Engineered glucose isomerase from Streptomyces sp. SK is resistant to Ca2+ inhibition and Co2+ independent

• Published in: Journal of industrial microbiology & biotechnology Vol. 39; no. 4; pp. 537 - 546
• Main Authors: Ben Hlima, Hajer, Aghajari, Nushin, Ben Ali, Mamdouh, Haser, Richard, Bejar, Samir
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.04.2012; Springer; Springer Verlag
• Subjects: Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - genetics; Aldose-Ketose Isomerases - metabolism; Amino Acid Sequence; Biocatalysis; Biochemistry; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Calcium - metabolism; Cobalt - metabolism; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Inorganic Chemistry; Kinetics; Life Sciences; Microbiology; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Sequence Alignment; Streptomyces - enzymology; Thermostability; Glucose isomerase; Site-directed mutagenesis; Calcium inhibition; Streptomycetaceae; Calcium; Enzyme; Site directed mutagenesis; Streptomyces; Glucose; Thermal stability; Actinomycetales; Resistance; Isomerases; Bacteria; Actinomycetes; Inhibition
• DOI: 10.1007/s10295-011-1061-1
• ISSN: 1367-5435

The role of two amino acid residues linked to the two catalytic histidines His54 and His220 in kinetics and physicochemical properties of the Streptomyces sp. SK glucose isomerase (SKGI) was investigated by site-directed mutagenesis and molecular modeling. Two single mutations, F53L and G219D, and a double mutation F53L/G219D was introduced into the xylA SKGI gene. The F53L mutation increases the thermostability and the catalytic efficiency and also slightly shifts the optimum pH from 6.5 to 7, but displays a profile being similar to that of the wild-type enzyme concerning the effect of various metal ions. The G219D mutant is resistant to calcium inhibition retaining about 80% of its residual activity in 10 mM Ca 2+ instead of 10% for the wild-type. This variant is activated by Mn 2+ ions, but not Co 2+ , as seen for the wild-type enzyme. It does not require the latter for its thermostability, but has its half-life time displaced from 50 to 20 min at 85°C. The double mutation F53L/G219D restores the thermostability as seen for the wild-type enzyme while maintaining the resistance to the calcium inhibition. Molecular modeling suggests that the increase in thermostability is due to new hydrophobic interactions stabilizing α2 helix and that the resistance to calcium inhibition is a result of narrowing the binding site of catalytic ion.