Climatic Chamber Stability Tests of Lipase-Catalytic Octyl-Sepharose Systems

Abstract

The application of the climatic chamber presented in this paper to assess the storage stability of immobilized lipases is a new approach characterized by the potential of unifying the study conditions of biocatalysts created in various laboratories. The data achieved from storing lipases in the climatic chambers may be crucial for the chemical and pharmaceutical industry. Our paper describes the developed protocols for immobilization via interfacial activation of lipase B from Candida antarctica (CALB) and lipase OF from Candida rugosa (CRL-OF) on the Octyl-Sepharose CL-4B support. Optimization included buffers with different pH values of 4–9 and a wide range of ionic strength from 5 mM to 700 mM. It has been shown that the optimal medium for the CALB immobilization process on the tested support is a citrate buffer at pH 4 and high ionic strength of 500 mM. Implementing new optimal procedures enabled the hyperactivation of immobilized CALB (recovery activity 116.10 ± 1.70%) under the applicable reaction conditions using olive oil as a substrate. Importantly, CALB storage stability tests performed in a climatic chamber under drastic temperature and humidity conditions proved good stability of the developed biocatalyst (residual activity 218 ± 7.3% of dry form, after 7 days). At the same time, the low storage stability of CRL OF in a climatic chamber was demonstrated. It should be emphasized that the use of a climatic chamber to test the storage stability of a dry form of the studied lipases immobilized on Octyl-Sepharose CL-4B is, to our knowledge, described for the first time in the literature.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives

“…As discussed in our previous papers [17,35], it is believed that the observed increase in catalytic activity may result from the elements of the CALB structure-including the lid. Additionally, it is thought that the increase in activity in an organic solvent after storing the tested lipase in an immobilized form at an extremely high temperature could be linked with, among others, changes in the enzyme's conformation.…”

“…A phosphate buffer (pH 7) has often been used to immobilize the CALB on octylagarose beads [39]. In our previous paper [35], we performed the immobilization of this lipase in a citrate buffer at pH 4. It should be noted that studies on the activity of the immobilized CALB on octyl-agarose described in the literature were most often conducted under aqueous conditions [40], whereas in the current study, we evaluated the lipase activity in the enantioselective esterification of (R,S)-flurbiprofen with methanol in an organic solvent.…”

“…In our previous paper [35], we extensively discussed, based on our own results and literature data, the influence of the buffer used for immobilization on lipase activity. It should be noted that in the previous publication, we conducted our studies in an aqueous medium (the hydrolysis of olive oil), while in this project we assessed the activity in an organic solvent (enantioselectivity).…”

“…Our previous papers [17,35] discussed the effect of storage in a climatic chamber and refrigerator on the stability of the CALB immobilized on octyl-agarose regarding its lipolytic activity assessed in an aqueous medium. The results we demonstrate in this paper are consistent with previous ones and confirm the observed phenomenon of an increase in the activity of the immobilized lipase after storage in a climatic chamber and a refrigerator.…”

“…Additionally, due to the potentially significant influence of the immobilization conditions on lipase catalytic activity, the impact of this process was also examined. It is worth mentioning that in previous studies [17,35], protocols concerning the immobilization of the CALB and CRL onto octyl-agarose have been introduced. The assessment of the lipolytic activity of the immobilized lipases conducted in an aqueous medium allowed the development of new, optimized catalytic systems.…”