Construction of Aspergillus niger Engineering Bacteria with High Yield and High Purity Prolyl Endopeptidase
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摘要: 利用基因工程技术，构建过表达黑曲霉来源带有自身信号肽的脯氨酰内肽酶黑曲霉重组菌株TH2-protAS和过表达以黑曲霉内源高效分泌的葡糖淀粉酶信号肽以及α-淀粉酶信号肽代替自身信号肽的脯氨酰内肽酶黑曲霉重组菌株TH2-SglaA-protA和TH2-SamyA-protA。SDS-PAGE结果显示，脯氨酰内肽酶在重组菌株中分泌表达，但是存在不同程度的糖基化。酶活检测结果表明，重组菌株TH2-protAS、TH2-SglaA-protA和TH2-SamyA-protA的最高酶活分别为1.70、2.19、1.91 U/mL。在重组菌株TH2-SglaA-protA的基础上，利用基因敲除技术，敲除了主要的背景蛋白酸稳定的α-淀粉酶，结合发酵条件优化，获得了高纯度的脯氨酰内肽酶。综合上述结果可得出结论：可在黑曲霉中同源高效分泌表达脯氨酰内肽酶；glaA信号肽和amyA信号肽明显增加黑曲霉中脯氨酰内肽酶的分泌表达量，且glaA信号肽的效果优于amyA信号肽；将基因敲除和发酵调控相结合，可以有效去除分泌的背景蛋白，获得高产高纯度脯氨酰内肽酶的菌株，该重组菌株具有明确的工业应用潜力。Abstract: Using genetic engineering technology,the recombinant strain TH2-protAS of Aspergillus niger which overexpressed the prolyl endopeptidase derived from Aspergillus niger with its own signal peptide and the glucoamylase signal peptide and α-Amylase signal peptide replaces its own signal peptide prolyl endopeptidase Aspergillus niger recombinant strains TH2-SglaA-protA and TH2-SamyA-protA. The results of SDS-PAGE showed that prolyl endopeptidase was secreted and expressed in the recombinant strain,but there were different degrees of glycosylation. The enzyme activity test results showed that the highest enzyme activities of recombinant strains TH2-protAS,TH2-SglaA-protA and TH2-SamyA-protA were 1.70,2.19,1.91 U/mL,respectively. On the basis of the recombinant strain TH2-SglaA-protA,gene knockout technology was used to knock out the main background protein acid-stable α-amylase,combined with optimization of fermentation conditions,to obtain high-purity prolyl endopeptidase. Based on the above results,it could conclude that prolyl endopeptidase could be secreted and expressed efficiently in Aspergillus niger. glaA signal peptide and amyA signal peptide significantly increased the secretion and expression of prolyl endopeptidase in Aspergillus niger,and the effect of signal peptide glaA was better than that of amyA signal peptide. The combination of gene knockout and fermentation regulation could effectively remove the secreted background protein and obtain a strain with high-yield and high-purity prolyl endopeptidase. This recombinant strain had a clear industrial application potential.
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[1] 康超.新型食品级脯氨酰内肽酶生产菌筛选、异源表达及分子改造研究[D].无锡:江南大学,2015. [2] Jaako K,Waniek A,Parik K,et al. Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro[J]. Journal of Cell Science,2016,129(20):3792-3802.
[3] Edens L,Dekker P,Van Der Hoeven R,et al. Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates[J]. Journal of Agricultural and Food Chemistry,2005,53(20):7950-7957.
[4] Lopez M,Edens L. Effective prevention of chill-haze in beer using an acid proline specific endoprotease from Aspergillus niger[J]. Journal of Agricultural and Food Chemistry,2005,53(20):7944-7949.
[5] Moreno Amador MdL,Arevalo-Rodriguez M,Duran EM,et al.A new microbial g-lutendegrading prolyl endopeptidase:Potential application in celiacdisease to reduce gluten immunogenic peptides[J]. PLoS One,2019,14(6):e0218346.
[6] Schulz K,Giesler L,Linke D,et al. A prolyl endopeptidase from Flammulina velutipes for the possible cegradation of celiac disease provoking toxic peptides in cereal proteins[J]. Process Biochemistry,2018,73:47-55.
[7] Benucci I,Caso M C,Bavaro T,et al. Prolyl endopeptidase from Aspergillus niger im-mobilized on a food-grade carrier for the production of gluten-reduced beer[J]. Food Control,2019,110:106987.
[8] Sui Y F,Ouyang L M,Schütze T,et al. Comparative genomics of the aconidial Aspergillus niger strain LDM3 predicts genes associated with its high protein secretion capacity[J]. Applied Microbiology and Biotechnology,2020,104(6):2623-2637.
[9] 姚善泾,蔡礼年,林东强.黑曲霉作为分泌蛋白细胞工厂的研究进展[J]. 化工学报,2019,70(10):3690-3703. [10] 许晓红. 高产脯氨酰内肽酶的黑曲霉菌株研究[D]. 广州:华南理工大学,2016. [11] Zhang H,Wang S,Li J,et al. The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background[J]. Microb Cell Fact 2016,15:68.
[12] 李杰,李双,于盛竹,等. 凤尾菇(Pleurotus sajor-caju)漆酶Lac4基因在黑曲霉中表达研究[J]. 东北农业大学学报,2017,48(4):7-14. [13] 江婷. 米曲霉脯氨酰内肽酶在毕赤酵母中的表达、酶学特性及其应用研究[D].无锡:江南大学,2016. [14] Xu Y,WangY H,Liu T Q,et al. The glaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger[J]. Biotechnol Lett,2018,40(6):949-955.
[15] Blumhoff M,Steiger M G,Marx H,et al. Six novel constitutive promoters for metabolic engineering of Aspergillus niger[J]. Applied Microbiology and Biotechnology,2013,97(1):259-267.
[16] Sasaki Yuka,Mitsui Ryosuke,Yamada Ryosuke,et al. Secretory overexpression ofthe endo-glucanase by Saccharomyces cerevisiae via CRISPR-delta-integrationand multiple promoter shuffling[J]. Enzymeand Microbial Technology,2019,121:17-22.
[17] 卢春霞,刘长彬,石国庆,杨华.基于密码子优化策略的牛妊娠相关糖蛋白9(bPAG9)的原核表达及纯化[J]. 江苏农业学报,2020,36(1):122-129. [18] 何明娟,刘宁,邹曙明,蒋霞云,潘迎捷,吴文惠.马槟榔甜蛋白基因(MabinlinⅡ)的密码子优化及其在大肠杆菌中的表达[J]. 食品工业科技,2019,40(18):78-84. [19] 李杰,张贺,王欣,等.多拷贝脂肪酶基因在黑曲霉中表达研究[J]. 东北农业大学学报,2018,49(4):49-58. [20] Yang H,Zhai C. Yu X,et al. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains[J]. Protein Expression and Purification,2016,122:38-44.
[21] 王义春,王龑,江均平,等.玉米赤霉烯酮降解酶多拷贝毕赤酵母菌株的构建及高效表达[J]. 生物工程学报,2020,36(2):372-380. [22] Sha C,Yu X W,Lin N X,et al. Enhancement of lipase r27RCL production in Pichiapastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase[J].Enzyme and Microbial Technology,2013,53(6-7):438-443.
[23] Damasceno L M,Anderson K A,Ritter G,et al. Co-overexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris[J]. Applied Microbiology and Biotechnology,2007,74(2):381-389.
[24] 高佳. 里氏木霉内质网蛋白折叠途径改造与异源蛋白BGLA和Lcc1的表达研究[D].济南:山东大学,2018. [25] Carvalho N D S P,Arentshorst M,Kooistra R,et al. Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger[J]. Applied Microbiology & Biotechnology,2011,89(2):357-373.
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