Cross-linked enzyme aggregates (CLEAs) with controlled particles: Application to Candida rugosa lipase
Introduction
Recently carrier-free enzyme immobilization approach has attracted more attention due to clear advantages: highly concentrated enzyme activity in the catalyst, high stability and the low production cost due to the exclusion of an additional carrier [1], [2], [3], [4]. Cao et al. [5] reviewed the various carrier-free enzyme immobilization approaches. Generally they are prepared by directly cross-linking different enzyme preparations such as dissolved enzyme, crystalline enzyme, spray-dried enzyme and physically aggregated enzyme, resulting in the formation of cross-linked dissolved enzymes (CLDEs), cross-linked enzyme crystals (CLECs), cross-linked spray-dried enzymes (CSDEs) and cross-linked enzymes aggregated (CLEAs), respectively. Thus, they are discriminated from each other only by the precursors used for cross-linking. Among these techniques, CLEA is a simpler one developed by Cao et al. in 2000 [6]. The procedure of the approach consists of the covalent cross-linking of a precipitated enzyme. The idea is from the concept that protein in general can be precipitated by agents such as inorganic salts or organic solvents without undergoing denaturation. This technique had been successfully applied in the preparation of carrier-free enzyme immobilization of aminoacylase and some lipases [7], [8], [9]. Nevertheless, how to control the lipase particle size in the CLEA process and the effect of particle size on the activity of the lipase had rarely been reported. In the paper, Candida rugosa lipase was precipitated to be cross-linking aggregate; some factors to influence the cross-linking lipase particle size including aggregation agent types, fraction of the aggregation agent and pH, were reported. The effect of particle size on the activity of the lipase and the enantioselectivity of the cross-linked lipase for kinetic resolution of ibuprofen with 1-propanol in isooctane at 30 °C were investigated.
Section snippets
Chemicals and enzyme
The lipase (E.C. 3.1.1.3) from C. rugosa was purchased from Sigma–Aldrich Company. The concentration of protein in the crude enzyme is 196.2 mg protein g−1 determined by UV detector at the wavelength of 280 nm. All other chemicals were of a reagent grade and obtained commercially.
General CLEA preparation
C. rugosa lipase (100 mg) was dissolved in phosphate buffer (2 ml, 0.1 M, pH 7). After centrifuge, the transparent enzyme solution was shifted to a crystallizer (50 ml) designed in our lab. (NH4)2SO4 (1.2 g), (NH4)2SO4 solution
Salt effect on particle size
It is possible to form aggregates by changing the hydration state of enzyme molecules or by altering the electrostatic constant of the solution by adding appropriate aggregation agents. Not all salts have the same effect on the lipase aggregation. It is well understood that the different salt exhibits different aggregation effect on the specific lipase. Therefore it is crucial to screen a suitable salt to be aggregation agent in the process of CLEAs. In the part, some salts were applied to be
Conclusions
Compared to other kind of immobilized enzyme, CLEA exhibits many advantages such as no solid support, easy preparation and only pure protein which might perform high activity and enantioselectivity. Many factors can affect the particle size of the protein in the precipitation process of C. rugosa lipase, including aggregation agent type and concentration, enzyme and glutaraldehyde concentration, and pH. Among these factors, enzyme and glutaraldehyde concentration plays a significant role in
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