菊粉外切酶的异源表达、纯化及酶学性质
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摘要
将菊粉降解菌Paenibacillus sp. Lfos16的菊粉外切酶基因克隆至表达载体p ET-28a (+),转入Escherichia. coli BL21(DE3)中实现了异源表达。利用镍柱亲和层析纯化重组菊粉外切酶,并进行SDS-PAGE检测。重组菊粉外切酶的表观分子质量为87 k Da,经纯化后,重组菊粉外切酶的比酶活为348.30 U/mg。重组菊粉外切酶作用菊粉和蔗糖的酶活分别为259.37 U/m L和592.16 U/m L,I/S值为0.438,且重组酶水解菊粉的主要产物为果糖。重组菊粉外切酶最适作用温度为40℃,且当温度低于30℃时酶活较稳定;最适作用pH为6。Ag~+、Cu~(2+)、Mn~(2+)、Zn~(2+)、Hg~(2+)、Fe~(3+)具有显著抑制作用。重组菊粉外切酶对菊粉的K_m为19. 28 mg/m L,Vmax为0.18 mg/(min·m L)。
The gene encoding an exo-inulinase was cloned into p ET-28 a( +) from an inulin-degrading Paenibacillus sp. Lfos16 and expressed in Escherichia coli BL21( DE3). The recombinant exo-inulinase was purified using nickel column affinity chromatography and verified by SDS-PAGE. The molecular weight of recombinant exo-inulinase was 87 k Da with the specific activity of 348.30 U/mg protein. Its activities on inulin and sucrose were 259.37 U/m L and 592.16 U/m L,respectively,with an I/S value of 0.438,and fructose was the main product from inulin. The recombinant exoinulinase had the optimal reaction temperature and p H of 40 ℃ and 6.0,respectively. It was stable when the temperature was lower than 30 ℃. It was significantly inhibited by Ag~+,Cu~(2+),Mn~(2+),Zn~(2+),Hg~(2+)and Fe~(3+). The Kmand Vmaxof recombinant exo-inulinase on inulin were 19.28 mg/m L and 0.18 mg/( min·m L),respectively.
The gene encoding an exo-inulinase was cloned into p ET-28 a( +) from an inulin-degrading Paenibacillus sp. Lfos16 and expressed in Escherichia coli BL21( DE3). The recombinant exo-inulinase was purified using nickel column affinity chromatography and verified by SDS-PAGE. The molecular weight of recombinant exo-inulinase was 87 k Da with the specific activity of 348.30 U/mg protein. Its activities on inulin and sucrose were 259.37 U/m L and 592.16 U/m L,respectively,with an I/S value of 0.438,and fructose was the main product from inulin. The recombinant exoinulinase had the optimal reaction temperature and p H of 40 ℃ and 6.0,respectively. It was stable when the temperature was lower than 30 ℃. It was significantly inhibited by Ag~+,Cu~(2+),Mn~(2+),Zn~(2+),Hg~(2+)and Fe~(3+). The Kmand Vmaxof recombinant exo-inulinase on inulin were 19.28 mg/m L and 0.18 mg/( min·m L),respectively.
引文
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[17]王东方,曹慧,陈冠军.黑曲霉菊糖酶酶学性质及酶解产物分析[J].食品工业,2015,36(9):168-171.
[18]李益民,高教琪,袁文杰,等.菊粉酶基因的异源表达、分离纯化及酶学性质[J].生物工程学报,2015,31(5):670-681.
[19]KOBAYASHI T,UCHIMURA K,DEGUCHI S,et al.Cloning and sequencing of inulinase andβ-fructofuranosidase genes of a deep-sea Microbulbifer species and properties of recombinant enzymes[J].Applied and Environmental Microbiology,2012,78(7):2 493-2 495.
[20]CHEN Xiao-ming,XU Xue-ming,JIN Zheng-yu,et al.Expression of an exoinulinase gene from Aspergillus ficuum in Escherichia coli and its characterization[J].Carbohydrate Polymers,2013,92(2):1 984-1 990.
[21]MA Jun-yan,CAO Hui-long,TAN Hai-dong,et al.Cloning,expression,characterization,and mutagenesis of a thermostable exoinulinase from Kluyveromyces cicerisporus[J].Applied Biochemistry and Biotechnology,2016,178(1):144-158.
[22]GAO Jian,XU You-yong,YANG Hong-mei,et al.Gene Cloning,Expression,and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9[J].Applied Biochemistry and Biotechnology,2014,173(6):1 419-1 430.
[2]LIU Yuan,ZHOU Shun-hua,CHENG Yu-rong,et al.Synergistic effect between the recombinant exo-inulinase and endo-inulinase on inulin hydrolysis[J].Journal of Molecular Catalysis B:Enzymatic,2016,128(1):27-38.
[3]RAMAPRIYA R,THIRUMURUGAN A,SATHISKUMART,et al.Partial purification and characterization of exoinulinase produced from Bacillus sp.[J].Journal of Genetic Engineering and Biotechnology,2018:S1687157×18300192.
[4]HEMANT K R,HEMANT S,NAVEEN K,et al.Continuous generation of fructose from Taraxacum officinale tap root extract and inulin by immobilized inulinase in a packed-bed reactor[J].Biocatalysis and Agricultural Biotechnology.2018,9(1):134-140.
[5]KANIKA C,ASHOK P,CHRISTIAN L,et al.Biocatalytic strategies for the production of high fructose syrup from inulin[J].Bioresource Technology,2018,260(1):395-403.
[6]刘成龙,周海岩,柳志强,等.葡萄糖异构酶及其在高果糖浆生产中的应用[J].氨基酸和生物资源,2014,36(3):12-19.
[7]FUSINITA V D E,JAN L.RF cloning:A restriction-free method for inserting target genes into plasmids[J].Journal of Biochemical and Biophysical Methods,2006,67(1):67-74.
[8]UNGER T,JACOBOVITCH Y,DANTES A,et al.Applications of the Restriction Free(RF)cloning procedure for molecular manipulations and protein expression[J].Journal of Structural Biology,2010,172(1):34-44.
[9]JOSHUA M J,ADAM E,MITCHELL R L,et al.Growth,ethanol production,and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y 50798 and NRRL Y 50799[J].Journal of Industrial Microbiology&Biotechnology,2016,43(7):927-939.
[10]YUAN Bo,HU Nan,SUN Juan,et al.Purification and characterization of a novel extracellular inulinase from a new yeast species Candida kutaonensis sp.nov.KRF1(T)[J].Applied Microbiology and Biotechnology,2012,96(6):1 517-1 526.
[11]TAREK M M,EHAB M A,ALY S A,et al.Immobilization and characterization of inulinase from Ulocladium atrum on nonwoven fabrics[J].Journal of Bioscience and Bioengineering,2014,39(5):785-794.
[12]RAM S S,KANIKA C,ASHOK P,et al.Purification and characterization of two isoforms of exoinulinase from Penicillium oxalicum BGPUP-4 for the preparation of high fructose syrup from inulin[J].International Journal of Biological Macromolecules.2018.
[13]HOUSSEINY M M.Production of an endoinulinase from Aspergillus niger AUMC 9375,by solid state fermentation of agricultural wastes,with purification and characterization of the free and immobilized enzyme[J].Journal of Microbiology,2014,52(2):389-398.
[14]张天祥,丁宁,杨春光,等.菊粉酶的研究进展[J].中国酿造,2016,35(11):21-25.
[15]JEZA S,MASEKO S B,Lin J.Purification and Characterization of Exo-Inulinase from Paenibacillus sp.d9 Strain[J].The Protein Journal,2018,37(1):70-81.
[16]YEDAHALLI S S.Expression of Exo-Inulinase Gene from Aspergillus niger 12 in E.Coli Strain Rosetta-Gami B(DE3)and Its Characterization[J].Biotechnology Progress,2016,32(3):629-637.
[17]王东方,曹慧,陈冠军.黑曲霉菊糖酶酶学性质及酶解产物分析[J].食品工业,2015,36(9):168-171.
[18]李益民,高教琪,袁文杰,等.菊粉酶基因的异源表达、分离纯化及酶学性质[J].生物工程学报,2015,31(5):670-681.
[19]KOBAYASHI T,UCHIMURA K,DEGUCHI S,et al.Cloning and sequencing of inulinase andβ-fructofuranosidase genes of a deep-sea Microbulbifer species and properties of recombinant enzymes[J].Applied and Environmental Microbiology,2012,78(7):2 493-2 495.
[20]CHEN Xiao-ming,XU Xue-ming,JIN Zheng-yu,et al.Expression of an exoinulinase gene from Aspergillus ficuum in Escherichia coli and its characterization[J].Carbohydrate Polymers,2013,92(2):1 984-1 990.
[21]MA Jun-yan,CAO Hui-long,TAN Hai-dong,et al.Cloning,expression,characterization,and mutagenesis of a thermostable exoinulinase from Kluyveromyces cicerisporus[J].Applied Biochemistry and Biotechnology,2016,178(1):144-158.
[22]GAO Jian,XU You-yong,YANG Hong-mei,et al.Gene Cloning,Expression,and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9[J].Applied Biochemistry and Biotechnology,2014,173(6):1 419-1 430.