Application of dhurrin for kinetics and thermodynamic characterization of linamarase (β-glucosidase) genetically engineered from Saccharomyces cerevisiae.

Abstract

Recombinant Saccharomyces cerevisiae cells at the stationary phase of growth were recovered, homogenized and centrifuged to obtain crude extracts designated as GELIN₀. Carboxy methyl cellulose, diethyl amino-ethyl-sephadex and diethyl amino-ethyl-cellulose were used to purify the crude extracts of GELIN₀ resulting in GELIN₁, GELIN₂ and GELIN₃, respectively. The ability of the enzyme extracts and a commercial native linamarase (CNLIN) to hydrolyse cyanogenic glucosides was challenged using dhurrin from sorghum as substrate. Precisely, the actions of commercial native linamarase (CNLIN) and the genetically engineered linamarase (β-glucosidase) from Saccharomyces cerevisiae on dhurrin as influenced by degree of its purification, pH (6.8 ) and temperature(30-45°C) were investigated and the data derived were applied for kinetics and thermodynamic characterization of the enzymes. Enzymic degradation kinetics of the dhurrin were evaluated using a 4 x 6 x 8 B/W design comprising of 4 enzyme types ( GELIN_o, GELIN₁ GELIN₂ and GELIN₃,, 6 temperatures (30, 32, 35, 37, 40, 45 °C) and 8 time intervals( 0-70 min.). Data obtained from the residual HCN with time were fitted into zero, first and second order kinetics models to derive reaction rate constant (Kmin^-1) values which were analyzed using the Arrhenius and absolute reaction rate models Thermodynamic parameters were obtained including; activation energies (E_a), frequency factor(K_o), enthalpy (ΔH ) and entropy (ΔS#) that characterized the reactions on dhurrin catalyzed by commercial native linamarase (CNLIN) and the genetically engineered linamarase (β-glucosidase) from Saccharomyces cerevisiae. The results showed that the best fitted order based on higher coefficient of linear regression (r²) values > 0.998 and linearity of curves was the first order kinetics model and not either zero or second order models. Kmin^-1 values ranged from GELIN_O- GELIN₃ 0.03-0.07 μmol/min while the derived D-values from K-values were in the range of 24-65 min. The frequency factors (K_o) increased with enzyme purity from GELIN₀ to GELIN₃ corresponding to K_o (min^-1) of 22.585 to 56.462. The energy of activation E_a (KJ/mol)generated 60.0995 to 150.6900 corresponding to enzymes GELIN₀ to GELIN₃ followed the same pattern with frequency factor for breaking of bonds in dhurrin molecules. At pH 6.8 CNLIN showed no action on dhurrin. The high correlation coefficient values of (r²= 0.97 to 0.99) indicated the best fit of the Arrhenius and the absolute reaction rate models. The entropy change (ΔS) increased with enzyme purity from 0.588 J/mol.deg. to 1.4625Jmol degree. The enthalpy change KJ/mol followed the same pattern whereby increases influenced by enzyme purity ranged from 1892 KJ/mol to 13104KJ/mol.

Keywords: kinetics, thermodynamic, characterization, dhurrin, genetically engineered β- glucosidase, Saccharomyces cerevisiae.