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首页> 外文期刊>BMC Biotechnology >In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli
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In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli

机译：筛选的铜绿假单胞菌伴侣依赖性脂肪酶在大肠杆菌中的体内功能表达

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Background Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. Results In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA . Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E. coli BL21/pET28a- lipAB and two dual expression plasmid systems E. coli BL21/pETDuet- lipA - lipB and E. coli BL21/pACYCDuet- lipA - lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a- lipAB and E. coli BL21/pETDuet- lipA - lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet- lipA - lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. Conclusions The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet- lipA - lipB was more suitable for the expression of active LipA than pET28a- lipAB and pETDuet- lipA - lipB . The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.
机译：背景技术微生物脂肪酶，特别是假单胞菌脂肪酶广泛用于生物技术应用。设计实验以获得高水平的活性脂肪酶是一项有意义的工作。假单胞菌脂肪酶功能过度表达存在一个限制因素，即分子伴侣对于有效折叠是必需的。如以前的报道，已经使用了几种方法来解决该问题。在这项工作中，来自大肠杆菌的铜绿假单胞菌的筛选菌株AB中的脂肪酶（LipA）及其伴侣蛋白（LipB）在大肠杆菌中用两个双重表达质粒系统过表达，无需体外即可增强活性脂肪酶LipA的产生重新折叠过程。结果在这项工作中，我们通过筛选程序筛选出了一种由脂肪酶生产的名为AB的菌株，该菌株在16S rDNA的基础上被鉴定为铜绿假单胞菌。从菌株获得的基因组DNA用于分离基因lipA（936bp）和脂肪酶特异性折叠酶基因lipB（1023bp）。成功构建了一个单表达质粒系统大肠杆菌BL21 / pET28a-lipAB和两个双表达质粒系统大肠杆菌BL21 / pETDuetlipa-lipB和大肠杆菌BL21 / pACYCDuet-lipA-lipB。比较了三种表达系统的脂肪酶活性，以选择最佳表达方法。在相同培养条件下，大肠杆菌BL21 / pET28a-lipAB和大肠杆菌BL21 / pETDuet-lipA-lipB表达的脂肪酶活性分别为1300 U / L和3200 U / L，而大肠杆菌BL21 / pACYCDuep-lipA-lipB表达的脂肪酶高达8500 U / L。脂肪酶LipA的最佳温度为30°C，最佳pH为9，具有很强的pH耐受性。活性LipA可以催化脂肪醇与脂肪酸之间的反应生成脂肪酸烷基酯，这意味着LipA可以催化酯化反应。最适合酯化的脂肪酸和醇底物分别是辛酸和己醇。结论不同质粒系统对活性LipA表达的影响明显不同。 pACYCDuet-lipA-lipB比pET28a-lipAB和pETDuet-lipA-lipB更适合表达活性LipA。 LipA显示出明显的酯化活性，因此具有潜在的生物催化应用。本文报道的表达方法可为需要伴侣蛋白的那些酶的表达提供参考。

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