Short Communication In situ immobilization of acid protease on mesoporous activated carbon packed column for the production of protein hydrolysates A. Ganesh Kumar a, * , K. Perinbam a , P. Kamatchi a , N. Nagesh b , G. Sekaran c a Department of Biomedical Engineering, Sathyabama University, Rajiv Gandhi Road, Chennai 600 119, India b Department of Molecular Biology, Centre for Cellular and Molecular Biology, Hyderabad, India c Department of Environmental Technology, Central Leather Research Institute, Chennai, India article info Article history: Received 1 April 2009 Received in revised form 2 September 2009 Accepted 3 September 2009 Available online 30 September 2009 Keywords: Immobilization Acid protease Mesoporous activated carbon Immobilized enzymes Protein hydrolysates abstract The mesoporous activated carbon (MAC) was used as a support material for in situ immobilization of acid protease (AP). The optimum temperature for the activities of both free and immobilized AP was found to be 50 °C. The catalytic efficiency of AP–MAC system has significantly been maintained for more than ten consecutive reaction cycles. The functional groups and surface morphology of the AP, MAC and AP–MAC were observed by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The production of protein hydrolysates was carried out from bovine serum albumin (BSA) using AP–MAC packed column and its properties were studied. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Protein hydrolysates are the main constituents of geriatric prod- ucts, high-energy supplements, enteral and parenteral solutions, and hypoallergenic foods. The enzymatic production of protein hydrolysates paves the way for increasing the nutritional properties of the hydrolysates (Neklyudov et al., 2000). The selection of the suitable immobilization matrix plays a vital role in enzyme immo- bilization (Andrzej et al., 2007) which ultimately increases the pro- duction of protein hydrolysates. The activated carbon assembles in many geometric forms with varied structural characteristics to suit the specific applications (Mirzarakhmetova et al., 2009). The prep- aration of protein hydrolysates with specific composition is not an easy task. It is of interest to use proteins with a high nutritional va- lue as a starting material for the preparation of protein hydroly- sates. Bovine serum albumin (BSA) fulfills this requirement because of its equilibrated amino acid composition. The acid prote- ases have been extensively used in pharmaceutical industry (Spelz- ini et al., 2005) and its immobilization appears to be a very beneficial one. In view of innumerable properties of enzyme immo- bilization, it was desirable to investigate the potential of the prote- ase immobilized on MAC for the production of protein hydrolysates. 2. Methods 2.1. Synthesis and characterization of the mesoporous activated carbon (MAC) The pre-carbonized rice husk carbon was activated, using ortho- phosphoric acid in the ratio 1:3.4 and followed by heating at 800 °C. The pore size distribution was determined using the BJH method and t-plot method (Gregg and Sing, 1982). The C, H, N content of the MAC was determined, using CHNS 1108 model analyzer. The XRD exper- iments were performed with a Philips X’pert diffractometer. 2.2. In situ – AP immobilization onto MAC and protease activity assay The AP was produced from tannery solid wastes as described by our previous observations (Ganesh Kumar et al., 2008). The column (0.5 cm inner diameter  50 cm length) was packed with MAC, and 1.0 g/L protease in 100 ml of phosphate buffer (pH 6.0) was applied at a flow rate of 1.0 ml/min and recirculated. The activity of prote- ase was determined using casein as the substrate (Ganesh Kumar et al., 2008). 2.3. Effect of temperature, pH and determination of kinetic parameters The effect of pH on the activity of free and immobilized AP was investigated in the pH range 5.5–7.5 at optimum temperature 37 °C using soluble casein in phosphate buffer. The effect of 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.09.014 * Corresponding author. Tel.: +91 44 24502430, +91 9444268156 (mobile); fax: +91 44 24502344. E-mail addresses: microganesh@yahoo.com (A. Ganesh Kumar), sistperinbam@ yahoo.co.in (K. Perinbam), kamatchi_3000@yahoo.com (P. Kamatchi), nagesh@ ccmb.res.in (N. Nagesh), ganesansekaran@hotmail.com (G. Sekaran). Bioresource Technology 101 (2010) 1377–1379 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech