Purification,characterization and cloning of an endo-1,4-B-glucanase fromRuminococcus sp. |
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Authors: | Sudeep Kumar Srivastava Arif Ali Sunil Khanna |
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Institution: | (1) Microbiology and Molecular Genetic Unit, Tata Energy Research Institute, 7 Jor Bagh, 110003 New Delhi, India;(2) Department of Biosciences, Faculty Of Natural Sciences, Jamia Millia Islamia, 110 025 New Delhi, India |
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Abstract: | Endoglucanase ofRuminococcus sp. is composed of seven active protein components when chromatographed on an ion exchange column (Q-Sepharose). Component I (endoglucanase A) did not bind to the column and was purified to homogeneity by molecular sieve chromatography. It had a mol. wt. of 22 000. Component II was fractionated into two active protein peaks (endoglucanase B and C) having mol. wt. of 225 000 and 10 000. The endoglucanase A had high affinity for CMC (Km 8 mg/ml). The temperature optimum of all three endoglucanase was between 40–45°C. The gene encoding for endolucanase activity was cloned inE. coli HB101 with pBR322. A 4.3 kilobaseBamH1 fragment encoding endoglucanase was hybridized toRuminococcus chromosomal DNA. |
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