Lipase for Biocatalysis

The recovered broth has to be formulated into a final product to comply with requirements appropriate to its final application. In formulation, whether for a solid or liquid product, different issues are addressed. Maintaining the activity of enzyme from the time of manufacture to the time of application through storage is one of the main factors tested during formulation. Other stability issues such as microbial stability and physical stability are important as well. Depending on the final application of the enzyme, the physical appearance can be customized (e.g., colored). A specific example of formulation is immobilization (see also Chapter 2). 

Lipases are a special class of esterases that hydrolyze fatty acid esters like triglycerides at lipid/water interfaces. In most lipases, a part of the enzyme molecule covers the active site with a short amphiphilic a-helix, called the lid. The side of the a-helical lid facing the catalytic site, as well as the protein chains surrounding the catalytic site are mostly composed of hydrophobic side chains. The lid in its closed conformation (i.e., in the absence of an interphase or organic solvent) prevents access of the substrate to the catalytic triad. Opening of the lid twists and exposes a large hydrophobic surface, and the previously exposed hydrophilic domain becomes buried inside the protein. 

All lipases show structural and functional similarities, regardless of the organism from which they were isolated. Hence, they all have a a-(i-hydrolase fold structure with a catalytic triad. However, small variations in the substrate binding site may have a strong effect on the catalytic properties and the stability of enzyme [1]. 

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The single most used lipase for biocatalysis is probably the Candida antarctica B-lipase (CALB) [42]. It is commercialized by Novozymes in liquid formulation as well as in immobilized form under the trade name Novozym 435 (previously SP 435). CALB has high activity on a wide range of substrates (it has some problems with very bulky substrates), often with outstanding selectivities. Formulated as Novozym 435 it is stable up to approx. 90 °C in solvents such as toluene (or solvent-free reaction mixtures). The A-lipase (CALA), currently only commercially available in liquid form, has attractive properties too, including even better thermostability and higher activity on sterically hindered substrates [43]. 

CaUeri, E. Temporini, C. Furlanetto, S. Loiodice, R 45. FracchioUa, G. Massolim, G. Lipases for biocatalysis Development of a chromatographic hioreactor. J. Pharm. 

Villeneuve, P., J. M. Muderhwa, J. Graille, and M. J. Haas. 2000. Customizing Lipases for Biocatalysis A Survey of Chemical, Physical and Molecular Biological Approaches. Journal of Molecular Catalysis B Enzymatic 9 (4-6) 113-148. 

Fig. 23.1 Microbial routes from natural raw materials to and between natural flavour compounds (solid arrows). Natural raw materials are depicted within the ellipse. Raw material fractions are derived from their natural sources by conventional means, such as extraction and hydrolysis (dotted arrows). De novo indicates flavour compounds which arise from microbial cultures by de novo biosynthesis (e.g. on glucose or other carbon sources) and not by biotransformation of an externally added precursor. It should be noted that there are many more flavour compounds accessible by biocatalysis using free enzymes which are not described in this chapter, especially flavour esters by esterification of natural alcohols (e.g. aliphatic or terpene alcohols) with natural acids by free lipases. For the sake of completeness, the C6 aldehydes are also shown although only the formation of the corresponding alcohols involves microbial cells as catalysts. The list of flavour compounds shown is not intended to be all-embracing but focuses on the examples discussed in this chapter... 

Lipases, which are noted for their tolerance of organic solvents, were obvious candidates for biocatalysis in ionic liquids. Indeed, stable microbial lipases, such as CaLB [8, 54, 55, 56] and Pseudomonas cepacia lipase (PcL) [28, 55, 57] were cat-alytically active in the ionic liquids of the l-alkyl-3-methylimidazolium and 1-alkylpyridinium families, in combination with anions such as [BF4], [PF6], [TfO] and [ Tf2N]. Early results were not always consistent, which may be caused by impurities that result from the preparation of the ionic liquid. Lipase-mediated transesterification reactions (Figure 10.3) in these ionic liquids proceeded with an efficiency comparable to that in tert-butyl alcohol [8], dioxane [57], or toluene... 

Enzymes have been used for biocatalysis in organic solvents since the early 1980s, and in ILs since 2000 [17]. As biological catalysts, enzymes accelerate reactions but do not affect the equilibrium distribution. Hence, hydrolytic enzymes that, as an example, under normal circumstances in aqueous solutions hydrolyze esters and amides, will, when placed in water-free conditions, also catalyze the reverse reaction, the condensation of an acid with an alcohol or an amine, to give esters and amides, respectively. Further, under water-free conditions hydrolytic enzymes can accept alternative nucleophiles to catalyze reactions such as transesterifications. An important industrial example of this is the lipase-catalyzed transesterification of triglycerides to obtain fats with a desired melting point [18]. 

Naik S, Basu A, Saikia R, et al. 2010. Lipases for use in industrial biocatalysis specificity of selected structural groups of lipases. J Mol Cat B Enzym 65 18-23. 

The most commonly used hydrolase for biocatalysis is lipase B from C. antarctica (CAL-B). The commercial material is usually Novozym 435, which is protein immobilized noncovalently on an acrylic resin. This immobilization is suitable for use in organic solvents, but in water, the lipase desorbs from the support. CAL-B is a recombinant protein from the yeast C. antarctica produced in a strain of the fungus Aspergillus oryzae [5]. CAL-B shows little or no interfacial activation and hydrolyzes long chain triglycerides only slowly. For this reason, it may be better classified as an esterase It shows high activity and enantioselertivity toward a... 

It was reported that PEGylated lipase entrapped in PVA cryogel could be conveniently used in organic solvent biocatalysis [279], This method for enzyme immobilization is more convenient in comparison to other types of immobilization that take advantage of enzyme covalent linkage to insoluble matrix, since the chemical step which is time consuming and harmful to enzyme activity is avoided. The application of this catalytic system to the hydrolysis of acetoxycoumarins demonstrated the feasibility of proposed method in the hydrolysis products of pharmaceutical interest and to obtain regioselective enrichment of one of the two monodeacetylated derivatives. 

A major cause of suboptimal activity in organic solvent results from the removal of essential water during enzyme dehydration. All enzymes require some water in order to retain activity through the provision of conformational flexibihty. Particularly in the case of lipases, the amount of water can be so low that it appears that none is required. For example, following the development of suitable techniques to analyse low water concentrations, it has been reported that the lipase from Rhizomucor miehei retains 30 % of its optimum activity with as little as two or three water molecules per molecule of enzyme.Owing to the apparent absence of water in some exceptional cases, the term biocatalysis in anhydrous solvent is commonly used, although in the vast majority of cases a monolayer of water is required for optimal activity (although this is often stUl well below its solubility limit in water-immiscible solvent). ... 

An interesting example of biocatalysis and chemical catalysis is the synthesis of a derivative of y-aminobutyric acid (GABA) that is an inhibitor for the treatment of neuropathic pain and epilepsy (Scheme 10.4). The key intermediate is a racemic mixture of cis- and trons-diastereoisomer esters obtained by a hydrogenation following a Horner-Emmons reaction. The enzymatic hydrolysis of both diaste-reoisomers, catalyzed by Candida antarctica lipase type B (CALB), yields the corresponding acid intermediate of the GABA derivative. It is of note that both cis- and trans-diastereoisomers of the desired enantiomer of the acid intermediate can be converted into the final product in the downstream chemistry [10]. 

Although many biochemical reactions take place in the bulk aqueous phase, there are several, catalyzed by hydroxynitrile lyases, where only the enzyme molecules close to the interface are involved in the reaction, unlike those enzyme molecules that remain idly suspended in the bulk aqueous phase [6, 50, 51]. This mechanism has no relation to the interfacial activation mechanism typical of lipases and phospholipases. Promoting biocatalysis in the interface may prove fruitful, particularly if substrates are dissolved in both aqueous phases, provided that interfacial stress is minimized. This approach was put into practice recently for the enzymatic epoxidation of styrene [52]. By binding the enzyme to the interface through conjugation of chloroperoxidase with polystyrene, a platform that protected the enzyme from interfacial stress and minimized product hydrolysis was obtained. It also allowed a significant increase in productivity, as compared to the use of free enzyme, and simultaneously allowed continuous feeding, which further enhanced productivity. 

Bioreactions. The use of supercritical fluids, and in particular C02, as a reaction media for enzymatic catalysis is growing. High diffusivities, low surface tensions, solubility control, low toxicity, and minimal problems with solvent residues all make SCFs attractive. In addition, other advantages for using enzymes in SCFs instead of water include reactions where water is a product, which can be driven to completion increased solubilities of hydrophobic materials increased biomolecular thermostability and the potential to integrate both the reaction and separation bioprocesses into one step (98). There have been a number of biocatalysis reactions in SCFs reported (99—101). The use of lipases shows perhaps the most commercial promise, but there are a number of issues remaining unresolved, such as solvent—enzyme interactions and the influence of the reaction environment. A potential area for increased research is the synthesis of monodisperse biopolymers in supercritical fluids (102). 

Thus the use and practice of biocatalysis at full scale has waxed and waned over the years. In the past, one factor limiting the use of biocatalysis has been the availability of a variety of enzymes and the time taken to refine/evolve enzymes for specific industrial apphcations. Hydrolytic enzymes such as lipases and proteases designed for other industrial uses such as detergents and food processing have always been available in bulk, and indeed used by process chemists. 

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