Glyoxylate Immobilization

Table 12 Results of reactions of ethyl glyoxylate with different alkenes, catalyzed by several bis(oxazoline)-copper complexes immobilized on Y zeolite...

A spectacular example of stability enhancement through immobilization has been reported for the enzyme catechol-2,3-dioxygenase.27 This enzyme, isolated from the thermophilic bacterium Bacillus stearothermophilus, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde (which can be monitored by absorbance at 375 nm). The soluble enzyme exhibits maximal activity at 50 °C, but following immobilization on glyoxyl agarose beads with a borohydride reduction step, the optimum reaction temperature shifted to 70 °C. At a total protein concentration of 0.010 mg/mL and a temperature of 55 °C, the half-life of the soluble enzyme was 0.08 h, while the enzyme-modified beads had a half-life of 68 h. This represents a 750-fold enhancement of stability that has been attributed to the prevention of subunit dissociation upon immobilization. 

Immobilization of glyoxylate represents a versatile means of gaining access to many biologically relevant compounds. Thus, reaction of potassium monoethyl tartrate with chloromethylated resin and subsequent oxidative cleavage of the resulting supported tartrate (549) with H5IO6 smoothly afforded the polymer-supported glyoxylate 550 in its hydrated form [397] (Scheme 113). 

D-p-Hydroxyphenyl glycine is a key raw material for the semisynthetic penicillins such as ampidllin and amoxycillin. It is also used in photographic developers. Racemic hydantoins are synthesized starting from phenol derivatives, glyoxylic acid and urea via the Mannich condensation (Fig. 19-28). The D-specific hydantoinase is applied as immobilized whole cells in a batch reactor. The unreacted L-hydantoins are readily racemized under the alkaline conditions (pH 8) of enzymatic hydrolysis, yielding quantitative conversion. This process enables the stereospecific preparation of various amino acids, such as L-tryptophane, L-phenylalanine, D-valine, D-alanine... 

By ionic adsorption Amino-glyoxyl-agarose (MANAE agarose), used as hydrophilic support, was prepared as described by Femandez-Lafiiente et al. [22], Standard immobilization procedure consisted of the addition of 1 g of the support to 10 ml of enzymatic solution containing Triton X-100 0.1% in phosphate buffer 5 mM pH 7. The mixture was shaken at 25°C and 250 rpm for 2 h, washed with distilled water and stored at 4°C. Periodically, samples of the suspensions and the supernatants were withdrawn and enzymatic activity was measured using pNPB as substrate as described above. 

The YLL lipase immobilized onto octadecyl-sepabeads was the most stable one in all conditions, presenting half-life ten times higher than the soluble preparation at pH 7.0. Palomo et al. [8] demonstrated that the interfacial adsorption on a hydrophobic resin (octadecyl-sepabeads) was the best immobilization technique for B. thermocatenulatus lipase. This immobilized preparation, retaining 100% of the initial activity at high temperature, is even better than the covalent immobilization by multipoint interaction on glyoxyl support, where the lipase retained only 80% activity. 

The results for the immobilization of CGTase from Thermoanaerobacter sp. in glyoxyl-silica, hydrophobic adsorption in octadecyl-sepabeads, and encapsulation into the sol-gel matrix are shown at Table 1. 

The activity recovery of the CGTase adsorbed in octadecyl-sepabeads was higher than that obtained with covalent attachment in glyoxyl-silica, but the latter support can be considered more appropriate for immobilization of this enzyme because it was weakly adsorbed into the octadecyl-sepabeads, being easily desorbed even at low ionic strength (25% desorbed with at 10 mM buffer). 

The thermal stability of an immobilized enzyme is another important factor for the selection of an immobilization method. A method that shifts the maximum cataljlic activity for a region of higher temperatures is undoubtedly preferred. Tardioli et al. [9] obtained greater thermal stability for a Thermoanaerobacter sp. CGTase covalently bound to glyoxyl-agarose the temperature range shift for the maximum activity was from 80 to 85 (for the free enzyme) to just above 90 °C (for the immobilized enzyme). 

Gonsalves LR, Femandez-Lafuente R, Guisan IM et al. (2002) The role of 6-aminopenicillanic acid on the kinetics of amoxiciUin enzymatic synthesis catalyzed by peniciUin G acylase immobilized onto glyoxyl agarose. Enzyme Microb Technol 31 464-471 Gorziglia G, Altamirano C, Conejeros R et al. (2002). Determination of kinetic parameters in the synthesis of ampicillin with immobilized penicillin acylase. Annals of the XV Chilean Congress of Chemical Engineering, Punta Arenas, October 2002, pp 107-112. 

Some results indicate that different attempts of FucA immobilization by covalent attachment provoked severe enzyme inactivation (Fessner and Walter 1997). FucA and DERA from E. coli and SHMT from Streptococcus thermophilus have been immobilized by multipoint covalent attachment to glyoxyl-agarose. Although this immobilization method had been very successful with many different enzymes (Guisdn et al. 1993), results obtained with these aldolases were dissimilar. For FucA, in spite of an immobilization yield of 80-90%, enzyme inactivation occurred during immobilization process and only 10-20% of activity was retained (Suau et al. 2005). On the other hand, SHMT immobilization yield was 100%, but the immobilized activity was lost during the sodium borohydride reduction step, probably due to the reduction of the Schiff base established between the cofactor (pyridoxal phosphate) and the aldolase. Finally, 100% of immobilization yield and 65% of retained activity in the immobilized derivative was achieved with DERA. 

Sequential batch synthesis of GOS with enzyme immobilized in glyoxyl-agarose 546 50 4.5 56 29 64... 

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