發(fā)布時間：2018-05-29 10:12

  本文選題：原兒茶酸3 + 4-雙加氧酶　； 參考：《華東理工大學(xué)》2017年碩士論文

【摘要】：原兒茶酸代謝途徑在芳香化合物的生物降解中起著關(guān)鍵性的作用,原兒茶酸3,4-雙加氧酶(P34O)是β-酮己二酸途徑中的原兒茶酸分支途徑催化芳香化合物的開環(huán)步驟的關(guān)鍵酶。首先本研究以根瘤菌屬(Rhizobium sp.LMB-1)、不動桿菌屬(Acinetobacter sp.LMB-5)的基因組為模板擴(kuò)增得到編碼P34O大小均約為1300 bp的基因片段。此外由重組菌LMB-1/proP34O和LMB-5/proP34O表達(dá)的兩重組P34O均由兩類亞基組成,分子量與預(yù)測大小接近。為了分析重組P34O的特性,考察了兩重組P34O的酶學(xué)特性差異,結(jié)果表明兩重組P34O的最適pH均為7.5,重組LMB-1/proP34O的P34O最適溫度為60℃,而重組LMB-5/proP34O 的 P34O的最適溫度為 50 ℃,在 60 ℃下孵育 450 min,LMB-1/proP34O的重組酶的相對活力緩慢降低,而LMB-5/proP34O的酶在60 ℃僅孵育15 min,酶活力降至20%。在本研究中,只有Fe3+對重組LMB-l/proP34O的P34O有激活作用,而Fe3+,Al3+和Mg2+都對重組LMB-5/proP34O的P34O有激活作用。此外,在芳香化合物鄰苯二甲酸酯(PAEs)的誘導(dǎo)下,兩株野生菌中編碼P34O的pcaG及pcaH基因的轉(zhuǎn)錄水平均有所提高,由此表明P34O的基因表達(dá)可受到一些芳香化合物的誘導(dǎo)。為進(jìn)一步提高酶的理化性質(zhì),以磁性納米顆粒Fe_3O_4為載體通過戊二醛共價交聯(lián)法固定LMB-1/proP34O的P34O并對其酶學(xué)性質(zhì)進(jìn)行研究。結(jié)果表明固定化酶比游離酶的最適溫度提高了10℃,而兩者最適pH均為7.5,且固定化酶表現(xiàn)出更優(yōu)越的熱穩(wěn)定性。此外,由反應(yīng)動力學(xué)參數(shù)測定結(jié)果知,固定化酶的Km,Vmax,Kcat值分別為159.29μM,19.16 μM/min和958/min,而游離酶的對應(yīng)的值分別為65.34 μM,12.29μM/min和615/min。同時,以磁性納米粒子Fe_3O_4為載體的固定化P34O重復(fù)利用10次仍保留64%的相對酶活。
[Abstract]:Protocatechuic acid metabolism pathway plays a key role in the biodegradation of aromatic compounds. Protocatechuic acid 3-dioxygenase (P34O) is the key enzyme in the catalytic ring opening of aromatic compounds by the protocatechuic acid branching pathway of 尾 -keto-adipic acid pathway. Firstly, the genome of Rhizobium sp. LMB-1 and Acinetobacter sp. LMB-5) was used as a template to amplify the genome of Rhizobium sp. LMB-5. The gene fragment encoding P34O was about 1300 BP in size. In addition, the two recombinant P34O expressed by LMB-1/proP34O and LMB-5/proP34O were composed of two subunits, and the molecular weight was close to the predicted size. In order to analyze the characteristics of recombinant P34O, the differences of enzymatic properties between the two recombinant P34O were investigated. The results showed that the optimum pH of the two recombinant P34O was 7.5, the optimum temperature of the recombinant P34O was 60 鈩，

本文編號：1950493