Influence of buffer and L-phenylalanine concentration in the Rhizomucor miehei lipase catalysed synthesis of L-phenylalanyl-D-glucose ester investigated through response surface methodology.

摘要

Reaction conditions for Rhizomucor miehei lipase (RML) catalysed synthesis of L-phenylalanyl-D-glucose using unprotected L-phenylalanine and D-glucose were optimized using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed involving 32 experiments of five variables (L-phenylalanine concentration in mmol, amount of RML in mg, pH, incubation period in h and buffer concentration in mM) at five levels. A second-order polynomial equation was developed in terms of linear, quadratic and cross product terms to study the effects of variables on esterification yields. A R2 value of 0.7 was obtained for this complex reaction. From the surface and contour plots it was found that higher yields were observed for a very narrow pH range of 4.5-6.5 at L-phenylalanine concentrations above 3 mmol. The extent of esterification decreased with increase in RML concentration for incubation periods below 60 h. However, longer incubation periods above 60 h, enhanced esterification at all RML concentrations. Lower conversions below 0.5 mmol required less than 3.5 mmol L-phenylalanine concentration and a broad range of buffer concentration from 0.1 mM (0.1 ml, 0.1 M buffer of pH 6.0) to 0.5 mM (0.5 ml, 0.1 M buffer of pH 6.0). However, higher conversions from 0.6 to 0.8 mmol required a buffer concentration above 0.25 mM (0.25 ml, 0.1 M buffer of pH 6.0) at L-phenylalanine concentrations above 3.5 mmol. An optimum predicted yield of 1.01 mmol for L-phenylalanyl-D-glucose at 3 mmol L-phenylalanine, 100 mg of RML, 24 h incubation period, 0.5 mM (0.5 ml of 0.1 M buffer), pH 4.8 acetate buffer was found to agree with 0.97 mmol obtained under these experimental conditions. Validation experiments carried out under random conditions also exhibited good correspondence between predicted and experimental yields.