| Bacillus pumilus UN-31-C-42 was obtained by mutations of strain BA(06), which is an alkaline protease producer and separated from life waste in ChengDu. The results of bith shaking and 100L fermentation experiments showed that the fermentation liquid has high dehairing and low collagen-degradation activity, indicating that the protease produced by this strain might be ideal enzyme agent for leather industry.When this strain was fermented, it produced alkaline protease after inoculation of 16h and the enzyme activity got the peak at 28h with 4200U/mL. Ammonium sulfate precipitation experiment showed that about 96% of alkaline protease was recovered in the 20-70% concentration. Although SDS-PAGE displayed that there were several protein bands in the sample after 16h fermentation, enzymatic active stain demonstrated that there was only one band during the whole fermentation period, indicating that there is only one kind alkaline protease in the fermentation liquid. The enzyme activity in fermentation liquid could be inhibited by PMSF and DFP. The fermentation liquor also showed good dehairing activity.The alkaline protease (named DHAP, dehairing alkaline protease) in the fermentation liquid was purified with hydrophobic interaction chromatography, ion exchange and gel filtration. The final yield was about 0.88% and the specific enzyme activity was increased 39-fold, with 17530 U/mg. The purified protease also displayed dehairing activity. In other words, DHAP can accomplish the whole process of dehairing by itself and without any other protease' s cooperation.DHAP had a pI of 9.0 and a-molecular weight of approximate 32,000 Dal ton. The protease has the maximal activity at pHIO and 55癈. The enzyme activity could be completely inhibited by PMSF and DFP, and partially inhibited by pepstatin A, EDTA and EGTA. Leupeptin, Benzamidine and hydrochloride Aprotin had no influence on the DHAP activity. Ca2*, Mg2+ and Na* could slightly increase, but Cu2+ and Zn2+ slightly inhibited protease activity. All these results demonstrated that DHAP is a serine protease.The first 20 amino acid residues of the purified DHAP were determined and the sequence is AQTVPYGIPQIKAPAVHAQG. The homologous analysis showed that the N-terminal sequence of DHAP is identical to that of serine protease from another B. pumilus strain TYO-67 and has high homology with those from other Bacillus species.Based on the homologous analysis, three pairs of primers were designed and synthesized. Three fragments, containing intact gene, whole and mature protease coding region ware amplified from total DNA of B. pumilus UN-31-O42 by polymerase chain reaction, respectively. After cloned into T-vector, the sequence of intact gene fragment with the size of 1.6 kb, was determined. The gene contains an open reading frame of 1149 nucleotides (Named Ap gene, encoding 383 amino acids), which contains a signal peptide consisting of 29 residues and a propeptide of 79 residues. The deduced 3 amino acid residues, D32, H64, and S221, were identical with 3 essential amino acids in the catalytic center of protease.Comparison of the entire derived peptide sequence with other serine protease revealed significant homology, especially with a reported gene from another B. pumilus strain TYO-67 (99% homology). The N-terminal amino acid sequence of deduced mature protease is identical to that of the purified alkaline protease. The sequence around these residues revealed that AP was a new member of the subtilisin family.The gene was expressed in Escherichia coli after cloned into pET-15b. SDS-PAGE showed the expressed product clearly, but no protease activity was detected.In order to express alkaline protease gene (AP gene) in Bacillus subtil is, the recombinant expression plasmid was constructed. This plasmid contains a promoter Bp53, also from B. pumilus UN-31-C-42, AP gene and the shuttle vector pSUGV4. After introduced into B. subtilis WB600, the transformants displayed the hydrolyzed zone on milk plate. The fermentation liquid of the transformant...
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