米曲霉酸性蛋白酶基因在毕赤酵母中的异源表达及酶学性质
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摘要
酸性蛋白酶作为一类重要的天冬氨酸蛋白酶,被广泛应用于食品、医药和皮革等领域。为推动酸性蛋白酶的研究及应用,通过对发酵豆制品样品进行宏基因组测序,从中获得米曲霉酸性蛋白酶基因pepA,在毕赤酵母GS115中进行异源表达,并对重组酶PepA进行酶学性质分析。结果显示毕赤酵母发酵上清液中酸性蛋白酶的活性为50.62 U/mL。SDS-PAGE验证PepA的分子量约为50 kDa,且发酵上清液几乎无杂蛋白。PepA的最适pH值为4.5,最适温度为50℃,Mn~(2+)和Cu~(2+)对其具有激活作用,而Fe~(3+)、Fe~(2+)与Ca~(2+)则具有抑制作用。上述研究结果可为米曲霉酸性蛋白酶的异源表达及其相关工业应用提供指导。
Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene(pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn~(2+) and Cu~(2+)enhanced the activity of PepA, whereas Fe~(3+), Fe~(2+) and Ca~2 had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.
Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene(pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn~(2+) and Cu~(2+)enhanced the activity of PepA, whereas Fe~(3+), Fe~(2+) and Ca~2 had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.
引文
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[3]Yu PB,Chen LL,Zhang B,et al.Study on improving the quality of rice wine Wheat Qu by making with two strains.Ferment Indust,2012,38(9):1-6(in Chinese).余培斌,陈亮亮,张波,等.双菌种制曲改善黄酒麦曲品质的研究.食品与发酵工业,2012,38(9):1-6.
[4]Zhao H,Zhao SX,Zhang W,et al.The study on the application of acid proteinase to ethanol fermentation.Ferment Indust,1997,23(2):26-28(in Chinese).赵华,赵树欣,张维,等.酒精发酵中应用酸性蛋白酶的研究.食品与发酵工业,1997,23(2):26-28.
[5]Xiao DG,Zhao H,Zhai JX,et al.Studies on application technology of acid protease in alcohol productionⅢ.The application experiment in alcohol production.Liquor-Making Sci Technol,2000(4):34-35,37(in Chinese).肖冬光,赵华,翟建新,等.酒用酸性蛋白酶在酒精生产中应用技术的研究Ⅲ.酒精生产应用试验.酿酒科技,2000(4):34-35,37.
[6]Arai S,Makoto A,Yamashita M,et al.Applying proteolytic enzymes on soybean.Agr Biol Chem,1971,35(4):552-559.
[7]Xu YS,Dai MJ,Zang JH,et al.Purification and characterization of an extracellular acidic protease of Pediococcus pentosaceus isolated from fermented fish.Food Sci Technol Res,2015,21(5):739-744.
[8]Lu C,Bian LQ,Liu XJ,et al.Effects of neutral and acid protease on growth performance in weaned piglets.Chin J Animal Nut,2009,21(6):993-997(in Chinese).卢晨,边连全,刘显军,等.中性和酸性蛋白酶对断奶仔猪生长性能的影响.动物营养学报,2009,21(6):993-997.
[9]Wu YL,Huo SY.Research on the determination of acidic protease activity in feed premixed with enzyme additive.J Agric Sci Technol,2009,11(1):52-55(in Chinese).武英利,霍韶瑜.饲用酶添加剂预混料中酸性蛋白酶活力测定方法研究.中国农业科技导报,2009,11(1):52-55.
[10]Chen Q.Anti-edema drug-swelling elimination.Shanxi Medical J,1985,24(2):112-113(in Chinese).陈铨.抗水肿新药--肿消净.山西医药杂志,1985,24(2):112-113.
[11]Li SP,Jiang QL,Bi CY.Studies and developments on the acid protease for leather processing.Shandong Sci,1996,9(4):62-65(in Chinese).李树品,蒋千里,毕春元.皮革用酸性蛋白酶的研究与开发.山东科学,1996,9(4):62-65.
[12]Lin SZ,Liu WC,Shan YZ.Application of enzyme mixtures in aquaculture feed.Fisher Sci,2009,28(12):798-800(in Chinese).林少珍,刘伟成,单乐州.酶制剂在水产养殖饲料中的应用.水产科学,2009,28(12):798-800.
[13]Xu DF.Breeding of novel fusant with higher activity of acid protease from aspergillus and its preliminary application in soy sauce fermentation[D].Guangzhou:South China University of Technology,2010(in Chinese).徐德峰.高活力酸性蛋白酶曲霉融合子的选育及其在酱油发酵中的初步应用[D].广州:华南理工大学,2010.
[14]Deng J,Xu J,Wu HC,et al.Screening of acid protease-overproducing stranins of Aspergillus oryzae.China Cond,2010,35(1):53-55,59(in Chinese).邓静,徐静,吴华昌,等.米曲霉高产酸性蛋白酶菌株的选育.中国调味品,2010,35(1):53-55,59.
[15]Cao XH,Zhang B,Lu MF,et al.A rapid method for screening excellent soy sauce producing Aspergillums oryza mutagenized by ion implanting.China Cond,2007,5(5):26-30(in Chinese).曹小红,张斌,鲁梅芳,等.离子注入诱变米曲霉及酱油优良生产菌株的快速筛选.中国调味品,2007,5(5):26-30.
[16]de Castro RJS,Sato HH.Advantages of an acid protease from Aspergillus oryzae over commercial preparations for production of whey protein hydrolysates with antioxidant activities.Biocatal Agric Biotechnol,2014,3(3):58-65.
[17]Srinubabu G,Lokeswari N,Jayaraju K.Screening of nutritional parameters for the production of protease from Aspergillus oryzae.E-J Chem,2007,4(2):208-215.
[18]Guo JP,Ma G.Optimization of solid-state fermentation medium for acid protease of Aspergillus oryzae.China Brewing,2009(8):125-128(in Chinese).郭继平,马光.响应面法优化米曲霉酸性蛋白酶的固态发酵培养基.中国酿造,2009(8):125-128.
[19]Lee SK,Wang JY,Choi SH,et al.Purification and characterization of Aspergillus oryzae LK-101salt-tolerant acid protease isolated from soybean paste.Food Sci Biotechnol,2010,19(2):327-334.
[20]Zhu TC,Li Y.Recent development of Pichia pastoris system:current status and future perspective.Chin JBiotech,2015,31(6):929-938(in Chinese).朱泰承,李寅.毕赤酵母表达系统发展概况及趋势.生物工程学报,2015,31(6):929-938.
[21]Ahmad M,Hirz M,Pichler H,et al.Protein expression in Pichia pastoris:recent achievements and perspectives for heterologous protein production.Appl Microbiol Biotechnol,2014,98(12):5301-5317.
[22]Damasceno LM,Chung JH,Batt CA.Protein secretion in Pichia pastoris and advances in protein production.Appl Microbiol Biotechnol,2012,93(1):31-39.
[23]Cos O,Ramón R,Montesinos JL,et al.Operational strategies,monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters:a review.Microb Cell Fact,2006,5:17.
[24]Daly R,Hearn MTW.Expression of heterologous proteins in Pichia pastoris:a useful experimental tool in protein engineering and production.J Mol Recognit,2005,18(2):119-138.
[25]Xiong AS,Peng RH,Li X,et al.Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris.Acta Bioch et Bioph Sin,2003,35(2):154-160(in Chinese).熊爱生,彭日荷,李贤,等.信号肽序列对毕赤酵母表达外源蛋白质的影响.生物化学与生物物理学报,2003,35(2):154-160.
[26]Zhan HJ,Liu BS,Reid SW,et al.Engineering a soluble extracellular erythropoietin receptor(EPObp)in Pichia pastoris to eliminate microheterogeneity,and its complex with erythropoietin.Protein Eng,1999,12(6):505-513.
[27]Qin HB,Li MF,Yang HJ.Heteroespression of cellobiohydrolase A gene of Aspergillus niger in Pachia pastoris.Biotechnology,2017,27(4):330-336(in Chinese).秦慧彬,李梦菲,杨洪江.黑曲霉纤维二糖水解酶基因cbhA在毕赤酵母中异源表达.生物技术,2017,27(4):330-336.
[28]Gomi K,Arikawa K,Kamiya N,et al.Cloning and nucleotide sequence of the acid protease-encoding gene(pepA)from Aspergillus oryzae.Biosci Biotechnol Biochem,1993,57(7):1095-1100.
[29]Nierman WC,Pain A,Anderson MJ,et al.Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.Nature,2005,438(7071):1151-1156.
[30]Yang H.Expression and characterization of acid protease from Aspergillus usamil in Pichia pastoris[D].Wuhan:Hubei University,2014(in Chinese).杨琥.宇佐美曲霉酸性蛋白酶在毕赤酵母中的表达及其酶学性质的研究[D].武汉:湖北大学,2014.
[31]Zhang L,Jiang Y,Zhang J,et al.Recombinant expression of bovine chymosin in Pichia pastoris.Chin J Biotech,2009,25(8):1160-1165(in Chinese).张莉,姜媛媛,张健,等.牛凝乳酶基因在毕赤酵母中的重组表.生物工程学报,2009,25(8):1160-1165.
[32]Cregg JM,Barringer KJ,Hessler AY,et al.Pichia pastoris as a host system for transformations.Mol Cell Biol,1985,5(12):3376-3385.
[33]Handelsman J,Rondon MR,Brady SF,et al.Molecular biological access to the chemistry of unknown soil microbes:a new frontier for natural products.Chem Biol,1998,5(10):R245-R249.
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