Fructosyltransferases a large proportion of non-catalytic mass. This

Fructosyltransferases
(EC 2.4.1.9) are enzymes that catalyse the transfer of fructose units from a
sucrose molecule to other in the process yielding higher fructooligosaccharide
(FOS) units (Maiorano et al. 2009).
Studies in industrial enzymology
has resulted in producing large-scale FOS by enzymatic processes through the
action of fructosyltransferases and or b-fructofuranosdases on sucrose
(Flores-Maltos et al. 2014).  Industrially, FOS production can be
classified under two broad categories namely the batch system, where the free
enzyme is used and a continuous system, in which immobilized cells and or
enzymes are used. Enzyme immobilization offers great potentials mostly in
bioreactors in which there is the possibility of high-enzyme load. This leads
to high volumetric productivities and a finite control on the extension of the
reaction itself and a simplification of the downstream process. Moreso, this
immobilization process offers instances where the biocatalysts are recovered
easily and ultimately re-used (Fernandez et al. 2005). The advantage enzyme immobilization offers are numerous as
there is tendency for the prevention of enzyme denaturation and substrate
inhibition. This is often useful when the process for immobilization is
automated (Sheldon, 2007; Fernandes, 2010).

Traditionally, the technique of enzyme
immobilization is carried out using a myriad of methods namely entrapment,
binding to a solid carrier and micro-encapsulation. This immobilization technique
is referred to as the carrier-type immobilization and it inevitably leads to
dilution of catalytic activity resulting from the introduction of a large
proportion of non-catalytic mass. This results in lower volumetric yield,
space-time yields and lower catalyst productivities (Cao et al. 2000). The other type(s) of immobilization
where there is no need for binding to a carrier is broadly termed cross-linked
enzyme aggregates (CLEA). Immobilization via cross-linking of enzyme
molecules with a bifunctional cross-linking agent is a carrier-free method and
the resulting biocatalyst essentially comprises of 100% active enzyme. Protein
cross-linking, through the reaction of glutaraldehyde with reactive amino
groups on the protein surface had been reported (Cao et al. 2003).  Cross-linked enzyme aggregates (CLEA) are
obtained from non-purified precipitated enzymes forming solid particles that
remain together through non-covalently binding (Tischer and Kasche et al. 1999). These CLEA remain
permanently insoluble after covalent binding with a cross-linking agent such as
glutaraldehyde. In a typical reaction model, it is known that CLEA exhibit
greater than 10-fold activity than their corresponding biocatalysts immobilized
using a carrier. Comparatively, the cross-linking agent, glutaraldehyde, has a
negligible molecular weight compared to the protein of interest to be
immobilized, hence, the resulting immobilized biocatalyst is perhaps 100%
target protein. In addition, CLEA is a heterogenous catalyst and hence it is
easily recovered from the reaction milieu by just low speed centrifugation (Par
et al. 2010). Also, CLEAs offer an
avenue whereby the proteins catalytic features combine readily with the
mechanical behaviour of industrially used biocatalyst.

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The advantages offered by the immobilization
of this FOS-producing enzyme using CLEA are numerous and they include pH and
thermal stability compared to the free enzyme, easy separation of products from
biocatalysts and a condition in which a continuous process can be
developed.  Overall, an increased
operational stability is envisaged. For the large-scale production of FOS,
FOS-producing enzymes has been immobilized on porous glass, porous silica
(Hayashi et al. 1993), ion-exchange
resins (Yun and Song, 1996), gluten (Chien et al. 2001), polymethacrylate (Ghazi et al. 2005), macroporous beads (Tanriseven and Aslan, 2005), calcium
alginate (Jung et al. 2011)

 The use of fructosyltransferase CLEA’s in the
synthesis and large-scale production of fructooligosaccharides has not been
fully exploited. The only recent report on the synthesis of
fructooligosaccharides involved the use of levansucrase from Bacillus subtilis (Ortiz-Soto et al. 2009). This implies that, the possibility
of the synthesis of fructooligosaccharides by using carrier-free immobilized
fructosyltransferase has not been fully exploited.

This study investigated the preparation of cross-linked
fructosyltransferase aggregates from an improved mutant strain of Aureobasidium pullulans NAC8 (Ademakinwa
and Agboola, 2016; Ademakinwa et al.
2017), developed a mathematical model for industrial scale up by optimizing the
conditions using statistical techniques and characterised the cross-linked
enzyme aggregates using microscopy and scanning electron microscopy energy
dispersive x-ray (SEM-EDAX). The structural changes that occurred after
crosslinking with glutaraldehyde were investigated using peak fitting analysis
of the amide I band of the FTIR spectra data. The re-usability of the CLEA was
also investigated over several reaction cycles. The reaction products of the
CLEA biocatalysis of sucrose were purified using activated charcoal and sugars
quantified using multivariate data analysis of the FTIR spectra data. Further
investigations into the use of the fructooligosaccharides as prebiotics were
investigated under anaerobic conditions with Lactobacillus sp as the choice probiotic strain.