The Penicillium strain used in this work was isolated
The Penicillium strain used in this work was isolated from Atlantic forest soil and previously screened as lipase producer (Tauk-Tornisielo et al., 2005). The immobilization was previously evaluated using hydrophobic supports, i.e. agarose based butyl-(But), phenyl-(Phe) and octyl-Sepharose (Oct), acrylic Toyopearl (Toyo), and macroporous Lewatit VP OC 1600 (Lew) and octadecyl Sepabeads (Sep) — to obtain highly active and stable biocatalysts. The properties of the immobilized enzyme were compared to the cyanogen bromide derivative which emulates the properties of the soluble enzyme, but without problems caused by molecular interaction. The derivatives were characterized and applied in reactions to obtain Omega-3 fatty acids and ethyl esters from fish oil, a very appreciated product in food and pharmaceutical industries (Turati et al., 2017). The aims of this work were to evaluate the culture conditions using pure and complex carbon sources for lipase production under submerged cultivations. The crude lipase was purified by using the interfacial activation strategy in hydrophobic interaction chromatography and the purified enzyme was biochemically characterized.
Materials and methods
SDS-PAGE Denaturing electrophoresis was carried out according to Hames (1987) with a 3.75% polyacrylamide stacking gel and 8–18% polyacrylamide gradient resolution gel. Gels were stained with 0.1% (w v−1) Coomassie Brilliant Blue R-250 (Hames, 1990).
Results and discussion
Conclusions In this work, the lipase production by Penicillium sp. section Gracilenta CBMAI 1583 was almost 2-fold increased by studying substrate and cultivation conditions. Initial purification studies revealed the fungus produces a 65.4 kDa enzyme with esterase activity and a 52.9 kDa enzyme with lipolytic activity. Lipase purification was successful using phenyl Sepharose chromatography under interfacial condition. The purified enzyme is optimally active in sonidegib australia pH (4.0) and high temperatures (70 °C). The enzyme presents low specificity, hydrolyzing p-nitrophenyl esters with chain-length from 10 to 18 carbons. Maximal activity was observed with p-nitrophenyl decanoate, suggesting a possible preference for intermediate-chain p-nitrophenyl esters. The observed characteristics indicate potential industrial application in processes that operate in acid pH, such as treatment of dairy and industry effluents, resolution of esters in the pharmaceutical industry or in the food industry. The stability of lipase in organic solvents also suggests that this enzyme is a candidate to act in organic synthesis reactions in non-aqueous media.
Acknowledgments The authors gratefully acknowledge to São Paulo Research Foundation - FAPESP, Brazil, for the scholarship granted to the first author and Spanish Ministry of Science and Innovation - MICINN, Spain (Project BIO-2012–36861).
Introduction Enzymes have higher selectivity, specificity and efficiency than chemical catalysts. Due to their properties and their green chemistry, biocatalysts are widely used in food, textile and pharmaceutical industry , . A high efficient biocatalyst for industrial applications must be stable in a wide range of temperatures and pHs and has to be easily separated and recovered from the product during the downstream process , , . Enzymes present thermal instability, susceptibility to attack by protease, activity inhibition, high sensitivity to pH and other denaturing agents, and cannot be separated at the end of reaction and reused. The immobilization of enzymes on solid supports, such as polymeric resins and inorganic materials , overcomes these drawbacks. In this context, nanoparticles (NPs), and in particular iron oxide NPs, have been used for the conjugation of enzymes thanks to their high surface area, high surface-volume ratio, low mass transfer limitation and their unique magnetic properties , . Indeed, iron oxide NPs can be manipulated by an external magnetic field , , and, as a consequence of Neel and Brown relaxation, they can produce heat under alternating magnetic field (AMF), a phenomenon known as magnetic hyperthermia . This has found application in biomedicine, where its use for cancer therapy and for controlling drug delivery is being extensively explored , , . However, the use of this property to improve biocatalytic processes is still almost totally unexplored.