棘孢青霉右旋糖酐酶基因克隆及其在毕赤酵母中的表达
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摘要
以右旋糖酐酶产生菌棘孢青霉F1001基因组为模板反转录合成右旋糖酐酶的c DNA(dex),基因全长1 866 bp,根据毕赤酵母密码子偏好性优化dex序列获得优化右旋糖酐酶基因(opt-dex),分别构建dex-p PICZαA和opt-dex-p PICZαA重组质粒,电击转入毕赤酵母X33中构建重组子。通过蓝色右旋糖酐T-2000平板以及摇瓶发酵筛选获得产右旋糖酐酶的重组酵母菌株。重组酶的酶学性质分析显示,重组酶分子质量65 k Da、最适p H 5.0、最适温度35℃,专一作用于α-1,6糖苷键。在摇瓶水平上对重组毕赤酵母表达条件进行优化,优化培养条件为培养温度25℃、初始p H 5.0、每24 h甲醇添加量1%(体积分数)、每24 h山梨醇添加量5 g/L、吐温-80添加量4 g/L、摇瓶装液量50 m L/500 m L锥形瓶,优化后的重组右旋糖酐酶分泌表达酶活力提高到240.74 U/m L。重组酵母X33是一株适合外源表达棘孢青霉右旋糖酐酶基因的工程菌,该重组酶可替代棘孢青霉右旋糖酐酶直接应用于工业生产催化制备右旋糖酐。
The dextranase-encoding gene(dex) was amplified by reverse transcription PCR from the genome of Penicillium aculeatum F1001. The gene consisted of 1 866 base pairs and encoded a protein of 622 amino acid residues. According to the codon usage bias of Pichia pastoris, optimized gene(opt-dex) was obtained. The expression recombinant plasmids dexp PICZαA and opt-dex-p PICZαA were constructed and then separately electro-transformed into P. pastoris X33 to form transformants. The dextranase-producing transformants were selected out using blue-dextran T-2000 specific plates and shake flask expression. The purified recombinant dextranase showed only one band about 65 k Da, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The recombinant dextranase reacted optimally at p H 5 and 35 ℃ and specifically cleaved α-1,6 glycosidic bonds. The shake-flask fermentation conditions of the recombinant strain were optimized as follows: temperature 25 ℃, initial p H 5.0, addition of 1%(V/V) methanol, 4 g/L Tween-80 and 5 g/L sorbitol at 24 h intervals, and 50 m L of medium contained in a 500-m L shake flask. Under the optimized conditions, the activity of dextranase was as high as 240.74 U/m L. In conclusion, this study indicates that P. pastoris X33 is suitable for heterologous expression of P. aculeatum dextranase and that the recombinant dextranase can be used as an alternative to the native dextranase in producing dextran for industrial application.
The dextranase-encoding gene(dex) was amplified by reverse transcription PCR from the genome of Penicillium aculeatum F1001. The gene consisted of 1 866 base pairs and encoded a protein of 622 amino acid residues. According to the codon usage bias of Pichia pastoris, optimized gene(opt-dex) was obtained. The expression recombinant plasmids dexp PICZαA and opt-dex-p PICZαA were constructed and then separately electro-transformed into P. pastoris X33 to form transformants. The dextranase-producing transformants were selected out using blue-dextran T-2000 specific plates and shake flask expression. The purified recombinant dextranase showed only one band about 65 k Da, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The recombinant dextranase reacted optimally at p H 5 and 35 ℃ and specifically cleaved α-1,6 glycosidic bonds. The shake-flask fermentation conditions of the recombinant strain were optimized as follows: temperature 25 ℃, initial p H 5.0, addition of 1%(V/V) methanol, 4 g/L Tween-80 and 5 g/L sorbitol at 24 h intervals, and 50 m L of medium contained in a 500-m L shake flask. Under the optimized conditions, the activity of dextranase was as high as 240.74 U/m L. In conclusion, this study indicates that P. pastoris X33 is suitable for heterologous expression of P. aculeatum dextranase and that the recombinant dextranase can be used as an alternative to the native dextranase in producing dextran for industrial application.
引文
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[17]王建荣,刘丹妮,夏雨,等.密码子优化及透明颤菌血红蛋白共表达提高耐热脂肪酶在毕赤酵母的表达[J].食品科学,2016,37(19):135-140.DOI:10.7506/spkx1002-6630-201619023.
[18]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72:248-254.
[19]BEN AZOUN S,KALLEL H.Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach[J].Microbiologyopen,2017,6(4):1-9.DOI:10.1002/mbo3.489.
[20]YANG Z,ZHANG Z.Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris:a review[J].Biotechnol Advance,2018,36(1):182-195.DOI:10.1016/j.biotechadv.2017.11.002.
[21]槐强强,贾禄强,丁健,等.通过在低细胞浓度下启动甲醇诱导、优化碳/能量代谢模式促进毕赤酵母表达Monellin[J].生物工程学报,2018,34(2):282-293.DOI:10.13345/j.cjb.170189.
[22]LIU Y,XUAN S,LONG C,et al.Screening,identifying of cellulosedecomposing strain L-06 and its enzyme-producing conditions[J].Chinese Journal of Biotechnology,2008,24(6):1112-1116.DOI:10.13345/j.cjb.2008.06.017.
[23]LOOSER V,BRüHLMANN B,BUMBAK F,et al.Cultivation strategies to enhance productivity of Pichia pastoris:a review[J].Biotechnology Advances,2015,33(6):1177-1193.DOI:10.1016/j.biotechadv.2015.05.008.
[24]汪汇慧,金虎,高敏杰,等.甲醇/山梨醇共混流加诱导改变毕赤酵母生产猪α-干扰素过程的代谢产能途径强化发酵性能[J].生物工程学报,2012,28(2):164-177.DOI:10.13345/j.cjb.2012.02.003.
[25]王丙莲,冯东,梁晓辉,等.甘油及甲醇补料策略对毕赤酵母表达猪α-干扰素的影响[J].激光生物学报,2016,25(6):553-558.DOI:10.3969/j.issn.1007-7146.2016.06.011.
[26]麻少莹.细丽毛壳菌发酵生产右旋糖酐酶的工艺条件优化及应用研究[D].南宁:广西大学,2014:20-50.DOI:10.7666/d.D524754.
[27]CHEN L,ZHOU X S,FAN W M,et al.Expression,purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris[J].Protein Expression and Purification,2008,58(1):87-93.DOI:10.1016/j.pep.2007.10.021.
[28]KOENIG D W,DAY D F.Induction of Lipomyces starkeyi dextranase[J].Applied&Environmental Microbiology,1989,55(8):2079.
[29]白仁惠,张云博,王春迪,等.里氏木霉Cel5A基因优化及其在毕赤酵母中的高效表达[J].生物工程学报,2016,32(10):1381-1394.DOI:10.13345/j.cjb.160017.
[30]王建荣,刘丹妮,夏雨,等.优化密码子及诱导温度提高雪白根霉脂肪酶在毕赤酵母中的表达[J].食品与发酵工业,2017,43(1):18-23.DOI:10.13995/j.cnki.11-1802/ts.201701004.
[2]ZHANG H B,GAN W W,ZHANG Y Q,et al.Synthesis of isomaltooligosaccharides by using recombinant dextransucrase and Hypocrea lixii dextranase[J].Journal of Chemical&Pharmaceutical Research,2013,5(11):49-53.
[3]KIM Y M,SEO M Y,KANG H K,et al.Construction of a fusion enzyme of dextransucrase and dextranase:application for one-step synthesis of isomalto-oligosaccharides[J].Enzyme&Microbial Technology,2009,44(3):159-164.DOI:10.1016/j.enzmictec.2008.10.007.
[4]THITARAM S N,CHUNG C H,DAY D F,et al.Isomaltooligosaccharide increases cecal bifidobacterium population in young broiler chickens[J].Poultry Science,2005,84(7):998.DOI:10.1093/ps/84.7.988.
[5]WANG X,CHENG H,LU M,et al.Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis[J].Biofouling,2016,32(10):1223-1233.DOI:10.1080/08927014.2016.1239722.
[6]OTSUKA R,IMAI S,MURATA T,et al.Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation[J].Microbiology&Immunology,2015,59(1):28-36.DOI:10.1111/1348-0421.12214.
[7]KANG H K,PARK J Y,AHN J S,et al.Cloning of a gene encoding dextranase from Lipomyces starkeyi and its expression in Pichia pastoris[J].Journal of Microbiology&Biotechnology,2009,19(2):172-177.DOI:10.4014/jmb.0802.100.
[8]ROCA H,GARCIA B,RODRIGUEZ E,et al.Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris[J].Yeast,1996,12(12):1187-1200.DOI:10.1002/(SICI)1097-0061(19960930)12:12<1187::AIDYEA986>3.0.CO;2-U.
[9]梁达奉.α-葡聚糖酶的基因工程菌构建、发酵及其应用研究[D].广州:广东工业大学,2011:20-50.DOI:10.7666/d.y2028007.
[10]黄曾慰,梁达奉,曾练强,等.朱黄青霉α-葡聚糖酶在毕赤酵母中的高效表达[J].广西科学,2014(6):614-618.DOI:10.13656/j.cnki.gxkx.2014.06.006.
[11]ABDEL-NABY M A,ISMAIL A M S,ABDEL-FATTAH A M,et al.Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase[J].Process Biochemistry,1999,34(4):391-398.DOI:10.1016/S0032-9592(98)00127-7.
[12]SZCZODRAK J,PLESZCZYNSKA M,FIEDUREK J.Penicillium notatum 1 a new source of dextranase[J].Journal of Industrial Microbiology and Biotechnology,1994,13(5):315-320.
[13]PLESZCZYNSKA M,ROGALSKI J,SZCZODRAK J,et al.Purification and some properties of an extracellular dextranase from Penicillium notatum[J].Mycological Research,1996,100(6):681-686.DOI:10.1016/S0953-7562(96)80198-5.
[14]ZHANG Y Q,LI R H,ZHANG H B,et al.Purification,characterization,and application of a thermostable dextranase from Talaromyces pinophilus[J].Journal of Industrial Microbiology&Biotechnology,2016,44(2):1-11.DOI:10.1007/s10295-016-1886-8.
[15]ERHARDT F A,STAMMEN S,J諶DENING H J.Production,characterization and(co-)immobilization of dextranase from Penicillium aculeatum[J].Biotechnology Letters,2008,30(6):1069-1073.DOI:10.1007/s10529-008-9659-8.
[16]张洪斌,吴定涛,黄丽君,等.一株产右旋糖酐酶青霉的分离及酶的纯化和性质[J].微生物学报,2011,51(4):495-503.DOI:10.13343/j.cnki.wsxb.2011.04.006.
[17]王建荣,刘丹妮,夏雨,等.密码子优化及透明颤菌血红蛋白共表达提高耐热脂肪酶在毕赤酵母的表达[J].食品科学,2016,37(19):135-140.DOI:10.7506/spkx1002-6630-201619023.
[18]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72:248-254.
[19]BEN AZOUN S,KALLEL H.Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach[J].Microbiologyopen,2017,6(4):1-9.DOI:10.1002/mbo3.489.
[20]YANG Z,ZHANG Z.Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris:a review[J].Biotechnol Advance,2018,36(1):182-195.DOI:10.1016/j.biotechadv.2017.11.002.
[21]槐强强,贾禄强,丁健,等.通过在低细胞浓度下启动甲醇诱导、优化碳/能量代谢模式促进毕赤酵母表达Monellin[J].生物工程学报,2018,34(2):282-293.DOI:10.13345/j.cjb.170189.
[22]LIU Y,XUAN S,LONG C,et al.Screening,identifying of cellulosedecomposing strain L-06 and its enzyme-producing conditions[J].Chinese Journal of Biotechnology,2008,24(6):1112-1116.DOI:10.13345/j.cjb.2008.06.017.
[23]LOOSER V,BRüHLMANN B,BUMBAK F,et al.Cultivation strategies to enhance productivity of Pichia pastoris:a review[J].Biotechnology Advances,2015,33(6):1177-1193.DOI:10.1016/j.biotechadv.2015.05.008.
[24]汪汇慧,金虎,高敏杰,等.甲醇/山梨醇共混流加诱导改变毕赤酵母生产猪α-干扰素过程的代谢产能途径强化发酵性能[J].生物工程学报,2012,28(2):164-177.DOI:10.13345/j.cjb.2012.02.003.
[25]王丙莲,冯东,梁晓辉,等.甘油及甲醇补料策略对毕赤酵母表达猪α-干扰素的影响[J].激光生物学报,2016,25(6):553-558.DOI:10.3969/j.issn.1007-7146.2016.06.011.
[26]麻少莹.细丽毛壳菌发酵生产右旋糖酐酶的工艺条件优化及应用研究[D].南宁:广西大学,2014:20-50.DOI:10.7666/d.D524754.
[27]CHEN L,ZHOU X S,FAN W M,et al.Expression,purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris[J].Protein Expression and Purification,2008,58(1):87-93.DOI:10.1016/j.pep.2007.10.021.
[28]KOENIG D W,DAY D F.Induction of Lipomyces starkeyi dextranase[J].Applied&Environmental Microbiology,1989,55(8):2079.
[29]白仁惠,张云博,王春迪,等.里氏木霉Cel5A基因优化及其在毕赤酵母中的高效表达[J].生物工程学报,2016,32(10):1381-1394.DOI:10.13345/j.cjb.160017.
[30]王建荣,刘丹妮,夏雨,等.优化密码子及诱导温度提高雪白根霉脂肪酶在毕赤酵母中的表达[J].食品与发酵工业,2017,43(1):18-23.DOI:10.13995/j.cnki.11-1802/ts.201701004.