A hyperthermostable multidomain GH family 3-glucosidase from Thermotoga naphthophila RKU- 10: Cloning, characterization and thermodynamic analysis

13^th International Conference on Metabolomics and Systems Biology

October 11-12, 2018 | Zurich, Switzerland

Ikram Ul Haq

Government College University, Pakistan

Posters & Accepted Abstracts: Metabolomics (Los Angel)

Abstract :

A novel gene (2,166 bp) was cloned from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10T and overexpressed a multidomain β-glucosidase protein (TnBglB) belonging to glycoside hydrolase family 3 (GH3) in Escherichia coli BL21 CodonPlus. An extracellular TnBglB enzyme with a molecular weight of 81kDa, was purified to homogeneity. Purified TnBglB showed peak activity at 85°C and pH 5.0 using p-nitrophenyl-β-D-glucopyranoside (PNPG) as substrate. Enzyme displayed high thermal stability over a broad range of temperature (60-90°C) and quite stable after 480 min incubation at 85°C. Enzyme activity was enhanced by 175% by the addition of 10mM Ca2+ cation and in the presence of short chain alcohol (10%v/v) activity was improved by 185%. Ki value of TnBglB for glucose and xylose inhibition was estimated 150 mM and 200 mM, respectively. Km, Vmax, kcat and kcat Km -1 values, towards PNPG as substrate, were 0.45mM, 153 mmolmg-1min-1, 1214285s-1 and 2698413, respectively. Thermodynamic parameters for PNPG hydrolysis by TnBglB like Î?H*, Î?G* and Î?S* were calculated at 85°C as 24.09kJ mol-1, 46.55kJ mol-1 and -62.74Jmol-1K-1, respectively. TnBglB displayed a half-life (t1/2) of 4.44 min at 94°C with denaturation parameters of enzyme including Î?H*D, Î?G*D and Î?S*D were 283.78 kJ mol-1, 108.69 kJ mol-1 and 0.477 kJ mol-1 K-1, respectively. TnBglB showed great affinity towards p-nitrophenyl substrates and cellobiose. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on TnBglB docking studies with its substrates. A hyperthermotolerant TnBglB with great catalytic efficiency and low product inhibition, also exhibited independence of various chemical inhibitors. All noteworthy features make TnBglB suitable candidate for industrial applications.

Biography :

E-mail: ikmhaq@yahoo.com