Study on characterization of chitinase from streptomyces

• Main Author: Nguyễn, Thanh Hương
• Other Authors: Dương, Văn Hợp
• Format: Theses and Dissertations
• Language: English
• Published: 2016
• Subjects: Enzym thủy phân; Thuốc kháng sinh; Chất kitin; Hóa sinh học; Đặc tính
• Online Access: http://repository.vnu.edu.vn/handle/VNU_123/15684
• Institution: Vietnam National University, Hanoi

In this study, a total of 500 Streptomyces strains isolated from soil in Hoang Lien Son national park (Sa Pa, Vietnam) were subjected to a screening for their chitinase activities. Through two screening steps, Streptomyces strain VN08-A0438 had the highest chitinase activity so it was selected for next studies. Taxonomical studies based on the morphology, physiological criteria and 16S rDNA gene sequencing indicated that strain VN08-A0438 was belonging genus Streptomyces and was proposed as Streptomyces chromofuscus. Besides that, selecting conditions for chitinase production from strain VN08-A0438 were studied, focused on some key factors on chitinase production: optimum temperature, pH, aeration, fermentation time, carbon and nitrogen sources. The culture grew well on medium with carbon source as glucose - 5 g, colloidol chitin 5 g and nitrogen source as (NH4)2SO4 - 2 g, at 35oC, pH 6.5 with shacking rate 200 rpm for 5 days. Chitinase from Streptomyces sp. VN08-A0438 was purified by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration. Treatment of chitinase (80% ammonium sulfate saturation) gave highest specific activity (40U/mg protein). The high chitinase activity was found in fractions from 45 to 70. The sample was concentrated by evaporation at room temperature to 10 folds, and loaded on SDS-PAGE and activity gel. Characterization of the partly purified chitinase was also checked, including effect of pH, temperature, and Thin layer chromatography (TLC) for detecting the enzymatic product. Enzyme was stable at pH 5-5.5 and 55oC. The TLC chromatogram showed that there were a number of three enzymes involved: endochitinase with chitobias and chitinooligosacharide as the main products, exochitinase with N-acetyl glucosamine and chitinooligosaccharide as the main products, chitobiase with N-acetyl glucozamin as the final products.