【摘要】：頭孢菌素C酰化酶是能將頭孢菌素C分子�；鶄�(cè)鏈水解形成7-氨基頭孢烷酸的酶。本研究將該酶基因轉(zhuǎn)化到大腸桿菌BL21(DE3)中,在優(yōu)化發(fā)酵培養(yǎng)基后,發(fā)酵酶活得到提高。在研究過程中,我們嘗試通過以下兩個途徑提高頭孢菌素C酰化酶的酶活。途徑一,利用透明顫菌血紅蛋白(VHb)能增強菌體對氧的攝取和利用能力,在微氧條件下促進細胞生長和代謝產(chǎn)物的合成,提高頭孢菌素C�；傅拿富�。本文將透明顫菌血紅蛋白基因(vgb)和頭孢菌素C酰化酶基因(CPCacy)組合在一起,轉(zhuǎn)化到大腸桿菌細胞中,探究VHb對CPCacy活性的影響。獲得轉(zhuǎn)化子pACYCDuet-1-vgb-CPCacy,其酶活比對照轉(zhuǎn)化子pACYCDuet-1-CPCacy高,但是依然比出發(fā)菌株pET-28a-CPCacy低。在對出發(fā)菌株產(chǎn)酶培養(yǎng)基優(yōu)化過程中,發(fā)現(xiàn)碳源成份甘油對酶活影響明顯,隨著甘油濃度增加,菌液OD600值出現(xiàn)類似二次生長的變化趨勢。在培養(yǎng)基成份中含有葡萄糖的情況下,菌體會優(yōu)先利用葡萄糖,此時其他碳源的利用會受到限制,若限制菌體對葡萄糖的攝取,可以促進其對其他碳源成份的有效利用。途徑二,通過P1噬菌體轉(zhuǎn)導(dǎo)的方法,置換大腸桿菌BL21(DE3)磷酸轉(zhuǎn)移酶系統(tǒng)中的葡萄糖特異性轉(zhuǎn)運蛋白ⅡCBGlc的編碼基因ptsG,減少菌體對葡萄糖的轉(zhuǎn)運利用,從而調(diào)動菌體其他代謝通路相關(guān)酶等蛋白的合成,提高菌體對培養(yǎng)基其他各種碳源成份的利用,增加發(fā)酵液的菌密度,提升外源蛋白的合成量,最終獲得更高的頭孢菌素C酰化酶合成量,提高發(fā)酵誘導(dǎo)后的酶活。獲得ptsG缺陷型菌株后,將四種表達載體pET-28a-CPCacy、pACYCDuet-1-CPCacy、pETDuet-1-CPCacy和pRSFDuet-1-CPCacy通過化學(xué)轉(zhuǎn)化法,轉(zhuǎn)入到無抗性、無ptsG基因的缺陷型BL21(DE3)菌株中,獲得四種工程菌。對比研究菌株ptsG基因缺陷對頭孢菌素C酰化酶活性的影響,發(fā)現(xiàn)四種工程菌的ptsG-型菌株的酶活均高于ptsG+型菌株的酶活,提升最為明顯的菌株為pRSFDuet-1-CPCacy(ptsG-),是其ptsG+型菌株的2.86倍。進一步的碳源成份優(yōu)化研究發(fā)現(xiàn),在LB培養(yǎng)基成份中,添加0.3%甘油或0.3%葡萄糖,均對四種工程菌的酶活有明顯的提升。在ptsG+型菌株中,添加葡萄糖成份對酶活的提升效果好于添加甘油的效果;而在ptsG-型菌株中,添加甘油成份對酶活的提升效果更加明顯。在菌株pACYCDuet-1-CPCacy(ptsG-)中,添加甘油成份后搖瓶發(fā)酵培養(yǎng)測得的酶活平均為4.197U/mL,是其ptsG+型菌株在未添加額外碳源的LB培養(yǎng)基中發(fā)酵測得酶活的6.59倍。本研究成功將ptsG-型菌株型BL21(DE3)用于表達頭孢菌素C�；傅鞍�,并且通過優(yōu)化碳源成份獲得明顯的效果,為研究提高大腸桿菌頭孢菌素C酰化酶的酶活開啟了一個新的途徑,同時,進一步優(yōu)化培養(yǎng)基的其他成份及培養(yǎng)條件等因素,有望更進一步提升CPCacy酶活,提高工業(yè)化發(fā)酵生產(chǎn)頭孢菌素C�；傅目尚行浴�
[Abstract]:Cephalosporin C acylase is an enzyme that hydrolyzes the acyl chain of cephalosporin C to 7-aminocephalic acid. In this study, the enzyme gene was transformed into Escherichia coli BL21 (DE3), and the fermentation enzyme activity was improved after optimization of fermentation medium. In the course of the study, we tried to improve the enzyme activity of cephalosporin C acylase through the following two ways. In the first way, (VHb) can enhance the ability of uptake and utilization of oxygen, promote cell growth and synthesis of metabolites, and increase the enzyme activity of cephalosporin C acylase. In this paper, the hemoglobin gene (vgb) and cephalosporin C acylase gene (CPCacy) were combined and transformed into Escherichia coli cells to investigate the effect of VHb on the activity of CPCacy. The enzyme activity of pACYCDuet-1-vgb-CPCacy, was higher than that of pACYCDuet-1-CPCacy, but still lower than that of the original strain pET-28a-CPCacy. During the optimization of the culture medium for enzyme production of the original strain, it was found that the carbon source glycerol had a significant effect on the enzyme activity, and with the increase of glycerol concentration, the OD600 value of the bacterial solution showed a similar trend of secondary growth. When the medium contains glucose, the bacteria will preferentially utilize glucose, and the utilization of other carbon sources will be restricted. If the uptake of glucose is restricted, the effective utilization of other carbon sources can be promoted. In the second way, the gene ptsG, encoding glucose specific transporter 鈪，

本文編號：2276187