The Penicillium strain used in this work
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 Caspase-4 Colorimetric Assay Kit 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 The ribokinase superfamily comprises kinases involved in several important metabolic reactions such as phosphorylation of sugars, vitamins, and nucleotides. Based on their phosphoryl donor and acceptor specificity the ribokinase superfamily can be split into three branches [1,2]. Two of them encompass ATP-dependent kinases, whereas the third one is constituted by ADP-dependent sugar kinases that transfer the β-phosphate of ADP to glucose or fructose-6-phosphate . All members of the ribokinase superfamily present a large domain formed by an α/β/α ribokinase-like fold. This domain possesses an N-terminal Rossmann-like motif that hosts the phosphoryl acceptor binding site and a C-terminal β-meander motif where the nucleotide-binding site (ATP or ADP) is located [4,5] (Fig. 1A and B). In most of these enzymes, an additional smaller domain acting as a lid or cap over the active site is also present . During evolution, proteins belonging to a superfamily often diverge via the introduction of point mutations into their sequences but can also evolve through the rearrangement of sequence stretches longer than one amino acid. Such rearrangements are called shuffling, recombination or permutation . Permutations are one of the most striking structural rearrangements where the alteration of the amino acid order at the sequence level leads to a change in the connectivity of secondary structure elements of a protein (change in topology) while preserving the tridimensional organization of these elements (same architecture). Two types of permutation have been described: circular permutation that can be envisioned as the result of the covalent linkage of the N and C termini and subsequent linearization of the circular protein by cleavage at a different peptide bond . This result in a new linear sequence only altered at the sites of cleavage and joining with a different order of secondary structural elements. At the gene level, mechanisms like whole-gene duplications followed by truncations at N and C terminus can give a circular permuted sequence . The other type is called non-cyclic permutation and refers to more complex topological changes. In this case, a partial duplication of a gene and deletion of terminal and internal element are proposed as a mechanism to give this permuted sequence [, , , ]. Also, algorithms have been developed to detect topological differences between two spatially similar protein structures. By this method, Dundas et al…  demonstrated the existence of previously unknown circular permutations and a naturally occurring non-cyclic permuted protein. Hence, permutation constitutes a common molecular mechanism for protein evolution through the generation of protein chains with different topologies, which can be identified by the existence of two homologs with the same architecture but different connectivity of their secondary structure elements. This is the case of the ADP and ATP-dependent kinases of the ribokinase superfamily. Although members of this superfamily present a conserved fold, careful inspection of the central β-sheet from the large domain of these homologous enzymes reveals that the ADP-dependent enzymes present a topological re-ordering of the secondary structural elements that produces an equivalent tertiary structure, which can be thought as a non-cyclic permutation of the β-meander region (Fig. 1).