Screening Of A Thermostable Alkaline Protease Producing Strain GXD9902 And Cloning Of Its AprN Gene

Alkaline proteases (or subtilisins, E.C.3.4.21.14) are a physiologically and commercially important group of enzymes. It has been apply universally to industrial of detergents, leather processing and silk. Besides these application, it has a wonderful prospect of application potential in medical purpose.. food processing., feeds., silver recovery,chemical industries and environment protection, as well as basic research. A thermostable alkaline protease producing strain, named GXD9902, was isolated from samples of the saline lands in the northwest of China. On the basis of observing of modality and the analysis of the 16S rDNA sequence, GX0992 was identified as Bacillus subtilis primarily. A 1.3 kb fragment which homologous to aprN gene encoding alkaline proteases of Bacillus subtilis, was amplified by PCR, using conservative sequence of aprN gene as primers. The 1.3 kb fragment was sequenced. A open reading frame(ORF) encoded 381 amino acids was identified from this fragment and was shared 90% homology with reported aprN gene. Two recombinant plasmids, named pUCM-A and pGEM-B, were obtained by ligatied the 1.3 kb fragment with pUCM-T and pGEM3zf(+) vector respectively and electroporated into E.coli strains DH5α. It was shown that this aprN gene can express in E.coli strains DH5α.