Journal of Molecular Catalysis B: Enzymatic 56 (2009) 34–40 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding Kayambu Kannan a , Raksh Vir Jasra a,b,∗ a Discipline of Inorganic Materials & Catalysis, Central Salt & Marine Chemicals Research Institute, CSIR G.B. Marg, Bhavnagar 364002, Gujarat, India b R&D Centre, Reliance Industries Limited, Vadodara Manufacturing Division, Vadodara 391346, Gujarat, India article info Article history: Received 31 October 2007 Received in revised form 9 April 2008 Accepted 11 April 2008 Available online 26 April 2008 Keywords: Alkaline serine endopeptidase Casein hydrolysis Enzyme immobilization SBA-15 MCF Enzyme reusability abstract An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activi- ties of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 ◦ C) than that of the free enzyme (55 ◦ C). Kinetic parameters, Michaelis–Menten constant (K m ) and maximum reaction velocity (V max ), were calculated as K m = 13.375, 11.956, and 8.698 × 10 -4 mg/ml and V max = 0.156, 0.163 and 0.17 × 10 -3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Alkaline proteases are important enzymes having diverse appli- cations in a wide variety of industries such as food, pharmaceutical, detergent, leather, silk, diagnostics, and for the recovery of silver from used X-ray films [1–4]. Alkaline serine endopeptidase from selected strain of Bacillus licheniformis, which hydrolyses casein, plays a vital role in dairy industries. Casein is a slowly digesting protein which is essential in building muscles in human body and is a major component of milk, whey and soy [5]. The industrial application of this enzyme requires specificity, stability at higher pH, temperature and reusability. Numerous techniques have been used for immobilization of free enzymes on solid support to obtain efficient biocatalysts [6]. Mesoporous silicas (MPSs) obtained by different templating methods demonstrate high potential as solid supports for enzyme immobilization [7–22]. These supports are environmentally acceptable, structurally more stable, and resis- tant to microbial attack. MPSs have a large specific surface area (∼1000 m 2 /g) and pore diameter, in the range of 2–50nm, which can be tuned to host the enzymes of varied size. As such, the ∗ Corresponding author. Tel.: +91 265 6693935; fax: +91 265 6693934. E-mail address: rakshvir@ril.com (R.V. Jasra). enzymes have considerable affinity towards the MPSs surfaces [7–14]. Besides, the MPSs surface can be modified with various anchor groups to covalently bind the enzyme molecules, which could reduce the enzyme leaching from the support during the recycling of the catalyst [14–22]. The uniform distribution of pores in MPSs favors the uniform loading of enzyme as well as facile diffusion of the substrate and product molecules inside the chan- nels. The loading of an enzyme and its activity depend upon the surface area and pore size of the MPSs [17–22]. Among the MPSs, SBA-15 and MCF have been shown to be efficient supports for covalent immobilization of -amylase, trypsin, chloroperoxi- dase, penicillin G acylase, organophosphorus hydrolase, glucose oxidase, glucoamylase, and invertase [8–11,14,19–22]. SBA-15 and MCF have pore sizes in the range of 9–25nm and they possess 600–800 m 2 /g surface area, which make them suitable to host the alkaline endopeptidase comfortably and also allow substrate and product molecules facile diffusion towards and from the active site of the enzyme [18,21]. The average size of the alkaline endopepti- dase is 4.7 nm as shown in Fig. 1, produced using Chem3D Pro 10.0 from RCSB enzyme database [23]. Alkaline endopeptidase immobilized on polymeric support has been evaluated [24–26] for stability, activity and reusability with reference to free enzyme. In the present study, covalent immobi- lization of alkaline serine endopeptidase has been done through amide bond formation on modified SBA-15 and MCF. Surface 1381-1177/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2008.04.007