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Protease secretion capacity and perforce analysis of recombinant Bacillus species

Çalık, Pınar
Kalender, N
Ozdamar, TH
Recombinant Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. subC gene was amplified from the chromosomal DNA of the wild-type Bacillus licheniformis by using PCR technology; thereafter, subC gene was first cloned into the pRS316 E. coli yeast shuttle plasmid, then sub-cloned into the pHV1431 E. coli-Bacillus shuttle vector, and transferred to the host Bacillus species, i.e. Bacillus licheniformis, Bacillus alvei, Bacillus firmus, Bacillus cereus, Bacillus subtilis, Bacillus badius, Bacillus sphaericus and Bacillus coagulans. pHV1431::subC was transferred to B. licheniformis and B. coagulans by electroporation, and to the other six hosts by free-transformation. Bioreactor experiments were conducted with the wild-type and recombinant Bacillus species in order to compare their production potentials. By cloning the pHV1431::subC, SAP activity increased, 2-fold in r-B. licheniformis, 26- fold in r-B. alvei, 71- fold in r-B. badius, 51- fold in r-B. cereus, 32- fold in r-B. firmus, 33- fold in r-B. subtilis, 105- fold in r-B. sphaericus and 14- fold in r-B. coagulans, in a defined medium with glucose as the sole carbon source at t=43 h of the fermentations. Physiological differences and similarities between the wild-type and recombinant Bacillus species are discussed.