Stabilization of the lipase of Hypocrea pseudokoningii by multipoint covalent immobilization after chemical modification and application of the biocatalyst in oil hydrolysis
Graphical abstract
Introduction
Lipases (EC 3.1.1.3) are widely used to catalyze the hydrolysis, alcoholysis, esterification and transesterification of carboxylic esters. They participate in several types of reactions and have a wide range of industrial application also, a growing number of researchers have reported their use [1], [2]. Therefore, in order to improve the lipases yield, immobilized lipases can be used. Nevertheless, one of the bottlenecks for the lipase industrial application is the high cost of biocatalysts. Methods of immobilization were introduced to improve the lipase stability and allow its use repeatedly [3].
Immobilized enzymes offer some operational advantages in relation to the soluble enzymes, what makes them more desirable for certain applications, as they provide greater stability against thermal and pH, keeping them more stable throughout the process. Immobilization also allows the recovery, recycling and removal of the enzyme from the reaction mixture, and they are able to adapt to several processes [4]. Therefore, immobilization is often the key to improve the operational performance of an enzyme [1], [5], [6].
The immobilization may occur through the adsorption or connection of the enzyme in an insoluble material using a multifunctional reagent through crosslinks, confinement in arrays formed by gel polymers or encapsulation through a polymeric membrane [7], [8].
Glyoxyl-supports have been described as a very adequate immobilization system to yield immobilized–stabilized proteins via multipoint covalent attachment. Many enzymes have been stabilized using this technique; among them are lipases from different sources, mainly fungi and yeast [9], [10], [11]. The success of the immobilization is the result of the formation of a rigid connection between the protein and the support.
One of the great possibilities for improving immobilization on multipoint supports is to conduct the enrichment of enzyme surfaces with amino groups. This process can to be done through of the chemical amination of the protein surface via reaction of the carboxylic groups, using group of Asp and Glu, with ethylenediamine after activation with carbodiimide [11], [12], [13], [14], [15], [16]. More recently, the chemical amination of lipases reversibly immobilized on hydrophobic supports, as Octyl–Sepharose, via interfacial activation has been reported to be a way of simplifying this chemical amination previous to its covalent attachment [11], [17].
In this work, amined lipases immobilized in Glyoxyl and Octyl supports showed capacity to hydrolysis of common and exotic oils with distinct application. Bacuri seed oil is used to make soap, cure skin diseases, as a remedy for the healing of wounds in animals and in the cosmetic industry. Murumuru is mainly used in cosmetics, soaps, creams and shampoos, and in the paint industry as secant. The fat can also be used in the manufacturing of butter [18], [19]. Cupuaçu oil is rich in essential nutrients and phytonutrients, antioxidants, vitamins and other substances with medicinal properties. The plant contains polyphenols, phytonutrients with antioxidant properties similar to those found in cocoa. It is also rich in theobromine, vitamins A and C and important fatty acids, amino acids, phosphorous, fibers, nutritional vitamins B1, B2 and B3 (niacin). Butter oil is used as it helps to improve skin elasticity, slow down aging and improve skin hydration capacity. It also protects against the harmful effects of UV-A and UV-B. The cupuaçu oil is also soothing to skin irritations such as eczema and dermatitis, and it reduces the appearance of wrinkles [20].
Hypocrea is a fungi genus of Hypocreaceae family and this genus is associated with Trichoderma. Due to changes in the code of nomenclature, the genus Trichoderma has been proposed for conservation over its teleomorph Hypocrea [21]. The lipase production for Hypocrea pseudokoningii was previously identified and characterized in our laboratory and the lipase was immobilized on ionic and hydrophobic supports [22], [23].
The aims of the present study were to immobilize and aminate the lipase of H. pseudokoningii. The chemical amination of the protein surface via reaction of the carboxylic groups was used, such as the side group of Asp and Glu, with ethylenediamine after activation with carbodiimide. The immobilization significantly increased thermal and solvent stabilities in relation to the free enzyme. This work shows that the lipase amination increased the yield of immobilization on Glyoxyl supports, allowing its use in biotechnological applications.
Section snippets
Enzyme production
H. pseudokoningii was isolated from the soil samples collected from the different regions of the São Paulo State—Brazil. The fungus was maintained at 30 °C in 1.8% Potato Dextrose Agar. A volume of 1.0 mL of a conidial suspension of H. pseudokoningii, final concentration of 105 spores/mL, was inoculated into 125 mL Erlenmeyer flasks containing 25 mL of liquid Adams medium [24], plus 1 mL olive oil. The liquid medium was composed by 0.2 (g/100 mL) yeast extract, 0.1 (g/100 mL) monobasic potassium
Immobilization of purified lipase from Hypocrea pseudokoningii
The pure lipase was immobilized (82%, Table 1) on Octyl–Sepharose; being this derivative 3-fold more activated compared with the control. The enzyme maintained its activation while the derivative was aminated on support. The enzyme was 99% desorbed from aminated Octyl–Sepharose support using 2% Triton X-100. During desorption, the enzyme retained twice-fold its activation in relation to initial activity of the purified and free enzymes.
After obtaining a solution of the purified and aminated
Discussion
Lipases possess a large hydrophobic region, which makes it easier for them to be immobilized on hydrophobic support. Lipases when immobilized on hydrophobic supports tend to get hyperactive; this is because the lipase keep the lid open and exposing the active site, which facilitates substrate input. The literature reports several studies that used columns or hydrophobic resins for lipase purification. As an example, we have the lipase immobilization from Aspergillus niger by using a highly
Conclusions
The amination of lipase allowed the immobilization on Glyoxyl–Agarose upon milder conditions. The multipoint immobilization was more thermo stable and more stable in organic solvents than the free enzyme. Glyoxyl–Agarose derivative had higher activity in all oils tested. Moreover, the results clearly indicate the improvement of derivatives in organic solvents, suggesting the enzyme has the potential for application in industrial processes.
Acknowledgements
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho de Desenvolvimento Científico e Tecnológico (CNPq process n° 406838/2013-5). This project is also part of the National Institute of Science and Technology of the Bioethanol (n° 574002/2008-1), CNPq—Ciência sem Fronteira (n° 242775/2012-8). JAJ and MLTMP are Research Fellows of CNPq. MGP and ACV are supported by CNPq. FDAF was recipient of a FAPESP Fellowship. We thank Ricardo F.
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2019, International Journal of Biological MacromoleculesCitation Excerpt :Thus, in some instances, the enzymes have been aminated (chemically and in some instances genetically) [17] to enhance the properties of the immobilized biocatalysts. Using lipases, this modification used to be performed in a reversible immobilization form of the enzyme (e.g., via interfacial activation on hydrophobic supports) [24–26], to simplify the process, but that possibility is not viable using other enzymes [27,28]. Among the enzymes with industrial relevance, proteases have a special interest [29–34].