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Candida antarctica lipase B (CALB) and Thermomyces lanuginose lipase (TLL) were co-immobilized on epoxy functionalized silica gel via an isocyanide-based multicomponent reaction. The immobilization process was carried out in water (pH 7) at 25 °C, rapidly (3 h) resulting in high immobilization yields (100%) with a loading of 10 mg enzyme/g support. The immobilized preparations were used to produce biodiesel by transesterification of palm oil. In an optimization study, response surface methodology (RSM) and central composite rotatable design (CCRD) methods were used to study the effect of five independent factors including temperature, methanol to oil ratio, t -butanol concentration and CALB:TLL ratio on the yield of biodiesel production. The optimum combinations for the reaction were CALB:TLL ratio (2.1:1), t -butanol (45 wt%), temperature (47 °C), methanol: oil ratio (2.3). This resulted in a FAME yield of 94%, very close to the predicted value of 98%. • Candida antarctica B and Thermomyces lanuginosa were simultaneously immobilized. • Immobilization was performed on silica via a multicomponent reaction. • Immobilization time decreased dramatically. • The thermal stability of derivatives were greatly improved. • All the derivatives were used to produce biodiesel from palm oil in high yield 94%. [ABSTRACT FROM AUTHOR]

• Two lipases were covalently immobilized on aldehyde-functionalized nanoparticles. • Leaching experiment confirmed covalent nature of the linkage. • The thermal and co-solvent stability of the immobilized derivatives were greatly improved. • All the derivatives were used to catalyze biodiesel production from waste cooking oil. • The effect of different parameters on biodiesel yield was studied by Response Surface Methodology Lipase from Rhizomucor miehei (RML) and Thermomyces lanuginosa lipase (TLL) were immobilized on silica core-shell magnetic nanoparticles (Fe 3 O 4 @SiO 2) produced by coating Fe 3 O 4 core with silica shell. The nanoparticles were functionalized with aldehyde groups followed by immobilization of RML and TLL by using a multi-component reaction in an extremely mild condition. Rapid immobilization of both enzymes (1.5−12 h) with high immobilization yields (81–100%) was observed. The maximum loading capacity of the support was determined to be 81 mg for RML and 97 mg for TLL. The thermal stability of the immobilized derivatives of RML and TLL were greatly improved by retaining 54 and 97 % of their initial activities at 65 °C, respectively. The immobilized preparations were used to produce biodiesel by transesterification of waste cooking oil. In an optimization study, Response Surface Methodology (RSM) and a central composite rotatable design (CCRD) were used to study the effect of amount of biocatalyst, temperature, reaction time, water adsorbent (wt.%) and ratio of t -butanol to oil (wt.%) on the yield of biodiesel production. Biodiesel production yield by immobilized TLL reached 93.1 % under optimal conditions while the maximum yield for RML was 57.5 %. Both immobilized derivatives showed high reusability after 5 cycles of the reaction. [ABSTRACT FROM AUTHOR]

Lipases from Candida antarctica B (nonspecific lipase) and Rhizomucor miehei (1,3-specific lipase) were simultaneously immobilized on epoxy functionalized silica gel under mild conditions. The results showed rapid and simple immobilization of 4–15 mg of CALB:RML (different ratios 4:1, 2:1, 1.5:1, 1:1) on 1 g of support after 6 h. The thermal stability of derivatives and also their stability in methanol were greatly improved compared to the single immobilized enzyme. All the derivatives were also used to catalyze the transesterification of palm oil with methanol to produce fatty acid methyl esters (FAMEs). Response surface methodology (RSM) and a central composite rotatable design (CCRD) was used to study the effects of five factors, reaction temperature, methanol/oil ratio, reaction time, t -butanol concentration and CALB:RML ratio 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 (2.5:1), t -butanol to oil (39.9 wt%), temperature (35.6 °C), methanol:oil ratio (5.9), reaction time 33.5 h. FAME yield of 78.3.5%, which was very close to the predicted value of 75.2%, was obtained. Verification experiment confirmed the validity of the predicted model. • Lipases from Candida antarctica B and Rhizomucor miehei were simultaneously immobilized on silica. • The thermal stability of derivatives and also their stability in methanol were greatly improved. • All the derivatives were used to catalyze the transesterification of palm oil with methanol to produce biodiesel. • The increasing the amount of CALB relative to RML resulted to improving the thermal and methanol stability and FAME yield. [ABSTRACT FROM AUTHOR]

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 multi-enzyme 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. [ABSTRACT FROM AUTHOR]

In the present work, lipase from Candida antarctica (CALB) was covalently immobilized on functionalized magnetic nanoparticles (MNPs) to catalyze biodiesel synthesis. Core-shell nanoparticles were synthesized by coating Fe 3 O 4 core with silica shell (Fe 3 O 4 @SiO 2 ). The nanoparticles functionalized with (3-glycidoxypropyl)trimethoxylsilane (GPTMS) were used as immobilization matrix. The protein binding efficiency on functionalized Fe 3 O 4 @SiO 2 was calculated as 84%, preserving 97% of specific activity of the free enzyme. Physical and chemical properties of the nanoparticles and the immobilized lipase were characterized by TGA, XRD, SEM, IR, TEM and DLS. Higher thermal stability and methanol tolerance for immobilized derivatives were obtained compared to the free enzyme. The immobilized lipase was then used to produce biodiesel by transesterification of waste cooking oil with methanol. In an optimization study, the effect of oil to methanol ratio, tert -butanol and molecular sieve as water adsorbent on the yield of biodiesel production were considered. Optimum oil to methanol ratio at 1:3 was observed for immobilized CALB in biodiesel production. Molecular sieve had a great effect on yield, with almost 100% conversion. The immobilized preparation of CALB also presented a good reusability, keeping 100% of its initial activity after 6 cycles of the reaction. [ABSTRACT FROM AUTHOR]

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, t -butanol 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. [ABSTRACT FROM AUTHOR]

Enzymatic production of biodiesel from canola oil using self-made covalently immobilized lipase from Candida antarctica (CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) on epoxy-functionalized silica as low cost catalyst was investigated. In this optimization study, the effect of water, t -butanol and water adsorbent content on the yield of fatty acid methyl ester (FAME) was considered. Complete conversion to FAMEs was achieved under optimum conditions for CALB immobilized on epoxy-functionalized silica (Silica-CALB); 30% (w/w) t -butanol by substrate weight, reaction time of 96 h, 50 °C and molar ratio of methanol to oil 3:1, which was added to the reaction mixture in three steps. In general by adding t -butanol to the reaction medium, the conversion of oil to FAME increased for RML immobilized on epoxy-functionalized silica (Silica-RML) and 50 wt.% t -butanol by substrate weight gave the best yield. TLL immobilized on epoxy-functionalized silica (Silica-TLL) reach to 100% yield at 10% t -butanol. Water suppresses the methanolysis reaction catalyzed by Silica-CALB but significantly increased FAME yield for Silica-TLL and Silica-RML. It was observed that at high water adsorbent amounts (more than 55%, w/w), conversion to FAME decreased possibly due to mass transfer limitation. The immobilized TLL was quite stable and can be reused for 16 cycles without significant loss in activity (95%). The immobilized preparations of RML and CALB also presented a good reusability, keeping 85% of their initial activities after 16 cycles of the reaction. [ABSTRACT FROM AUTHOR]

In the present work, lipases from Candida antarctica (CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) were covalently immobilized on epoxy-functionalized silica. The immobilized lipases were used to produce biodiesel by transesterification of canola oil with methanol. It was found that lipases immobilized on silica provided biocatalyst derivatives with lower cost compared with the cost of commercially available Novozym 435. Thermal stability of the immobilized derivatives and the influence of methanol on the catalytic activity were also evaluated. Optimum oil to methanol ratio at 1:3 was observed for CALB and RML in biodiesel production; the corresponding fatty acid methyl ester (FAME) yields obtained after 96 h were 68% and 45% at 50 °C respectively. The lipase from T. lanuginosus immobilized on epoxy-functionalized silica displayed particularly high catalytic ability regarding reaction rate and final yield. TLL also gave high FAME contents in the reaction mixture with up to 6 molar equivalents of methanol to oil (98%). The immobilized TLL was quite stable and can be reused for 16 cycles without significant loss in activity (5%). The immobilized preparations of RML and CALB also presented a good reusability, keeping 85% of their initial activities after 16 cycles of the reaction. [ABSTRACT FROM AUTHOR]