High-level Soluble Expression Of Lipase LipA From Burkholderia Sp. ZYB002 In Escherichia Coli

Burkholderia sp.lipase LipA was an kind of extracellular lipase secreted by Burkholderia sp.ZYB002 strain which isolated from oil contaminated soil by researchers from our laboratory.LipB was the specific foldase of lipase LipA which can help LipA to form an active conformation.The lipase gene lipA and its specific foldase gene lipB were cloned,and the recombinant expression strain E.coli BL21(DE3)-pACYCDuet-lipA/lipB was constructed.The soluble expression of lipase gene lipA in E.coli was realized,but the level of soluble expression was low.In order to enhance the soluble expression level of lipase gene lipA in E.coli,the expression system for lipA/lipB was redesigned.Firstly,the recombinant expression vectors of lipase gene lipA and its specific foldase gene lipB were redesigned and constructed.Secondly,a series of conditions suitable for the soluble expression of lipA in E.coli were optimized,such as the host strains of Escherichia coli,the concentration of inducer,the time and temperature for induced expression.Thirdly,the recombinant lipase LipA produced by the best high-yielding recombinant strain E.coli Origami 2(DE3)pETDuet-lipB/lipA was purified by HisTrapHP affinity chromatography and its basic enzymatic properties were analyzed.The results of the experiments were as follows:1.Optimization of the expression system of lipase gene lipA.Series of recombinant expression vectors were successfully constructed which lipA and lipB were inserted into different multiple clone sites on the same expression vector or on different expression vectors.The results indicated that the co-expression plasmid pETDuet-lipB/lipA(pETDuet-1 with insertion of lipB at MCS1 and lipA at MCS2)was most beneficial to the soluble expression of lipase gene lipA.E.coli Origami 2(DE3)was more suitable for the soluble expression of lipase gene lipA than E.coli BL21(DE3).2.Optimization of fermentation conditions for the soluble expression of lipase LipA.The yield of LipA produced by E.coli Origami 2(DE3)pETDuet-lipB/lipA was enormously increased to about 123 U/OD600 which cultivated and induced by IPTG(at a final concentration of 0.10 mmol/L)at 20? for about 40 h.3.Purification and enzymatic characterization of recombinant lipase LipA.Under the above optimum fermentation conditions,the recombinant strain E.coli Origami 2(DE3)pETDuet-lipB/lipA was fermented,and the lipase LipA was purified by the HisTrapTM HP affinity chromatography and the basic enzymatic characterization of lipase LipA were analyzed.The experimental results show that the lipase LipA produced by the recombinant strain E.coli Origami 2(DE3)pETDuet-lipB/lipA cultivated in the conditions which mentioned above can been one-step purif1ed only through HisTrapTM HP affinity chromatography;The specific activity of lipase LipA in hydrolyzing pNPL was 664.74 U/mg;And the recovery rate of enzyme activity of lipase LipA was 74.55%in the process of purification and the purification fold was 3.11 times;The most suitable temperature and pH of lipase LipA to hydrolyze pNPL was 40? and pH 8.0;The half-life of lipase LipA at 40? was 36.54 minutes.