Screening Of A New Bacillus Pumilus Strain Producing Dehairing Protease And The Cloning And Expression Of Its Alkaline Protease Gene

The leather industry causes severe environmental pollution owing to the use of various chemicals and the release of a variety of detrimental materials, despite making significant contribution to the economy. The conventional dehairing process involves the use of high proportions of lime and sulfide, which contributes 80-90% of the total pollution in the leather industry. The application of protease to dehairing would eliminate the need for hazardous chemicals, and thus reduces the pollution load.Based on the enzymatic specificity for dehairing requirements, attempts have been made to develop novel enzymes. The samples collected from proteinaceous wastes were spread on casein plates for screening protease producers. One hundred and seventy-one colonies with clear zones formed by the hydrolysis of casein were obtained. Six isolates exhibited protease activity as higher than 500 U/mL. The fermentation supernatant of these 6 strains was used to remove hairs from skins. The protease produced by isolate BA06 showed prominent dehairing quality comparing with other samples. Based on the morphological, physiological characteristics and 16S rDNA sequence analysis, the strain BA06 was identified as Bacillus pumilus. The secreted protease content in the BA06 culture supernatant was further examined by casein-SDS-PAGE and the result revealed that there was only one perceptible hydrolyzed casein band with molecular weight of around 30 kDa. To increase the protease productivity of BA06, the strain was subjected to a combined mutagenesis of UV, NTG and Co⁶⁰-γ-rays irradiation. Two mutant strains UN-31-C-42 and SCU11, showing alkaline protease activity of 4,200 and 6,000 U/ml, respectively, were obtained. The sequence alignment showed that the sequences of alkaline protease gene from these three strains were identical.The DNA coding for the pro- and mature peptide of alkaline protease (Apr) from B. pumilus was cloned and inserted into pET32a to test the expression in Escherichia coli. The recombinant plasmid expressed protease in the form of Trx-pro-Apr, which appeared as inclusion body. The fused protein was not folded correctly even with refolding process. The alkaline protease gene was further expressed in Bacillus. The native promoter of alkaline protease gene, named Papr, was first cloned from B. pumilus through thermal asymmetric interlaced PCR. P43 promoter was then cloned from B. subtilis. The Papr and P43 promoters were fused with the coding region of alkaline protease gene, respectively, resulted in the expression plasmids pSUBpWAp and pP43AP for Bacillus. The two plasmids were transformed into B. subtilis WB600 and protease activity was detected extracellularly. The plasmid pSUBpWAp was also introduced into B. pumilus UN-31-C-42. However, the level of protease production from the engineered strain was not improved, compared with the parent strain, although the plasmid was stable in the host cells. The plasmid pSUBpWAp was found structural instable in B. subtilis WB600. The plasmid deletion was frequently occurred in the host cell during cultivation, and sequentially, no protease activity was detected extracellularly. We deduced that the plasmid instability was associated with the over-expression of heterogeneous protease gene directed by Papr promoter in B. subtilis WB600. In addition, the plasmid instability was further influenced by the properties of the host strain.In order to further improve the properties of alkaline protease to satisfy the applications in leather industry, the alkaline protease gene was applied to directed evolution. In such process, the protease-coding gene was subjected to random mutagenesis and a library with large number of mutated genes should be constructed in B. subtilis. Thus a simple and efficient method for transforming library DNA into B. subtilis is highly required. To determine whether there are differences among E. coli strains for producing multimeric plasmid, the plasmid DNA was prepared from E.coli BL21 (DE3), DH5α, JM109 and Top10. The result revealed that an adequate amount of plasmid multimers were formed in E. coli DH5αand BL21 (DE3), which resulted in a 10-fold increase in transformation frequency of B. subtilis. Special emphasis was made on two parameters of practical importance in transformation of B. subtilis: the time of exposure of competent cells to DNA and the recovery of the transformed cells. Under the optimized transformation procedure, the transformation frequency of B. subtilis WB600 by plasmid pSUGV4 was usually 1-2×10⁴ transformants /μg plasmid, with the highest rate of 6×10⁴/μg DNA. Further more, some other Bacillus strains could be transformed by using this method.The coding region of alkaline protease gene and its 3' flanking terminator region from B. pumilus were subjected to random mutagenesis by error-prone PCR. The mutant library was firstly established in E. coli, and then introduced into competent B. subtilis WB600. The cells of B. subtilis library were spread on skim-milk plates and the mutants with increased protease activity were judged according to their halo-forming activity. One candidate mutant, termed MAp-37, was selected by its highly enlarged clear zone on skim-milk plates comparing with that of wild-type gene. However, the extracellular protease activity of MAp-37 was markedly lower than wild-type. DNA sequencing revealed that MAp-37 possessed 16 nucleotide mutations, 12 in coding region and 4 in terminator region. The mutations in coding region resulted in two amino acid substitutions in propeptide and two in mature peptide. To analyze the contribution of each mutation, we then combined the mutated and wild-type alkaline protease gene in different ways and the expressions of the recombinant genes were detected. The results suggested that the synergistic effect of mutations in propeptide, mature peptide and terminator was critical to the special property of MAp-37.