No abstract

The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL-CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA' observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 μmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL-SOY-CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL-SOY-CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12-fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2-fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL-SOY-CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier-free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910-920, 2018.

Due to the heterogeneity of oils, the use of mixtures of lipases with different activity for a large number of glycerol-linked carboxylic acids that compose the substrate has been proposed as a better alternative than the use of one specific lipase preparation in the enzymatic synthesis of biodiesel. In this work, mixtures of lipases from different sources were evaluated in their soluble form in the ethanolysis of soybean oil. A mixture of lipases (50% of each lipase, in activity basis) from porcine pancreas (PPL) and Thermomyces lanuginosus lipase (TLL) gave the highest fatty acid ethyl ester (FAEE) yield (around 20 wt.%), while the individual lipases gave FAEE yields 100 and 5 times lower, respectively. These lipases were immobilized individually by the cross-linked enzyme aggregates (CLEAs) technique, yielding biocatalysts with 89 and 119% of expressed activity, respectively. A mixture of these CLEAs (also 50% of each lipase, in activity basis) gave 90.4 wt.% FAEE yield, while using separately CLEAs of PPL and TLL, the FAEE yields were 84.7 and 75.6 wt.%, respectively, under the same reaction conditions. The mixture of CLEAs could be reused (five cycles of 6 h) in the ethanolysis of soybean oil in a vortex flow-type reactor yielding an FAEE yield higher than 80% of that of the first batch.

Eversa® Transform 2.0 has been launched to be used in free form, but its immobilization may improve its performance. This work aimed to optimize the immobilization of Eversa® Transform 2.0 by the crosslinked enzyme aggregates (CLEAs) technique, using almost all the available tools to improve its performance. Several variables in the CLEA preparation were optimized to improve the recovered activity, such as precipitant nature and crosslinker concentration. Moreover, some feeders were co-precipitated to improve the crosslinking step, such as bovine serum albumin, soy protein, or polyethyleneimine. Starch (later enzymatically degraded) was utilized as a porogenic agent to decrease the substrate diffusion limitations. Silica magnetic nanoparticles were also utilized to simplify the CLEA handling, but it was found that a large percentage of the Eversa activity could be immobilized on these nanoparticles before aggregation. The best CLEA protocol gave a 98.9% immobilization yield and 30.1% recovered activity, exhibited a porous structure, and an excellent performance in the transesterification of soybean oil with ethanol: 89.8 wt% of fatty acid ethyl esters (FAEEs) yield after 12 h of reaction, while the free enzyme required a 48 h reaction to give the same yield. A caustic polishing step of the product yielded a biodiesel containing 98.9 wt% of FAEEs and a free fatty acids content lower than 0.25%, thus the final product met the international standards for biodiesel. The immobilized biocatalyst could be reused for at least five 12 h-batches maintaining 89.6% of the first-batch yield, showing the efficient catalyst recovery by applying an external magnetic field.

The performance of the previously optimized magnetic cross-linked enzyme aggregate of Eversa (Eversa-mCLEA) in the enzymatic synthesis of biolubricants by transesterification of waste cooking oil (WCO) with different alcohols has been evaluated. Eversa-mCLEA showed good activities using these alcohols, reaching a transesterification activity with isoamyl alcohol around 10-fold higher than with methanol. Yields of isoamyl fatty acid ester synthesis were similar using WCO or refined oil, confirming that this biocatalyst could be utilized to transform this residue into a valuable product. The effects of WCO/isoamyl alcohol molar ratio and enzyme load on the synthesis of biolubricant were also investigated. A maximum yield of around 90 wt.% was reached after 72 h of reaction using an enzyme load of 12 esterification units/g oil and a WCO/alcohol molar ratio of 1:6 in a solvent-free system. At the same conditions, the liquid Eversa yielded a maximum ester yield of only 34%. This study demonstrated the great changes in the enzyme properties that can be derived from a proper immobilization system. Moreover, it also shows the potential of WCO as a feedstock for the production of isoamyl fatty acid esters, which are potential candidates as biolubricants.

No abstract

No abstract

No abstract