Process Biochemistry 46 (2011) 1486–1491 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Purification, characterization and immobilization of urease from Momordica charantia seeds Bankapalli Leela Krishna, Abhay Narayan Singh, Sanjukta Patra ∗ , Vikash Kumar Dubey ∗ Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781039, India article info Article history: Received 12 January 2011 Received in revised form 15 March 2011 Accepted 30 March 2011 Keywords: Chitosan Immobilization Momordica charantia Purification Urease abstract Ureases are used in beverage, fertilizer and pharmaceutical industries. We report purification, characteri- zation and a simple method for immobilization of urease form Momordica charantia seeds. The purification of the enzyme is a two steps process involving anion exchange chromatography using DEAE–Sepharose matrix followed by size exclusion chromatography. The fold purification of the enzyme was found to be 6.5. The estimated molecular mass and K m of the enzyme were 174.5 kDa and 34 ± 0.5 mM, respectively. The purified urease was immobilized on chitosan beads activated with glutaraldehyde. The activation of chitosan beads and immobilization were confirmed by Fourier Transform Infrared (FTIR) spectrometry and Scanning Electron Microscope (SEM), respectively. Moreover, Energy Dispersive X-ray (EDX) analysis shows increased percentage of nitrogen which further confirms immobilization. The efficiency of immo- bilization was found to be approximately 97%. The immobilized enzyme retains 60% activity till fourteen successive batches of enzymatic reactions which makes it a highly stable product. Comprehensively, the enzyme shows desirable properties for various applications. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Urease (EC 3.5.1.5) catalyses the hydrolysis of urea at a rate 10 14 times faster than the uncatalysed reaction, yielding ammo- nia and unstable carbamic acid. Hydrolysis of one molecule of urea results in the release of one molecule of ammonia and one molecule of carbamate [1]. Urease from seeds has been purified to homogeneity from different plant sources with distinct stabil- ity, biochemical and serological properties [2–4]. Jack bean urease is the first enzyme ever, to be purified and crystallized for use in blood urea estimation [5–8]. Ureases are the only nickel contain- ing metalloenzyme identified so far in plants which require two Ni 2+ metals for activating the substrate and water for the reac- tion [2]. Urease is responsible for recycling metabolically derived urea in plant. Urease plays an important role in germination and in seedling’s nitrogen metabolism. Urease activity in the leaf is also important if foliar application of urea is considered as a fertil- izer. It has been reported that foliar treatment with urea increases the nutritional quality of leaf. In silkworm, mulberry leaf urease activity could be directly related to silk production. Normally these insects feed on mulberry leaf, so the urease produced from leaf act on urea in stomach and release ammonia which is reabsorbed ∗ Corresponding authors. Tel.: +91 361 2582203/2213; fax: +91 361 2582249. E-mail addresses: sanjukta@iitg.ernet.in (S. Patra), vdubey@iitg.ernet.in (V.K. Dubey). and used for silk protein synthesis in the insect. Ureases from soil are involved in crop yield by exploiting urea as fertilizer. Urease of many pathogenic organisms also have been purified and character- ized including Vibrio parahaemolyticus [9]. Ureases are usually homo or hetero-oligomer. Urease from pigeon pea exists as trimer or hexamer in its native form [10]. Molecular weight of urease from pigeon pea was found to be 540 kDa (hexamer). Similarly urease from Morus alba was found to be 175 kDa (homo dimer) [11]. Follmer found canatoxin as the isoform of urease possessing insecticide activities [12]. The insecticidal activities of plant ureases including canatoxin are inde- pendent of ureolytic activity [13]. Ureases are extensively used for various purposes [14]. The enzyme is used in quantitative determination of urea in pathologi- cal conditions as well as determination of blood urea concentration. Acid ureases are used for elimination of urea from alcoholic bev- erages. This is done to prevent the formation of ethyl carbamate which is known to be carcinogenic. Likewise, urease conductomet- ric biosensors are used for detection of heavy metals in ground water. Immobilized ureases have wide applications in estimation of blood urea level and construction of biosensor [15,16]. Momordica charantia (Family: Cucurbitaceae) is a herb and widely grown in India. It has been widely used in medical treat- ment for Diabetes Mellitus from long time. Various parts of plant have been shown as highly beneficial in treatment for Anorexia, Piles, Hangovers, Pyrrohea, Blood impurities, Diarrhea, Jaundice, Kidney stones, Leprosy, Laxative, Blood disorders like Blood boils, 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.03.022