Glyoxyl-agarose

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. 

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2 h, washed with distilled water and stored at 4°C. Periodically, samples of the suspensions and the supernatants were withdrawn and enzymatic activity was measured using pNPB as substrate as described above.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 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). 

Table 4.1 Stabilization of Enzymes by Multi-Point Covalent Attachment to Glyoxyl-Agarose...

Pedroche J, Yust MM, Mateo C et al. (2007) Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports correlation between enzyme-support linkages and thermal stability. Enzyme Microb Technol 40 1160-1166... 

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. 

Illanes A, Cabrera Z, WUson L et al. (2003) Synthesis of cephalexin in ethylene glycol with glyoxyl-agarose immobUised peniciUin acylase temperature and pH optimisation. Proc... 

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... 

---