Manipulation of nanofiber-based β-galactosidase nanoenvironment for enhancement of galacto-oligosaccharide production

Abstract

The nanoenvironment of nanobiocatalysts, such as local hydrophobicity, pH and charge density, plays a significant role in optimizing the enzymatic selectivity and specificity. In this study, Kluyveromyces lactis β-galactosidase (Gal) was assembled onto polystyrene nanofibers (PSNFs) to form PSNF-Gal nanobiocatalysts. We proposed that local hydrophobicity on the nanofiber surface could expel water molecules so that the transgalactosylation would be preferable over hydrolysis during the bioconversion of lactose, thus improve the galacto-oligosaccharides (GOS) yield. PSNFs were fabricated by electro-spinning and the operational parameters were optimized to obtain the nanofibers with uniform size and ordered alignment. The resulting nanofibers were functionalized for enzyme immobilization through a chemical oxidation method. The functionalized PSNF improved the enzyme adsorption capacity up to 3100 mg/g nanofiber as well as enhanced the enzyme stability with 80% of its original activity. Importantly, the functionalized PSNF-Gal significantly improved the GOS yield and the production rate was up to 110 g/l/h in comparison with 37 g/l/h by free β-galactosidase. Our research findings demonstrate that the localized nanoenvironment of the PSNF-Gal nanobiocatalysts favour transgalactosylation over hydrolysis in lactose bioconversion.

Interfacial Biocatalytic Performance of Nanofiber-Supported β-Galactosidase for Production of Galacto-Oligosaccharides

“…It can be concluded that the interface from the nanofiber and the newly developed PSNF-Gal recirculating column reactor provide favourable nanoenvironments that maximize the biocatalytic performance. More importantly, comparing to static feed as reported in our previous study [7], the process in the recirculating column bioreactor enhances the lactose bioconversion from 40% to 88% and the GOS yield from 110 to 160 g/L. The superiority of the disc reactor in this study was further demonstrated by its higher yield per area than that in the spiral reactor.…”

“…For our nanofiber/β-galactosidase hybrids, although minor cracks are noticed on 2 h treated nanofibers, all enzyme-loading nanofibers have a nearly identical thickness, which means that a thin layer of enzyme coating uniformly covers the PSNF surface ( Figure 1e,f at 20 µm and 50 µm magnification, respectively). This thin layer is formed by the enzyme molecule interaction with functional groups distributed on the nanofiber surface that is characterized by our previous reports [7,26]. A similar layer of homogenous antibody coating was recently reported on electrospun polyethersulfone nanofibrous membrane due to hydrophobic interactions [28].…”

“…Nanomaterial carriers allow the creation of a nanoenvironment that favours desirable biochemical kinetics and selectivity of enzyme molecules for maximal reaction efficiencies. We have reported that incorporation of β-galactosidase on functionalized polystyrene nanofiber can provide a nanoenvironment that favours the enzymatic bioreaction to maximize galacto-oligosaccharide production [7]. Functionalized nanocarriers enable assembling enzyme in an ordered structure besides providing a high enzyme payload attributed by its large surface area [8].…”

“…Recent studies reported that enzymes could be immobilized on various nanofibers made from cellulose polymers [15], polystyrene [7], poly(ε-caprolactone) [16], polyaniline [17], chitosan [18], polyethylene oxide [19], polylactic acid [20], and polyacrylonitrile [21]. Nanofiber is fabricated using an electrospinning device, and the size of fibrous mats ranges from nanometers to a few micrometres.…”

Development of a thiol‐ene based screening platform for enzyme immobilization demonstrated using horseradish peroxidase

“…For this reason different approaches have been investigated to achieve surface modifications, which can lead to improved biocatalytic activity of immobilized enzymes. Surface functionalizations with charged, hydrophilic, or hydrophobic moieties as well as functional groups, to which enzymes can bind covalently, have been investigated. Direct attachment to the surface via covalent binding leads to structurally confined and rigid immobilized enzymes with strong interactions between the surface and the enzyme .…”