Biocatalysis enzyme immobilization

Chapters are also included on yeast-mediated stereoselective biocatalysis, stereoselective synthesis of steroids, chemo-enzymatic synthesis of enantiopure arylpropionic acids, supercritical carbon dioxide as a solvent in enzyme catalysis, state-of-the-art techniques in enzyme immobilization, biocatalysis by polyethylene glycol-modified enzymes, and enzymatic deprotection techniques in organic synthesis. 

Macroporous substrates with interconnected voids can be used as platforms for biomacromolecule separation and enzyme immobilization. These assemblies are likely to find application in biocatalysis and bioassays. The inorganic framework can provide a robust substrate, while their large and abundant pores allow the transportation of biomolecules. The availability of various morphologies for macroporous materials provides another level of control over the function of the hybrids. 

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. 

Because enzymes are insoluble in organic solvent, mass-transfer limitations apply as with any heterogeneous catalyst. Water-soluble enzymes (which represent the majority of enzymes currently used in biocatalysis) have hydrophilic surfaces and so tend to form aggregates or stick to reaction vessel walls rather than form the fine dispersions that are required for optimum efficiency. This can be overcome by enzyme immobilization, as discussed in Section 1.5. 

Pharmaceutical production generally uses multipurpose equipment, and so entrapment behind a membrane would require significant capital expenditure on specialized equipment. In spite of this, the use of membrane reactors in biocatalysis represents an efficient method of enzyme immobilization, given the large molecular weight difference between enzymes (10-150 kDa) and most substrates (300-500 Da). The reader is referred to some recent reviews on the topic. 

Another example in which biocatalysis is combined with analysis is the system reported by Honda et al. [436]. A microreaction system, consisting of an enzyme-immobilized microreactor, for optical resolution of racemic amino acids was devel-... 

Despite the limitations, the great success of enzyme immobilization in diagnostics, pharmaceutical, food and chemical industries is undeniable12, 3. The decision whether one should use a soluble enzyme preparation or an immobilized enzyme does not have a universal solution and can be decided only on a case by case basis. Ordinarily, if the cost of an enzyme represents a significant portion of the overall cost or if isolation of the final product is complicated by the presence of the soluble protein, the cost of immobilization can be offset by the gains in productivity and improved product quality. The intent of this section is to describe, in general terms with illustrative examples, the features and considerations of these broad classes of enzyme immobilization as they impact their application to biocatalysis. Detailed experimental protocols are available in the original literature and exemplary protocols for these methods are offered in many excellent reviews and texts 14L... 

Keywords Biocatalysis Immobilized enzymes Immobilized biocatalyst ... 

Electrocatalysis is an important application of electron-induced processes. In electrocatalysis the catalyst has at first to accept chaige(s) from the electrode, and thereafter catalysis can take place. Enzyme-immobilized electrodes are tj ical examples used for various biosensors as well as for investigation of fundamental biocatalysis. The enzymatic active center is often located inside a protein molecule, so that mediation of charges from the electrode surface to the active center is important. Viologens, metallocenes and other metal complexes have been used as such mediators. 

We here report on the design, synthesis, characterization, and applications of template-synthesized nanotube membranes. We will first briefly review the synthesis of the template-synthesized nanotube membranes. Some details of differential-surface chemistry on nanotnbes and nanotubes for bioextraction and biocatalysis will be presented. We discuss in details the drug detoxification nsing functionalized nanotnbes and apoenzyme-immobilized, enzyme-immobilized, and antibody and DNA-immobilized nanotubes for enantiomeric and DNA separations, biocatalysis, and bioextractions. ... 

Membranes represent good supports for enzyme immobilization because they enable the integration of biocatalysis and separation. Often, the available commercial membranes require modifications to make them suitable for enzyme immobilization. Different immobilization techniques can be used on such suitable membranes, depending on many factors mainly related to enzymes and membrane properties and bioprocess performances. 

Examples of immobilized biocatalysts. Enzyme immobilization clearly imparts many benefits to biocatalysts. A primary advantage is the opportunity to tailor an immobilization matrix for a specific application, use, or set of conditions. Such a process has been, and continues to be, undertaken for a host of biocatalysts and a broad array of applications. The authors describe herein one instance of adapting immobilization techniques and chemistry to a specific application of biocatalysis. Although the specifics and details of this particular effort may not be applicable to every and all uses of enzyme catalysis, some detail is provided to convey to the reader those issues that need to be considered when one attempts to immobilize enzymes for a particular task. 

Roessl U, Nahalka J, Nidetzky B. Carrier-free immobilized enzymes for biocatalysis. Biotechnol Lett 2010 32 341-50. 

Another category of enzymatic transformations in multiphase systems is enzymes immobilized on the reactor wall as presented in Table 10.4. Enzymes are advantageously used in immobilized form because this strategy allows for increased volumetric productivity and improves stability. Continuous mode of operation is employed in these systems. The approaches commonly used for immobilization in conventional multiphase biocatalysis can also be employed in microreactors such as covalent methods, cross-linked enzyme aggregates (CLEA), and adsorption methods. The experimental setups can either be chip-type reactors with activated charmel surface walls where enzyme binds, or enzyme immobilized monolith reactors, where a support is packed inside a capillary tube. 

Zhou Z, Hartmann M (2012) Recent progress in biocatalysis with enzymes immobilized on mesoporous hosts. Top Catal 55 1081-1100... 

The final common method for employing enzymes in biocatalysis involves their prior immobilization on a solid support by covalent or noncovalent bonds. In many cases, this modification deaeases their catalytic effidendes, and it can also introduce mass transport limitations. Minimizing both of these issues by optimizing both the nature of the solid support and the enzyme/support connection is an area of intensive current research. Some enzymes are available commerdally in immobilized form. The best-known example is Novoz5mi 435, which is a trade name for Candida antarctica lipase B adsorbed on a polymeric support. 

Fig. 6.67 Classification of the methods for enzyme immobilization. (From A lllanes, R. Fernandez-Lafuente, J.M. Guisan, L Wilson, Heterogeneous enzyme kinetics, in A lllanes (Ed.), Enzyme Biocatalysis, Springer, 2008, pp. 155—203. Copyright 2008 Springer).

Some of the industrial biocatalysts are nitrile hydralase (Nitto Chemicals), which has a productivity of 50 g acrylamide per litre per hour penicillin G amidase (Smith Kline Beechem and others), which has a productivity of 1 - 2 tonnes 6-APA per kg of the immobilized enzyme glucose isomerase (Novo Nordisk, etc.), which has a productivity of 20 tonnes of high fmctose syrup per kg of immobilized enzyme (Cheetham, 1998). Wandrey et al. (2000) have given an account of industrial biocatalysis past, present, and future. It appears that more than 100 different biotransformations are carried out in industry. In the case of isolated enzymes the cost of enzyme is expected to drop due to an efficient production with genetically engineered microorganisms or higher cells. Rozzell (1999) has discussed myths and realities... 

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