The gene encoding an exo-inulinase was cloned into pET-28a(+) 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 kDa with the specific activity of 348.30 U/mg protein. Its activities on inulin and sucrose were 259.37 U/mL and 592.16 U/mL, respectively, with an I/S value of 0.438, and fructose was the main product from inulin. The recombinant exo-inulinase had the optimal reaction temperature and pH of 40 ℃ and 6.0, respectively. It was stable when the temperature was lower than 30 ℃. It was significantly inhibited by Ag+, Cu2+, Mn2+, Zn2+, Hg2+ and Fe3+. The Km and Vmax of recombinant exo-inulinase on inulin were 19.28 mg/mL and 0.18 mg/(min·mL), respectively.
MA Jun
,
AN Qikun
,
TANG Wenzhu
. Heterelogous expression, purification and characterization of an exo-inulinase from Paenibacillus sp. Lfos 16[J]. Food and Fermentation Industries, 2019
, 45(4)
: 25
-30
.
DOI: 10.13995/j.cnki.11-1802/ts.018537
[1] 于春,张春红,刘秋,等. 灰平链霉菌Streptomyces griseoplanus S501产外切菊粉酶的分离纯化及酶学性质研究[J]. 食品与发酵工业, 2015, 41(8): 42-47.
[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, SATHISKUMAR T, 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 and β-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.
