Carrier-free biocatalysts

Cao L, van Rantwijk F, Sheldon RA (2000) Cross-linked enzymes aggregates a simple and effective method for the immobilization of penicillin acylase. Org Lett 2 1361-1364 Cao L, van Langen LM, van Rantwijk F et al. (2001) Cross-linked aggregates of penicillin acylase robust biocatalysts for the synthesis of P-lactam antibiotics. 1 Mol Catal B Enzym 11 665-670 Cao L, van Langen LM, Sheldon RA (2003) Immobilised enzymes carrier-bound or carrier-free Curr Opin Biotechnol 14 1-8... 

Immobilization can help to increase the operational stability of a biocatalyst, which is a particularly important aspect of preparative and industrial-scale biotransformations. The following immobilization techniques have been reported for EHs carrier-free approaches, adsorption or covalent attachment of the enzyme to a support, whole-cell encapsulation and entrapment, and immobilized metal ion affinity binding via genetically engineered His tags. The latter technology can be used for both one-step extraction from cellular crude extracts and facile immobilization of the recombinant EH. 

Enzymes are an attractive tool in asymmetric catalysis and efficiently complement traditional chemical methods [32,33]. The use of biocatalysts makes it possible to carry out chemical transformations without the need for laborious protection and deprotection steps [34]. Immobilized enzymes are preferred over free enzymes in solution, due to the possibility of repeated use, higher resistance to denaturing effects, and easy separation. The use of a structured support material could be an interesting alternative for conventional particulate enzyme carriers. When optimizing the use of immobilized enzymes, the immobilization method chosen is a very important factor to consider [35]. In this study, a reaction in an organic medium is considered most enzymes do not readily dissolve in organic media, and the enzyme will not detach from the support. This makes physical adsorption a very suitable technique to prepare a biocatalyst for use in an organic medium... 

Nano/microporous cellulose (NMC) prepared after removal of lignin from wood cellulose was found suitable for the development of cold pasteurization" processes acting as a biofilter for cell removal. It was also used successfully as biocatalyst in food fermentations acting as both cell immobilization carrier and as promoter of biochemical reactions, even at extremely low temperatures. The cumulative surface area of the NMC pores was found to be 0.8 to 0.89 m g" as indicated by porosimetry analysis. This surface is relatively small compared with other porous materials such as y-alumina however, using a natural organic material is attractive from the point of view that it is safer for bioprocess applications and is better accepted by consumers. The NMC/immobilized yeast biocatalyst increased the fermentation rate and was more effective at lower temperatures compared with free cells. Furthermore, the activation energy E, of fermentation was found to be 28% lower than that of free cells, indicating that it is an excellent material to promote the catalytic action of cells for alcoholic fermentation. 

Immobilized enzymes are defined as enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, which can be used repeatedly and continuously. This definition is applicable to the enzymes as well as aU types of biocatalysts such as cellular organelles, microbial cells, plant cells, and animal cells. In some cases, these biocatalysts are bound to or within insoluble supporting materials (carriers) by chemical or physical binding. In other cases, biocatalysts are free, but confined to limited domains or spaces of supporting materials (entrapment). 

An ample number of in vivo biochemical processes rely on redox reactions, as such reactions stay at the heart of many metabolic pathways, such as cellular respiration and photosynthesis. For this purpose Nature has developed a broad portfolio of enzymes (biocatalysts) that are able to ubiquitously catalyze those reactions in a highly efficient and selective way, dealing with the complexity of molecules and metabolism, and thus adapting life to different and challenging environments. In some of these cases, the problematic of the electron transport has been sorted out by introducing organic cofactors - as electron carriers - whereas in other cases, other smart alternatives have been evolved to set up natural cofactor-free redox processes [1.2]. 

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