Lipase immobilization physical adsorption

Abstract An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t /2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction. 

Immobilization of lipases on membranes have also been described and several bioreactors were developed (see review, Balcao, Paiva Malcata, 1996). The immobilization can be done by simple physical adsorption of the lipase on hydrophobic hollow fibers or flat sheets where polypropylene types are the preferred e.g. Accnrel or Celgard) (Bouwer, Cupenus Derksen, 1997). 

Immobilization of Yamnvia Upolytica Lipase— a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques... 

This work shows that lipase from Y lipolytica does not undergo hyperactivation phenomenon that is largely observed for lipases from other sources when immobilized in hydrophobic supports [12, 13], However, immobilization by physical adsorption showed to be the key for the immobilization process and especially in very high hydrophobic supports that are by far, the best way to get an optimal compromise between activity and stability. Our results also showed that YLL lost activity when immobilized by multipoint covalent attachment. 

Wang et al. (2006) immobilized the lipase enzyme from Candida rugosa by physical adsorption on the surface of polysulfone (PSF) composite nanofibrous membranes. PVP and PEG were used as additives, aiming to tailor the surface properties of the PS nanofibers, increasing their hydrophilicity. The results showed that the activity of the immobilized lipase increased with the content of PVP or PEG, whereas the adsorbed amount of lipase was unchanged. These results were attributed to the enrichment of PVP or PEG at the nanofiber surface. In another study, lipase from Pseudomonas cepacia was immobilized by physical adsorption onto electrospun PAN fibers and used for the conversion of (S)-glycidol with vinyl n-butyrate to glycidyl n-butyrate in isooctane (Sakai et al. 2010). 

The drawbacks of previous methods can be overcome by a combining two of any immobilization methods (Kanwar et al., 2004 Yadav and Jadhav, 2005 Zarcula et al., 2009). Ursoiu et al. (2011) deposited immobilized C. antarctica lipase B via sol-gel entrapment on support material. Yadav and Jadhav (2005) tested the preimmobilization of C. antarctica lipase B on hexagonal mesoporous silica using physical adsorption, followed by encapsulation in calcium alginate beads, which resulted in a reusable lipase with no leaching even after the fourth reuse. 

In addition to chemical bonding, the enzymes were also applied onto nanofibers simply via physical adsorption. Polyacrylonitriles-2-methacryloyloxyethyl phosphoryl choline (PANCMPC) nanofiber was reported to have high biocompatibility with enzymes because of the formation of phospholipid microenvironment on the nanofiber surface. Lipase on the nanofibers showed a high immobilization rate, strong specific activity and good activity retention. 

Mesoporous materials with controlled porosity and functionality have been used to immobilize biomolecules, e.g., proteins. Enzymes of small or medium size such as cytochromes, oxidases, peroxidases, lipases or proteases, can be immobilized via physical adsorption, encapsulation, or chemical binding. Such immobilization was found to maintain some activity of the biomolecule, with applications in the biosensor field. Recently, some developments have been reported in the immobilization of redox proteins in mesoporous transparent electrodes for... 

Traditional techniques such as physical adsorption and covalent linkage onto solid supports, entrapment in polymer matrices, and microencapsulation have long been used for immobilizing such enzymes as lipases, proteases, hydantoinases, acylases, amidases, oxidases, isomerases, lyases, and transferases [12-18]. Encapsulation and adsorption have also proved their utility in the immobilization of bacterial, fungal, animal, and plant cells [12-21]. However, as biocatalysis applications have grown, so the drawbacks and limitations of traditional approaches have become increasingly evident. The forefront issues now facing bioimmobilization are indicated in Table 1. 

Fig. 2. Gas adsorption/desorption isotherms obtained for pure silica gel (PS), silanized and activated silica (SPS), and immobilized derivatives in silica gels (ADS, silica with lipase physically adsorbed CB1, silica with lipase covalently bonded CB2, silica with lipase covalently bonded in presence of PEG EN1, silica with entrapped lipase EN2, silica with lipase entrapped in presence of PEG). (O),Adsorption ( ), desorption.

Adsorption, hnkage, bonding of the enzyme to an insoluble support and entrapment of the enzyme in polymeric gels or encapsulation have all been used as ways to immobilise lipases [38]. To be successful, various parameters of the immobilization support need to be considered such as its mechanical strength, chemical and physical stability, its hydrophilicity/lipophilicity and the relevant loading capacity of the enzyme [39]. 

Adsorption is a commonly used method to immobilize lipase. Several noncovalent interactions are involved in this immobilization, including nonspecific physical... 

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