Purification and characterization of the CelB endoglucanase from Streptomyces lividans 66 and DNA sequence of the encoding gene.
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ABSTRACT:
SUBMITTER: Wittmann S
PROVIDER: S-EPMC201545 | biostudies-other | 1994 May
REPOSITORIES: biostudies-other
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Purification and characterization of the CelB endoglucanase from Streptomyces lividans 66 and DNA sequence of the encoding gene.
Wittmann S S Shareck F F Kluepfel D D Morosoli R R
Applied and environmental microbiology 19940501 5
The endoglucanase CelB isolated from culture filtrates of Streptomyces lividans IAF9 has an M(r) of 36,000. With carboxymethyl cellulose as the substrate, the Vmax and Km values are 110 IU/mg of enzyme and 1.3 mg/ml, respectively. Comparison of primary amino acid sequences classifies CelB in the H family of cellulases. ...[more]
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Project description:The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.
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Project description:The gene encoding an alpha-L-arabinofuranosidase (abfA) was homologously cloned in Streptomyces lividans and its DNA sequence was determined. The enzyme was purified from the cytoplasm of the hyperproducing clone S. lividans IAF116. Its M(r) was estimated by gel filtration and found to be approx. 380,000. Since SDS/PAGE indicated a native protein of M(r) 69,000, it can be concluded that the native protein consists of several subunits of that size. The pI value was 4.6. The kinetic constants determined with p-nitrophenyl alpha-L-arabinofuranoside as substrate were a Vmax of 180 units/mg of protein and a Km of 0.6 mM. The specific activity of the purified enzyme on this substrate was 153 units/mg of protein. Optimal enzyme activity was obtained at 60 degrees C and pH 6.0. The enzyme cleaved p-nitrophenyl alpha-L-arabinofuranoside, but had no activity on a variety of other p-nitrophenyl glycosides, except on p-nitrophenyl beta-D-xylopyranoside. The enzyme showed no activity on oat-spelts (Avena sativa) xylan or arabinogalactan, but acted on beet (Beta) arabinan or arabinoxylan. Hydrolysis occurred on arabino-oligoxylosides obtained from oat-splets xylan after digestion with xylanases. Since S. lividans normally does not secrete arabinofuranosidase, this enzyme may play a role in the assimilation of arabinose moieties from arabinose-containing xylo-oligosaccharides generated by beta-xylosidases or xylanases.
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