棘孢曲霉β-葡萄糖苷酶的乳酸乳球菌表达
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摘要
构建棘孢曲霉β-葡萄糖苷酶食品级分泌表达载体并在乳酸乳球菌MG1363(L.lactis MG1363)中实现表达。通过PCR扩增L.lactis MG1363基因组中分泌信号肽Usp45、质粒pLEB590的乳链菌肽(Nisin)抗性基因NisI和质粒pPIC9k-Abgl的棘孢曲霉β-葡萄糖苷酶基因Abgl,PCR方法连接后获得片段Usp45-Abgl-NisI,将其克隆到质粒pMD19中,CaCl2法转化到E.coli DH5α,测序鉴定后将该片段连接到大肠杆菌-乳酸菌穿梭质粒pMG36e中并转化到E.coli XL1-Blue,得重组子E.coli XL1-Blue/pMG36e-Usp45-Abgl-NisI。通过PCR方法敲除质粒pMG36e-Usp45-Abgl-NisI中红霉素抗性基因以构建食品级分泌载体pMG36N-Usp45-Abgl-NisI,将其电转到L.lactis MG1363中。转化子E.coli XL1-Blue/pMG36e-Usp45-Abgl-NisI能将β-葡萄糖苷酶分泌到胞外使七叶苷平板显色,L.lactis MG1363/pMG36N-Usp45-Abgl-NisI能在20 IU/mL Nisin上生长,经RT-PCR验证β-葡萄糖苷酶在乳酸乳球菌中实现表达。表明分泌型表达载体构建成功,为其在乳酸乳球菌中实现食品级活性表达奠定基础。
The food-grade secretion expression vector of β-glucosidase from Aspergillus aculeatus was constructed and expressed in Lactococcus lactis MG1363.Secretion signal peptide Usp45 from the L.lactis MG1363 genome,Nisin resistance genes NisI from plasmid pLEB590 and Abgl from plasmid pPIC9k-Abgl were amplified by PCR and ligated to construct the Usp45-Abgl-NisI fragment.The fragment was subcloned into plasmid pMD19 and then transformed into E.coli DH5α by CaCl2 method.It was identified by sequencing and inserted into the E.coli-Lactic acid bacteria shutter vector pMG36e.The recombinant strain E.coli XL1-Blue/pMG36e-Usp45-Abgl-NisI was obtained by transformation.Food-grade secretion expression vector pMG36N-Usp45-Abgl-NisI,in which erythromycin resistance gene of plasmid pMG36e-Usp45-Abgl-NisI was knocked out by PCR method,was constructed and then electrotransformed into L.lactis MG1363.β-Glucosidase produced by E.coli XL1-Blue was active on the chromogenic substrate aesculin.L.lactis MG1363/pMG36N-Usp45-Abgl-NisI could grow on the plates containing 20IU/mL Nisin.β-Glucosidase expressed in L.lactis MG1363 was verified by RT-PCR.
The food-grade secretion expression vector of β-glucosidase from Aspergillus aculeatus was constructed and expressed in Lactococcus lactis MG1363.Secretion signal peptide Usp45 from the L.lactis MG1363 genome,Nisin resistance genes NisI from plasmid pLEB590 and Abgl from plasmid pPIC9k-Abgl were amplified by PCR and ligated to construct the Usp45-Abgl-NisI fragment.The fragment was subcloned into plasmid pMD19 and then transformed into E.coli DH5α by CaCl2 method.It was identified by sequencing and inserted into the E.coli-Lactic acid bacteria shutter vector pMG36e.The recombinant strain E.coli XL1-Blue/pMG36e-Usp45-Abgl-NisI was obtained by transformation.Food-grade secretion expression vector pMG36N-Usp45-Abgl-NisI,in which erythromycin resistance gene of plasmid pMG36e-Usp45-Abgl-NisI was knocked out by PCR method,was constructed and then electrotransformed into L.lactis MG1363.β-Glucosidase produced by E.coli XL1-Blue was active on the chromogenic substrate aesculin.L.lactis MG1363/pMG36N-Usp45-Abgl-NisI could grow on the plates containing 20IU/mL Nisin.β-Glucosidase expressed in L.lactis MG1363 was verified by RT-PCR.
引文
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[11]Nanyan Noreen,Wei Yeng Hooi,Ali Baradaran,et al.Lactococcus lactis M4,a potential host for the expression of heterologous proteins[J].Microbial Cell Factories,2011,10:28
[12]Arno Wegkamp,Wietske van Oorschot,Willem M de Vos,et.al.Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis[J].Applied and Environmental Microbiology,2007:2673-2681
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[14]Igor Mierau,Michiel Kleerebezem.10 years of the nisin-controlled gene expression system(NICE)in Lactococcus lactis[J].Applied Microbiology and Biotechnology,2005,68:705-717
[2]韦斌如,刘端玉,韩双艳,等.棘孢曲霉β-葡萄糖苷酶1在毕赤酵母中的表达及烷基糖苷的催化合成[J].高等学校化学学报,2012,7(33):1498-1504WEI Bin-ru,LIU Duan-yu,HAN Shuang-yan,et al.Expression of Aspergllus aculeatusβ-Glucosidase 1 gene in Pichia pastoris and its application on the synthsis of alkyl glucoside[J].Chemical Journal of Chinese Universities,2012,7(33):1498-1504
[3]Reiichiro Sakamoto,Jinshu Kanamoto,Motoo Arai,et al.Purification and physicochemical properties of threeβ-glucosida ses from Aspergillus aculeatus No.F-50[J].Agricultural and Biological Chemistry,1985,49:1275-1281
[4]李远华.β-葡萄糖苷酶的研究进展[J].安徽农业大学学报,2002,29:421-425LI Yuan-hua.The research progress ofβ-glucosidase[J].Journal of Anhui Agricultural University,2002,29:421-425
[5]Udo Wegmann,Aldert Zomer,Girbe Buist,et al.Complete genome sequence of the prototypelactic acidbacterium Lactoco ccus lactis subsp.cremoris MG1363[J].Journal of Bacteriology,2007,189:3256-3270
[6]Yves Le Loir,Vasco Azevedo,Sergio C Oliveira,et al.Protein secretion in Lactococcus lactis:anefficient way to increase the overall heterologous protein production[J].Microbial Cell Factories,2005,4:2
[7]Martien van Asseldonk,Willem M de Vos,Guus Simons.functional analysis of the Lactococcuslactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologousα-amylase[J].Molecular and General Genetics MGG,1993,240:428-434
[8]T Takala,P Saris.A food-grade cloning vector for lactic acid bacteria based on the nisinimmunity gene nisI[J].Applied Microbiology and Biotechnology,2002,59:467-471
[9]Xiaobo Liang,Zhizeng Sun,Jin Zhong,et al.Adverse effect of nisin resistance protein onnisin-induced expression system in Lactococcus lactis[J].Microbiological Research,2010,165:458-465
[10]Helge Holo,Ingolf F Nes.High-frequency transformation,by electroporation of Lactococcuslactis subsp.cremoris grown with glycine in osmotically stabilized media[J].Applied and Environmental Microbiology,1989,55:3119-3123
[11]Nanyan Noreen,Wei Yeng Hooi,Ali Baradaran,et al.Lactococcus lactis M4,a potential host for the expression of heterologous proteins[J].Microbial Cell Factories,2011,10:28
[12]Arno Wegkamp,Wietske van Oorschot,Willem M de Vos,et.al.Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis[J].Applied and Environmental Microbiology,2007:2673-2681
[13]Prithy Rupa,Vicente Moneero,Bruce N Wilkie.Expression of bioactive porcine interferon-gammaby recombinant Lactococc uslactis[J].Veterinary Microbiology,2008,129:197-202
[14]Igor Mierau,Michiel Kleerebezem.10 years of the nisin-controlled gene expression system(NICE)in Lactococcus lactis[J].Applied Microbiology and Biotechnology,2005,68:705-717