Improvement of enzyme stability for alkyl esters synthesis in miniemulsion systems by using media engineering

Abstract

BACKGROUND Oil‐in‐water miniemulsions have been suggested to display potential for application as a ‘green’ system for enzymatic alkyl ester synthesis. Still, a realistic assessment of the feasibility of this approach for practical applications requires further insight, namely the relation between enzyme stability and reaction operational conditions. The objective of this work is within such scope. Accordingly, it extends previous research on esterification catalyzed by Fusarium solani pisi cutinase, by addressing medium‐chain length substrates, and aims to contribute to the optimization of reaction conditions and towards the design of an efficient set‐up. RESULTS The esterification yield was significantly enhanced (e.g. 50% increase for octyl octanoate system) through the correction of the initial pH of the reaction medium to pH 6. Under such environment, conformational changes of the enzyme were negligible after 24 h, resulting in improved enzyme stability for all systems under study. Maximum esterification rate (3.59 mmol min‐1) and product yield (91%) were achieved for octyl decanoate synthesis. Fed‐batch mode of operation allowed for the production of 1 mol L‐1 hexyl octanoate, while retaining enzyme activity over 5 days. Consecutive batch runs allowed for the cumulative production of 1.1 mol L‐1 hexyl octanoate after four cycles. CONCLUSIONS Medium engineering enabled the use of miniemulsion systems for the synthesis of medium‐chain alkyl esters and increase in the final concentration of the intended product. Through a fed‐batch mode the enzyme stability and reutilization were tuned allowing long‐term utilization of the free enzyme which could not be achieved through a repeated batch system. © 2017 Society of Chemical Industry

Biodegradable Polyester Synthesis in Renewed Aqueous Polycondensation Media: The Core of the New Greener Polymer-5B Technology

“…The maximum POS molecular weight of 3550 g mol −1 for CRL was observed at a concentration of 0.1 M. In the case of BCL and PBL, the maximum POS molecular weight of 3850 and 6950 g mol −1 , respectively, was detected at a concentration of 0.5 M. Based on these data, an increase in the monomer concentration above 0.1 M showed a high inhibition effect on CRL. Besides the maximum polymer weight observed for the same monomer concentration (0.5 M) for BCL and PBL, PBL presented higher monomer selectivity with regard to the higher mo- Enzyme activity depends on the pH of the reaction media, which in the case of a miniemulsion system is determined by the continuous (water) phase [40]. A comparison of the polymer molecular weight at pH 3.3 (pH of the original miniemulsion) and miniemulsions with the corrected pH value was made ( Figure 2A and Table 2).…”

“…Polycondensation in batch operation mode allows flexibility and adaptability to obtain different polyesters [54]. However, the inhibition effect of the high substrate concentrations on lipase activity in the miniemulsion, as well as in an organic solvent, was previously observed for the esterification [38][39][40]55]. To overcome the inhibition effect of high substrate concentration on enzyme activity, the reactions can be carried out in fed-batch operation mode, which means the stepwise addition of substrates [55].…”

“…Aqueous miniemulsions (oil-in-water (o/w)) are systems characterized by oil droplets in the range between 200 and 300 nm, dispersed in a continuous water phase, which contributes to about 80% (w/w) [37]. Several reactions have been carried out in miniemulsion systems using enzymes by our research group, such as high-added-value ester synthesis using various lipases and cutinases [38][39][40], resolution of enantiomers with Candida antarctica lipase B (CALB) [41], triglyceride hydrolysis, and glycerolysis with Pseudomonas cepacia [42]. More recently, aldol reactions were performed using Rhizopus arrhizus lipase [43].…”

A Middle-Aged Enzyme Still in Its Prime: Recent Advances in the Field of Cutinases

“…An alternative reaction system for the esterification of medium-chain esters by FsC was developed, creating an oil-in-water mini-emulsion system consisting of 80% water, which led to yields of ca. 85% for the synthesis of hexyl octanoate (HxOc) after 24 h [163]. The same group, in order to make the process feasible, tried to increase the final product concentration by keeping the substrate concentration constant in a fed-batch mode for five days, yielding 1 M of HxOc.…”

One-Step Purification of Recombinant Cutinase from an E. coli Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand

“…Recombinant cutinase has been produced and used over years in our lab as a modelenzyme for a variety of studies [16,17]. The protocol used for its purification is a wellestablished but time-consuming process that involves an osmotic shock, an acidic precipitation, and two anion exchange chromatographic steps, followed by dialysis [18].…”