Immobilization of Biotechnologically Important Candida rugosa Lipase onto Commercial Matrices
Abstract
The continual search for alternative environmentally cleaner technologies in industrial processes has led to an increase in the use of enzymatic processes globally. However, due to their physical characteristics they require immobilization in order to remain effective. The objective of this study was to investigate the immobilization of the biotechnologically important and commercially available Candida rugosa lipase (CRL) by physical interfacial adsorption onto a number of matrices to act as biocatalysts. Five different types of support were tested: i) macroporous silica (synthetic inorganic), ii) polyhydroxybutyrate (natural organic), iii) polypropylene (synthetic organic), iv) polymethacrylate (synthetic organic), and v) polystyrene-divinylbenzene (synthetic organic). Results generated during this study showed that from the group of materials tested, polystyrene-divinylbenzene gave the best results with the highest amount of immobilized protein (8.10 ± 0.31 mg/g) and a good immobilization yield (90.35% ± 1.53%). The efficiency of protein immobilization was found to be highest when carried out at pH4.5, which is close to the isoelectric point of the enzyme.
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Introduction
Over recent decades, the search for green / environmentally friendly / cleaner technologies (balanced with the need to remain economically viable) for use in almost all industrial chemical processes has seen the increase in the use of enzymatic methodologies to achieve these targets. This has resulted in the enzyme market experiencing a high growth rate; it is estimated that in the period 2018-2023, the global enzyme market will grow at an annual rate of 4.9% and rise from US $5.5 Billion in 2018 to US$7 Billion in 2023 [1].Within the global enzyme market, there are six groups of enzymes which stand out: proteases, carbohydrases, lipases, phytases, polymerases and nucleases.
The interest in these biocatalysts goes beyond their ability to simply increase the rate a chemical reaction (i.e. the role of a traditional catalyst); they offer alternatives for many chemical processes due to their unique properties of biodegradability, specificity and selectivity in reactions [1-3].
Of specific interest in this study, Lipases are extremely versatile enzymes and are used in a variety of applications: in addition to simple hydrolysis reactions they can catalyse esterification and transesterification reactions and are also frequently used in the cosmetics, food, flavouring, beverage, paper, lubricant, detergent, effluent treatment, biosensor and biodiesel industries[2-4].
The industrial application of lipases (and other enzymes) in large-scale processes is often limited by the high cost and stability issues faced under industrial conditions; they are fragile structures which are sensitive to high temperature, variations in pH and various solvents. In addition, it can be difficult to separate them from some reaction systems, which limits their recovery and re-use which in turn may also lead to the contamination of the final product [5,6]
In order to overcome some of these limitations, the use of enzymes that are immobilized is a strategy that has been adopted to protect the enzyme, however in order to remain as an efficient active biocatalyst, the selection of an appropriate support material and application protocol are key factors [7]. Support can be from various origins: organic / inorganic / natural / synthetic and may also have a variety of porosities (porous, microporous, mesoporous, macroporous).
Conclusion
The results obtained during this study indicate that polystyrene-divinylbenzene is a very good material for a support structure in the immobilization of Candida rugosa lipase, especially when carried out at pH4.5, which is close to its isoelectric point (pH 4.2). In addition, the physical properties of polystyrene-divinylbenzene allowed the Candida rugosa lipase to be immobilized preferentially within the support where the biocatalyst is protected from variations in a reaction environment, reducing the possibility of denaturation. Additional future work we focus on the stability of the prepared biocatalyst when applied in biotransformation reactions.