Immobilization of a thermostable alpha-amylase onto reactive membranes: kinetics characterization and application to continuous starch hydrolysis


Bayramoglu G., YILMAZ M. , Arica M.

FOOD CHEMISTRY, vol.84, no.4, pp.591-599, 2004 (Peer-Reviewed Journal) identifier identifier

  • Publication Type: Article / Article
  • Volume: 84 Issue: 4
  • Publication Date: 2004
  • Doi Number: 10.1016/s0308-8146(03)00283-8
  • Journal Name: FOOD CHEMISTRY
  • Journal Indexes: Science Citation Index Expanded, Scopus
  • Page Numbers: pp.591-599
  • Keywords: polymer membrane, alpha-amylase, immobilization, starch hydrolysis, enzyme kinetics, enzyme reactor, COVALENT IMMOBILIZATION, INVERTASE, GLUCOAMYLASE, METHACRYLATE, STABILITY

Abstract

Epoxy groups containing porous membranes were prepared by UV-initiated photopolymerisation of hydroxyethylmethacrylate (HEMA) and glycidyl methacrylate (GMA). Epoxy supports could provide multipoint covalent attachment of enzymes, therefore, to stabilize their three-dimensional structure. alpha-Amylase was immobilized onto the poly(HEMA-GMA-1-3) membranes by means of the amide linkage formation between the amino groups of alpha-amylase and the epoxy groups of the support. The alpha-amylase immobilization capacity of the membranes was increased as the GMA ratio increased in the membrane structure. The retained activity of the immobilized alpha-amylase was 76% with poly(HEMA-GMA-2) membrane. The decrease in activity of the immobilized a-amylase could be considered to be due to reduced conformational flexibility of the immobilized alpha-amylase molecules for binding its large substrate, starch, as a result of the covalent immobilization. The immobilized alpha-amylase has more resistance to temperature inactivation than that of the free form. The optimum pH value of alpha-amylase was not affected by the immobilization reaction, but the pH profile was broadened for the immobilized enzyme. Kinetic parameters were determined for immobilized alpha-amylase as well as for the free enzyme. The values of the Michaelis constant K-m of alpha-amylase, were significantly larger (ca. 2.3 times) upon immobilization, indicating decreased affinity of the enzyme for its substrate, whereas V-max was smaller for immobilized a-amylase. In a 120 h continuous operation at 35 degreesC only 4% of immobilized alpha-amylase activity was lost. The operational inactivation rate constant (k(opi)) of the immobilized a-amylase with 2% starch was 8.06x 10(-6) min(-1). (C) 2003 Elsevier Ltd. All rights reserved.