Enhanced extracellular chitinase production in <em>Pseudomonas fluorescens</em>: biotechnological implications

Abstract

Chitin is an important renewable biomass of immense commercial interest. The processing of this biopolymer into value-added products in an environmentally-friendly manner necessitates its conversion into N-acetyl glucosamine (NAG), a reaction mediated by the enzyme chitinase. Here we report on the ability of the soil microbe Pseudomonas fluorescens to secrete copious amounts of chitinase in the spent fluid when cultured in mineral medium with chitin as the sole source of carbon and nitrogen. Although chitinase was detected in various cellular fractions, the enzyme was predominantly localized in the extracellular component that was also rich in NAG and glucosamine. Maximal amounts of chitinase with a specific activity of 80 µmol NAG produced mg–1 protein min–1 was obtained at pH 8 after 6 days of growth in medium with 0.5 g of chitin. In-gel activity assays and Western blot studies revealed three isoenzymes. The enzyme had an optimal activity at pH 10 and a temperature range of 22–38 ℃. It was stable for up to 3 months. Although it showed optimal specificity toward chitin, the enzyme did readily degrade shrimp shells. When these shells (0.1 g) were treated with the extracellular chitinase preparation, NAG [3 mmoles (0.003 g-mol)] was generated in 6 h. The extracellular nature of the enzyme coupled with its physico-chemical properties make this chitinase an excellent candidate for biotechnological applications.

Keywords

• biotechnology
• extracellular chitinase
• microbial system
• N-acetylglucosamine
• shrimp shell degradation