米曲霉乳糖酶基因在乳酸克鲁维酵母中的表达
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摘要
为获得高活性的乳糖酶制剂,根据米曲霉乳糖酶基因序列以及宿主细胞乳酸克鲁维酵母GG799密码子的偏爱性,对米曲霉乳糖酶基因进行优化。将已优化的米曲霉乳糖酶lacA基因片段插入到乳酸克鲁维酵母GG799表达载体pKLAC1中,构建重组载体p KLAC1-lacA,用电脉冲法将SacⅡ酶线性化的重组质粒转入乳酸克鲁维酵母GG799中。经过5 mmol/L酵母碳基础培养基(yeast carbon base,YCB)活性筛选,全细胞聚合酶链式反应(polymerase chain reaction,PCR)鉴定,最后获得了一株多拷贝整合的基因工程菌株lacA-1。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectrophoresis,SDS-PAGE)和薄层层析分析(thin-layer chromatography,TLC),该重组菌株可胞外分泌乳糖酶,并具有生物活性。摇瓶发酵培养120 h,酶活力最高达到120.65 U/mL。本研究成功地将米曲霉乳糖酶基因在乳酸克鲁维酵母GG799中表达,获得的重组菌株lacA-1可胞外分泌的乳糖酶并有较高的乳糖酶活性,试验结果为利用乳酸克鲁维酵母GG799表达系统规模化生产乳糖酶奠定基础。
To obtain lactase preparations with high activity,a lactase gene from Aspergillus oryzae was synthesized based on the preference of Kluyveromyces lactis GG799 codon. The synthesized lactase gene from Aspergillus oryzae was cloned into the expression vector pKLAC1,resulting in pKLAC1-lacA.The pKLAC1-lacA was linearized by SacⅡand transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by yeast carbon base(YCB)plates containing 5 mmol/L acetamide. A multi-copy recombinant strain lacA-1 was obtained by whole-cell polymerase chain reaction(PCR) verification. The recombinant strain secreted extracellular lactase with biological activity by sodium dodecyl sulfate polyacrylamide gelelectrophoresis(SDS-PAGE) and thin-layer chromatography(TLC)analysis. The lactase activity of the culture supernatant was 120.65 U/mL after 120 hours shaking flask cultivation. This research successfully expressed recombinant lactase gene from A. oryzae in K. lactis GG799 and the expressed protein showed high activity,which provided the basis for the study on the large-scale production of recombinant lactase gene using K. lactis GG799 expression system.
To obtain lactase preparations with high activity,a lactase gene from Aspergillus oryzae was synthesized based on the preference of Kluyveromyces lactis GG799 codon. The synthesized lactase gene from Aspergillus oryzae was cloned into the expression vector pKLAC1,resulting in pKLAC1-lacA.The pKLAC1-lacA was linearized by SacⅡand transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by yeast carbon base(YCB)plates containing 5 mmol/L acetamide. A multi-copy recombinant strain lacA-1 was obtained by whole-cell polymerase chain reaction(PCR) verification. The recombinant strain secreted extracellular lactase with biological activity by sodium dodecyl sulfate polyacrylamide gelelectrophoresis(SDS-PAGE) and thin-layer chromatography(TLC)analysis. The lactase activity of the culture supernatant was 120.65 U/mL after 120 hours shaking flask cultivation. This research successfully expressed recombinant lactase gene from A. oryzae in K. lactis GG799 and the expressed protein showed high activity,which provided the basis for the study on the large-scale production of recombinant lactase gene using K. lactis GG799 expression system.
引文
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[15]Qu P,Zhu J,Liu L,et al.Functional identification of a putative beta-galactosidase gene in the special lac gene cluster of Lactobacillus acidophilus[J].Current Microbiology,2010,60(3):172-178
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[19]徐龙龙,刘思彤,任永成,等.骆驼凝乳酶原基因在乳酸克鲁维酵母中的重组表达[J].中国食品学报,2017,17(12):72-78
[20]刘思彤,徐龙龙,衣泽浩,等.人溶菌酶基因在乳酸克鲁维酵母中的表达[J].现代食品科技,2017,33(3):105-109
[21]惠华英,黄莺,雷志丹,等.乳糖酶活性检测方法研究进展[J].中国微生态学杂志,2016,28(9):1103-1106
[22]赵从波,章淑艳,罗同阳,等.响应面分析法优化乳糖酶发酵培养基[J].中国乳品工业,2015,42(4):38-41
[23]姚乾元.薄层层析法及其应用[J].化学通报,1962,8(2):457-463
[24]吴琼,孙洋,谷伟,等.薄层层析法检测乳糖水解产物的研究[J].食品与机械,2011,27(6):135-137
[25]Juers DH,,Mattews BW,Huber RE.Lac Zβ-galactosidase:Structure and function of an enzyme of historical and molecular biological importance[J].Protein Science,2012,21:1792-1807
[26]Brüssow H.Nutrition,population growth and disease:a short history of lactose[J].Environ Microbiol,2013,15(8):2154-2161
[27]Qianqian Zhao,Fei Liu,Zhongwen Hou,et al.High level production ofβ-Galactosidase exhibiting excellent milk-lactose degradation ability from Aspergillus oryzae by codon and fermentation optimization[J].Applied Biochemistry and Biotechnology,2014,172(6):2787-2799
[2]赵晓慧,苏佳,史玉东,等.牛乳工业中乳糖酶的应用研究进展[J].食品科技,2016,41(4):228-231
[3]陶东亚,苏述平.儿童乳糖不耐受症的诊断与治疗进展[J].现代医药卫生,2018,34(6):879-882
[4]俞栋,叶环.婴儿乳糖不耐受病因研究及防治进展[J].预防医学,2018,30(8):803-805
[5]贺璐,龙承星,刘又嘉,等.微生物乳糖酶研究进展[J].食品与发酵工业,2017,43(6):268-273
[6]van Casteren WH,Eimerman M,van den Broek LA,et al.Purifieation and charaeterization ofβ-galactosidase from Aspergillus aculeatus with activity towards(modified)expoly sacchrids from Lacotococcus Lctis subsp.Cremoris B39 and B891[J].Carbohydrate Research,2000,329(1):75-85
[7]Martin DB,Souza CG,Simoes DA,et al.Theβ-galactosidase activity in Kluyveromyces marxianus CBS6556 decreases by high concentrations of galactose[J].Current Microbiology,2002,44:379-382
[8]张智敏,庄淼,金锋杰.米曲霉基因工程技术的进展[J].生物技术通报,2018,34(9):1-6
[9]徐金利.产乳糖酶酵母菌株的遗传改良和乳糖酶的发酵生产[D].北京:中国海洋大学,2012
[10]欧军.米曲霉生产乳糖酶的研究[D].哈尔滨:东北林业大学,2008
[11]李淑娟.黑曲霉乳糖酶基因的克隆及其在毕赤酵母中的表达[D].保定:河北林业大学,2007
[12]张伟,范云六,姚斌.亮白曲霉乳糖酶基因在毕赤酵母中的高效分泌表达及酶学性质研究[J].微生物学报,2005,45(2):247-252
[13]侯重文,朱梓昂,张秀华,等.毕赤酵母工程菌产乳糖酶发酵条件优化的研究[J].食品与药品,2016,18(5):305-311
[14]张霞.基因工程技术生产乳糖酶的研究[D].北京:北京化工大学,2015
[15]Qu P,Zhu J,Liu L,et al.Functional identification of a putative beta-galactosidase gene in the special lac gene cluster of Lactobacillus acidophilus[J].Current Microbiology,2010,60(3):172-178
[16]Chen W,Chen H,Xia Y,et al.Production,purification,and charaterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillues stearorhermophilus[J].Journal of Dairy Science,2008,91(5):1751-1758
[17]刘波,马清钧,吴军.乳酸克鲁维酵母表达外源蛋白研究进展[J].生物技术通讯,2007,18(6):1039-1042
[18]唐南筠,霍克克,李育阳.乳酸克鲁维酵母高拷贝整合载体的构建及应用[J].生物化学与生物物理学报,1996,28(5):540-546
[19]徐龙龙,刘思彤,任永成,等.骆驼凝乳酶原基因在乳酸克鲁维酵母中的重组表达[J].中国食品学报,2017,17(12):72-78
[20]刘思彤,徐龙龙,衣泽浩,等.人溶菌酶基因在乳酸克鲁维酵母中的表达[J].现代食品科技,2017,33(3):105-109
[21]惠华英,黄莺,雷志丹,等.乳糖酶活性检测方法研究进展[J].中国微生态学杂志,2016,28(9):1103-1106
[22]赵从波,章淑艳,罗同阳,等.响应面分析法优化乳糖酶发酵培养基[J].中国乳品工业,2015,42(4):38-41
[23]姚乾元.薄层层析法及其应用[J].化学通报,1962,8(2):457-463
[24]吴琼,孙洋,谷伟,等.薄层层析法检测乳糖水解产物的研究[J].食品与机械,2011,27(6):135-137
[25]Juers DH,,Mattews BW,Huber RE.Lac Zβ-galactosidase:Structure and function of an enzyme of historical and molecular biological importance[J].Protein Science,2012,21:1792-1807
[26]Brüssow H.Nutrition,population growth and disease:a short history of lactose[J].Environ Microbiol,2013,15(8):2154-2161
[27]Qianqian Zhao,Fei Liu,Zhongwen Hou,et al.High level production ofβ-Galactosidase exhibiting excellent milk-lactose degradation ability from Aspergillus oryzae by codon and fermentation optimization[J].Applied Biochemistry and Biotechnology,2014,172(6):2787-2799