Fructosyltransferases with a cross-linking agent such as glutaraldehyde.

Fructosyltransferases(EC 2.4.1.9) are enzymes that catalyse the transfer of fructose units from asucrose molecule to other in the process yielding higher fructooligosaccharide(FOS) units (Maiorano et al. 2009).

Studies in industrial enzymologyhas resulted in producing large-scale FOS by enzymatic processes through theaction of fructosyltransferases and or b-fructofuranosdases on sucrose(Flores-Maltos et al. 2014).  Industrially, FOS production can beclassified under two broad categories namely the batch system, where the freeenzyme is used and a continuous system, in which immobilized cells and orenzymes are used. Enzyme immobilization offers great potentials mostly inbioreactors in which there is the possibility of high-enzyme load. This leadsto high volumetric productivities and a finite control on the extension of thereaction itself and a simplification of the downstream process. Moreso, thisimmobilization process offers instances where the biocatalysts are recoveredeasily and ultimately re-used (Fernandez et al.

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2005). The advantage enzyme immobilization offers are numerous asthere is tendency for the prevention of enzyme denaturation and substrateinhibition. This is often useful when the process for immobilization isautomated (Sheldon, 2007; Fernandes, 2010).

Traditionally, the technique of enzymeimmobilization is carried out using a myriad of methods namely entrapment,binding to a solid carrier and micro-encapsulation. This immobilization techniqueis referred to as the carrier-type immobilization and it inevitably leads todilution of catalytic activity resulting from the introduction of a largeproportion 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 immobilizationwhere there is no need for binding to a carrier is broadly termed cross-linkedenzyme aggregates (CLEA). Immobilization via cross-linking of enzymemolecules with a bifunctional cross-linking agent is a carrier-free method andthe resulting biocatalyst essentially comprises of 100% active enzyme. Proteincross-linking, through the reaction of glutaraldehyde with reactive aminogroups on the protein surface had been reported (Cao et al. 2003).

 Cross-linked enzyme aggregates (CLEA) areobtained from non-purified precipitated enzymes forming solid particles thatremain together through non-covalently binding (Tischer and Kasche et al. 1999). These CLEA remainpermanently insoluble after covalent binding with a cross-linking agent such asglutaraldehyde. In a typical reaction model, it is known that CLEA exhibitgreater than 10-fold activity than their corresponding biocatalysts immobilizedusing a carrier.

Comparatively, the cross-linking agent, glutaraldehyde, has anegligible molecular weight compared to the protein of interest to beimmobilized, hence, the resulting immobilized biocatalyst is perhaps 100%target protein. In addition, CLEA is a heterogenous catalyst and hence it iseasily recovered from the reaction milieu by just low speed centrifugation (Paret al. 2010). Also, CLEAs offer anavenue whereby the proteins catalytic features combine readily with themechanical behaviour of industrially used biocatalyst.The advantages offered by the immobilizationof this FOS-producing enzyme using CLEA are numerous and they include pH andthermal stability compared to the free enzyme, easy separation of products frombiocatalysts and a condition in which a continuous process can bedeveloped.

  Overall, an increasedoperational 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-exchangeresins (Yun and Song, 1996), gluten (Chien et al. 2001), polymethacrylate (Ghazi et al. 2005), macroporous beads (Tanriseven and Aslan, 2005), calciumalginate (Jung et al.

2011) The use of fructosyltransferase CLEA’s in thesynthesis and large-scale production of fructooligosaccharides has not beenfully exploited. The only recent report on the synthesis offructooligosaccharides involved the use of levansucrase from Bacillus subtilis (Ortiz-Soto et al. 2009). This implies that, the possibilityof the synthesis of fructooligosaccharides by using carrier-free immobilizedfructosyltransferase has not been fully exploited. This study investigated the preparation of cross-linkedfructosyltransferase aggregates from an improved mutant strain of Aureobasidium pullulans NAC8 (Ademakinwaand Agboola, 2016; Ademakinwa et al.2017), developed a mathematical model for industrial scale up by optimizing theconditions using statistical techniques and characterised the cross-linkedenzyme aggregates using microscopy and scanning electron microscopy energydispersive x-ray (SEM-EDAX). The structural changes that occurred aftercrosslinking with glutaraldehyde were investigated using peak fitting analysisof the amide I band of the FTIR spectra data. The re-usability of the CLEA wasalso investigated over several reaction cycles.

The reaction products of theCLEA biocatalysis of sucrose were purified using activated charcoal and sugarsquantified using multivariate data analysis of the FTIR spectra data. Furtherinvestigations into the use of the fructooligosaccharides as prebiotics wereinvestigated under anaerobic conditions with Lactobacillus sp as the choice probiotic strain.

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