Lipase immobilization cross-linking

Among biocatalysts, hydrolases like lipases and proteases are the most popular. There are several types of biocatalysts in commercial products. Immobilized lipases and cross-linking enzymes are briefly described in this section. 

The photopolymer solution used for the immobilization of lipase on a FET contained 10% PVP and 0.3% BASC (7, 13). A lipase-immobilizing soludon was prepared by dissolving 150 mg of lipase and 100 mg of bovine serum albumin in 1 mL of the photopolymer soludon. A photolithographic lipase membrane was formed on one of two FET elements, similar to the glucose oxidase and urease membranes, except that the development was done in water. After the development, the lipase-immobilized membrane was immersed in a 3% glutaraldehyde soludon for the addidonal chemical cross-linking of the protein molecules. 

The application of enzymes as catalysts in organic chemistry is closely linked to their immobilization. Indeed, many enzymes are only available in an immobilized form. The immobilized enzymes can be used as received, greatly easing their application. Numerous of these readily available immobilized enzymes are now the working horses of biocatalysis. This has even led to the incorrect use of the abbreviation of an enzyme name for a specific enzyme preparation, that is CALB for the immobilized form of Candida antarctica lipase B on cross-linked polymethacrylate (also known as Novozym 435). Vice versa the commercial name of an enzyme preparation-Amano PS-has taken the place of the enzyme (Burkhdderia cepacia lipase on dextrin or diatomaceous earth). Surprisingly, often no attention is paid to the fact that the enzyme is immobilized [1]. 

The non-aqueous lipase system for flavor esters developed by our group used components and preparative techniques for enzyme immobilization, that would not only be cost effective and simple but also meet regulatory requirements. The enzyme could have been immobilized by a number of methods however for the Intended application only (i) adsorption (11) ionic bonding or (lii) glutaraldehyde cross-linking would be... 

Immobilized enzymes are currently the object of considerable interest. This is due to the expected benefits over soluble enzymes or alternative technologies. The number of applications of immobilized enzymes is increasing steadily [5]. Occasionally, however, experimental investigations have produced unexpected results such as a significant reduction or even an increase in activity compared with soluble enzymes. Thus, cross-linked crystals of subtihsin showed 27 times less activity in the aqueous hydrolysis of an amino acid ester compared to equal amounts of soluble enzyme [6]. On the other hand, in the application of hpo-protein lipase in the solvent-mediated synthesis of esters there was a 40-fold increase in activity using immobihzed or otherwise modified enzyme preparations as compared to enzyme powder [7]. 

Lipases act in nature on oil-water interfaces and often have hydrophobic domains on their surface. Hence, immobilization by adsorption to a hydrophobic carrier is often a simple and effective way to immobilize Upases. A wide range of different hydrophobic support materials is commercially available, including synthetic acrylic, divinylbenzene-styrene or polypropylene polymers. An example of the latter is Accurel MP 1000, which is available from Membrana. Novozym 435 is immobilized on Lewatit VP OC 1600, a divinylbenzene-cross-linked poly(methyl methacrylate) resin produced by Lanxess (previously Bayer). 

Cross-linked crystals of Candida rugosa lipase are highly efficient catalysts for resolving racemic esters, whereas PPL immobilized in microemulsion-based gel has been used to hydrolyze one of the polyphenolic perpropanoates. Actually, symmetrical diacetates are readily cleaved to provide the monoesters by PPL. ... 

One interesting technology uses lipases in the form of cross-linked enzyme crystals (CLECs) (Margolin 1996). This immobilization method does not use any solid support and the lipase specific activity (units of activity/g of immobilized catalyst) of the immobilized lipase derivative can be enhanced by 10-fold because there is no inert support, that usually represent more than 90% of the catalyst weight in the case of carrier-bound enzymes. These cross-linked crystals have been used for the chiral resolution of commercially important organic compounds, such as ibuprofen. 

HUal N, NigmatuUin R, Alpatova A (2004) Immobilization of cross-linked lipase aggregates within microporous polymeric membranes. J Membrane Sci 238(1-2) 131-141 Hiol A, Jonzo MD, Rugani N et al. (2000) Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme Microb Technol 26 421 30... 

Hilal N, Nigmatullin R, Alpatova A (1995), Immobilization of cross-linked lipase aggregates within microporous polymeric membranes , J. Memb. Sci., 238,131-141. 

Lipases enantioselectivity can be improved physically, chemically, or genetically. Physically, lipases can be modified by cross-linking, crystallization, and immobilization that involves attaching the lipase onto an insoluble solid support. Immobilization enhances the stability of lipase and facilitates lipase recovery. Details of lipase immobilization methods are discussed in Chapter 3. 

With lipase in immobilized form, it will not be in direct contact with any external hydrophobic interface that may inactivate soluble proteins. For example, in the presence of an organic solvent the immobilized lipase may be in contact but will not be soluble, thus preventing inactivation from occurring. This was investigated by Nawani et al. (2006), who compared the catalytic properties of Bacillus sp. lipases immobilized by different techniques. In their study, stability was improved with an optimum temperature of 5°C higher than that of the soluble lipase. This was reported after immobilization by adsorption on silica and HP-20 beads followed by cross-linking with gluteraldehyde on HP-20. Montero et al. (1993) compared the stability... 

Enzymes were incorporated into nanofibers viaelectrospinning, and subsequent crosslinking the enzymes incorporated effectively prevented their leaching. In the presence of PEO or PVA, casein and lipase were electros-pun into ultra-thin fibers. After crosslinking with 4,4 -methylenebis(phenyl diisocyanate) (MDI), the fibers became insoluble, and the lipase encapsulated exhibited 6 times higher hydrolysis activity towards olive oil than that of the films cast from the same solution. The cross-linked enzymes in nanofibers showed very high activity and stability. For example, the immobilized a-chymotrypsin in a shaken buffer solution maintained the same activity for more than two weeks. 

Wu, Y, Wang, Y, Luo, G., and Dal, Y. (2009). In situ preparation of magnetic Fe304-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Biores. Technol., 100, 3459-3464. 

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