This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van der Laan, J C Articles by Quax, W J Search for Related Content PubMed PubMed Citation Articles by van der Laan, J C Articles by Quax, W J Agricola Articles by van der Laan, J C Articles by Quax, W J

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1991 April; 57(4): 901-909

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene.

Royal Gist-brocades N.V., Research and Development, Delft, The Netherlands.

ABSTRACT

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.

This article has been cited by other articles:

• Gilmour, R., Messner, P., Guffanti, A. A., Kent, R., Scheberl, A., Kendrick, N., Krulwich, T. A. (2000). Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively Alkaliphilic Bacillus pseudofirmus OF4 Lead to Characterization of an S-Layer Protein with a Role in Alkaliphily. J. Bacteriol. 182: 5969-5981 [Abstract] [Full Text]  
• Rao, M. B., Tanksale, A. M., Ghatge, M. S., Deshpande, V. V. (1998). Molecular and Biotechnological Aspects of Microbial Proteases. Microbiol. Mol. Biol. Rev. 62: 597-635 [Abstract] [Full Text]