Secretory Expression Of Reconbinant Thermobifida Fusca Cutinase In Escherichia Coli

Cutinase (EC 3.1.1.74) is a hydrolytic enzyme, which is capable of hydrolyzing various longchain and shortchain fatty acid esters, as well as insoluble cutin. It is considered to be the smallest hydrolytic enzyme that can degrade cutin in theα/βhydrolytic enzyme family. As a multifunctional enzyme, cutinase has been widely used in many industries, for instance, the food industry, chemical engineering, detergent engineering, papermaking industry, and the textile industry recently. Cutinase is believed to be able to break horny layer in the cotton fiber, and wipe off pectine, protein and other impurities to get a better scouring effect of the textile. In our preliminary study, we first identified and expressed a bacterial cutinase Tfu₀883 from Thermobifida fusca, and found it very potential in industrial application with a great thermal stability, a wonderful tolerance with high temperature and extreme pH. Based on the study above, the present study is dedicating in enhance the extracellular production of cutinase in E.coli with optimized extracellular secretional strategies, which can be illustrated as follows:1. An outer membrane lipoprotein—lpp was knocked out successfully, and a further fermentation of the lpp defective strain was carried out to investigate the cutinase production.According to the importance of the permeability of cell membranes in the protein transportation across membranes, lpp was deleted to increase the membrane permeability. A further fermentation study found that lpp knock-out has an apparent influence on the first period of fermentation, and shorten the fermentation cycle from 60h-72h to 36-48h;2. To enhance the secretion of cutinase and find a possible solution for the inclusion body formed inside the cell, protein secretion chaperones—SecA and SecM-SecA were co-expressed with cutinase.Translocation across the inner membrane of E. coli is a complicated process, involving several transportation chaperones such as SecA, SecYEG. To make it more twisting, the expression of SecA is strictly regulated by its upper stream monitor—SecM. The inner membrane translocation issue is thus a systematic project requiring patient and detailed study. In the present study, recombinant plasmids of pSTV/secA and pSTV/secM-secA were constructed to be expressed together with our targeting protein—pET/cutinase 0883. The results showed that there were still inclusion bodies formed in the cytoplasm, indicating that the secretion capacity may not match the expression strength, which can not be resolved by simply co-expressing the chaperones of SecA or SecM-SecA;3. A TIR mutagenesis was carried out to investigate the potential of cutinase extracellular production under a reduced expression strength.A degeneracy mutagenesis in the TIR (Translational Initial Region) was carried out on the recombinant plasmid pTRC99A/cutinase 0883, introducing a series of expression strengths, from one tenth to four times of the control. A mutant with half translational strength was selected and applied to expression vector pET/cutinase 0883 by a site-directed mutagenesis. It is observed that in the fermentation under 37℃of translation-decreased strain, the extracellular production ratio of cutinase was apparently increased with the fermentation cycle shortened, indicating a further application potential of the translation-decreased producing strain;4. The fermentation of the lpp defective strain with a decreased translation level was further optimized through adding chemical additives.According to the mechanism of membrane-across transportation, several metal ions, electron receivers, and surface active agents are investigated to evaluate their influence on the extracellular production of cutinase. It was found that addition of some electron receivers, such as Fe3+ and NO3-, had barely positive influence on the enhancement of the permeability of inner membrane. TritonX-100, however, was found to have a remarkable influence on the permeability of cell membranes. The final optimized concentration of TritonX-100 is 0.1%, which dramatically increase the extracellular ratio of cutinase, while having no significant influence on the growth condition of producing strain. The extracellular enzyme activity and ratio were elated to 80U/mL and 60%, 2times that of the initial strain.