Characterization of immobilized biocatalyst

Ballesteros, A., van Beynum, G., Bomd, O. and Buchholz, K., Guidelines for the characterization of immobilized biocatalysts. Enzyme Microb. TechnoL, 1983, 5, 304-307. 

At a glance, the rapprochement between biochemistry and polymer chemistry seems to have played an important role in the methodological development of preparations for immobilized biocatalysts. A number of articles on the preparation and characterization of immobilized biocatalysts, together with their applications in a variety of fields besides synthetic chemical reactions - chemical and clinical analysis, medicine, and food processing, for example - have already been published. These results have been reviewed by many of the pioneers in this and related fields [1-20]. The technology for immobilizing enzymes and cells is believed to be relatively mature at this point. In addition, the nature of immobilized biocatalysts has become somewhat more transparent to us. The key now is to come up with new uses and new systems which can fulfill specific needs [21]. 

Immobilized enzymes are not restricted to bioanalytical applications. Increasingly they attract a huge amount of interest in industrial organic chemistry due to their excellent stereo - and enantioselectivity. Moreover, they work under mild conditions of temperature, pH and pressure. Therefore, the determination of kinetic constants is of great interest. They allow quantitative characterization of immobilized biocatalyst preparations and facilitate comparisons between different materials and procedures for biocatalyst immobilization. 

Fig. 14. Optimal procedure for the kinetic characterization of immobilized biocatalysts by flow microcalorimetry...

Buchholz K, Klein J (1987) Characterization of immobilized biocatalysts. In Mosbach K (ed) Methods in Enzymology, vol. 135. Immobilized Enzymes and Cells, Part B. Academic Press, Orlando, p 3... 

K. Buchholz, Characterization of Immobilized Biocatalysts, DECHEMA Monographic 84, VCH Weinheim, 1979. 

The refences below include review articles and research articles on specific topics, len, B.R., Charles, M. and Coughlin, R.W. (1979) Fluidized immobilized enzyme reactor for the hydrolysis of cornstarch to glucose. Biotechnol. Bioeng., 21, 689-706. ichholz, K. (1979) Characterization of immobihzed biocatalysts. Dechema-Monograph, 84, 208-223. 

Gemeiner P, Stefuca V, Welwardova-Vikartovska A (1996) Screening and design of immobilized biocatalysts through the kinetic characterization by flow microcalorimetry. In Wijfels RH, Buitelaar RM, Bucke C, Tramper J (eds) Immobilized Cells Basics and Applications. Elsevier Science BV, Amsterdam, pp 320-327 Grau C (1993), PhD-thesis, University of Hannover, Germany... 

Flow microcalorimetry, which makes many rapid and accurate measurements of the activity of immobilized biocatalysts, provides a tool for researchers that can be used to discriminate between different preparatives of immobilized biocatalysts. Table 3 shows previous experiments where the characterization of kinetic properties by flow micro calorimetry was used to compare different techniques of purified enzyme immobilization [27, 30, 31, 35] as well as the immobilization of enzymes fixed in cells [28,29,40]. More details can be found in our recent review article [41]. 

The complexity of the physical and catalytic properties of immobilized biocatalysts and the difficulty in comparison of effectiveness based on literature descriptions has led to the publication of guidelines for the characterization of immobilized bio-catalysts1351. The authors suggest that description of parameters listed in Table 6-4 should be the minimum required for characterization of an immobilized preparation. 

Biological catalysts in the form of enzymes, cells, organelles, or synzymes that are tethered to a fixed bed, polymer, or other insoluble carrier or entrapped by a semi-impermeable membrane . Immobilization often confers added stability, permits reuse of the biocatalyst, and allows the development of flow reactors. The mode of immobilization may produce distinct populations of biocatalyst, each exhibiting different activities within the same sample. The study of immobilized enzymes can also provide insights into the chemical basis of enzyme latency, a well-known phenomenon characterized by the limited availability of active enzyme as a consequence of immobilization and/or encapsulization. 

Reaction engineering helps in characterization and application of chemical and biological catalysts. Both types of catalyst can be retained in membrane reactors, resulting in a significant reduction of the product-specific catalyst consumption. The application of membrane reactors allows the use of non-immobilized biocatalysts with high volumetric productivities. Biocatalysts can also be immobilized in the aqueous phase of an aqueous-organic two-phase system. Here the choice of the enzyme-solvent combination and the process parameters are crucial for a successful application. 

Keywords Flow microcalorimetry, Immobilized biocatalysts, Reaction rate monitoring, Investigation of kinetic properties, Characterization of bioaffinity systems. 

Enzymes are characterized by unusual specific activities and remarkably high selectivities. They are effective catalysts at relatively low temperatures and ambient pressure. The primary driving force for efforts to develop immobilized forms of these biocatalysts is cost, especially when one is comparing process alternatives involving either conventional inorganic catalysts or soluble enzymes. Immobilization can permit conversion of labile enzymes into forms appropriate for use as catalysts in industrial processes—production of sweeteners, pharmaceutical intermediates, and fine chemicals—or as biosensors in analytical applications. Because of their high specificities, immobilized versions of enzymes are potentially useful in situations where it is necessary to obtain high yields of the desired product... 

The morphologic characterization of the immobilized enzyme is important to correlate the biocatalyst performance with porous structure parameters. BET analysis, which is usually based on N2 isothermal adsorption at 77 K, allows determining the solid-specific surface area, total pore volume, pore size distribution, and mean pore diameter. It is not recommended for solids with a low specific surface area (<5 m g ). Table 2 shows the specific smface area, mean pore diameter, and total pore volume determined by BET for the pure sol-gel silica matrix having TEOS as the precursor and the same matrix with the encapsulated CGTase. 

Ospina S, Lopez-Munguia A, Gonzalez R et al. (1992) Characterization and use of a penicillin acylase biocatalyst. J Chem Technol Biotechnol 53 205-214 Ospina S, Barzana E, Ramirez O et al. (1996) Effect of pH in the synthesis of ampicilfinby penicillin acylase. Enzyme Microb Technol 19 462-469 Palazzi E, Converti A (2001) Evaluation of diffusional resistances in the process of glucose isomerization to fructose by immobilized glucose isomerase. Enzyme Microb Technol 28 246-252 Pan JL, Syu MJ (2004) A thermal study on the use of immobilized penidllin G acylase in the formation of 7-amino-3-deacetoxy cephalosporanic acid from cephalosporin G. J Chem Technol Biotechnol 79(10) 1050-1056... 

Reddy V.L., Reddy P.L., Wee Y.-J. and Reddy O.V.S. Production and characterization of wine with sng-arcane piece immobilized yeast biocatalyst. Food and Bioprocess Technology 4 (1) (2011) 142-148. 

Reddy V.L., Reddy H.K.Y., Reddy P.A.L. and Reddy V.S.O. Wine production by novel yeast biocatalyst prepared by immobilization on watermelon (Citrullus vulgaris) rind pieces and characterization of volatile compounds. Process Biochemistry 43 (7) (2008) 748-752. 

The aim of this chapter is to give a detailed overview of the characterization of biocatalysts and the development of membrane bioreactors, in particular, the main aspects of biocatalyst kinetics and their immobilization/ entrapment, either within the porous membrane structure, or on its surface. Thansport models that can help to predict the behaviour of membrane bioreactors, as well as the most relevant theoretical models and operating parameters, are presented below. This data is then analysed in order to ascertain how to improve effectiveness and productivity of the membrane bioreactors. Some relevant fields of application are also discussed in order to show the potential of such systems. 

Numerous hydrolaseotalyzed KRs of various secondary alcohols were performed in continuous-flow mode (Figure 9.8 and Table 9.6). The bioimprinting effect in sol-gel immobilization of various lipases (Lipase AK, Lipase PS, CaLB, and CrL) was studied (116]. The performance of the immobilized biocatalysts were characterized by enantiomer selective acylation of various racemic secondary alcohols in two different multisubstrate systems (mix A rac-23a,c-e and mix B rac-23b and roc-23i) in batch and continuous-flow mode. The synthetic usefiilness of the best biocatalysts was demonstrated by the KR of racemic l-(thiophen-2-yl)ethanol (rac-23j) in batch and continuous-flow systems [116]. 

Various lipase immobilization methods were tested with different silica matrices, and the immobilized enzyme samples were examined by morphologic, physicochemical, and biochemical characterization methods. The results allowed correlation of the activity-coupling yield of different immobilization methods in relation to the incorporation of lipase in the silica gels and showed that the most active biocatalyst resulted from the encapsulation of commercial CRL in the presence of PEG. 

Contrary to the Fuji process, BASF described the characterization and cloning of an L-specific pantolactone hydrolase from Agrobacterium tumefa-ciens [103,104]. This enzyme exclusively opens up the undesired lactone l-1 12, providing a more direct route to d-1 12 (Scheme 35, right side). In addition, this new process is expected to be much more robust toward the competing spontaneous chemical hydrolysis, which could theoretically cause a diminished optical yield in the Fuji process. The enzymatic resolution of d/l-112 in repeated batches with membrane filtration techniques provided d-1 12 in 50% yield and with 90-95% ee. By immobilization onto Eupergit C it was possible to obtain a stable biocatalyst which was easy to use in repeated batch reactions. 

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