K.paraultunense角蛋白酶的分离纯化及酶学性质研究
|
摘要
将角蛋白降解菌Keratinibaculum paraultunense(K.paraultunense)所产角蛋白酶的粗酶液,依次通过硫酸铵沉淀、DEAE-Sepharose-FF阴离子交换以及Superdex 75凝胶色谱层析等分离纯化,最终得到一种电泳纯的角蛋白酶。其纯化倍数为14.55,回收率为7.24%。SDS-PAGE分析结果表明该酶的相对分子质量约为29 KDa。酶学性质研究表明,该酶最适作用温度为80℃;最适pH为7.5,在pH 6.0～9.0范围内酶活力较为稳定;当以酪蛋白为底物时,该酶的动力学常数Km为4.37 mg·mL~(-1),Vm为14 556 U·(mg·min)~(-1),而当以羊毛角蛋白为底物时,该酶的动力学常数Km为15.66 mg·mL~(-1),Vm为2610 U·(mg·min)~(-1);Ca~(2+)对酶活有明显的促进作用,但苯基甲基磺酰氟(PMSF)对酶活有较强的抑制作用。
The purpose of this study was to separate and purify the keratinase fermented by K.paraultunense,and explore its corresponding enzymatic properties.Keratinase was first extracted from fermentation broth of K.paraultunense by the precipitation of ammonium sulfate,and further purified by the DEAE-Sepharose-FF anion exchange and Superdex-75 Gel filtration chromatography.It was found that the keratinase was purified up to 14.55 times with a recovery of 7.24%,and the molecular weight of the purified keratinase is about 29 KDa identified by SDS-PAGE.Furthermore,the optimal reaction temperature of as prepared keratinase is around 80℃;the optimal reaction pH of this keratinase is 7.5,and its activity is stable in the pH range of 6.0-9.0.Its kinetic constants of Km and Vm for casein as a substrate are 4.37 mg·mL~(-1) and 14 556 U·(mg·min)~(-1),respectively.Meanwhile its Km and Vm for wool keratin are 15.66 mg·mL~(-1) and 2610 U·(mg·min)~(-1),respectively.In addition,Ca~(2+) could greatly enhance the activity of keratinase,but its activity was strongly inhibited by phenyl methyl sulfonyl fluoride(PMSF).
The purpose of this study was to separate and purify the keratinase fermented by K.paraultunense,and explore its corresponding enzymatic properties.Keratinase was first extracted from fermentation broth of K.paraultunense by the precipitation of ammonium sulfate,and further purified by the DEAE-Sepharose-FF anion exchange and Superdex-75 Gel filtration chromatography.It was found that the keratinase was purified up to 14.55 times with a recovery of 7.24%,and the molecular weight of the purified keratinase is about 29 KDa identified by SDS-PAGE.Furthermore,the optimal reaction temperature of as prepared keratinase is around 80℃;the optimal reaction pH of this keratinase is 7.5,and its activity is stable in the pH range of 6.0-9.0.Its kinetic constants of Km and Vm for casein as a substrate are 4.37 mg·mL~(-1) and 14 556 U·(mg·min)~(-1),respectively.Meanwhile its Km and Vm for wool keratin are 15.66 mg·mL~(-1) and 2610 U·(mg·min)~(-1),respectively.In addition,Ca~(2+) could greatly enhance the activity of keratinase,but its activity was strongly inhibited by phenyl methyl sulfonyl fluoride(PMSF).
引文
[1]Mazotto A M,Ascheri J L R,Godoy R L D O,et al.Production of feather protein hydrolyzed by B.subtilis,AMRand its application in a blend with cornmeal by extrusion[J].LWT-Food Science and Technology,2017,84(10):701-709.
[2]Rajput R,Gupta R.Thermostable keratinase from Bacillus pumilus KS12:production,chitin crosslinking and degradation of Sup35NM aggregates[J].Bioresource Technology,2013,133(2):118-126.
[3]Calin M,Constantinescu-Aruxandei D,Alexandrescu E,et al.Degradation of keratin substrates by keratinolytic fungi[J].Electronic Journal of Biotechnology,2017,28(3):101-112.
[4]Ramakrishna M R,Sathi K R,Ranjita Y C,et al.Effective feather degradation and keratinase production by Bacillus pumilus GRK for its application as bio-detergent additive[J].Bioresource Technology,2017,243(11):254-263.
[5]贾文睿,丁家琳,张宗才,等.碱性角蛋白酶用于兔皮脱毛的研究[J].皮革科学与工程,2017,27(2):44-48.
[6]候燕燕,鲁凤娟,李晓广,等.芽孢杆菌胞内丝氨酸蛋白酶ISPr的克隆表达及脱毛应用[J].皮革科学与工程,2018,28(3):20-23.
[7]Sanghvi G,Patel H,Vaishnav D,et al.A novel alkaline keratinase from Bacillus subtilis DP1 with potential utility in cosmetic formulation[J].International Journal of Biological Macromolecules,2016,87(3):256-262.
[8]Elhoul M B,Jaouadi N Z,Rekik H,et al.Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea,DZ50[J].International Journal of Biological Macromolecules,2016,92(7):299-315.
[9]王月.角蛋白酶生产菌筛选、发酵优化及其酶学性质研究[D].江南大学,2014.
[10]刘建新,姚大伟,刘艳海,等.嗜热地衣芽胞杆菌Y6角蛋白酶的异源表达与表征[J].农业生物技术学报,2018,26(2):302-310.
[11]羊希,李霞,余康,等.角蛋白降解菌Keratinibaculum paraultunense对废弃羽毛的发酵特性研究[J].中国酿造,2018,37(8):101-106.
[12]Hmidet N,Ali N E H,Zouari-Fakhfakh N,et al.Chicken feathers:a complex substrate for the co-production ofα-amylase and proteases by B.licheniformis NH1[J].Journal of Industrial Microbiology and Biotechnology,2010,37(9):983-990.
[13]Sedmak J J,Grossberg S E.A rapid,sensitive,and versatile assay for protein using Coomassie brilliant blue G250[J].Analytical Biochemistry,1977,79(1-2):544-552.
[14]张荣先.Brevibacillus parabrevis角蛋白酶的表征、异源表达及其分子改造[D].江南大学,2017.
[15]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2001.
[16]秦日甜,谢占玲.镰刀菌Q7-31T果胶酶PGL1的分离纯化、酶学性质鉴定及结构分析[J].生物技术通报,2018,34(04):157-166.
[17]Hamada S,Kubota K,Sagisaka M.Purification and characterization of a novel extracellular neutral metalloprotease from Cerrena albocinnamomea.[J].Journal of General and Applied Microbiology,2017,63(1):51-57.
[18]Ferrareze P A G,Correa A P F,Brandelli A.Purification and characterization of a keratinolytic protease produced by probiotic Bacillus subtilis[J].Biocatalysis and Agricultural Biotechnology,2016,7(2):102-109.
[19]闵杰.金黄杆菌来源的角蛋白酶的分离纯化和酶学性质研究[D].浙江大学,2008.
[20]舒伟学.角蛋白酶高产菌株的选育及其产酶研究[D].西北农林科技大学,2017.
[21]Gegeckas A,Gudiukait R,Citavicius D.Keratinolytic proteinase from Bacillus thuringiensis AD-12[J].International Journal of Biological Macromolecules,2014,69(3):46-51.
[22]朱耀霞.芽孢杆菌角蛋白降解菌及其胞外角蛋白酶的性质、应用研究[D].山东大学,2018.
[23]Pawar V A,Prajapati A S,Akhani R C,et al.Molecular and biochemical characterization of a thermostable keratinase from Bacillus altitudinis,RBDV1[J].3 Biotech,2018,8(2):107-113.
[24]Chaturvedi V,Bhange K,Bhatt R,et al.Production of kertinases using chicken feathers as substrate by a novel multifunctional strain of Pseudomonas stutzeri,and its dehairing application[J].Biocatalysis and Agricultural Biotechnology,2014,3(2):167-174.
[25]Zhang R X,Gong J S,Su C,et al.Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis,CGMCC10798[J].International Journal of Biological Macromolecules,2016,93(11):843-851.
[26]熊丹,王睿,李志强,等.非离子表面活性剂对超临界CO2下酶催化蛋白质水解的影响[J].皮革科学与工程,2010,20(2):31-36.
[27]孔纤,曾运航,张文华,等.角蛋白酶的脱毛能力及对胶原水解作用的评价[J].中国皮革,2016,45(7):25-30.
[28]曹竹,陈坚,高海军,等.生物反应工程原理[M].北京:清华大学出版社,2011:14-16.
[2]Rajput R,Gupta R.Thermostable keratinase from Bacillus pumilus KS12:production,chitin crosslinking and degradation of Sup35NM aggregates[J].Bioresource Technology,2013,133(2):118-126.
[3]Calin M,Constantinescu-Aruxandei D,Alexandrescu E,et al.Degradation of keratin substrates by keratinolytic fungi[J].Electronic Journal of Biotechnology,2017,28(3):101-112.
[4]Ramakrishna M R,Sathi K R,Ranjita Y C,et al.Effective feather degradation and keratinase production by Bacillus pumilus GRK for its application as bio-detergent additive[J].Bioresource Technology,2017,243(11):254-263.
[5]贾文睿,丁家琳,张宗才,等.碱性角蛋白酶用于兔皮脱毛的研究[J].皮革科学与工程,2017,27(2):44-48.
[6]候燕燕,鲁凤娟,李晓广,等.芽孢杆菌胞内丝氨酸蛋白酶ISPr的克隆表达及脱毛应用[J].皮革科学与工程,2018,28(3):20-23.
[7]Sanghvi G,Patel H,Vaishnav D,et al.A novel alkaline keratinase from Bacillus subtilis DP1 with potential utility in cosmetic formulation[J].International Journal of Biological Macromolecules,2016,87(3):256-262.
[8]Elhoul M B,Jaouadi N Z,Rekik H,et al.Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea,DZ50[J].International Journal of Biological Macromolecules,2016,92(7):299-315.
[9]王月.角蛋白酶生产菌筛选、发酵优化及其酶学性质研究[D].江南大学,2014.
[10]刘建新,姚大伟,刘艳海,等.嗜热地衣芽胞杆菌Y6角蛋白酶的异源表达与表征[J].农业生物技术学报,2018,26(2):302-310.
[11]羊希,李霞,余康,等.角蛋白降解菌Keratinibaculum paraultunense对废弃羽毛的发酵特性研究[J].中国酿造,2018,37(8):101-106.
[12]Hmidet N,Ali N E H,Zouari-Fakhfakh N,et al.Chicken feathers:a complex substrate for the co-production ofα-amylase and proteases by B.licheniformis NH1[J].Journal of Industrial Microbiology and Biotechnology,2010,37(9):983-990.
[13]Sedmak J J,Grossberg S E.A rapid,sensitive,and versatile assay for protein using Coomassie brilliant blue G250[J].Analytical Biochemistry,1977,79(1-2):544-552.
[14]张荣先.Brevibacillus parabrevis角蛋白酶的表征、异源表达及其分子改造[D].江南大学,2017.
[15]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2001.
[16]秦日甜,谢占玲.镰刀菌Q7-31T果胶酶PGL1的分离纯化、酶学性质鉴定及结构分析[J].生物技术通报,2018,34(04):157-166.
[17]Hamada S,Kubota K,Sagisaka M.Purification and characterization of a novel extracellular neutral metalloprotease from Cerrena albocinnamomea.[J].Journal of General and Applied Microbiology,2017,63(1):51-57.
[18]Ferrareze P A G,Correa A P F,Brandelli A.Purification and characterization of a keratinolytic protease produced by probiotic Bacillus subtilis[J].Biocatalysis and Agricultural Biotechnology,2016,7(2):102-109.
[19]闵杰.金黄杆菌来源的角蛋白酶的分离纯化和酶学性质研究[D].浙江大学,2008.
[20]舒伟学.角蛋白酶高产菌株的选育及其产酶研究[D].西北农林科技大学,2017.
[21]Gegeckas A,Gudiukait R,Citavicius D.Keratinolytic proteinase from Bacillus thuringiensis AD-12[J].International Journal of Biological Macromolecules,2014,69(3):46-51.
[22]朱耀霞.芽孢杆菌角蛋白降解菌及其胞外角蛋白酶的性质、应用研究[D].山东大学,2018.
[23]Pawar V A,Prajapati A S,Akhani R C,et al.Molecular and biochemical characterization of a thermostable keratinase from Bacillus altitudinis,RBDV1[J].3 Biotech,2018,8(2):107-113.
[24]Chaturvedi V,Bhange K,Bhatt R,et al.Production of kertinases using chicken feathers as substrate by a novel multifunctional strain of Pseudomonas stutzeri,and its dehairing application[J].Biocatalysis and Agricultural Biotechnology,2014,3(2):167-174.
[25]Zhang R X,Gong J S,Su C,et al.Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis,CGMCC10798[J].International Journal of Biological Macromolecules,2016,93(11):843-851.
[26]熊丹,王睿,李志强,等.非离子表面活性剂对超临界CO2下酶催化蛋白质水解的影响[J].皮革科学与工程,2010,20(2):31-36.
[27]孔纤,曾运航,张文华,等.角蛋白酶的脱毛能力及对胶原水解作用的评价[J].中国皮革,2016,45(7):25-30.
[28]曹竹,陈坚,高海军,等.生物反应工程原理[M].北京:清华大学出版社,2011:14-16.