Chitinolytic enzyme production and genetic improvement of a new isolate belonging to Streptomyces anulatus

Abstract

Thirty bacterial isolates were obtained from different sources and sites at Jeddah, Saudi Arabia, on chitin agar medium; 9 of the 30 isolates were cultured in liquid medium containing chitin as sole carbon and nitrogen sources. Isolate SM21, which was isolated from shrimp shells, showed the best growth and chitinase production in liquid medium. According to its morphological, physiological and biochemical characteristics, SM21 belongs to the genus Streptomyces and was identified as Streptomyces anulatus SM21. Identification was confirmed using 16S rDNA analysis. The chitinase enzyme was precipitated with 80% NH4SO4 and purified using DEAE-cellulose ion exchange chromatography followed by Sephadex G-100 gel filtration. The molecular weight determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis was 28 kDa. Genetic improvement using the protoplast fusion technique was carried out between the identified Streptomyces isolate and Streptomyces coelicolor SM1. These two species, which have different resistance profiles to streptomycin and tetracycline (400 μg/ml and 10 μg/ml, respectively), were used in an intraspecific protoplast fusion using PEG 6000. The percentage of real protoplasts that could regenerate successfully was 71% for S. coelicolor SM1 and 80% for S. anulatus SM21. Out of three recombinant fusants obtained, one (named Fu3) showed higher chitinase production compared to both parents (5 fold increase).