Aims:  To purify and characterize an exo‐acting chitinolytic enzyme produced from a Gram‐negative bacterium Pseudomonas fluorescens JK‐0412. Methods and Results:  A chitinolytic bacterial strain that showed confluent growth on a minimal medium containing powder chitin as the sole carbon source was isolated and identified based on a 16S ribosomal DNA sequence analysis and named Ps. fluorescens JK‐0412. From the culture filtrates of this strain, a chito‐oligosaccharides‐degrading enzyme was purified to apparent homogeneity with a molecular mass of 50 kDa on SDS–PAGE gels. The kinetics, optimum pH and temperature, and substrate specificity of the purified enzyme (named as NagA) were determined. Conclusions:  An extracellular chitinolytic enzyme was purified from the Ps. fluorescens JK‐0412 and shown to be an exo‐type β‐N‐acetylglucosaminidase yielding GlcNAc as the final product from the natural chito‐oligosaccharides, (GlcNAc)n, n = 2–5. Significance and Impact of the Study:  As NagA is secreted extracellularly in the presence of colloidal chitin, Ps. fluorescens JK‐0412 can be recognized as a potent producer for industry‐level and cost‐effective production of chitinolytic enzyme. This enzyme appears to have potential applications as an efficient tool for the degradation of chitinous materials and industry‐level production of GlcNAc. To the best of our knowledge, this is the first report on an exo‐type chitinolytic enzyme of Pseudomonas species.

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