ptsG基因缺陷型大肠杆菌对头孢菌素C酰化酶基因表达的影响
发布时间：2018-10-17 09:10
【摘要】：头孢菌素C酰化酶是能将头孢菌素C分子酰基侧链水解形成7-氨基头孢烷酸的酶。本研究将该酶基因转化到大肠杆菌BL21(DE3)中,在优化发酵培养基后,发酵酶活得到提高。在研究过程中,我们尝试通过以下两个途径提高头孢菌素C酰化酶的酶活。途径一,利用透明颤菌血红蛋白(VHb)能增强菌体对氧的摄取和利用能力,在微氧条件下促进细胞生长和代谢产物的合成,提高头孢菌素C酰化酶的酶活。本文将透明颤菌血红蛋白基因(vgb)和头孢菌素C酰化酶基因(CPCacy)组合在一起,转化到大肠杆菌细胞中,探究VHb对CPCacy活性的影响。获得转化子pACYCDuet-1-vgb-CPCacy,其酶活比对照转化子pACYCDuet-1-CPCacy高,但是依然比出发菌株pET-28a-CPCacy低。在对出发菌株产酶培养基优化过程中,发现碳源成份甘油对酶活影响明显,随着甘油浓度增加,菌液OD600值出现类似二次生长的变化趋势。在培养基成份中含有葡萄糖的情况下,菌体会优先利用葡萄糖,此时其他碳源的利用会受到限制,若限制菌体对葡萄糖的摄取,可以促进其对其他碳源成份的有效利用。途径二,通过P1噬菌体转导的方法,置换大肠杆菌BL21(DE3)磷酸转移酶系统中的葡萄糖特异性转运蛋白ⅡCBGlc的编码基因ptsG,减少菌体对葡萄糖的转运利用,从而调动菌体其他代谢通路相关酶等蛋白的合成,提高菌体对培养基其他各种碳源成份的利用,增加发酵液的菌密度,提升外源蛋白的合成量,最终获得更高的头孢菌素C酰化酶合成量,提高发酵诱导后的酶活。获得ptsG缺陷型菌株后,将四种表达载体pET-28a-CPCacy、pACYCDuet-1-CPCacy、pETDuet-1-CPCacy和pRSFDuet-1-CPCacy通过化学转化法,转入到无抗性、无ptsG基因的缺陷型BL21(DE3)菌株中,获得四种工程菌。对比研究菌株ptsG基因缺陷对头孢菌素C酰化酶活性的影响,发现四种工程菌的ptsG-型菌株的酶活均高于ptsG+型菌株的酶活,提升最为明显的菌株为pRSFDuet-1-CPCacy(ptsG-),是其ptsG+型菌株的2.86倍。进一步的碳源成份优化研究发现,在LB培养基成份中,添加0.3%甘油或0.3%葡萄糖,均对四种工程菌的酶活有明显的提升。在ptsG+型菌株中,添加葡萄糖成份对酶活的提升效果好于添加甘油的效果;而在ptsG-型菌株中,添加甘油成份对酶活的提升效果更加明显。在菌株pACYCDuet-1-CPCacy(ptsG-)中,添加甘油成份后摇瓶发酵培养测得的酶活平均为4.197U/mL,是其ptsG+型菌株在未添加额外碳源的LB培养基中发酵测得酶活的6.59倍。本研究成功将ptsG-型菌株型BL21(DE3)用于表达头孢菌素C酰化酶蛋白,并且通过优化碳源成份获得明显的效果,为研究提高大肠杆菌头孢菌素C酰化酶的酶活开启了一个新的途径,同时,进一步优化培养基的其他成份及培养条件等因素,有望更进一步提升CPCacy酶活,提高工业化发酵生产头孢菌素C酰化酶的可行性。
[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，
本文编号：2276187
[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，
本文编号：2276187
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