High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers.
High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers.
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DOI：
10.1186/s12896-018-0427-0
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发表时间：
2018-03-20
影响因子：
3.5
通讯作者：
Schiraldi C
中科院分区：
文献类型：
--
作者：
Restaino OF;Borzacchiello MG;Scognamiglio I;Fedele L;Alfano A;Porzio E;Manco G;De Rosa M;Schiraldi C
热稳定性磷酸三酯酶样内酯酶（PLLs）能够降解有机磷，并有可能作为生物修复工具和生物清除剂。但目前其高产率的生产仍然是限制其工业应用的一个问题。本研究旨在建立一种高效生产和纯化两种原核表达的重组PLL的生物技术。大肠杆菌中分离到的野生型SacPox、来自酸热硫化叶菌的野生型SacPox和来自硫磺硫化叶菌的三重突变SsoPox C258 L/I261 F/W263 A。为了实现这一目标，研究了新的诱导方法以提高酶的产量，建立了高细胞密度发酵策略以达到越来越高的酶产量，达到22-L规模，研究了下游列车以满足高效工业纯化工艺的要求。摇瓶中的生理学研究表明，使用半乳糖作为诱导剂使酶浓度增加至4.5倍，与通过用IPTG诱导获得的生产相比。优化高细胞密度补料分批策略，两种酶的产量和生产率进一步提高了26倍，SacPox达到2300 U·L-1和47.1 U·L-1·h-1，SsoPox C258 L/I261 F/W263 A达到8700 U·L-1和180.6 U·L-1·h-1，并且发酵过程导致从2.5到22.0L的可扩展性。在从细胞中产生和提取后，首先通过热沉淀步骤纯化酶，其条件通过响应面方法优化。随后在100和5 KDa截留膜上进行超滤过程，最终纯度和两种酶的总回收率为70.0 ± 2.0%，适合于工业应用。本文首次建立了SacPox和SsoPox C258 L/I261 F/W263 A重组酶的高产生物技术生产工艺。通过将新的半乳糖诱导方法与高细胞密度补料分批发酵策略相结合来提高酶的产量。设计了一种高效的酶纯化方案，将热沉淀步骤与随后的基于膜的超滤过程耦合。本文的在线版本（10.1186/s12896-018-0427-0）包含补充材料，可供授权用户使用。
Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L− 1 and 47.1 U·L− 1·h− 1 for SacPox and to 8700 U·L− 1 and 180.6 U·L− 1·h− 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process. The online version of this article (10.1186/s12896-018-0427-0) contains supplementary material, which is available to authorized users.
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影响因子：
2.1
作者：
Horne, I;Qiu, XH;Oakeshott, JG
通讯作者：
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影响因子：
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影响因子：
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影响因子：
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影响因子：
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