Three enzymes, α-amylase, glucoamylase and invertase, were immobilized on acid activated montmorillonite K 10 via two independent techniques, adsorption and covalent binding. The immobilized enzymes were characterized by XRD, N2 adsorption measurements and 27Al MAS-NMR spectroscopy. The XRD patterns showed that all enzymes were intercalated into the clay inter-layer space. The entire protein backbone was situated at the periphery of the clay matrix. Intercalation occurred through the side chains of the amino acid residues. A decrease in surface area and pore volume upon immobilization supported this observation. The extent of intercalation was greater for the covalently bound systems. NMR data showed that tetrahedral Al species were involved during enzyme adsorption whereas octahedral Al was involved during covalent binding. The immobilized enzymes demonstrated enhanced storage stability. While the free enzymes lost all activity within a period of 10 days, the immobilized forms retained appreciable activity even after 30 days of storage. Reusability also improved upon immobilization. Here again, covalently bound enzymes exhibited better characteristics than their adsorbed counterparts. The immobilized enzymes could be successfully used continuously in the packed bed reactor for about 96 hours without much loss in activity. Immobilized glucoamylase demonstrated the best results.
Glucoamylase was immobilized on acid
activated montmorillonite clay via two different procedures
namely adsorption and covalent binding. The
immobilized enzymes were characterized by XRD,
NMR and N2 adsorption measurements and the
activity of immobilized glucoamylase for starch
hydrolysis was determined in a batch reactor. XRD
shows intercalation of enzyme into the clay matrix
during both immobilization procedures. Intercalation
occurs via the side chains of the amino acid residues,
the entire polypeptide backbone being situated at the
periphery of the clay matrix. 27Al NMR studies
revealed the different nature of interaction of enzyme
with the support for both immobilization techniques.
N2 adsorption measurements indicated a sharp drop in
surface area and pore volume for the covalently bound
glucoamylase that suggested severe pore blockage.
Activity studies were performed in a batch reactor. The
adsorbed and covalently bound glucoamylase retained
49% and 66% activity of the free enzyme respectively.
They showed enhanced pH and thermal stabilities. The
immobilized enzymes also followed Michaelis–Menten
kinetics. Km was greater than the free enzyme that was
attributed to an effect of immobilization. The immobilized
preparations demonstrated increased reusability
as well as storage stability.
Glucoamylase from Aspergillus Niger was immobilized on montmorillonite clay (K-10) by two procedures, adsorption and covalent binding. The immobilized enzymes were characterized using XRD, surface area measurements and 27Al MAS NMR and the activity of the immobilized enzymes for starch hydrolysis was tested in a fixed bed reactor (FBR). XRD shows that enzyme intercalates into the inter-lamellar space of the clay matrix with a layer expansion up to 2.25 nm. Covalently bound glucoamylase demonstrates a sharp decrease in surface area and pore volume that suggests binding of the enzyme at the pore entrance. NMR studies reveal the involvement of octahedral and tetrahedral Al during immobilization. The performance characteristics in FBR were evaluated. Effectiveness factor (η) for FBR is greater than unity demonstrating that activity of enzyme is more than that of the free enzyme. The Michaelis constant (Km) for covalently bound glucoamylase was lower than that for free enzyme, i.e., the affinity for substrate improves upon immobilization. This shows that diffusional effects are completely eliminated in the FBR. Both immobilized systems showed almost 100% initial activity after 96 h of continuous operation. Covalent binding demonstrated better operational stability.