Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

Abstract

AbstractsBackgroundThe lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems.ResultsTo produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded in vivo into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1a and BL21/pELipA-LipB3a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%.ConclusionsOur findings d...

Biomimetic CO2 capture using a highly thermostable bacterial α-carbonic anhydrase immobilized on a polyurethane foam

“…There is however a search for CAs, which are more stable and more active in the operative conditions and, in addition, enzymes that could be produced in large amounts at low purification costs. With the technologies available on the market it is possible to produce large amounts of enzymes at moderate costs as happens, for example, for the enzymes utilized in detergents plants [32][33][34] . Generally, CAs isolated from mammals or prokaryotes are active at physiological temperatures and are, as many enzymes, quite unstable under or extreme conditions, such as high temperature, high concentrations of salts, etc.…”

Classification of lipolytic enzymes and their biotechnological applications in the pulping industry

“…Proteins utilizing this pathway contain a unique twin arginine translocation motif in their signal sequence (Tjalsma et al 2000). In general, active expression of lipases from Pseudomonas and Burkholderia requires the presence of a chaperone protein known as the lipasespecific foldase (Lif), for precise folding of the lipase (Quyen et al 2012;Wu et al 2012). A text book example would be that of the cold-active lipase gene isolated from Psychrobacter sp., which was expressed in E. coli BL21 yielding a specific activity of 66.51 U/mg.…”

First co-expression of a lipase and its specific foldase obtained by metagenomics

“…Some success has been obtained using this approach [ 44 ]-[ 47 ]. Secondly, in vivo folding can be used, in which traditional molecular biology techniques are used to co-express the foldase with the lipase [ 46 ]. However, in the case of the metagenomic approach, the co-expression is more complicated, since it requires the cloning of both the lipase and foldase on the same DNA fragment.…”