Study On Gene Expression Of Aspergillus Aculeatus β-glucosidase And Tetragenococcus Halophilus Superoxide Dismutase In Lactococcus Lactis

Lactococcus lactis is a kind of food-grade bacterium which is widely used in food field.Protein expression was affected by low oxygen resistance and density during fermentation inLactococcus lactis. We constructed food-grade secretion expression vector with β-glucosidasefrom Aspergillus aculeatus and expression vector with SOD from Tetragenococcus halophilusby using E.coli-Lactic acid bacteria shutter vector pMG36e and expressed in L. lactisMG1363by electrotransformed.Part one：Construction and expression of the vector pMG36N-Usp45-Abgl-NisI in L.lactisMG1363Secretion signal peptide Usp45from the L. lactis MG1363genome, Nisin resistancegenes NisI from plasmid pLEB590and Abgl from plasmid pPIC9k-Abgl were amplified byPCR and ligated to construct the Usp45-Abgl-NisI fragment. The fragment was subcloned intoplasmid pMD20and then transformed into E.coli DH5α by CaCl2method. It was identifiedby sequencing and inserted into vector pMG36e. The recombinant strain E.coliXL1-Blue/pMG36e-Usp45-Abgl-NisI was obtained by transformed. Food-grade secretionexpression vector pMG36N-Usp45-Abgl-NisI, which knocked out erythromycin resistancegene of plasmid pMG36e-Usp45-Abgl-NisI by PCR method, was constructed and thenelectrotransformed into L. lactis MG1363.The transcription and expression of β-glucosidasewas observed via SDS-PAGE、activity analysis and RT-PCR. β-Glucosidase produced byE.coli XL1-Blue was active on the chromogenic substrate aesculin. L.lactisMG1363/pMG36N-Usp45-Abgl-NisI could grow on plates containing20IU/ml Nisin.β-Glucosidase was expressed in L.lactis MG1363verified by SDS-PAGE and RT-PCR.Thefood-grade secretion expression vector pMG36N-Usp45-Abgl-NisI was constructedsuccessfully. β-Glucosidase can be expressed in recombinant L.lactis MG1363/pMG36N-Usp45-Abgl-NisI and made the foundation for the biological activities in L.lactis.Part two：Construction and expression of the vector pMG36e-SOD in L. lactis MG1363SOD from the T. halophilus genome was amplified by PCR and was subcloned into plasmid pMD18, then transformed into E.coli DH5α by CaCl2method. It was identified bysequencing and inserted into vector pMG36e. The recombinant strain E.coliDH5α/pMG36e-SOD was obtained by transformed. Then electrotransformed vectorpMG36e-SOD into L. lactis MG1363. The transcription and expression of SOD was observedvia SDS-PAGE、activity analysis and RT-PCR. The activity of recombinant L.lactisMG1363/pMG36e-SOD was120.95U/mg prot compared with negative controlled L.lactisMG1363/pMG36e50.06U/mg prot in the cytoplasm, which indicated SOD had experessedbut the activity is low. The vector pMG36e-SOD was constructed successfully. SOD can beexpressed in recombinant L.lactis MG1363/pMG36e-SOD, which endowed the character thatmore insensitive to O2and improved the density of strain.