Immobilized Forms of the Ophiostoma piceae Lipase for Green Synthesis of Biodiesel. Comparison with Eversa Transform 2.0 and Cal A.

In this work, we analyzed the suitability of a versatile recombinant lipase, secreted by Ophiostoma piceae (OPEr) and produced in Pichia pastoris, as a catalyst of the synthesis of biodiesel. The enzyme was immobilized by five covalent procedures and by hydrophobicity on functionalized nanoparticles of magnetite or of a novel Zn/Mn oxide named G1. Then, they were tested for green production of biodiesel by solventless enzymatic transesterification of discarded cooking oil and methanol (1:4) at 25 °C. The results were compared with those shown by free OPEr and the commercial lipases Eversa® and Cal A®. Several preparations with immobilized OPEr produced high synthesis yields (>90% transesterification), comparable to those obtained with Eversa®, the commercial enzyme designed for this application. Three of the biocatalysts maintained their catalytic efficiency for nine cycles. The process catalyzed by AMNP-CH-OPEr was scaled from 500 µL to 25 mL (50 times), improving its efficiency.

Sustainable and Green Synthesis of Stanol Esters from Oil Wastes.

The recombinant lipase ofOphiostoma piceae (OPEr) is characterized by its prominent sterol esterase activity. The protein was immobilized on magnetic nanoparticles, giving four enzyme variants that have been tested in solvent-free transesterification of methyl oleate and sitostanol. The yields of stanol esters reached 85%, and the catalysts can be reused. Stanol esters were also obtained in a two-step cascade reaction; a mixture of fatty acid methyl esters was enzymatically synthesized from cooking oil wastes and then used for stanol transesterification. An 85% conversion was achieved in 2 h from the second cycle onward, maintaining the activity over 5 cycles. The biocatalysts can be safely used since they don't release toxic compounds for HeLa and A549 cell lines. These procedures comply with the principles of green chemistry and contribute to the sustainable production of these nutraceuticals from secondary raw materials, like the lipid fraction from industrial or agricultural residues.

Enzymatic glycosylation of bioactive acceptors catalyzed by an immobilized fungal ß-xylosidase and its multi-glycoligase variant.

A recombinant ß-xylosidase (rBxTW1) from the ascomycete Talaromyces amestolkiae and a mutant derived from it, with mostly synthetic activity, have been immobilized as magnetic cross-linked enzyme aggregates (mCLEAs). The mCLEAs of rBxTW1 kept the excellent hydrolytic and O-transxylosylating activities of the free enzyme and had improved thermal and pH stability. The mCLEAs of the mutant also maintained or improved the catalytic properties of the soluble enzyme, synthetizing the O-xylosides of vanillin and (-)-epigallocatechin gallate, and the N- and S-xyloside of 3,5-dibromo-1,2,4-triazole and thiophenol, respectively. The mCLEAs were recyclable across 4 cycles of synthesis of the O-xylosides through a green and highly selective process. The magnetic properties of the scaffold used for immobilization may allow the easy recovery and reuse of the biocatalyst even from reactions containing insoluble lignocellulosic biomass.

Effect of the Immobilization Strategy on the Efficiency and Recyclability of the Versatile Lipase from Ophiostoma piceae.

The recombinant lipase from Ophiostoma piceae OPEr has demonstrated to have catalytic properties superior to those of many commercial enzymes. Enzymatic crudes with OPEr were immobilized onto magnetite nanoparticles by hydrophobicity (SiMAG-Octyl) and by two procedures that involve covalent attachment of the protein (mCLEAs and AMNP-GA), giving three nanobiocatalysts with different specific activity in hydrolysis of p-nitrophenyl butyrate (pNPB) and good storage stability at 4 °C over a period of 4 months. Free OPEr and the different nanobiocatalysts were compared for the synthesis of butyl esters of volatile fatty acids C4 to C7 in reactions containing the same lipase activity. The esterification yields and the reaction rates obtained with AMNP-GA-OPEr were in general higher or similar to those observed for the free enzyme, the mCLEAs-OPEr, and the non-covalent preparation SiMAG-Octyl-OPEr. The time course of the esterification of the acids C4 to C6 catalyzed by AMNP-GA-OPEr was comparable. The synthesis of the C7 ester was slower but very efficient, admitting concentrations of heptanoic acid up to 1 M. The best 1-butanol: acid molar ratio was 2:1 for all the acids tested. Depending on the substrate, this covalent preparation of OPEr maintained 80-96% activity over 7 cycles, revealing its excellent properties, easy recovery and recycling, and its potential to catalyze the green synthesis of chemicals of industrial interest.

Optimization of lipase-catalyzed synthesis of ß-sitostanol esters by response surface methodology.

The esters of ß-sitostanol and fatty acids are known for their effect as cholesterol-lowering agents. In this work, the efficiency of three lipases as biocatalysts of the esterification of ß-sitostanol and C16 and C18 fatty acids was compared. The sterol esterase of Ophiostoma piceae (OPEr) yielded the highest esterification rates and was selected for further optimization of the reaction. The effects of four parameters (temperature, enzymatic dosage, acyl donor concentration, and reaction time) on ester synthesis were investigated and the process conditions were optimized using response surface methodology (RSM). The best conditions for esterification for each fatty acid were predicted using a second-order model, and experimentally validated. Very high esterification efficiencies (86-97%) were observed using the predicted values for the four variables. This approach was shown to be suitable for optimizing the enzymatic production of ß-sitostanol esters, which represents a green alternative to the chemical synthesis of these dietary complements.

Green synthesis of ß-sitostanol esters catalyzed by the versatile lipase/sterol esterase from Ophiostoma piceae.

ß-sitostanol esters, used as dietary complement for decreasing cholesterol absorption, have been synthesized at 28°C via direct esterification or transesterification catalyzed by the versatile lipase/sterol esterase from the ascomycete fungus O. piceae. Direct esterification was conducted in biphasic isooctane: water systems containing 10mM ß-sitostanol and lauric or oleic acid as acyl donors, reaching 90% esterification in 3h with the recombinant enzyme. The use of molar excesses of the free fatty acids did not improve direct esterification rate, and the enzyme did not convert one of the two fatty acids preferentially when both were simultaneously available. On the other hand, solvent-free transesterification was an extremely efficient mechanism to synthesize ß-sitostanyl oleate, yielding virtually full conversion of up to 80mM ß-sitostanol in 2h. This process may represent a promising green alternative to the current chemical synthesis of these esters of unquestionable nutraceutical value.