Simplified Method of Preparing Colloidal Chitin Used For Screening of Chitinase- Producing Microorganisms

摘要

A simplified and efficient method of preparing colloidal chitin from inexpensive crab shell flakes was developed. It modifies some steps of existing techniques to provide significant saving in effort and materials for colloidal chitin preparation. In colloidal chitin preparation, unless the initial chitin flakes are ground to a fine powder, it becomes difficult to later separate the chitin chunks from the precipitated colloidal chitin. The modified technique reported here involves the use of simple everyday lab materials to extract colloidal chitin from crab shell flakes, without the need of powdering the chitin flakes to a uniformly fine size. Utility of the colloidal chitin obtained was shown by using it in plate assays to screen for extracellular chitinase producers, and labeling it with Remazol Brilliant Blue (RBB) for chitinase assays. Introduction Chitin is widespread in both terrestrial and aquatic environments, as a component of invertebrate exoskeletons, fish scales, and cell walls of many fungi. Only cellulose is more globally abundant as a biological polymer (reviewed by Shahidi et al., 1999; and Zaku et al., 2011). Chitin is the most abundant biopolymer in marine environments (Souza et al., 2011). A variety of microorganisms produce extracellular chitinases that can break down chitin (reviewed by Howard et al., 2003; Gohel et al., 2006; and Dahiya et al., 2006). To isolate such microorganisms, chitin is often used in solidified agar media, where clearing zones surrounding microbial colonies indicate production of extracellular chitinase. Because chitin is not readily water soluble, chitin is often chemically modified to form colloidal chitin, with a small particle size that is more readily manipulated to obtain homogenous distribution in agar media, compared to use of physically modified, finely ground chitin that can be difficult to obtain. Lingappa and Lockwood (1961; 1962) devised a colloidal chitin preparation method that was later modified by Hsu and Lockwood (1975). These are referred to in this paper as Lockwood protocols. The Lockwood protocols have been widely used for isolation of chitinase-producing culturable microorganisms. Modifications of the Lockwood protocols have been developed and used by various researchers since (Gomez Ramirez et al., 2004; O’Risal, 2008), depending on their research needs. No matter the specific goals, modification of protocols to save time or materials is of continuing interest to researchers. In our studies to characterize a variety of culturable microorganisms for chitinase activity, we arrived at a modified version of the Hsu and Lockwood protocol (1975) combining it with some steps from the protocol of Gomez Ramirez et al. (2004) to save effort and materials. We report in this note on a modified method for colloidal chitin preparation from crab shell flakes that save effort and materials compared to some previous methods. Materials and Methods A modified protocol of Hsu and Lockwood (1975) was used for the preparation of colloidal chitin. Crab shell flakes (Neptune’s Harvest, MA, USA) were manually ground in a mortar and pestle for 5 minutes, then sieved through the top piece of 130 mm two piece polypropylene Buchner filter. Twenty grams of the sieved crab shell flakes were then treated with 150 ml of ~12M concentrated HCl (BDH Aristar) in a 1000 ml beaker. The HCl was added slowly with continuous stirring with the use of a glass pipette for 5 minutes, followed by stirring for 1 minute at an interval of every 5 minutes for 60 minutes in a chemical fume hood at room temperature (25 ° C). The chitin- HCl mixture was then passed through 8 layers of cheesecloth to remove large chitin chunks. The clear filtrate obtained (100 ml) was then treated with 2 liters of ice cold distilled water to allow precipitation of colloidal chitin. This was incubated overnight under static conditions at 4°C to facilitate better precipitation of colloidal chitin. This was later passed through two