Composites of Crosslinked Aggregates of Eversa® Transform and Magnetic Nanoparticles. Performance in the Ethanolysis of Soybean Oil

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.

Immobilization of Eversa® Transform via CLEA Technology Converts It in a Suitable Biocatalyst for Biolubricant Production Using Waste Cooking Oil

“…Up to date, Eversa has been mainly utilized for biodiesel production using oils and fats and short-chain alcohols [ 26 , 74 , 75 ]; there is no evidence of its use in the synthesis of biolubricants [ 24 ]. This may be due to the poor performance of liquid Eversa in this reaction.…”

“…Another point that still needs to be evaluated is the scale-up of the process and what devices will be adequate to efficiently separate the magnetic CLEAs at large scales. At the lab scale, the Eversa-mCLEA could be rapidly, efficiently, and easily recovered and reused (at least five times) using a neodymium magnet [ 26 ]. Aiming to scale-up the magnetic separation processes, interesting pilot-scale magnetic separators have been reported, such as “rotor-stator” high-gradient magnetic separators [ 77 , 78 , 79 , 80 ].…”

“…The immobilization of Eversa was carried out according to the methodology described previously [ 26 ]. A solution containing 21 mL of a PEI solution (50 mg/mL), 32 mL of liquid Eversa (32.75 ± 1.75 mg protein/mL and 127.63 ± 12.88 U est /mL), and 17 mL of sodium phosphate buffer (5 mM and pH 7.0) was prepared.…”

“…Eversa ® Transform 2.0 was initially launched to be used as a liquid biocatalyst in the production of biodiesel [ 23 , 24 ]. However, it has been shown that if the enzyme is properly immobilized, the enzyme performance could be improved in diverse applications, such as biodiesel [ 25 , 26 , 27 ], free fatty acid (FFA) [ 28 ], or glyceride [ 29 ] production, among others process [ 24 ]. Eversa is a genetically modified industrial evolution of the lipase from Thermomyces lanuginosus (TLL) expressed in Aspergillus oryzae by Novozymes A/S [ 24 , 30 ].…”

“…Magnetic CLEAs of several enzymes have been reported [ 46 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. Specifically, CLEAs of Eversa containing magnetic nanoparticles (Eversa-mCLEA) showed good performance and reusability in the production of biodiesel [ 26 ].…”