Abstract
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.
Highlights
The current concern about global warming and the reduction of fossil fuel sources have driven the search for less polluting and renewable fuels [1,2,3,4,5,6]
We evaluated the transesterification of soybean oil with ethanol at 40 ◦ C, and increasing the enzyme load (4, 7, and 12 unit of esterification (Uest) /g oil, equivalent to 3.8, 6.6, and 11.3% of liquid Eversa, w/woil ) to reach the maximum fatty acid ethyl esters (FAEEs) yield in a shorter reaction time
NextThus, we evaluated the transesterification of soybean oil were performed directly with the biocatalysts, and in others an adjustment of the water content in the with ethanol at 40 °C
Summary
The current concern about global warming and the reduction of fossil fuel sources have driven the search for less polluting and renewable fuels [1,2,3,4,5,6]. The production of biodiesel at a large scale uses acids or alkalis as catalysts [4,8], but the replacement of chemical catalysts by enzymatic ones has been widely studied [6,7,9,10,11]. Catalysts 2020, 10, 817 cost and the longer reaction time compared to the chemical route [13,14,15,16] Another problem of lipases is that they have substrate specificity, one specific lipase may not recognize the high number of likely substrates contained in an oil, which is an heterogeneous substrate [17,18]. A similar behavior was observed for the FFAs content, fourth and fifth batches, respectively). Part of the activity loss could be derived from the release of non-magnetic fragments from the magnetic CLEAs after breaking the CLEA particle in the stirring or some non-magnetic fragments from the magnetic CLEAs after breaking the CLEA particle in the manipulation [44]
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