EFFECT OF VITREOSCILLA HEMOGLOBIN CO-EXPRESSION DRIVEN BY DIFFERENT PROMOTERS ON THE HETEROLOGOUS EXPRESSION OF D-AMINO ACID OXIDASE

• Main Authors: Wen-shan Lin, 林文山
• Other Authors: I-Ching Kuan
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/25580102137499673455

碩士 === 大同大學 === 生物工程學系(所) === 94 === D-amino acid oxidase (DAO), a flavoenzyme, using flavin adenine dinucleotide (FAD) as its prosthetic group is a key enzyme for the production of 7-aminocephalosporanic acid which is the precursor for synthetic cephalosporins. Vitreoscilla hemoglobin is a protein that Beggiatoaceae product in the little oxygen environment(lower than 10% of the oxygen content). The main function of VHb is picking and fetching the external oxygen, and supply oxygen to the terminal oxidase (subunit I). The function of oxygen is to be electron accepter. This research has constructed two groups of plasmids that VHb and DAO display together.(1) The plasmid pSV-RTD or pSV-TVD are using promoter S10 to urge VHb gene and using promoter T7 to urge Rhodosporidium toruloides or Trigonopsis variabilis DAO cDNA gene.(2) The plasmid pNV-RTD or pNV-TVD are using promoter nar to urge VHb gene and using promoter T7 to urge R. toruloides or T. variabilis DAO cDNA gene. While put 30 ml LB broth into 125 ml bottle to execute culture and to lead expression, the activity of R. toruloides DAO behave with VHb that urge by promoter S10 is 1.7 times than R. toruloides DAO behave without VHb. But under the same condition, T. variabilis DAO behave with VHb or not has no effect to its ativity. When using TB replace LB broth, the activity of R. toruloides and T. variabilis DAO that behave with VHb are both advance 1.3 times. Adjust the ratio of the LB broth and the volume of bottle to 2：5 or 3：5, the activity of R. toruloides DAO that behave with VHb is 2.9 and 8.9 times than R. toruloides DAO behave IV without VHb. Check the influence of bacterium concentration to DAO expression, the improvement effect of VHb only appears in higher bacterium concentration(OD600=1.5). When use promoter nar to urge VHb gene, the activity of R. toruloides DAO behave with VHb is 1.6 times than without VHb. In the same cultivation condition, VHb espression is not improve T. variabilis DAO expression and activity. Using promoter S10 to urge VHb gene, the peak activity of R. toruloides DAO appear in 6th hour after inducing, and then decline. Using promoter nar to urge VHb gene, the peak activity of R. toruloides DAO maintain 20 hours or more after inducing.