Kamarulzaman Kamaruddin

Response surface methodological study on lipase-catalyzed synthesis of amino acid surfactants

Lipozyme (Rhizomucor miehei lipase) was used to catalyze the acylation of the amino acid L-lysine (L) with the free fatty acids, palmitic (PA) and oleic (OA) acids, to synthesize N-e-palmitoyllysine and N-e-oleoyllysine, respectively. Response surface methodology (RSM) based on a five-level, five-variable design was employed, firstly, for studying the interactive effects of various parameters on the reactions, and secondly, for their optimization. Simultaneously increasing temperature and solvent hydrophobicity, fatty substrate concentration or enzyme amount improved yields in both reactions, as did increasing solvent hydrophobicity and substrate concentration or enzyme amount, and substrate concentration and enzyme amount together. Increasing desiccant amount in very non-polar solvents, at very high levels of enzyme, and in very concentrated substrate solutions led to higher yields in the PA reaction but compromised the OA reaction. The optimum conditions predicted for the two reactions were: temperature, 69.3°C (PA) and 56.6°C (OA); solvent log P=3.46 (PA) and log P=3.50 (OA); fatty substrate concentration, 98.0 mM (PA) and 99.9 mM (OA); enzyme amount, 186 mg (PA and OA); molecular sieves, 160 mg (PA) and 80 mg (OA). Reactions under optimized conditions yielded 16.1% of N-e-palmitoyllysine and 33.1% of N-e-oleoyllysine.

Optimization of the enzyme-catalyzed synthesis of amino acid-based surfactants from palm oil fractions

The feasibility of using palm oil fractions as cheap and abundant sources of raw material for the synthesis of amino acid surfactants was investigated. Of a number of enzymes screened, the best results were obtained with the immobilized enzyme, Lipozyme. The effects of temperature, solvent, incubation period, fatty substrate/amino acid molar ratio, enzyme amount, and water removal on the reactions were analyzed and compared to those on reactions with free fatty acids and pure triglycerides as fatty substrates. All reactions were most efficient when carried out at high temperatures (70-80 degrees C) in hexane as a solvent. However, while reactions with free fatty acids proceeded better when a slight excess of the free fatty acids over the amino acids was used, reactions with triglycerides and palm oil fractions were best performed at equimolar ratios. Also, the addition of molecular sieves slightly enhanced reactions with free fatty acids but adversely affected reactions with triglycerides and palm oil fractions. Although reactions with palm oil fractions took longer (6 d) to reach equilibrium compared to reactions with free fatty acids (4 d) and pure triglycerides (4 d), better yields were obtained. Such lipase-catalyzed transacylation of palm oil fractions with amino acids is potentially useful in the production of mixed medium- to long-chain surfactants for specific applications.

Effects of buffer properties on cyclodextrin glucanotransferase reactions and cyclodextrin production from raw sago (Cycas revoluta) starch.

Results from the present study have shown that the ionic species of buffers, pH values and reaction temperature can affect the enzyme unit activities and product specificity of Toruzyme® (Novo Nordisk A/S Bagsvaerd, Denmark) CGTase (cyclodextrin glucanotransferase). Applying a similar reaction environment (acetate buffer, pH 6.0; temperature, 60 °C), the CGTase was found to be capable of producing pre dominantly β‐cyclodextrin from either raw or gelatinized sago (Cycas revoluta) starch. Changing the buffer from acetate to phosphate reduced the yield of β‐cyclodextrin from 2.48 to 1.42 mg/ml and also affected the product specificity, where production of both α‐ and β‐cyclodextrins were more pronounced. The decrease in the production of cyclodextrins in phosphate buffer was significant at both pH 6.0 and 7.0. However, changing the buffer to Tris/HCl (pH 7.0) showed a significant increase in β‐cyclodextrin production. Increasing the ionic strength of sodium acetate and Tris/HCl buffers at pH 6.0 and 7.0 to equivalent ionic strength of phosphate buffers showed no significant effects on cyclodextrin production. Higher yield of cyclodextrins at pH 7.0 when Tris/HCl was used might be due to the binding of chloride ions at the calcium‐binding sites of the CGTase, resulting in the shift of the optimum pH close to physiological environment, leading to an increase in the activities and specificity.