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Recombinant Aspergillus japonicus cysteine-S-conjugate β-lyase to releases thiol aroma compounds

學生姓名: 張伸維
指導教授: 林泓廷
學期: 111下
摘  要: Volatile thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), 4- mercapto-4-methyl-pentan-2-one (4MMP) are powerful aromatic compounds, fungi rely on its metabolic versatility plays an important role in the food industry process, so the purpose of this study is to screen out the cysteine-S-conjugated β-lyase genes related to the release of volatile thiols from mold species, through recombinant protein technology allows it to be expressed in large quantities to generate the target protein, and explores the ability of this protein to produce sulfur-containing aroma compounds. Cysteine-S-conjugated β-lyase mainly exists in the form of tetramer, which is an enzyme containing pyridoxal 5′-phosphate, PLP cofactor, it can catalyze β-elimination reaction of cysteine-S-conjugated electron-withdrawing groups on sulfur, the final products are pyruvate, ammonium and sulfur-containing compounds, which contribute greatly to the aroma , in view of their high concentration and low threshold in food, but in the process of natural fermentation, the conversion rate of enzymes to convert odorless precursors into aroma compounds is very low, so it needs to be improved by transformation, through Prokaryotes produce a large number of target enzymes to elevate convertion efficiency. After using the protein database Uniprot and BLAST protein alignment tools for comparison, Aspergillus japonicus was selected as the gene source of this enzyme. After DNA molecular extraction and PCR amplification, which express in the E. coli C43 system, induce and purify the protein by using ÄKTA fast protein liquid chromatography, FPLC. Based on the above results, through subsequent quantitative, qualitative and enzyme activity analysis tests, the protein identity of the recombinant enzyme can be identified and compared, also can test its ability to produce sulfur compounds. Expecting to apply this technology in the spice market, providing a new option for the production of spices.
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