Purification of a novel cysteine protease, procerain B, from Calotropis procera with distinct characteristics compared to procerain Abhay Narain Singh a , Anil Kumar Shukla a , M.V. Jagannadham b , Vikash Kumar Dubey a, * a Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India b Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India 1. Introduction Bioinformatics analysis of the human and mouse genomes indicates at least 2% genes of the genome codes for proteases [1] which indicates physiological role of proteases. Proteases have therapeutic and industrial applications as well. The annual sales of proteases accounts 60% of the total world enzyme market and estimated to reach 220 billion US$ by the year 2009 [2]. Plant cysteine proteases are used in industry owing to their high temperature stability and broad substrate specificity [3]. Plant genomes encode hundreds of proteases, but little is known about what roles they play in the life of a plant. Proteases are thought to be involved in a range of biological processes, including senescence, implicated in perception, signaling and execution leading to plant defense [4]. Few other cysteine proteases from the plant sources have been purified and characterized. As usefulness of the proteases depends on its unique cleavage site, stability as well as optimum activity condition search of a novel protease with unique properties is always on. Calotropis procera (family Asclepiadaceae) is a tropical plant that has been widely used in Indian traditional medicinal system for the treatment of various diseases namely leprosy, ulcers, tumors, piles and diseases of the spleen, liver and abdomen [5]. Various parts of the plant show anti-microbial, anti-inflammatory, antipyretic and anti-malarial activities [6,7]. The latex of the plant shows antidiabetic, hepatoprotective, antiarthritic, cytotoxic and anticancerous properties [8]. Preliminary screening of the latex of the plant showed very high proteolytic activity. We have earlier reported purification and characterization of a novel protease procerain, from the latex of the plant [9]. Here we report purification of another protease from the plant, which we named as procerain B, with distinct properties and cleavage site. 2. Materials and methods 2.1. Materials Superficial incisions on the C. procera yielded milk like latex. Fresh latex of the plant was collected. CM-Sepharose FF was purchased from GE Healthcare. Azocasein, DTNB (5,5 0 -dithiobis-[2-nitro benzoic acid]), DTT (dithiothreitol), GuHCl (guanidine hydrochloride), urea, o-phenanthroline, EDTA (ethylenediaminetetraacetic acid), EGTA (glycol-bis(2-aminoethylether)-N,N,N 0 ,N 0 -tetraacetic acid), leupeptin, SBTI (soybean trypsin inhibitor), NEM (N-ethylmaleimide), b-mercaptoethanol, PMSF (phenylmethanesulfonylfluoride), acrylamide,N,N-methylene bisacrylamide, Coo- massie brilliant blue R-250, E-64 (l-trans-epoxysuccinylleucylamide (4-guanidino) butane-N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl] agimatine), are obtained from Sigma Chemical Co., USA. Sodium tetrathionate (Na 2 S 4 O 6 2H 2 O) was Process Biochemistry 45 (2010) 399–406 ARTICLE INFO Article history: Received 21 May 2009 Received in revised form 8 September 2009 Accepted 22 October 2009 Keywords: Cysteine protease ELISA Food industry Substrate specificity Calotropis procera ABSTRACT Proteases have applications in food, detergent and pharmaceutical industries. A novel protease has been purified from the latex of Calotropis procera and characterized. As another cysteine protease, procerain, is reported from the same source, the newly purified enzyme was named as procerain B. The enzyme shows distinct properties compared to procerain, in terms of cleavage recognition site, immunological properties and other physical properties like molecular weight, isoelectric point, etc. The newly purified enzyme shows a broad optimum pH (6.5–8.5) as well as broad optimum temperature (40–60 8C). Additionally, the enzyme retains its activity where most of other proteases are not active. Moreover, the enzyme appeared to be very efficient in hydrolysis of blood stain and may have potential application in detergent industries. Simple and economic purification of procerain B, together with easy availability of latex, makes the large-scale production of procerain B possible, thus enables to explore various industrial as well as biotechnological applications. ß 2009 Elsevier Ltd. All rights reserved. Abbreviations: BAPA, NR-benzoylarginine p-nitroanilide; DFP, diisopropylfluoro- phosphate; DTNB, 5,5 0 -dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EGTA, glycol-bis(2-aminoethylether)-N,N,N 0 ,N 0 - tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; GuHCl, guanidine hydrochloride; NEM, N-ethyl-maleimide; PCMB, p-chloromercurybenzoate; PMSF, phenyl-methanesulfonyl fluoride; SBTI, soybean trypsin inhibitor; TCA, trichlor- oacetic acid; TFA, trifluoroacetic acid; TEMED, N,N,N,N-tetramethylethylenedia- mines. * Corresponding author. Tel.: +91 361 2582203; fax: +91 361 2582249. E-mail address: vdubey@iitg.ernet.in (V.K. Dubey). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.10.014