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Lipase from Rhizomucor miehei (RML) and lipase B from Candida antarctica (CALB) were covalently immobilized onto epoxy-functionalized silica. In this study, we developed a multienzyme system to produce biodiesel with waste cooking oil and methanol. To increase the biodiesel production yield, a mixture of 1,3-specific lipase (RML) and nonspecific lipase (CALB) was used. Response Surface Methodology (RSM) and a central composite rotatable design (CCRD) was used to study the effects of four factors, CALB:RML ratio, ratio of t-butanol to oil (wt.%), water adsorbent content (wt.%) and reaction time on the fatty acid methyl esters (FAME) yield. A quadratic polynomial equation was obtained for methanolysis reaction by multiple regression analysis. The optimum combinations for the reaction were CALB:RML ratio (3:1), t-butanol to oil (10 wt.%), water adsorbent content (22.5 wt.%) at the reaction time of 10 h. FAME yield of 91.5%, which was very close to the predicted value of 95.6%, was obtained. Verification experiment confirmed the validity of the predicted model.

The enzymatic production of biodiesel by methanolysis of canola oil was studied using self-made biocatalysts. Mesoporous SBA-15 nanoparticles were prepared, characterized and functionalized by 3-glycidyloxypropyl trimethoxysilane. Lipases from Candida antarctica (CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) were covalently immobilized onto SBA-epoxy. Thermal stability and the influence of methanol concentration on the catalytic activity were also evaluated.higher thermal stability and methanol tolerance for immobilized derivatives were achieved compared to the free enzyme. In an optimization study, the effect of water, tbutanol and blue silica gel as water adsorbent on the yield of FAME was considered.For the SBA-RML catalysed reaction, water had little effect in increasing FAME yield, but when 20 wt% water by substrate weight was added to the SBA-TLL catalysed reaction, the methyl ester content reached nearly complete conversion (98% FAME). t-Butanol had a great effect on yield, with almost complete conversion for SBA-RML and SBA-TLL. The effect of blue silica gel also was investigated. The immobilized TLL was quite stable and can be reused for 20 cycles without significant loss in activity (6%). RML and CALB also presented a good reusability, keeping 95% of their initial activities after 7 and 15 cycles of the reaction.

Rapid and high capacity immobilization of Rhizomucor miehei lipase on aldehyde-functionalized supports was performed under mild condition via a multi component reaction. The mechanism of immobilization reaction was determined as the Ugi reaction.

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