Purification and partial characterization of an intracellular NADH:quinone oxidoreductase from Phanerochaete chrysosporium

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Abstract
Summary: In order to study the genetic control of chitinolytic activity in Streptomyces, chitinase genes were cloned from S. lividans TK64 into the multicopy plasmid pIJ702 and their expression monitored in their natural host by measuring increases in chitinase productivity. Four independent clones were obtained, and the plasmids named pEMJ1, pEMJ5, pEMJ7 and pEMJ8. Restriction endonuclease digestion showed that although two of the plasmids (pEMJ7 and pEMJ8) shared a common DNA fragment, there were no substantial similarities between the inserts of plasmids pEMJ1, pEMJ5 and pEMJ7. This was confirmed by DNA-DNA hybridization studies. Four chitinases (A, B, C, and D) were identified, with molecular masses of 36, 46, 65, and 41 kDa, respectively. Production of chitinases A and B was specified by the plasmids pEMJ1 and pEMJ5, respectively. Genes for the other two chitinases (C and D) were carried by plasmid pEMJ7. Although significant differences were observed between chitinases A, B, and C in terms of optimum pH for activity and mode of digestion of substrates, chitinases C and D were very similar in these respects. Cloned genes were also expressed in S. coelicolor M130 and in Escherichia coli.

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