| Cutinase is a multi-functional enzyme, which could catalyse the hydrolysis ofpolyesters, insoluble triglycerides and soluble esters, the esterification of a variety of organicacids and alcohols as well as the transesterification of esters and alcohols; therefore, it haspotential usage in textile, detergent, ester synthesis, environmental protection, and otherindustries. However, there’s no commercialized enzyme preparation to date. The productionof cutinase is still at the stage of laboratory research.In our previous work, it was found that the cutinase from Thermobifida fusca exhibitedsuperior thermostability and broad pH stability, thus it was particularly suitable for applicationin the pre-treatment process of textile industry. In addition, in order to high-efficient express T.fusca cutinase, the encoding gene of cutinase was identified, and it was extracellularlyexpressed in Escherichia. coli BL21(DE3) via Sec pathway of type II secretion systemfollowed by preliminary culture condition optimization in the shake flask. In the present paper,the extracellularly overexpression of T. fusca cutinase and the application of facilitatingsecretion of other recombinant proteins were studied based on the secretion mechanism andthe property of T. fusca cutinase itself. The main results are as follows:(1) Expression of cutinase mediated by type II secretion system: The fermentation ofrecombinant E. coli BL21(DE3) extracellularly expressing cutinase via Sec pathway wasperformed in3L fementor. Results showed that different induction temperature and addingglycine had no obvious influence on the secretion of cutinase. The highest cutinase activity inthe culture supernatant was238.8U mL-1, which was only13.6%of the total activity(extracellular and intracellular activity), and the productivity was7.5U mL-1h-1. In addition,the E. coli BL21(DE3) expressing cutinase via Tat pathway was constructed, and when it wascultivated in the shake flask, the secretion level was lower than that mediated by Sec pathway.When treating the cells expressing cutinase mediated by Sec pathway, the highest percentageof intracellular cutinase which could release out of the cells was only24.8%. In order toimprove the secretion of cutinase, site-directed mutagenesis of hydrophobic residues (Y60、L90、I178、F209、I213) near the catalytic center or substrate binding site was performed,however, it failed to work efficiently.(2) Expression of cutinase mediated by type I secretion system: The recombinant E. coliBL21(DE3) expressing cutinase via type I secretion system was constructed. The cultivationin shake flasks showed that cutinase activity in the culture supernatant was2.5times that fromSec pathway. In addition, the signal peptide HlyAs is not cleaved after translocation andremained in the final product, thus, the recombinant cutinase-HlyAs was purified, and furtheranalysis showed that the purified cutinase-HlyAs had enzymatic properties and applicationcapability similar to that of wild-type cutinase. Moreover, optimization of culture conditionsof the recombinant E. coli BL21(DE3) was performed in3L fementor. Utilizing the inductionstrategy of0.04mmol L-1IPTG and0.5g L-1h-1lactose, the cutinase activity in the culturesupernatant reached596.6U mL-1, which was45.8%of the total activity. The productivitywas19.2U mL-1h-1,2.6times that from type II secretion system. (3) Expression of cutinase without mediation of signal peptide: T. fusca cutinase washeterologously expressed in E. coli BL21(DE3) without mediation of any signal peptide(referred as cutinaseNS). Unexpectedly, the extracellular secretion proportion was as high as92.5%of the sum of all the activity. Biochemical characterization of purified cutinaseNSsuggested that it had rather similar enzymatic properties to that of wild-type cutinase. In a3Lfermenter cultivation of the recombinant E. coli BL21(DE3), the cutinase activity in theculture supernatant reached1063.5U mL-1,69.8%of the total activity, and the productivitywas40.9U mL-1h-1, which was2.1and5.5times of that from type I and type II secretionsystem, respectively. To further increase the expression level, the gene of cutinaseNSwasoptimized, and the optimized gene was expressed in E. coli BL21(DE3). In3L fermentercultivation, the cutinase activity in the culture supernatant reached1765.7U mL-1, which was1.7times in contrast to the expression level of the original gene, the secretion proportion was87.0%, and the productivity was64.2U mL-1h-1. The yield of cutinase obtained in the presentstudy represents the highest production reported to date.(4) Exploring the underlying mechanism for “secretion” of cutinase without mediation ofsignal peptide: The recombinant E. coli BL21(DE3) producing inactive mutantcutinaseNSS130A was constructed, and SDS-PAGE analysis showed that, the majority ofinactive enzyme was located in cytoplasm. In addition, it was found that T. fusca cutinase wascapable of catalyzing the hydrolysis of phosphatidyl ethanolamine, the main component of E.coli membrane phospholipids. When compared to the cells harboring pET-20b(+) andexpressing the inactive mutant cutinaseNSS130A, the cells expressing cutinaseNSexhibitedirregular morphology and increased membrane permeability, but no obvious cell lysis. Basedon these results, the underlying mechanism of the extracellular “secretion” of cutinaseNSwasprobably that cutinaseNScould partially hydrolyze the phospholipids in the membrane, leadingto the enhanced membrane permeability, which eventually resulted in non-specifically releaseof cutinaseNSfrom cytoplasm into culture medium, and it was further verified by the releaseinto culture medium of other cytosolic proteins (D-xylose isomerase and trehalose synthase)with co-expression with cutinaseNS.(5) Enhancing extracellular secretion of recombinant secretory proteins with co-expression of cutinaseNS: The two secretory enzymes, xylanase and α-amylase attached withpelB signal peptide, were co-expressed with cutinaseNS. In shake flask fermentation, theproductivity of the co-expression system was7.9and2.0-fold that of the control withoutco-expression, respectively, and the improved extracellular secretion was not related with celllysis. Purification and N-terminal amino acids sequencing of the target proteins in the culturesupernatant showed that their N-terminal amino acids were corresponding to that of theirindividual mature proteins, indicating the correct excision of pelB signal peptide. Moreover,the two target proteins without a signal peptide were also co-expressed with cutinaseNS.Results suggested that all of the expressed proteins were in the form of inclusion bodies.Based on these results, the present study provided a novel strategy for enhancing extracellularsecretion of recombinant secretory proteins. |
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