Gene Cloning and Expression of an Alkaline Serine Protease with Dehairing Function from Bacillus pumilus

Abstract

A new gene (named AP gene) encoding an alkaline serine protease with dehairing function was cloned from Bacillus pumilus UN-31-C-42 and its nucleotide sequence was determined. The expression of AP gene was induced with IPTG in Escherichia coli after the mature protease region was cloned into pET15b and SDS-PAGE showed expressed product clearly, but no alkaline protease activity was detected. In order to express the AP gene in B. subtilis, a recombinant expression plasmid was constructed which contained a promoter Bp53 (also from B. pumilus), the AP gene and an E. coli-B. subtilis shuttle vector pSUGV4. This plasmid was introduced into B. subtilis WB600 and the transformant displayed the hydrolyzed zone on a milk plate. The expressed product can be easily detected with SDS-PAGE and the fermentation fluid of the transformant showed low alkaline protease activity and dehairing activity. This is the first report of a gene cloned from B. pumilus, encoding an alkaline serine protease, which can alone accomplish the whole dehairing process.

Single amino acid mutation alters thermostability of the alkaline protease from <italic>Bacillus pumilus</italic>: thermodynamics and temperature dependence

“…The recombinant plasmid pSUBpAp [19], hosting the full-coding region of DHAP, was served as the template for site-directed mutagenesis. The mutagenesis was performed following the QuikChange method (Stratagene, Cedar Creek, USA) according to manufacturer's instruction with minor modification.…”

“…DHAP is encoded by the strain of B. pumilus BA06, and consists of 382 amino acids with 107 amino acids at the N-terminus as pre-pro-sequence [19]. The active form of DHAP is a single chain composed of 275 amino acids [19].…”

“…The active form of DHAP is a single chain composed of 275 amino acids [19]. Since DHAP belongs to the subtilisin family Q, it has the conserved catalytic triad (Asp32, His64, and Ser221) and oxyanion hole (Asn155) [10].…”

A new organic solvent tolerant protease from Bacillus pumilus 115b

“…Protease from B. pumilus 115b is tolerant to organic solvents and as far as we know, is the first of such protease reported from this Bacillus species. Meanwhile, alkaline serine protease from B. pumilus UN-31-C-42 was reported to posses a high dehairing activity and low collagen-degradation [15] while alkaline serine protease from B. pumilus TYO-67 was used in coagulating soybean milk [20]. Both proteases were not reported to be organic solvent tolerant enzymes.…”

Draft Genome Sequence of Bacillus pumilus BA06, a Producer of Alkaline Serine Protease with Leather-Dehairing Function

“…B. pumilus BA06, isolated from the proteinaceous soil, is able secrete a major alkaline serine protease that shows good potential in leather dehairing (8,17). The gene encoding this protease was cloned and expressed in B. subtilis (WB600) (11). In order to get deep insights into transcriptional regulation and for comparative genome analysis, we sequenced the genome of B. pumilus BA06 using Illumina HiSeq 2000 at Shenzhen Huada Genomics Institute (China).…”