以不同驅動子共表現透明颤菌血紅蛋白對於D型胺基酸氧化酵素異源表現的影響

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

林文山(Wen-shan Lin)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

D-amino acid oxidase (DAO)是一種以FAD為輔基的flavoenzyme，為製造頭孢菌素類抗生素(cephalosporins)前驅物 7-aminocephalosporanic acid的重要酵素。Vitreoscilla hemoglobin (VHb)是透明颤菌處於微氧環境(低於10%的含氧量)下所產生的蛋白質，VHb的主要功能是擷取外界的氧氣，供應給終端氧化酶，作為電子接受者。 本研究建構了兩組VHb和DAO共同表現的重組質體，分別為(1)以啟動子S10驅動VHb基因和以啟動子T7驅動Rhodosporidium toruloides 或 Trigonopsis variabilis DAO cDNA基因的pSV-RTD或pSV-TVD (2)以啟動子nar驅動VHb基因和以啟動子T7驅動R. toruloides 或 T. variabilis DAO cDNA基因的pNV-RTD或pNV-TVD。使用30 ml LB broth於125 ml平底搖瓶中進行培養和誘導表現時，在以S10啟動的VHb共同表現下，R. toruloides DAO活性是無VHb表現的1.7倍，但在此培養條件下，VHb存在與否對T. variabilis DAO的表現卻沒有影響。以TB取代LB broth進行培養時，則在VHb存在下，R. toruloides 和T. variabilis DAO活性皆約提升1.3倍。調整LB broth和搖瓶的體積比成2:5或3:5，可使有VHb共同表現的R. toruloides DAO活性分別提升為無VHb表現的2.9和8.9倍。檢視誘導表現時菌液濃
II
度的影響發現，VHb的提升效應只出現在較高的誘導菌液濃度(OD600=1.5)。 以啟動子nar驅動VHb共同表現時，R. toruloides DAO活性是無VHb表現的1.6倍。同樣培養條件下，VHb的存在亦未提升T. variabilis DAO表現。相較於以啟動子S10驅動時，R. toruloides DAO的活性高峰於誘導後6小時出現即下降，以啟動子nar驅動VHb時，可使R. toruloides DAO的誘導表現高峰持續維持至誘導後20小時以上。

關鍵字

D型胺基酸氧化酵素 ； 透明颤菌

並列摘要

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.

並列關鍵字

VITREOSCILLA HEMOGLOBIN ； D-AMINO ACID OXIDASE

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