Evaluation of Styrene-Divinylbenzene Beads as a Support to Immobilize Lipases

Abstract

A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL ® CHP20P, has been compared to octyl-Sepharose ® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase ® Ultra, a commercial artificial phospholipase. The immobilization mechanism on both supports was similar: interfacial activation of the enzymes versus the hydrophobic surface of the supports. Immobilization rate and loading capacity is much higher using MCI GEL ® CHP20P compared to octyl-Sepharose ® (87.2 mg protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase ® Ultra). The thermal stability of all new preparations is much lower than that of the standard octyl-Sepharose ® immobilized preparations, while the opposite occurs when the inactivations were performed in the presence of organic co-solvents. Regarding the OPEN ACCESSMolecules 2014, 19 7630 hydrolytic activities, the results were strongly dependent on the substrate and pH of measurement. Octyl-Sepharose ® immobilized enzymes were more active versus p-NPB than the enzymes immobilized on MCI GEL ® CHP20P, while RML became 700-fold less active versus methyl phenylacetate. Thus, the immobilization of a lipase on this matrix needs to be empirically evaluated, since it may present very positive effects in some cases while in other cases it may have very negative ones.

Immobilization of Proteins in Poly-Styrene-Divinylbenzene Matrices: Functional Properties and Applications

“…The resulting thermal stability greatly decreased, while the stability versus organic solvents was greatly increased. The effect of the immobilization on this support on the hydrolytic activities greatly depended on the substrate and enzyme, in some cases the octyl-Sepharose lipases were 100 fold more active than the new one, in other cases the opposite were observed [96].…”

“…The situation reversed in the presence of organic solvents, where this new preparation became the most stable [94]. Very recently the use of MCI GEL CHP20P has been reported to immobilize the lipases from T. lanuginusus and R. miehei, and also the artificial chimeric phospholipase Lecitase Ultra [96]. Again, the immobilization rate and yield were higher using this support than the standard one used for comparison (in this case octyl-agarose) [86], reaching values as high as 200 mg/ml of packed wet support.…”

“…Recently, a large activity on this kind of reactions has been performed using MCI GEL CHP20P, a commercially available PSD support [94,96]. In a first report lipase B from C. antarctica, immobilized on MCI GEL CHP20P was compared with the commercial Novozym 435 as biocatalysts for the esterification of acetic acid and n-butanol [13].…”

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

“…However, this mechanism of action causes lipases to become an exception among water soluble proteins: while the surface of the closed form is fairly hydrophilic, the open form has a huge hydrophobic pocket. This permits that the lipases may suffer interfacial activation with different hydrophobic surfaces: a drop of oil (Verger, 1997) or a hydrophobic support (Bastida et al, 1998, FernandezLafuente et al, 1998, Garcia-Galan et al, 2014 (Fernandez-Lorente et al, 2008, Fernández-Lorente et al, 2007 and to purify it .…”

“…and new enzyme loaded, enabling theo reuse ofe the support. Full lipase immobilization on these supports may proceed in few minutes (even using 1 g of support/50 ml of lipase solution), with increments on enzyme activity that may become even 100 fold for some substrates while in other cases it may produce a decrease in activity (Garcia-Galan et al, 2014). Lipase stability uses tends to be increase for thousand thousand-fold factors using octyl agarose or Sepabeads decaoctyl (Palomo et al, 2002)., this This has been attributed to the importance of the stabilization of the open form of the lipases, that has a more compact and ordered structure (Peters et al, 1996).…”