Aug 31, 2000·Applied and Environmental Microbiology·K A ErlandsonC A Batt
Apr 27, 2016·Journal of Basic Microbiology·Bilqees FatimaIkram-Ul Haq
Jan 11, 2001·Biochimica Et Biophysica Acta·B S HartleyM Rangarajan
Jun 1, 1996·Microbiological Reviews·S H BhosaleV V Deshpande
Oct 14, 2021·Critical Reviews in Biotechnology·Renan Yuji MiyamotoLeticia Maria Zanphorlin

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