Biodegradation of chitinous substances and chitinase production by the soil actinomycete Streptomyces rimosus

Abstract

A chitinolytic actinomycete was isolated from agricultural soil in the centre of Poland. It was identified as Streptomyces rimosus. Identification was based on standard biochemical tests and on 16S rRNA gene sequence analysis. The degradation of chitinous substances (shrimp shell waste, crab chitin powder and chitosan) was examined on the basis of oxygen consumption by applying OxiTop (WTW). The enzyme catalyzed the hydrolysis of the disaccharide 4-MU-(GlcNAc)3 most efficiently and was therefore classified as an endochitinase. The chitinase was purified from culture medium by fractionation with ammonium sulfate and affinity chromatography. The purified proteins were subjected to identification by mass spectrometry. Based on analysis of the resultant fragments of the amino acid sequence of chitinase produced by the examined bacteria, degenerate primers for PCR were designed. The results of our research prove that the substances which are metabolised by S. rimosus the most efficiently are chitosan and the shrimp shell waste. Chitinolytic activity examination showed that shrimp shell waste was the finest inducer of chitinase synthesis. The molecular mass of the purified enzyme was 63 kDa. The highest activity of chitinase was obtained at the temperature of 40°–45 °C. Optimal pH for chitinase activity was 7.0. The activity of the purified chitinase was stabilized by Mg2+ ions. The activity of the enzyme was inhibited by Hg2+ and Pb2+ ions. Chitinase inhibited the growth of the fungal phytopathogens Fusarium solani and Alternaria alternata. The nucleotide sequence of the amplified gene fragment proved to be similar to the sequence of chitinase-encoding genes of the glycoside hydrolases family 18.