Immobilization of Lipase from Penicillium sp. Section Gracilenta (CBMAI 1583) on Different Hydrophobic Supports: Modulation of Functional Properties.

Daniela Turati,Jose Guisan,Gloria Fernandez-Lorente,Eleonora Carmona,Wilson Morais Júnior,César Terrasan,Sonia Moreno-Perez,Benevides Pessela

doi:10.3390/molecules22020339

Abstract

Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p-nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lipase and hence different functional properties.

Highlights

Lipases constitute a class of enzymes that catalyze the hydrolysis of ester bonds from long chain triacylglycerol with low solubility in water, giving themMolecules 2017, 22, 339; doi:10.3390/molecules22020339 www.mdpi.com/journal/moleculesMolecules 2017, 22, 339 the property to catalyze at an oil/water interface [1]
Immobilization of lipases occurs via a peculiar mechanism known as interfacial activation [7,8], This naturally-occurring mechanism was used as a tool to immobilize several microbial lipases in many different hydrophobic supports proving to be a simple and efficient method that can allow selective purification, due to lipase adsorption even at low ionic strength, giving rise to purified-stabilized derivatives [9,10,11,12,13,14,15,16]
The Penicillium sp. (CBMAI 1583) lipase was hydrophobically immobilized by performing incubation during 90 min for Butyl Sepharose (But), Phenyl Sepharose (Phe), and Octyl Sepharose (Oct) and 120 min for Toyo, Lewatit VP OC 1600 (Lew), and Sep supports

Summary

Introduction

Lipases (triacylglycerol acyl hydrolase, EC 3.1.1.3) constitute a class of enzymes that catalyze the hydrolysis of ester bonds from long chain triacylglycerol with low solubility in water, giving themMolecules 2017, 22, 339; doi:10.3390/molecules22020339 www.mdpi.com/journal/moleculesMolecules 2017, 22, 339 the property to catalyze at an oil/water interface [1]. Enzyme immobilization is a primordial requirement to overcome common bottlenecks which hinder the large-scale application of biocatalysts at industrial level. This way, the immobilization technique/protocol should be designed in such a way to improve enzyme properties, in relation to activity, selectivity, performance in organic solvents, pH tolerance, heat stability, or the functional stability [4,5,6]. In the presence of a hydrophobic surface, the lid changes its position exposing the hydrophobic pocket to the medium, easing substrate access to the active site This open form is quite unstable in aqueous and homogenous medium, but becomes stabilized by adsorption on hydrophobic surfaces, which can be originated from a substrate or hydrophobic supports. A limitation of this type of biocatalyst, is the enzyme release from the support which can occur in the presence of some reaction products, an important factor that should be taken in consideration during the selection of an industrial lipase biocatalyst [17]

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