【作者】 黄君；

【导师】 梁运祥；

【作者基本信息】 华中农业大学 ， 发酵工程， 2008， 硕士

【摘要】 内切β-1,4-葡聚糖酶(EC3.2.1.4)是一种重要的工业用酶,广泛应用于饲料工业,酿造业等领域。随着生产的发展,β-1,4-葡聚糖酶需求量日益增加。本实验的主要目的就是,从分泌热稳定性的β-1,4-葡聚糖酶的黑曲霉高产菌株中,克隆该基因,利用基因工程手段实现高效表达,满足工业生产的需求。实验首先是利用RT-PCR技术,提取黑曲霉(Aspergillus niger)L3的总RNA进行反转录,并通过PCR扩增得到去除天然信号肽的β-1,4-葡聚糖酶基因egⅠ,将其插入到毕赤酵母表达载体pPIC9k上,使之位于α-因子信号肽下游,并与之同框,构建重组表达质粒pPIC9k-egⅠ,电击转化毕赤酵母GS115,经MM、MD、CMC-Na和G418平板筛选,得到重组毕赤酵母工程菌1-1#和1-5#,在摇瓶发酵水平上,以0.5%的β-葡聚糖为底物检测,酶活分别达到1 456 U/mL和1 928 U/mL。重组酶最适pH为5.0,最适反应温度为70℃,在70℃时保温30 min后仍然具有90%以上的活力。为了进一步提高表达量,根据毕赤酵母偏爱的密码子优化来源于A.niger L3的内切β-1,4-葡聚糖酶的DNA序列,共改变193个碱基,G+C%由54%下降到44.22%。设计了14对寡聚核苷酸引物,采用重叠延伸PCR三步法获得全长基因序列共改变。将其插入到毕赤酵母表达载体pPIC9K上,构建重组表达质粒pPIC9k-syn-egⅠ,电击转化毕赤酵母GS115,经MM、MD、CMC-Na和G418平板以及摇瓶筛选,得到重组毕赤酵母工程菌2-7#。摇瓶发酵条件下,以0.5%的葡聚糖和1%的CMC-Na为底物检测,酶活分别达到3 658 U/mL和591.9 U/mL;采用50 L发酵罐进行发酵,酶活则分别达到65 649.6 U/mL和39 728.32 U/mL。更多还原

【Abstract】 Endo-β-1,4-glucanase(EC3.2.1.4),as an important industrial enzyme,has been widely used in the many fields such as brewing and animal feed industry.With the developing of production,the need for it is increasing day and day.The purpose of this research is to clone thermostable endo-β-1,4-glucanase from a strain of Aspergillus niger, which effectively secreted EGⅠ,to improve the expression level with the methods of molecular biology and gene engineering and meet the need in industrial production.The gene encoding endo-β-1,4-glucanase from Aspergillus niger L3 was isolated with RT-PCR method.egⅠwas eliminated its native signal peptide by PCR and inserted into the pPIC9K vector of Pichia pastoris in reading frame withα-factor secreting signal peptide sequence to construct the recombinant plasmid pPIC9K-egⅠ.The recombinant plasmid pPIC9K-egⅠwas transformed into P.pastoris GS115 with electroporation.Two of recombinant P.pastoris stains 1-1# and 1-5# were obtained by screening with MM,MD, CMC-Na and G418 plates and the activity of recombinant EGⅠcould reach to 1456 U/mL and 1928U/mL respectively.The optimal temperature for the recombinant EGⅠwas 70℃and the optimal pH was 5.0.To improve expression efficiency in recombinantβ-1,4-glucanase in P.pastoris,the egⅠgene from A.niger L3 was modified with codon optimization.A total of 193 nucleotides are changed,the G+C ratio was decreased from 54%to 44.22%.14 pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egⅠgene was synthesized using PCR-basic three-step DNA synthesis method.The modified syn-egⅠgene was inserted into the pPIC9K vector to construct the recombinant plasmid pPIC9K-syn-egⅠ.The recombinant plasmid pPIC9K-syn-egⅠwas transformed into P. pastoris GS115 with electroporation.A recombinant P.pastoris stains 2-7# was obtained by screening with MM,MD,CMC-Na and G418 plates.The activity of recombinant EGⅠat shaking flask level could reach to 3658 U/mL and 591.9 U/mL with 0.5%barleyβ-glucan and 1%CMC-Na as substrate,respectively.At 50 L fermentor level,theβ-1, 4-glucanase activity was 65649.6 and 39728.32 U/mL with 0.5%barleyβ-glucan and 1% CMC-Na as substrate.更多还原