| Transglutaminases (EC2.3.2.13, TGase) can form ε-(γ-glutamyl)-lysine peptide chainbridges by crosslinking between a γ-carboxyamide group of glutamine in proteins anddifferent amino receptors, which have been widely used in food engineering. Nowadays,Streptomyces is the main source of TGase enzymes in commercialization. However, its yieldis always so poor just depending on wild strains fermentation, which limits its application inindustries. With the development of food grade protein expression system, researchers fromboth home and abroad have focused on the construction of food grade recombinant strains forhigher TGase production.Pro-TGase from S. hygroscopicus WSH03-13TGase was extracellularly expressed inEscherichia coli previously in our lab, which was processed to produce active enzyme byremoval of its N-terminal pro-peptide by dispase. In this study, in order to construct foodgrade recombinant strains for higher TGase production, pro-TGase from S. hygroscopicus wasextracellularly expressed in Yarrowia lipolytica, and fermentation conditions of recombinantstrains and the enzymatic properties of recombinant enzyme were examined. The main resultsare listed as following:(1) Secretory expression of the S. hygroscopicus pro-TGase in Yarrowia lipolytica andfermentation optimizationBased on S. hygroscopicus TGase soluble expression depending on pro region, thepro-TGase gene from S. hygroscopicus was obtained by PCR and cloned to the expressionvector pINA1296(a pBR-based mono-copy integrative vector) and pINA1297(anauto-cloning multi-copy integrative vector), yeilding recombinant strainspINA1296/pro-TGase and pINA1297/pro-TGase. They were transformed into Y. lipolyticaPo1g and Po1h strains, respectively. On the basis of pINA1297/pro-TGase, site-directedmutagenesis was conducted by a PCR method to detect the effect of N-linked glycosylationon pro-TGase production, yielding pINA1297/pro-TGase derivatives N160Q, N355Q andN160/355Q. Following activation by dispase, the activities of N160Q, N355Q andN160/355Q were equivalent to100%,147%, and57%of that from wild type pro-TGase.In order to enhance TGase production, the medium composition was determined bysingle factor optimization of highly N355Q expresstion. When cultured in shaker flasks withthe medium consisted of15g/L glycerol,20g/L yeast extract,2.64g/L NH4Cl,0.32g/LKH2PO4,0.25g/L MgSO4,3.34×10-4g/L Vitamin B1at pH8.0and28°C for120h, the yieldof N355Q after activation by dispase corresponded to8.47U/mL of TGase. When cultured in3L fermenter with flowing glycerol short time, the yield of N355Q was corresponded to35.00U/mL of TGase. Compared with the highest enzyme activity in shaker flasks, N355Qactivity after the activation in3L fermenter increased4times.(2) Expression of active TGase from S. hyroscopicus in Y. lipolyticaTo avoid the recombinant pro-TGase activation in vitro, pINA1297was used to constructthe co-expression vectors of the S. hygroscopicus TGase and its pro-region contained onepromoter and two promoters respectively. The co-expression vectors were transformed into Y. lipolytica and then cultured in PPB medium. Sodium dodecyl sulfatepolacrylamide gelelectrophoresis (SDS-PAGE) analysis and measurement of enzyme activity indicated thatthere was no active TGase expression. Then the processing site of Kex2endoprotease wasinserted between TGase and its pro-region by site-directed mutagenesis on the basis ofpINA1297/pro-TGase, yielding recombinant vectors pINA1297/pro-kex-TGase, N160Q-kex,N355Q-kex and N160/355Q-kex. These vectors were transformed into Y. lipolytica and thencultured in PPB medium in shaker flasks. After24h, secreted by above recombinant strains,TGase activity reached0.60U/mL,0.89U/mL,0.67U/mL,1.06U/mL respectively. Activityassay and SDS-PAGE analysis showed that total pro-TGase production decreased and just apart of pro-TGase were actived into active TGase.(3) Purification and characterization of the recombinant TGaseThe recombinant pro-TGase from Yarrowia lipolytica/(pINA1297/pro-TGase) and itsthree mutants were purified by Fractogel EMD SO3-ion exchange chromatography. Then westudied their characterization after activation.The specific activity and Kmof mutants wereboth decreased compared with wild type. In fact, N160/355Q possessed the lowest specificactivity which was3.11U/mg and N160Q possessed the lowest Kmvalue which was18.63mmol/L. All activated mutants exhibited an optimal pH activity profile (pH5.0-6.0) similar tothat of recombinant wild type TGase. When incubated at different pHs, the activatedpro-TGase and N355Q retained highest residual activities for pH8.0, while N160Q andN160/355Q do so for pH7.0. The activated N160/355Q showed its highest activity at40°C,and the other activated zymogens displayed highest activities at45°C, and deglycosylationdecreased thermal stability of recombinant TGase expressed by Y. lipolytica. Circulardichroism analysis showed that the α-helix and β-sheet content were not changed by thesemodifications. Fluorescence spectra analysis indicated that site-directed mutagenesis ofN-linked glycosylation did not remarkably affect the tertiary structure of the pro-TGase. |
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