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首页> 外文期刊>Journal of microbiology and biotechnology >Bioproduction of trans-10,cis-12-Conjugated Linoleic Acid by a Highly Soluble and Conveniently Extracted Linoleic Acid Isomerase and an Extracellularly Expressed Lipase from Recombinant Escherichia coli Strains
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Bioproduction of trans-10,cis-12-Conjugated Linoleic Acid by a Highly Soluble and Conveniently Extracted Linoleic Acid Isomerase and an Extracellularly Expressed Lipase from Recombinant Escherichia coli Strains

机译：通过高度溶于和方便地提取的亚油酸异构酶和来自重组大肠杆菌菌株的细胞外表达脂肪酶的反式-10，CIS-12-缀合的亚油酸的生物处理

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The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10, c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.
机译：丙酮痤疮杆菌多不饱和脂肪酸异构酶（PAI）的低溶解度和高成本回收是高附加值缀合的亚油酸（CLA）的生物生产中的关键问题。为了改善重组PAI的溶解性，用PAI共同表达了六个伴侣蛋白。腹股沟蛋白引入从29％达到97％显着改善了PAI溶解度，活性增加57.8％。 DNAKJ-GRPE和腹圈蛋白的组合表达将活性增加11.9％。相比之下，DNAKJ-GRPE蛋白的共表达明显将活性降低了57.4％。质粒pTF16含有TIG基因和含有TIG和GROEL-GROES基因的PG-TF2对PAI表达没有明显的影响。然后将裂解蛋白E引入重组大肠杆菌中以进行细胞自分解系统。通过悬浮在蒸馏水中的裂解细胞，从细胞质中释放出总细胞内pai的35％活性。通过优化释放条件，PAI回收率进一步提高至81％。来自Rhizopus oryzae的脂肪酶也在大肠杆菌中表达，细胞外活性为110.9 u / ml。通过使用自由的PAI和脂肪酶作为催化剂，建立了从葵花籽油生产CLA的联合系统。在优化条件下，T-10，C-12-CLA的最大滴度达到9.4克/升。这项工作提供了一种有效和低成本的策略，以改善重组细胞内PAI的溶解性和恢复，以进一步大规模生产CLA。

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