Metallic/bimetallic magnetic nanoparticle functionalization for immobilization of a-amylase for enhanced reusability in bio-catalytic processes Vishal Singh a , Kanak Rakshit a , Shweta Rathee a , Stanzin Angmo a , Shimayali Kaushal a , Pankaj Garg c , Jong Hoon Chung c , Rajat Sandhir d , Rajender S. Sangwan b , Nitin Singhal a,⇑ a National Agri-food Biotechnology Institute (NABI), C-127, I Floor, Phase-VIII, Industrial Area, SAS Nagar, Mohali 160071, Punjab, India b Center for Innovative and Applied Bioprocessing (CIAB), C-127, II Floor, Phase-VIII, Industrial Area, SAS Nagar, Mohali 160071, Punjab, India c Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea d Department of Biochemistry, Panjab University, Chandigarh 160014, India highlights  Bimetallic based magnetic nanoparticles have been developed to immobilize the enzyme.  Enhanced reusability in bio-catalytic processes has been discussed.  Activity and kinetic parameters have been calculated and compared.  The properties of immobilized amylase were enhanced than the free enzyme. graphical abstract article info Article history: Received 10 March 2016 Received in revised form 2 May 2016 Accepted 3 May 2016 Available online 5 May 2016 Keywords: a-Amylase Magnetic nanoparticles Enzyme immobilization Kinetics abstract Novel magnetic nanoparticles coated with silica and gold were synthesized for immobilization of a-amylase enzyme and characterized with Fourier transform infrared spectroscopy, transmission elec- tron microscopy. Effect of various limiting factors such as substrate concentration, temperature, and pH on the catalytic activity of enzyme was investigated. The optimum pH for free and immobilized enzyme was found unaffected (7.0), whereas optimum temperature for the enzyme activity was increased from 60 °C for free enzyme to 80 °C for immobilized counterpart. The gains in catalytic attri- butes concomitant to ease of recovery of the enzyme reflect the potential of the approach and the product to be useful for the enzymatic bioprocessing. The Michaelis–Menten constant (K m ) value of the immobi- lized a-amylase was higher than that of free a-amylase, whereas maximum velocity (V max ), and turn over number (K cat ), values were almost similar. Immobilized a-amylase maintained 60% of the enzyme activity even after recycling ten times. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction a-amylase (1,4-a-D-glucan-glucanhydrolase, EC. 3.2.1.1) catalyzes hydrolysis of internal a-1, 4-glycosidic linkages of starch resulting in formation of low molecular weight products such as maltotriose and maltose from amylose, or maltose, glucose and ‘‘limit dextrins” from amylopectin. Human salivary a-amylase con- tains aromatic residues, Trp58, Trp59, Tyr151 and Phe256 in its active site that play a crucial role in determining substrate binding, catalytic reaction and reaction rate. The free enzyme uses in bio- processing has many shortcomings that include instability, poor http://dx.doi.org/10.1016/j.biortech.2016.05.002 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. E-mail address: nitin@nabi.res.in (N. Singhal). Bioresource Technology 214 (2016) 528–533 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech