Enzyme preparation whole cells

The results presented in Tables 3 and 4 deserve some comments. First, a variety of enzymes, including whole-cell preparations, proved suitable for the resolution of different hydroxyalkanephosphorus compounds, giving both unreacted substrates and the products of the enzymatic transformation in good yields and, in some cases, even with full stereoselectivity. Application of both methodologies, acylation of hydroxy substrates rac-41 and rac-43 or the reverse (hydrolysis of the acylated substrates rac-42 and rac-44), enables one to obtain each desired enantiomer of the product. This turned out to be particularly important in those cases when a chemical transformation OH OAc or reverse was difficult to perform. As an example, our work is shown in Scheme 3. In this case, chemical hydrolysis of the acetyl derivative 46 proved difficult due to some side reactions and therefore an enzymatic hydrolysis, using the same enzyme as that in the acylation reaction, was applied. Not only did this provide access to the desired hydroxy derivative 45 but it also allowed to improve its enantiomeric excess. In this way. 

H. G. Davies, R. H. Green, D. R. Kelly and S. M. Roberts, Biotransformations in Preparative Organic Chemistry The Use of Isolated Enzymes and Whole Cell Systems, 1989... 

Roberts SM, Preparative biotransformations the employment of enzymes and whole-cells in synthetic organic chemistry, J. Chem. Soc., Perkin Trans., 1 157-169, 1998. 

Another favorable aspect of stirred batch reactors is the fact that they are compatible with most forms of a biocatalyst. The biocatalyst may be soluble, immobilized, or a whole-cell preparation in the latter case a bioconversion might be performed in the same vessel used to culture the organism. Recovery of the biocatalyst is sometimes possible, typically when the enzyme is immobilized or confined within a semi-permeable membrane. The latter configuration is often referred to as a membrane reactor. An example is the hollow fiber reactor where enzymes or whole cells are partitioned within permeable fibers that allow the passage of substrates and products but retain the catalyst. A hollow-fiber reactor can be operated in conjunction with the stirred tank and operated in batch or... 

The most prevalent catalysts are homogeneous metal complexes mostly with chiral diphosphine ligands, isolated enzymes and whole cell preparations, whereas... 

Many applications rely on enzymes being retained by membranes, aggregated by cross-linking, or immobilized by encapsulation. These techniques are often simple and inexpensive, but typically also generate a poorly defined immobilized enzyme. The immobilization can involve isolated enzymes or whole cell preparations. Sweetzyme IT, an immobilized glucose isomerase produced by Novozymes is an example of the latter, in which the cells are cross-linked by glutaraldehyde (GA) and extruded to produce dry, solid particles [32]. 

The term encapsulation has been used to distinguish entrapment preparations in which the biocatalyst environment is comparable to that of the bulk phase and where there is no covalent attachment of the protein to the containment medium (Fig. 6-1 D)[21J. Enzymes or whole cells may be encapsulated within the interior of a microscopic semi-permeable membranes (microencapsulation) or within the interior of macroscopic hollow-fiber membranes. Liposome encapsulation, a common microscopic encapsulation technique, involves the containment of an enzyme within the interior of a spherical surfactant bilayer, usually based on a phospholipid such as lecithin. The dimensions and shape of the liposome are variable and may consist of multiple amphiphile layers. Processes in which microscopic compart-mentalization (cf. living cells) such as multienzyme systems, charge transfer systems, or processes that require a gradient in concentration have employed liposome encapsulation. This method of immobilization is also commonly used for the delivery of therapeutic proteins. 

Davies, H.G., Green, R.H., Kelly, D.R., and Roberts, S.M. (eds) Biotransformations in Preparative Organic Chemistry the Use of Isolated Enzymes and Whole Cell Systems in Synthesis. Academic Press London, 1989. 

Biocatalysis is a key route to both natural and non-natural polysaccharide structures. Research in this area is particularly rich and generally involves at least one of the following three synthetic approaches 1) isolated enzyme, 2) whole-cell, and 3) some combination of chemical and enzymatic catalysts (i.e. chemoenzymatic methods) (87-90). Two elegant examples that used cell-fi-ee enzymatic catalysts were described by Makino and Kobayashi (25) and van der Vlist and Loos (27). Indeed, for many years, Kobayashi has pioneered the use of glycosidic hydrolases as catalysts for polymerizations to prepare polysaccharides (88,91). In their paper, Makino and Kobayashi (25) made new monomers and synthesized unnatural hybrid polysaccharides with regio- and stereochemical-control. Van der Vlist and Loos (27) made use of tandem reactions catalyzed by two different enzymes in order to prepare branched amylose. One enzyme catalyzed the synthesis of linear structures (amylose) where the second enzyme introduced branches. In this way, artificial starch can be prepared with controlled quantities of branched regions. 

Biotransformations involve the use of isolated enzymes or intact microbial cells for the highly selective transformations of organic molecules for cutting edge preparative organic syntheses. Ideally, the use of biocatalysts (i.e. enzymes or whole cells) in the industrial preparation of useful compounds would be economical and environmentally friendly. Readers wishing a more thorough discussion on this topic are referred to a recently published article [85]. 

The activity of bacitracin production was the highest when whole cells were immobilized in the gel prepared with 5 % total acrylamide (95 % of acrylamide monomer and 5 % BIS). On the other hand, no effect of BIS content on production of bacitracin was observed. However, the best productivity of bacitracin by immobilized whole cells was only 20-25 % of that by washed cells. These results suggest that the lower rate of bacitracin production is mainly caused by the inactivation of enzymes in whole cells with polymerization reagents (especially AA and APS) and may be partly due to hindered diffusion of the substrates and/or products through the gel. 

Richter ER (1993) Biosensors applications for dairy food industry. J Dairy Sci 76(10) 3114-3117 Roberts S (1998) Preparative biotransformations the employment of enzymes and whole-cells in synthetic organic chemistry. J Chem Soc Perkin Trans 1 157-170 Roberts S (2000) Preparative biotransformations. J Chem Soc Perkin Trans 1 611-633 Roig M, Kennedy J (1992) Perspectives of chemical modification of enzymes. Critic Rev Biotechnol 12 391 12... 

DSM has developed an industrial process for the preparation of (D)- and (L)-amino acids, which is based on the enantioselective hydrolysis of racemic amino acid amides using amidases, for example from Pseudomonasputida. It is often not necessary to isolate the pure enzyme standardised whole-cell or crude enzyme preparations can be used instead. It is noteworthy that in some cases the enzyme activity can be increased up to ten-fold by the addition of magnesium salts. The enzymes accommodate a broad spectrmn of substrates with considerable selectivity. Typical products are (L)-phenylalanine and (L)-homophenyl-alanine. 

There are many different ways to exploit enzymes and micro-organisms so as to undertake a diverse range of biotransformations. By having the capability of carrying out isolated-enzyme and whole-cell bioconversions, the whole of this range is available to the experimentalist. Such bioconversions can be used to prepare new intermediates for the synthesis of important and valuable fine chemicals. These aspects will be dealt with in the next chapters. At this time (February, 1994), research and development work is split approximately equally into three categories ... 

Davies HG, Green RH, Kelly DR, Roberts SM (1989) Biotransformations in preparative orgaitic chemistry the use of isolated enzymes and whole-cell systems in synthesis. Academic, London, pp 1-23... 

Immobilization is the method of cultivation of microorganisms that allows a repeated use of biocatalysts (be it enzyme or whole cells), creating prerequisites for the production of valuable products in an automated continuous mode. The most considerable problem in using biocatalysts is related to mass transfer. In aerobic systems, low solubility of oxygen in carriers, especially in some gels and polymers, can decrease the effectiveness of biocatalyst action. In this respect, propionic acid bacteria, which do not require aeration, show certain advantages over aerobic cultures. At present, about eight different processes that use immobilized enzymes and cells have found industrial applications. These are mainly one-or two-step processes used in the manufacture of foods and pharmaceutical preparations (Vorobjeva et al, 1978). An essential characteristic of a biocatalyst is productivity. 

In Chapters 13 and 14 of this book the applications of conventional chemical catalysts were described. The use of enzymes or whole cells as catalysts for chemical transformations is well known. They can bring about various reactions at ambient temperature and pressure and afford high reaction velocities. In fact, enzymatic reaction sequences may be designed to give the ideal efficiency embodied in the second law of thermodynamics. Thus, hundreds of compounds that are very difficult to prepare by purely chemical methods may be obtained quite readily and economically with the help of enzymes. Until recently, most laboratory investigations and manufacturing processes employed soluble enzymes in dilute aqueous solutions. Before use, the required enzyme must be obtained from biological sources as a concentrated extract. It is not uncommon for a particular type of cell to contain many proteins in addition to the one desired. Therefore, the purification and concentration of enzymes in preparation for use is a very cumbersome process. When used in solution, enzyme catalysts are invariably lost after each batch operation. The use of immobilized enzymes and whole cells has been proposed as a means that could eliminate such losses and preserve hard won stocks of specialized enzymes. 

As mentioned already, the use of organic solvents for the HNL-catalyzed addition of HCN to carbonyl compounds was decisive for many investigations concerning optically active cyanohydrins. Several variations for the practical performance of the HNL-c alyzed preparation of (R)- and (S)-cyanohydrms have been developed in recent years. Instead of pure organic solvents, a biphasic system (water/organic solvent) can be used for the reaction whereby HCN can be prepared in situ from sodium cyanide and acetic acid [20] or by transcyanation with acetone cyanohydrin [21]. It is possible to replace isolated enzymes by whole cells, e.g., by almond and apple meal instead of PaHNL or by Sorghum shoots instead of SbHNL [21,22]. 

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