Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase

Abstract

This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance.

A holistic carrier-bound immobilization approach for unspecific peroxygenase

“…When comparing their results with the ones shown before ( Table 1 ) a similar behavior can be noted: the loss of enzyme performance (in both cases around 20-fold) is well-balanced by the increase in the enzyme stability. Recently, the same enzyme was chosen by Carballares and coworkers to investigate the effect of the pH in ion exchange immobilization procedures on cationic supports; after an in-depth analysis, different activity assays proved that the best results are achieved with MANAE agarose ( Carballares et al, 2021 ). As it also emerges from the experiments conducted (see Table 2 ), high immobilization yield value can be achieved via ionic immobilization.…”

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

“…5C). In the case of ion exchange, the use of an optimized support coated with an ionic polymeric bed has proved to be a way to make the enzyme adsorption stronger, reducing release 108,193,211,247 (Fig. 5C).…”

“…The actual situation is that an improperly designed immobilized enzyme biocatalyst may have a far worst performance than the soluble enzyme, including lower activity and stability. 200,211,212 And it may even be that retention of the immobilized enzyme under the required operational conditions is not achieved, as will be discussed later.…”

Broadening the Biocatalytic Toolbox—Screening and Expression of New Unspecific Peroxygenases

“…Nonetheless, the results presented here increase the toolbox of UPOs available for genetic engineering and heterologous expression—a general requirement for the industrial application of this enzyme type. The described expression of new UPOs, the (evolutionary) optimization of the enzymes [ 6 , 9 , 24 , 29 , 30 , 31 ], as well as process-engineering approaches [ 32 , 33 , 34 , 35 , 36 , 37 ] will certainly make UPOs a key player in technical oxy-functionalization in the future.…”