Cloning of aMicrobispora bisporacellobiohydrolase gene inStreptomyces lividans

✍ Scribed by P. Hu; T. Chase; D. E. Eveleigh

• Publisher: Springer
• Year: 1993
• Tongue: English
• Weight: 780 KB
• Volume: 38
• Category: Article
• ISSN: 1432-0614
• DOI: 10.1007/bf00182802

✦ Synopsis

The cellobiohydrolase II (CBHII) of Microbispora bispora, originally cloned in Escherichia coli, was subcloned into Streptomyces lividans using shuttle vectors pSKN 01 and pSKN 02. The enzyme was secreted from Streptomyces, whereas it was intracellular in E. coli. The yields of CBHII produced by S. fividans transformants were 15-20-fold higher than those produced by E. coli transformants. The optimal pH of M. bispora native cellobiohydrolase and the cloned enzyme from S. lividans is 6.5. The thermal and pH stability of CHBII produced in M. bispora, E. coli and S. lividans were compared. Enzyme produced in E. coli was inactivated more rapidly (k = 0.252 rain -1 at 90 ° C; 90% inactivation after 10rain vs. 0.119rain -1 for the others). CBHII was monitored following electrophoretic separation by reaction with a monoclonal antibody. The apparent molecular mass of the protein produced from the S. lividans clone was 93 kDa, the same as that of the native enzyme, but that of the enzyme produced in E. coil was smaller (82 kDa).