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

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 ( GELINo, 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 (Ea), frequency factor(Ko), 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 GELINO- 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 (Ko) increased with enzyme purity from GELIN0 to GELIN3 corresponding to Ko (min-1) of 22.585 to 56.462. The energy of activation Ea (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

Arrhenius And Absolute Reaction Rate Models for Thermodynamic Characterization of Linamarase (Β-Glucosidase) using Linamarin Substrate

Thermodynamic characterization of linamarase (β-glucosidase)influenced by linamarin substrate purification, pH and temperature were investigated. In the study, 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 resulting in GELIN1, GELIN2 and GELIN3, respectively. Commercial native linamarase (CNLIN) was purchased and used as control. The ability of the GELIN extracts(β-glucosidase) and the commercial native linamarase (CNLIN) to hydrolyse cyanogenic glucosides was challenged using linamarin extracted from cassava as substrates. Degradation of linamarin was evaluated at optimum pH 6.8 using a 4 × 6 × 8 between and within factorial design comprising of 4 enzyme types (GELIN0, GELIN1, GELIN2 and GELIN3), and 6 temperatures (25, 27, 29, 31, 33, 35 oC, respectively) and 8 time intervals (0, 10, 20, 30, 40, 50, 60 and 70 min.). Data obtained from residual hydrocyanic acid with time were fitted with zero, first and second order kinetics, respectively, to determine the best fit order (based on r2 and linearity). Arrhenius and absolute reaction rate models were applied to obtain activation energies (Ea), frequency factor (K0) and enthalpy (ΔH#), entropy (ΔS#), respectively, that characterized the reactions. The results indicated that the degradation of linamarin by GELIN at the optimum pH 6.8 was best described by first order kinetics, Arrhenius and absolute reaction rate models showing high coefficient of linear regression (r2>0.996) with reaction rate constant increasing from 0.0252 -0.0923 min-1 with enzyme purification ranging for GELIN0 – GELIN3. Frequency factor (Ko), Ea, ΔH# and ΔS# values decreased with enzyme purification. Activation energy (Ea) values for the degradation of linamarin (GELIN0 – GELIN3) ranged from 60.9 to 91.7 kJ/mol. Enthalpy values varied from 58 to 89 kJ/mol while ΔS# values varied from -92.8 to 4.1 J/mol. deg.) indicating spontaneous and irreversible degradation reactions which suggest a possible use of the purified linamarase (β-glucosidase) in detoxification process for foods containing linamarin.Keywords: purified linamarase, rate models, linamarin, Saccharomyces cerevisiae, cassav