1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

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.

Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System

“…The crosslinking mechanism generally involves reactive primary amino groups of the protein (epsilon-amino group of Lys residues and N terminus); some proteins may not possess a high amount of this residue on the surface, so that in these cases, the CLEA preparation is not straightforward. This has been solved by using a feeder (which can be a Lys-rich protein [186][187][188][189][190][191][192][193] or an aminated polymer [194,195]), by a chemical amination of the enzyme [196][197][198] or by using an alternative crosslinking group in the enzyme (e.g., carboxylic groups) [199]. Usually, the crosslinking reagent is glutaraldehyde [182,184] due to its good properties as crosslinking reagent [200,201].…”

Combi-CLEAs of Glucose Oxidase and Catalase for Conversion of Glucose to Gluconic Acid Eliminating the Hydrogen Peroxide to Maintain Enzyme Activity in a Bubble Column Reactor

“…Another likely problem is when one enzyme is poor in Lys groups, and intermolecular crosslinking of the enzyme aggregate is difficult. This may be solved using a feeder protein or an aminated polymer, or by chemically aminating the enzyme [14,[41][42][43][44][45][46][47][48][49][50][51][52][53]. The feeders also reduce the enzyme volumetric load, and as a consequence, the substrate diffusional problems.…”

Evaluation of Strategies to Produce Highly Porous Cross-Linked Aggregates of Porcine Pancreas Lipase with Magnetic Properties

“…This wide lipase specificity, added to their excellent chemo-, regio- and enantioselectivities and/or specificities, have been exploited for several important biotechnological applications in the pharmaceutical, food and agrochemical industries [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. Among lipases, porcine pancreas lipase (PPL) is widely used in biotransformation reactions in organic media for several industrial applications because of its high selectivity, high solvent tolerance, high catalytic activity, and thermal stability at high temperatures under low water concentrations [ 8 , 13 , 14 , 15 , 16 ].…”