| Summary: | 碩士 === 國立中正大學 === 化學工程研究所 === 105 === Oil crops are raw materials for manufacturing biodiesel by transesterification. After oil extraction, the residues in oil crops are rich in protein, starch, cellulosic fiber and other polysaccharides, which still have economic values for recycling. To develop a variety of enzymes for converting the remaining residues to animal feeds and improving the palatability of animal feeds is helpful for reducing the overall manufacturing cost of biodiesel.
We have previously showed that the alkaliphilic gram-positive bacterium Bacillus halodurans can secrete thermophilic and alkaliphilic xylanases. As indicated in the gene bank, this microorganism is also able to synthesize a variety of proteolytic enzymes. In the present work, the asp gene encoding for extracellular alkaline serine protease from Bacillus halodurans Thonburi was cloned using pET-15b as the plasmid vector and over-expressed in E. coli BL21 (DE3). The recombinant protein was found in the extracellular space without the formation of inclusion body. The keratinolytic activities were assayed on the recombinant proteolytic enzyme, which will be used for the enzymatic treatment of residues from oil crops to produce animal feeds.
The recombinant protein encoded by asp was found mostly in the culture medium. As the keratinolytic activity was assayed on the recombinant proteolytic enzyme encoding by asp, the proteins in the medium contributed about 89.38±3.12% % of total enzymatic activity obtained from the recombinant cell. Results suggested that the original signal peptide worked well for protein secretion in the E. coli system. In addition, this extracellular alkaline serine protease was confirmed to be a keratinase. Using soluble keratin as the substrate, the enzymatic activity in the medium was determined as 1124.6 ± 314.6 U/ml with a specific activity of 388.7 ± 96.8 U/mL suggesting that the extracellular proteolytic enzyme is a keratinase and very potential for application.
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