Ceylon Journal of Science 48(3) 2019: 243-249 DOI: http://doi.org/10.4038/cjs.v48i3.7648 RESEARCH ARTICLE Chitinases from pitcher fluid of Nepenthes distillatoria R.W.K.M. Senevirathna1, V.N. Seneviratne2 and S. Rajapakse1,* 1Department of Molecular Biology and Biotechnology, Faculty of Science, University of Peradeniya, Peradeniya 20400, Sri Lanka 2Department of Chemistry, Faculty of Science, University of Peradeniya, Peradeniya 20400, Sri Lanka Received: 06/12/2018 ; Accepted: 12/06/2019 Abstract: Chitinases belong to a diverse family of enzymes ribonucleases, peptidases, esterases, and chitinases (Moran among many other hydrolytic enzymes of the pitcher fluid of and Clarke, 2010). Chitinases are a particularly interesting Nepenthes sp. There are no existing studies on identification of group of plant enzymes because their substrate chitin is not chitinases in Nepenthes distillatoria the only representative of the present in plant tissues (Rottloff et al., 2011). The induction genus Nepenthes in Sri Lanka. In this study an assay procedure of chitinases in plants following fungal infection is well was developed and optimized to detect the chitinolytic activity in documented (Wang et al., 2002). Therefore, chitinases the pitcher fluid of N. distillatoria using colloidal Chitin Azure are thought to be necessary in plant defense against as the substrate. Crude Nepenthes pitcher fluid was subjected phytopathogenic fungi, due to their ability to hydrolyze to DEAE Cellulose chromatography in an attempt to partially and degrade chitin (Van Loon et al., 2006). Chitinases purify and separate chitinases. The optimum pH, temperature and are widely used in the fields of industry, agriculture and incubation time for the chitinolytic activity were 5.0, 27 °C and medicine for numerous important functions. This is because 30 min, respectively. DEAE Cellulose Chromatography revealed the chitinases have the ability of direct digestion of chitin the presence of more than one type of chitinases within the pitcher biopolymers into low molecular weight chitooligomers juice. Further studies are required to characterize and confirm the (Yuli et al., 2004). Also, elicitor action of chitinases can origin/s of these chitinases. be used for controlling diseases which are caused by fungi and harmful insects (Mathivanan et al., 1998; Mendonsa et Keywords: Carnivorous plant, Chitin, Chitinase assay, Colloid al., 1996). Chitin Azure, Pitcher plant. Chitin is a linear polysaccharide of β-1, INTRODUCTION 4-N-acetylglucosamine monomers (Shibuya and Minami, 2001; Van Loon et al., 2006) and is a major constituent of The ability to catch and digest prey, mainly insects, the outer skeleton of insects, cell wall of fungi as well as allows carnivorous plants growing in very low nutrient in the internal structures of most of the other invertebrates habitats to acquire mainly nitrogen and phosphorus and (Theis and Stahl, 2004; Kramer and Muthukrishnan, 2009). other mineral nutrients. Plants of the genus Nepenthes grow in nutrient poor habitats and have evolved modified For the conventional chitinase assay procedures, leaves called pitchers to capture insect preys (Ellison insoluble chitin which is covalently bound to dyes, tritium- and Adamec, 2011). Nepenthes belong to monotypic labeled chitin or colloidal chitin are used as substrates of family Nepenthaceae. According to the classification of chitinases (Gomez-Ramırez et al., 2004; Spindler, 1997). angiosperms, at least 583 species of carnivorous plants In these methods the chitinase activity is determined by can be recognized belonging to 20 genera dispersed in 12 measuring the quantity of reducing sugars (e.g., chito- families falling under 5 orders. Nepenthes which belongs oligosaccharides) (Shen et al., 2010). However, the major to order Caryophyllales comprises approximately 90 problem associated with this system was that those results species (Givnish, 2015). Nepenthes distillatoria is the can be affected by other reducing sugars present in the only representative of the genus Nepenthes in Sri Lanka reaction medium (Imoto and Yagishita, 1971; Shen et al., and it is endemic to the island (Dassanayake and Fosberg, 2010; Hackman and Goldberg, 1964; Liu et al., 2009). 1980). According to the IUCN red list, N. distillatoria can Conversely, a special type of chitin substrate was introduced be recognized as a vulnerable plant species (IUCN, 2000). for the chitinase assay, known as colloidal Chitin Azure. It is mainly distributed in the South West and South-East This is a kind of chitin which is covalently coupled with regions of Sri Lanka (Dassanayake and Fosberg, 1980). Remazol brilliant violet 5R dye and the resulting products can be measured at 560 nm in a colorimeter. In addition, To facilitate the prey digestion and make the nutrients most significantly the assay is not affected by reducing available, carnivorous plants possess a variety of sugars of the medium (Gomez-Ramırez et al., 2001). endogenous enzymes including proteases, phosphatases, *Corresponding Author’s Email: [email protected] https://orcid.org/0000-0002-0014-5811 This article is published under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 244 Ceylon Journal of Science 48(3) 2019:243-249 There are many studies carried out for identification, the assay procedure was carried out as follows. characterization and purification of proteases present in For detection of optimum pH, a series of buffer the pitcher fluid of Nepenthes sp. (Thornhill et al., 2008; solutions in the range of pH 1.0 to pH 7.0 were used. The Plachno et al., 2006; Fukusaki and Kobayashi, 2002; optimization of incubation time was done by incubating the Athauda et al., 2004; Takahashi et al., 2005; Stephenson reaction mixtures at varying time intervals ranging from and Hogan, 2006; Hatano and Hamada, 2008), but there 10 min to 60 min while for determination of optimum are only limited research carried out for identification incubation temperature, four different temperatures (4 °C, and purification of chitinases (Eilenberg et al., 2006; 27 °C, 37 °C and 50 °C) were used. Ishisaki et al., 2012) and there are no studies conducted for identification of chitinases in N. distillatoria. The foremost Then the enzyme activity was calculated using the reason for limited research is the unavailability of a soluble following equation, and an inexpensive substrate to analyze chitinase activity (Shen et al., 2010; Gomez-Ramırez et al., 2004). In this study for the first time, chitinolytic activity of the pitcher fluid of N. distillatoria was detected using Where, dispersible, soluble colloid Chitin Azure as the substrate. Following the detection of chitinolytic activity, attempts - were made to partially purify and characterize the chitinases of the pitcher fluid. The optimum pH and optimum temperature for chitinase activity of the crude pitcher fluid Comparison of the chitinase activity of the crude open as well as for the fractions obtained after anion exchange and closed pitcher fluid chromatography were determined. Moreover, the thermal stability of the chitinase activity of the different partially After carrying out the optimized chitinase assay procedure purified fractions was investigated. The remarkable mentioned in section 2.3 for both unpurified OP and CP stability in the major fraction among the three fractions fluids, the activity data were subjected to normality testing obtained after anion exchange chromatography over a long followed by GLM and LS means-pdiff mean separation period of incubation time seems to indicate that they have procedure using the statistical package, SAS 9.1.3 (SAS evolutionally well adapted to their original habitat. Institute, NC, Cary, USA). MATERIALS AND METHODS Purification and analysis of chitinase(s) using ion exchange chromatography Collection of the crude sample All purification procedures were performed at 4 °C. The The open (OP) and closed (CP) crude pitcher fluids of N. crude pitcher fluid (100 ml) was dialyzed in about 2000 distillatoria were collected from Hakurugala forest patch ml of 0.1 M sodium phosphate buffer at pH 7.0. The at Ruwanwella, Sri Lanka. They were clearly labeled and proteins in the dialyzed Nepenthes fluid were absorbed to 0 brought into the laboratory and kept at -20 C until use. DEAE cellulose (pre equilibrated with the same buffer) by Preparation of the colloidal Chitin Azure substrate batch adsorption and the gel was placed in a 5 ml column. The column was washed with 100 ml of 0.1 M sodium The substrate Colloidal Chitin Azure was prepared phosphate buffer at pH 7.0 and the proteins were eluted according to the procedure given in Shen et al (2010). with the same buffer containing 0.5 M NaCl. Five milliliter fractions were collected and the chitinase activity in each Assay procedure for chitinase activity fraction was measured. The assay was carried out for the crude OP and CP fluid Then a 2nd DEAE cellulose chromatography was carried samples separately. Each test and control experiments were out as follows. Five grams of DEAE cellulose powder was conducted in replicates. For the test samples, 1000 µL of suspended in a little volume of phosphate buffer (0.1 M, pH 50 mM sodium phosphate buffer solution at pH 5.0, 200 7.0). Column (10 ml) was evenly packed on to a syringe µL of colloidal CA substrate and 100 µL of unpurified using a glass rod and a Pasteur pipette. The packed column open or closed crude pitcher fluid were added to 1.5 ml was equilibrated with 50 ml of phosphate buffer (0.1 M, centrifuge tube. For the controls, similar to test samples, pH 7.0). Then 400 ml each of crude open pitcher fluid was buffer solution and substrate were added except the crude added to the top of the column and was collected at the pitcher fluid. The samples were incubated for 3 hrs at 37 bottom of the syringe. The sample collected at the bottom °C.