Enhanced activity and stability of α-amylase immobilized on alumina

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Abstract

α-amylase was immobilized on alumina via adsorption. The support and the immobilized enzymes were characterized using XRD, IR spectra and N2 adsorption studies. The efficiency of immobilized enzymes for starch hydrolysis was tested in a batch reactor. The effect of two different calcination temperatures on properties of the support as well as on immobilization was studied. From XRD, IR and N2 adsorption studies it was confirmed that the enzyme was getting adsorbed only on the external surface of the support. pH, buffer concentration and substrate concentration had a significant influence on the activity of immobilized enzyme. Km for immobilized α-amylase was found to be higher than the free enzyme, which may be due to interparticle diffusional mass transfer restrictions. The immobilized enzymes showed enhanced pH stability than the free enzyme.

Introduction

Biotechnology is currently considered as a useful alternative to conventional process technology in industrial and analytical fields. The demand for industrial enzymes is ever increasing owing to their applications in a wide variety of processes. Enzymatic reaction can be favored over chemical catalysis under such circumstances where thermal degradation of labile compounds is minimized and use of chemicals with a potential for pollution can be avoided [1]. However, the common drawbacks associated with the enzymatic processes are the inhibition of enzyme at high concentration of substrate or product [2] and instability of enzyme under the reaction condition [3] as well as in repetitive usage [4]. Immobilized enzymes are preferred over the native ones owing to their multiple and repetitive use. In addition, the reaction product is not contaminated with the enzyme (especially useful in the food and pharmaceutical industries). Furthermore, the immobilized enzyme has a longer half-life and predictable decay rate. Amylases see a great deal of use in food and fermentation industries. The hydrolysis of starch to low-molecular weight products using α-amylase is widely applied in the food, paper, textile, distillery, and brewing industries. Hydrolysis of starch can be achieved using either acid or enzyme catalysts [5]. α-Amylase (EC.3.2.1.1;1,4-d-glucan glucanohydrolase) hydrolyses α-1,4 glucosidic bonds in amylase, amylopectin and glycogen in an endo fashion but the α-1, 6 glucosidic linkages in the branched polymers are not hydrolysed [6]. Enzymes are immobilized by physical adsorption, ionic binding, covalent binding, cross-linking and entrapment methods [7]. In the past few decades, many immobilization methods and carrier materials have been investigated [8], [9]. There have been many reports about immobilization of α-amylase used for the hydrolysis of starch and production of maltose. Organic polymeric carriers are the most widely studied materials because of the presence of rich functional groups, which provide essential interactions with the enzymes. However, the organic supports suffer a number of problems such as poor stability towards microbial attacks and organic solvents and disposal issues. In contrast, inorganic materials such as silica gels, alumina, and layered double hydroxides are known to be thermally and mechanically stable, non-toxic, and highly resistant against microbial attacks and organic solvents [10]. Inorganic supports provide better stability characteristics. Porous silica is found to be good medium for immobilization [11]. Various enzymes have been immobilized on clays [12], [13]. α-Amylase has been immobilized to collagen [14], Kaolin [15] and sand. Bacterial amylase has been covalently bound to silica carriers using glutaraldehyde or titanium chloride [16]. Solid acid supports can be used to immobilize enzymes since the acidic sites can act as centres of immobilization via the amino groups of enzymes. Reports on immobilization of enzymes to metal oxides are limited.

In the present study, the hydrolysis of starch to low molecular weight carbohydrates is carried out using α-amylase immobilized on alumina via adsorption. The procedures involved in adsorption are quite simple, making it one of the most widely used methods of enzyme immobilization. This property of reversibility of binding has often been used for the economic recovery of the support. The support and the immobilized enzymes were characterized using XRD, IR spectra and N2 adsorption studies. The activity for starch hydrolysis was studied in a batch reactor. The effect of different calcination temperatures on the properties of support before and after immobilization was studied. The effect of pH on activity and stability of free and immobilized enzymes were studied. The effect of buffer concentrations on immobilization was also studied. The kinetics of the reaction was determined at various substrate concentrations and the kinetic parameters (Km and Vmax) were calculated from the Hanes–Woolf plot.

Section snippets

Materials

α-Amylase from Bacillus subtilis was procured from Sigma–Aldrich Chemicals Pvt. Ltd., Bangalore. Aluminium nitrate was purchased from BDH Chemicals, Mumbai and ammonium hydroxide was from Qualigens Fine Chemicals, Mumbai. Starch was obtained from SRL Chemicals, Mumbai. All other chemicals were of highest purity commercially available.

Preparation of the catalyst

Sufficient amount of ammonium hydroxide (1:1 v/v in deionized water) was slowly added to 0.1 M aluminium nitrate under vigorous stirring at a temperature of 70 °C

Characterization studies

The XRD pattern of A-1 (Fig. 1) shows two peaks at 2θ = 32° for the single phase material together with two peaks at 46° and 67° which are the characteristic peaks corresponding to the (2 2 0), (4 0 0) and (4 4 0) reflections of γ-Al2O3 phase [19]. In the case of A-2, the same distinct peaks as that of A-1 were present but with higher intensity and an additional peak appeared at 2θ = 37° which is one of the characteristic peak of γ-alumina. The planes corresponding to the gamma phase are more prominent

Conclusions

α-Amylase was immobilized on alumina via adsorption. From XRD and N2 adsorption studies it was confirmed that the enzyme is getting adsorbed only on the external surface of the support. In XRD, there was a decrease in the intensities of the peaks and a slight broadening after adsorption of α-amylase on AA-1 and AA-2. The surface area also showed a decrease after adsorption. IR studies reveal that the enzyme is adsorbed via the OH groups, as there is disappearance of the bands in the OH region.

Acknowledgements

The authors thank Sophisticated Instruments Facility, Indian Institute of Science, Bangalore and Sree Chithra Tirunal Institute for Medical Sciences and Technology, Thiruvanthapuram, for providing NMR and IR analysis.

References (34)

  • M. Markweghanke et al.

    Enzyme Microb. Technol.

    (1995)
  • L. Cong et al.

    J. Biotechnol.

    (1995)
  • W. Tischer et al.

    Biotechnology

    (1999)
  • L. Cao et al.

    J. Mol. Catal. B: Enzyme

    (1999)
  • G. Sanjay et al.

    Catal. Commun.

    (2005)
  • O.H. Lowry et al.

    J. Biol. Chem.

    (1951)
  • H. Tumturk et al.

    Food Chem.

    (2000)
  • J.M. Dominguez et al.

    Appl. Catal. A: Gen.

    (2000)
  • J.S. Church et al.

    Appl. Catal.

    (1993)
  • D. Tanyolac et al.

    Biochem. Eng. J.

    (1998)
  • D. He et al.

    Biochem. Eng. J.

    (2000)
  • K.F. Tipton et al.

    Method. Enzymol.

    (1979)
  • G. Bayramoglu et al.

    Food Chem.

    (2004)
  • R.A. Sheldon

    Chirotechnology: Industrial Synthesis of Optically Active Compounds

    (1993)
  • V.V. Mozhaev et al.

    Eur. J. Biochem.

    (1989)
  • L. Giorno et al.

    J. Chem. Technol. Biotechnol.

    (1995)
  • F.W. Schenck et al.

    Starch Hydrolysis Products: Worldwide Technology, Production and Applications

    (1992)
  • Cited by (0)

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