High performance liquid chromatography analysis and quantitation of chitinolytic products of CDC group EF-4a

Abstract

A chitinolytic organism was cultured from a lotic habitat in central Indiana. It was isolated on the basis of its hydrolytic activity on chitin agar. It was subsequently identified as EF-4a. The chitinases of the EF-4a were isolated, purified and concentrated. A colloidal chitin suspension was then incubated with the enzyme. Samplings were taken at one minute, one hour and 24 hours. The samplings were filtered through a Swinney filtration unit and immediately after applied to a Beckman Ultrasil Amino column. It was eluted with acetonitrile: water (75:25) at a flow rate of 2 ml/min. The absorbance at 214 nm was monitored using the Beckman 160 UV detector. The chromatographs were analyzed for product elaboration. It was observed that EF-4a produced soluble, extracellular enzymes. The enzymes were a chitinase-chitobiase complex. The enzymes' action on chitin was immediate. There were varying reaction products including G1cN, GlcNAc, chitobiose, chitotriose and higher order oligomers. There was also chromatographic evidence suggesting a and B anomers of these saccharides.