Xylanase covalent binding onto amidated pectin beads: Optimization, thermal, operational and storage stability studies and application.

Amidated pectin-polyethylene imine-glutaraldehyde (AP-PEI-GA) immobilizer was prepared. The ideal protocol that should be adopted during the immobilizer preparation was investigated via Box-Behnken design (BBD), and it comprised processing the AP beads with 3.4 % (w/w) PEI solution of pH 9.65 followed by 5.96 % (v/v) GA solution. The obtained AP-PEI-GA immobilizer was efficient, and it acquired 3.03 U.g-1 of immobilized xylanase (im-xylanase) activity. The computed Km and Vmax values for AP-PEI-GA im-xylanase were 16.67 mg.ml-1 and 20 g.ml-1.min-1, respectively. Through covalent coupling to AP-PEI-GA, Aspergillus niger xylanase thermodynamic properties T1/2 and D-values were increased by 2.05, 3.08, and 1.35 at 40, 50, and 60 °C, respectively. ΔHd and ΔGd for AP-PEI-GA im-xylanase at 40, 50, and 60 °C were higher than those for free form emphasizing more resistance to thermal denaturation. Im-xylanase showed 100 % activity for 20 successive cycles and hydrolyzed different agro-industrial wastes into reducing sugar and xylooligosaccharides (XOS) with more efficiency on pea peel (PP). AP-PEI-GA im-xylanase, PP weight, and hydrolysis time that should be adopted to obtain the highest reducing sugar and XOS yield were optimized through central composite design (CCD). Extracted XOS showed prebiotic and anti-oxidant activities.

Treated calcium pectinate beads for the covalent immobilization of ß-d-galactosidase.

The calcium pectinate (CP) gel beads were treated with polyethyleneimine (PEI) and glutaraldehyde (GA). This treatment greatly enhanced the beads' mechanical strength. Moreover, it enabled the CP beads to covalently immobilize enzymes, such as ß-d-galactosidase (ß-gal). The central composite design (CCD) was applied to optimize the PEI/GA treatment while employing the observed activity of the immobilized ß-gal as a response. The CCD predicted that treating the CP beads with a 3.49% PEI solution of pH 10.55, followed by a 5.66% GA solution in 0.1M phosphate buffer pH 7.02 would allow for the immobilization of 6.25U/g gel of the ß-gal. The verification experiment run at these optimum conditions offered 6.285±0.22U/g gel immobilized ß-gal which was in close agreement with the predicted value. The reusability study revealed that the ß-gal immobilized onto the treated CP beads retained 79.34% of its initial activity after being used for fourteen times.