Keratinolytic protease genes in Bacillus pumilus strain C4 - Enhancement of extracellular protease production and molecular screening

Abstract

Optimization of extracellular protease production by Bacillus sp. C4was carried out using a twostep strategy One Variable at A Time (OVAT). On the basis of the results obtained from OVAT experiments, 1XSG medium supplemented with 0.2% yeast extract was the best medium formula for maximum protease production. An overall of 1.8-fold increase in protease production was achieved in the optimized medium as compared to the non-optimized one. The optimum predicted production conditions were as follows: beef extract (0.3%), peptone (0.5%), yeast extract (0.2%), glucose (0.1%), KCl (0.2%), FeSO4 (1mM), MgSO4 (2mM), MnCl2 (0.1mM), and CaCl2 (1 mM) and an incubation period of 24 hours. It was also found out that under the optimal conditions the extracellular protease production started four hours (04 hours) earlier than that produced in the nonoptimized conditions. A keratinolytic protease gene (ker1 gene) was isolated from a keratin-degrading Bacillus sp. C4 using PCR amplification and degenerate primers. Ker1 gene consists of an open reading frame of 1,152 bps, encoding a prepropeptide (polypeptide) consists of 383 amino acidres residues. The BLASTp analysis showed that the deduced amino acid sequence of ker1 gene has identity to many serine proteases from different Bacillus pumilis. This indicated that ker1 gene encodes one of the serine protease from Bacillus sp. C4. The three residues, found in all subtilisins, at the active site that form the catalytic triad (Asp-140, His-172 and Ser-329) were conserved in the deduced protein sequence of the ker1 gene. The ker1 gene was subsequently submitted to the NCBI GenBank (accession number: KX184831). Genome sequencing and analysis of the Bacillus sp. C4 was performed as an alternative approach to investigate further keratinase genes involved in keratin degradation. Bacillus sp. C4 genome was constructed as a single circular chromosome containing 3,659,360 bps with an average G+C content of 41.4% and no plasmids were detected. Annotation results indicated that Bacillus sp C4 belongs to the B. pumilus group of organisms. NCBI Prokaryotic genome annotation pipeline predicted a total of 3,698 genes, from which 3,596 proteincoding sequences (CDS), 71 RNA genes and 31 pseudogenes. The RNA coding genes predicted include 60 tRNAs, 10 rRNAs, and 1 non-coding RNA (ncRNA). The genome was found to be 99.6 % complete when analyzed. This whole-genome Bacillus pumilus C4 sequence has been deposited at NCBI GenBank under the accession number CP011109. The bioproject number was PRJNA278012. Eighty one candidate genes for peptidase were found overall in the draft genome. Out of them six (06) genes were annotated as putative peptidase S8 in which two genes were10 predicted also as subtilisins based on BASys annotation. The first one is the Ker1 gene (accession number: KX184831) previously isolated in this study. The second one is a homolog to Ker1 gene and was named Ker2 gene. This gene was subsequently submitted to NCBI GenBank (Accession Number: KX184832). The ORF of this gene has an equal amount of base pairs as Ker1 gene. The two genes had a gene sequence similarity of 67% using BLASTn at NCBI. The deduced amino acid sequence of ker2 gene of B. pumilus C4 contained 383 amino acid residues and the three residues at the active site that form the catalytic triad in all subtilisins are Asp-140, His-172 and Ser-329. Results of PCR amplification, de novo sequencing, and genome annotation indicated that the protease producer and keratin degrading Bacillus pumilus C4 has at least two genes encoding subtilisin-like serine proteases. The broad substrate specificity and keratinolytic activity towards both type of keratin of this strain could originate from the presence of this high number of putative peptidases and proteases. Identification of some protease enzymes produced upon growing Bacillus pumilus C4 on optimized medium was done using nLC-ESI-MS/MS followed by Mascot database search. Mascot Search results indicated that the partial purified protease fraction in fact consists of two enzymes, corresponding to the previously identified genes Ker1 and Ker2 of the B. pumilus strain C4. This confirmed that Bacillus pumilus C4 produced at least two protease enzymes that could be related to its remarkable keratinilytic activity towards both type of keratins.