肉桂链霉菌PLD基因原核表达载体构建及表达条件优化
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摘要
本文致力于研究一种被广泛关注的重要工业用途的酶,即链霉菌(Stv. Cinnamoneum)来源的磷脂酶D。它对多种磷脂均具有水解和转磷脂活性,可高效、持续转化磷脂酰胆碱(phosphatidylcholine,PC)生成磷脂酰乙醇胺(phosphatidylethanolamine,PE)。因此磷脂酶D是很有前景的催化剂,可合成具有各样生理功能的多种极性头基的新型磷脂,广泛应用于食品、化妆品和医药工业。然而,磷脂酶D因在微生物中的低产率已严重羁绊了其在理论及应用酶学方面的深入研究。
在构建分泌磷脂酶D表达系统前,就已有许多研究学者试图用大肠或毕赤酵母作为宿主菌在细胞内分泌表达磷脂酶D。然而,研究发现磷脂酶D对这些宿主细胞有极强的细胞毒性,一旦表达了有活性的磷脂酶D就会立即引发严重的细胞溶解,这很可能是活性磷脂酶D将宿主细胞膜中的磷脂降解所致。因此,很有必要研究磷脂酶D在原核宿主中分泌表达的可行性,为工业化生产提供一定的理论依据。本研究利用本身携带信号肽的pET-26b (+)、pEZZ18质粒作为表达载体,E.coli w+ Rosetta、E.coli JM105作为宿主菌,Stv. Cinnamoneum来源的磷脂酶D基因作为扩增目的片段构建了原核表达系统,并成功的在细胞周质表达出具可溶活性的磷脂酶D。并进一步优化了诸如,诱导剂浓度、葡萄糖浓度和培养基组成等可影响磷脂酶D表达产量、活性的各种实验参数。最终,构建的pEZZ18-PLD/ JM105、pET26b(+) -PLD/W+ Rosetta原核表达系统在葡萄糖添加终浓度均为0.5%,温度分别为18℃、28℃,IPTG浓度为0.5mM,OD600值分别为0.75、0.85时目的蛋白PLD的表达含量达到最高,分别为0.8mg/L、1.1mg/L;活性则分别达到0.62×103 U/L、0.86×103 U/L。
本研究构建的磷脂酶D原核表达系统使表达的磷脂酶D产量、活性都得到了进一步提高,虽然不够理想,但仍可为磷脂酶D基础理论研究和磷脂工业化生产提供一定的借鉴和参考。
Stv.Cinnamoneum PLD is an industrially important enzyme, exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. Furthermore, it was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food, cosmetics and pharmaceutical industries. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far.
Before establishing the expression system for secretory PLD, many researchers had been attempted to use E. coli or Pichia pastoris as a host cell for intracellular and secretory production of PLD. However, they found that PLD is extremely cytotoxic for these host cells, causing severe cell lysis immediately upon expression of active PLD, which likely degrades phospholipids contained in the membrane of the host cells. In this research, combating pET-26 b (+)、pEZZ18 vectors, with using E.coli w+ Rosetta、E.coli JM105 as a host for transformation of the PLD gene form Stv. Cinnamoneum. We have succeeded in constructing an expression system for secretory PLD. Furthermore PLD secretion was improved by optimize the experimental parameters includes inductor concentration, glucose concentration, constituents of culture medium, and so on.
The experiment result in pEZZ18-PLD/ JM105 and pET26b(+) -PLD/W+ Rosetta Prokaryotic expression transformers have been constructed in the experiment reach a climax in produced the PLD level and PLD activity: 0.62×103 U/L, 0.86×103 U/L; 0.8mg/L, 1mg/L when glucose concentration 0.5%, cultivation temperature 18℃and 28℃separately, concentration of inductor 0.5mM, OD600 0.75 and 0.85 separately.Our PLD-production system should promote both basic studies on PLD and its application to industrial phospholipid synthesis. Although this heterologous expression system is not more efficient in production of secretory PLD, it can be refer to the phospholipid synthesis in an industrial scale.
在构建分泌磷脂酶D表达系统前,就已有许多研究学者试图用大肠或毕赤酵母作为宿主菌在细胞内分泌表达磷脂酶D。然而,研究发现磷脂酶D对这些宿主细胞有极强的细胞毒性,一旦表达了有活性的磷脂酶D就会立即引发严重的细胞溶解,这很可能是活性磷脂酶D将宿主细胞膜中的磷脂降解所致。因此,很有必要研究磷脂酶D在原核宿主中分泌表达的可行性,为工业化生产提供一定的理论依据。本研究利用本身携带信号肽的pET-26b (+)、pEZZ18质粒作为表达载体,E.coli w+ Rosetta、E.coli JM105作为宿主菌,Stv. Cinnamoneum来源的磷脂酶D基因作为扩增目的片段构建了原核表达系统,并成功的在细胞周质表达出具可溶活性的磷脂酶D。并进一步优化了诸如,诱导剂浓度、葡萄糖浓度和培养基组成等可影响磷脂酶D表达产量、活性的各种实验参数。最终,构建的pEZZ18-PLD/ JM105、pET26b(+) -PLD/W+ Rosetta原核表达系统在葡萄糖添加终浓度均为0.5%,温度分别为18℃、28℃,IPTG浓度为0.5mM,OD600值分别为0.75、0.85时目的蛋白PLD的表达含量达到最高,分别为0.8mg/L、1.1mg/L;活性则分别达到0.62×103 U/L、0.86×103 U/L。
本研究构建的磷脂酶D原核表达系统使表达的磷脂酶D产量、活性都得到了进一步提高,虽然不够理想,但仍可为磷脂酶D基础理论研究和磷脂工业化生产提供一定的借鉴和参考。
Stv.Cinnamoneum PLD is an industrially important enzyme, exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. Furthermore, it was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food, cosmetics and pharmaceutical industries. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far.
Before establishing the expression system for secretory PLD, many researchers had been attempted to use E. coli or Pichia pastoris as a host cell for intracellular and secretory production of PLD. However, they found that PLD is extremely cytotoxic for these host cells, causing severe cell lysis immediately upon expression of active PLD, which likely degrades phospholipids contained in the membrane of the host cells. In this research, combating pET-26 b (+)、pEZZ18 vectors, with using E.coli w+ Rosetta、E.coli JM105 as a host for transformation of the PLD gene form Stv. Cinnamoneum. We have succeeded in constructing an expression system for secretory PLD. Furthermore PLD secretion was improved by optimize the experimental parameters includes inductor concentration, glucose concentration, constituents of culture medium, and so on.
The experiment result in pEZZ18-PLD/ JM105 and pET26b(+) -PLD/W+ Rosetta Prokaryotic expression transformers have been constructed in the experiment reach a climax in produced the PLD level and PLD activity: 0.62×103 U/L, 0.86×103 U/L; 0.8mg/L, 1mg/L when glucose concentration 0.5%, cultivation temperature 18℃and 28℃separately, concentration of inductor 0.5mM, OD600 0.75 and 0.85 separately.Our PLD-production system should promote both basic studies on PLD and its application to industrial phospholipid synthesis. Although this heterologous expression system is not more efficient in production of secretory PLD, it can be refer to the phospholipid synthesis in an industrial scale.
引文
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