Chitinolytic activity of highly halotolerant Streptomyces tendae against Fusarium oxysporum PTK2

Abstract

Fifteen (15) highly halotolerant Streptomyces isolates were isolated from saline soil of Teachers College garden in Riyadh city. Chitin nitrate agar medium containing 20% (w/v) NaCl was used for the isolation purpose. These chitinolytic Streptomyces isolates were purified and sub-cultured on chitin nitrate broth medium containing 20% NaCl (w/v). The dry weight and absorbance (585 nm) were measured for each isolate to determine the best chitinolytic isolate. The best chitinolytic isolate was symbolized HS-5 which in turn was identified as S. tendae using morphological, physiological and biochemical characteristics as well as 16S rRNA gene partial sequence. The later revealed that there was 98% similarity between isolate HS-5 and S. tendae M23 which has accession number HM594286.1 as in Gene Bank. Optimization was studied to produce the maximum yield of chitinase enzyme. High chitinase productivity was obtained at 1.6 × 10⁷ CFU ml^-1 inoculum size, 3rd day incubation period, 35°C incubation temperature, 8.5 pH value and eventually with casein; was the best nitrogen source. Chitinase enzyme was precipitated from the filtrate of S. tendae at 50% saturated ammonium sulphate and purified using sephadex G200 column chromatography. Chitinase enzyme was separated at 20 KDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by protein gel electrophoresis, and stained by coomassie blue dye. Amino acids of chitinase enzyme and their concentrations were determined using High-performance liquid chromatography (HPLC) device.

Key words: Chitinolytic activity, enzyme purification, halophytic actinomycetes, soil salinity.