Optimized production of biodiesel from waste cooking oil by lipase immobilized on magnetic nanoparticles.

Biodiesel, a non-toxic and biodegradable fuel, has recently become a major source of renewable alternative fuels. Utilization of lipase as a biocatalyst to produce biodiesel has advantages over common alkaline catalysts such as mild reaction conditions, easy product separation, and use of waste cooking oil as raw material. In this study, Pseudomonas cepacia lipase immobilized onto magnetic nanoparticles (MNP) was used for biodiesel production from waste cooking oil. The optimal dosage of lipase-bound MNP was 40% (w/w of oil) and there was little difference between stepwise addition of methanol at 12 h- and 24 h-intervals. Reaction temperature, substrate molar ratio (methanol/oil), and water content (w/w of oil) were optimized using response surface methodology (RSM). The optimal reaction conditions were 44.2 °C, substrate molar ratio of 5.2, and water content of 12.5%. The predicted and experimental molar conversions of fatty acid methyl esters (FAME) were 80% and 79%, respectively.

Alkyl-substituted methoxysilanes enhance the activity and stability of D-amino acid oxidase encapsulated in biomimetic silica.

Several alkyl-substituted methoxysilanes were evaluated as potential activity and stability enhancing agents for biomimetic silicification of Rhodosporidium toruloides D-amino acid oxidase (RtDAO). When methyl-substituted silanes along with tetramethoxysilane were used as silicic acid precursors for polyallylamine (PAA)--or R5 peptide-catalyzed silicic encapsulation, the RtDAO activity increased with the degree of substitution and the molar ratio up to 15 % of methyl-substituted silanes added. In the presence of 15 mol% trimethylmethoxysilane, the specific activities of encapsulated RtDAO catalyzed by PAA and R5 increased by 1.4- and 4.8-fold, respectively. For PAA-catalyzed encapsulation, a 2.4-fold increase occurred with 30 mol% n-propyltrimethoxysilane; this modification increased the T (m) value by 10 °C and gave a threefold longer half-life in the presence of 10 mM H(2)O(2) as compared to the encapsulation using tetramethoxysilane only.

Optimization of lipase production by Burkholderia sp. using response surface methodology.

Response surface methodology (RSM) was employed to optimize the extracellular lipase production by Burkholderia sp. HL-10. Preliminary tests showed that olive oil, tryptone and Tween-80 exhibited significant effects on the lipase production. The optimum concentrations of these three components were determined using a faced-centered central composite design (FCCCD). The analysis of variance revealed that the established model was significant (p < 0.01). The optimized medium containing 0.65% olive oil (v/v), 2.42% tryptone (w/v) and 0.15% Tween-80 (v/v) resulted in a maximum activity of 122.3 U/mL, about three fold higher than that in basal medium. Approximately 99% of validity of the predicted value was achieved.

Activity enhancement and stabilization of lipase from Pseudomonas cepacia in polyallylamine-mediated biomimetic silica.

Triacylglycerol lipase from Pseudomonas cepacia and Fe(3)O(4) magnetic nanoparticles were encapsulated simultaneously within biomimetic silica through the catalysis of polyallylamine. The encapsulation efficiency reached 96% with an activity recovery of 51%. After 5 h at 37°C, the activities of the free and encapsulated lipases decreased by 77 and 16%, respectively. Addition of 10 and 15 mol% trimethylmethoxysilane to tetramethoxysilane during encapsulation doubled the lipase activity while inclusion of 50 and 60 mol% γ-(methacryloxypropyl)-trimethoxysilane tripled the activity. Thus, such encapsulation not only stabilized P. cepacia lipase but also could enhance the activity by varying silane additives.

Surfactant-assisted in situ transesterification of wet Rhodotorula glutinis biomass.

In situ transesterification of oleaginous microbes with short chain alcohol has been developed as a renewable process for the production of biodiesel. Dry biomass is often a requisite for the process to avoid the adverse effect of water on the productivity. As a consequence, large amount of energy consumption is required for prior biomass drying. In this study, the wet biomass of Rhodotorula glutinis, an oleaginous yeast, was used directly in in situ transesterification without biomass drying. The reaction conditions were optimized for the production of fatty acid methyl esters (FAME) and the effects of adding different surfactants were also studied. The highest FAME yield of 110% was achieved with a methanol loading of 1:100 at 90°C for 8 h as catalyzed by 0.36 M H2SO4, and the FAME content was 97%, which meets the 96.5% specified in both European biodiesel standards and Taiwanese biodiesel standards. The addition of 50 mM 3-(N,N-dimethylmyristylammonio)propanesulfonate (3-DMAPS, a zwitterionic surfactant) improved the FAME yield from 69% to 83%, which was obtained with a low methanol loading of 1:10 at 90°C for 10 h. Hence, the production of FAME with wet biomass under optimized reaction conditions was as effective as that with the dry form. This clearly indicates that using wet R. glutinis as the feedstock is feasible for the production of biodiesel by in situ transesterification.

Stabilization of D-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles.

D-amino acid oxidase from Rhodosporidium toruloides was immobilized onto glutaraldehyde-activated magnetic nanoparticles. Approximately four enzyme molecules were attached to one magnetic nanoparticle when the weight ratio of the enzyme to the support was 0.12. After immobilization, the T(m) was increased from 45 degrees C of the free form to 55 degrees C. In the presence of 20 mM H2O2, the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h.

Stabilization of native and double D-amino acid oxidases from Rhodosporidium toruloides and Trigonopsis variabilis by immobilization on streptavidin-coated magnetic beads.

Double D: -amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k(cat)/K(M)) of immobilized DAOs toward D: -alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward D: -alanine was decreased by 56%. After immobilization, the T(m) value for RtDAO was shifted 15 degrees C higher to 60 degrees C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5-8 degrees C to 56, 60 and 60 degrees C, respectively. In the presence of 10 mM H(2)O(2), immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin-streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.

Coexpression of Vitreoscilla hemoglobin reduces the toxic effect of expression of D-amino acid oxidase in E. coli.

Expression of the gene (daao) encoding D-amino acid oxidase (DAAO) in Escherichia coli typically results in a marked decrease of cell viability, and it has generally been assumed that the consumption of intracellular D-alanine by DAAO is responsible for this effect. Vitreoscilla hemoglobin (VHb) gene (vgb) was coexpressed with Rhodosporidium toruloides D-amino acid oxidase in E. coli BL21(DE3) and BL21(DE3)pLysS, expression hosts differing in the stringency of suppressing basal transcription. Not only was the toxic effect of DAAO on cell growth relieved but also the pronounced cell lysis of BL21(DE3)pLysS caused by the expression of DAAO was prevented by coexpressing VHb with DAAO. As a result of the higher cell density achieved, DAAO activity about 1.5-fold higher than that of DAAO-expressing control strains could be obtained by DAAO/VHb-coexpressing strains. The relieving effect of VHb on DAAO toxicity resulted from its oxygen-binding ability. The low availability of free intracellular oxygen reduced DAAO activity and consequently its toxicity.

Subunit fusion of two yeast D-amino acid oxidases enhances their thermostability and resistance to H2O2.

D-amino acid oxidases from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) are both yeast homodimeric flavoenzymes. Two of their cDNA genes were connected by a hexanucleotide linker and heterologously expressed in E. coli to produce the corresponding double DAOs (dRtDAO and dTvDAO) with two subunits fused into a single polypeptide. The specific activities of double DAOs remained similar to those of native dimeric DAOs, although the catalytic efficiencies (k(cat)/K(M)) were decreased due to higher K(M) values. The T(m) value for dRtDAO was shifted 5 degrees C higher while that for dTvDAO was increased only by 2 degrees C, in comparison with the corresponding native counterparts. In the presence of 10 mM H(2)O(2), dRtDAO and dTvDAO exhibited half-lives of about 60 and 40 min, respectively, which were 2- and 1.5-fold, respectively, longer than their native DAOs. These yeast DAOs can therefore be thermally and oxidatively stabilized by linking their subunits together.