Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum

• Published in: Journal of industrial microbiology & biotechnology 2014-10, Vol.41 (10), p.1581-1589
• Main Authors: Xu, Heng, Shen, Dong, Wu, Xue-Qiang, Liu, Zhi-Wei, Yang, Qi-He
• Format: Article
• Language: English
• Subjects: Acids; Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - genetics; Aldose-Ketose Isomerases - isolation & purification; Amino Acid Substitution; Analysis; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Biocatalysis; Biochemistry; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biosynthesis; Biotechnology; catalytic activity; Chromatography; Cloning; cobalt; Corn syrup; DNA polymerase; E coli; Enzymatic activity; enzyme activity; Enzyme Stability; Enzymes; Fundamental and applied biological sciences. Psychology; gel electrophoresis; Genes; Genetic Engineering; Genetics and Molecular Biology of Industrial Organisms; Glucose; Glucose - chemistry; Half-Life; high fructose corn syrup; High Fructose Corn Syrup - chemistry; Hydrogen-Ion Concentration; Inorganic Chemistry; Kinetics; Life Sciences; magnesium; Microbiology; Microorganisms; Mutagenesis; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - isolation & purification; Mutants; Mutation; Plasmids; Protein expression; Proteins; Studies; thermal stability; Thermoanaerobacterium - enzymology; Thermoanaerobacterium saccharolyticum; tryptophan
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-014-1478-4

A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co²⁺ or Mg²⁺ for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.