Immobilization of commercial lipase onto different supports: characterization and application in esterification reaction
Abstract
The current preference of costumers for natural and healthy products is increasing the employment of biotechnological processes that use enzymes, and the synthesis of esters is an example of this change. However, enzymes are high-cost product, which stimulates research in finding solutions that make them more economically attractive, like immobilization. This work aimed to use different protocols for immobilizing lipase and its application in ester synthesis. The results showed that Pseudomonas fluorences lipase (AKL) was the most efficient for immobilization among other studied lipases (Pseudomonas fluorences lipase (AKL), Pseudomonas cepacia lipase (PSL), Hog Pancreas lipase (PHL), Pancreas Porcinas lipase (PPL), and Mucor Javanicus lipase (MJL)), with hydrolytic activity of 3323.6 U/g. Both immobilization methods (physical adsorption and entrapment) showed promising results towards hydrolytic activity. The best immobilization by adsorption was obtained using AKL onto PHB (polyhydroxybutyrate), with 698.61 U/g of hydrolytic activity. For entrapment, AKL also presented the best result, with 247.30 U/g of hydrolytic activity. For the synthesis of ester, after a 60 h-reaction using the immobilized derivatives by physical adsorption, the esterification yield was 74.26 %. In terms of hydrolytic activity, the employed protocols were very promising and encourage the continuity of this study towards the optimization of processes using industrial lipases.
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Introduction
In the drive for alternative solutions for production, reduction of costs, and application of environmental friendly production, the replacement of chemical catalysts for biological catalysts has been growing over the time due to the attractive characteristics of enzyme for industries, such as: high specificity an selectivity, low toxicity, purity of the product, reduced environmental impact, easy handling, reducing energy cost by lowering the process temperature, use of mild pH values, among others [1,2,3]. Even if the enzyme demand is high, not all types of enzymes are produced in industrial scale because of the difficulty in finding high yield, activity, and stability enzymes [4].
In order to bypass the difficulties of using enzymes industrially, the development of immobilization techniques revolutionized and expanded the employment of enzymes, because the immobilization process allows them to be reused many times [5]. The most known and applied immobilization methods are: covalent binding [6], physical adsorption [7], entrapment (or encapsulation) in polymeric matrix [8], reticulation [9], and bio selective adsorption [10].
The entrapment of enzymes in porous matrices promotes the creation of a protector microenvironment for the enzymatic structure, which improves its operational stability [11]. The pores formed in the enzyme-support complex allow small molecules to diffuse easily and reach the active site of the entrapment enzyme, which enables it to keep its activity even after immobilization [8]. In physical adsorption, the molecules of enzyme adhere to the support surface through hydrophobic interactions, and, because of that, the adsorbed enzymes usually are resistant to proteolysis and aggregation [12, 13].
Conclusion
At the end of this work it is possible to consider that both immobilization methods presented satisfactory results as the enzymes were able to increase their hydrolytic activity under several conditions. The lipase immobilized by physical adsorption demonstrates the potential of PHB in natura to be used as support for lipase immobilization. Free and immobilized lipases had similar operating conditions, and the the PHB immobilized AKL showed the best percentage yield of isoamyl acetate formation after 60h-reaction (74.26%).