【作者】 王霁昀；

【导师】 陈卫；

【作者基本信息】 江南大学 ， 食品科学， 2009， 硕士

【摘要】 β-半乳糖苷酶(EC.3.2.1.23)俗称乳糖酶,该酶能水解β-1,4糖苷键在乳品加工中常用于水解牛奶中的乳糖制备低乳糖奶,可有效缓解人体乳糖不耐症,促进乳品工业的发展。耐热β-半乳糖苷酶,或称高温乳糖酶,以其良好的热稳定性和较高温度下的水解特性使之具有常温乳糖酶无法比拟的优越性。近年来,高温乳糖酶尤其是细菌来源的中性、耐热乳糖酶成为研究的热点。本文围绕一种来源于嗜热脂肪芽孢杆菌的耐热β-半乳糖苷酶(bgaB),从基因克隆、表达及分泌等方面进行了较为深入的研究。枯草芽孢杆菌是一种食品级微生物,在食品生产中有很好的应用前景。本文选用实验室已构建的含有不同信号肽的枯草芽孢杆菌质粒,将bgaB基因插入到四种质粒表达单元中进行重组表达,其中重组菌株WB600-pMAwprA-bgaB摇瓶发酵的最高酶活达到0.37 U/mL,实现了bgaB在枯草芽孢杆菌中的表达。本文尝试了bgaB在毕赤酵母中的表达。构建pPIC9k-bgaB重组质粒,电转化到毕赤酵母GS115中构建重组转化子,用含G418的YPD平板筛选多拷贝转化子,并验证重组转化子的正确性后,用甲醇诱导发酵表达目的基因。结果在发酵后的菌液破壁上清液中检测到耐热乳糖酶的酶活,最高活性为0.26 U/mL,SDS-PAGE电泳也在70 kD处检测到目的条带,表明bgaB基因在毕赤酵母中得到了表达。乳酸克鲁维酵母表达系统也是近年来研究的热点,本文构建重组质粒pKLAC1-bgaB,电转化到乳酸克鲁维酵母GG799中构建重组转化子,验证重组转化子正确性后进行发酵表达。结果在发酵后的菌液破壁上清液中检测到耐热乳糖酶的酶活,最高活性为0.068 U/mL,SDS-PAGE电泳也在70 kD处检测到目的条带,表明bgaB基因在乳酸克鲁维酵母中得到了表达。本文研究了bgaB基因在三种不同表达系统中的表达情况,结果表明bgaB基因在枯草芽孢杆菌中的表达效果要优于真核表达系统,达到了0.371 U/mL,并首次实现了bgaB基因在真核表达系统中的表达。更多还原

【Abstract】 β-Galactosidase (EC.3.2.1.23), also called lactase which can hydrolyzeβ-1,4-D-galactosidic linkage, are industrially important for their applications in producing lactose-free milk which can eliminate the lactose-intolerance of human beings.Compared to the mesophilic enzyme from Kluyveromyces lactis, thermostableβ-galactosidases from bacterium with optimum pH value near neutral are preferable with their good thermophilicity and thermostability in practice to prevent microbial contamination, to save energy and equipment and, to make operations within one shift. In this paper, the gene bgaB from Bacillus stearothermophilus encoding the thermostableβ-galactosidase was cloned and expressed in several systems. The enzyme properties and the fermentation conditions of the recombinant strains were also discussed in details.Bacillius subtilis is a food grade expression system. bgaB gene was inserted into the four different shuttle expression vectors and the recombinant plasmids were transformed into Bacillius subtilis WB600 by electroporation. Recombinant strains were obtained through LB plate screening with kanamycin. The enzyme activity of the recombinant strain WB600 pMAwprA-bgaB reached to 0.37 U/mL, and the SDS-PAGE showed that the molecular weight was about 70 kDa.In this work,bgaB gene was inserted into the shuttle expression vector pPIC9k to construct recombinant plasmid pPIC9k-bgaB. The recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. Transformants were obtained through MD plate and G418 plate screening. After the induction of methanol, the enzyme activity of recombinant Pichia pastoris strain reached to 0.26 U/mL, and the SDS-PAGE showed that the molecular weight was about 70 kDa.BgaB gene was inserted into the shuttle expression vector pKLAC1 to construct recombinant plasmid pKLAC1-bgaB. The recombinant plasmid was transformed into Kluyveromyces lactis GG799 by electroporation. Transformants were screened. After fermentation, the enzyme activity of recombinant Kluyveromyces lactis strain reached to 0.068 U/mL, and the SDS-PAGE showed that the molecular weight was about 70 kDa.In this work, the expression of bgaB was studied in three different expression systems. The results showed that the expression of bgaB in Bacillus subtitles system was better than eukaryotic expressing system, the enzyme activity reached to 0.371 U/mL, and this work realized the expression of bgaB in eukaryotic expression system for the first time.更多还原