内切菊粉酶可以有效地水解菊粉得到低聚果糖(inulooligosaccharides,IOS)。低聚果糖是一种强效益生元,对人体健康有益,具有广泛应用价值。前期研究获得了Lipomyces starkeyi NRRL Y-11557来源的内切菊粉基因inu3B,并在大肠杆菌中进行了克隆表达。为进一步提高该酶催化活力,利用同源建模预测INU3B的三维结构,结合分子对接技术确定底物结合口袋附近的氨基酸,将筛选的关键氨基酸分别突变成丙氨酸,获得突变体M46A,比酶活力达到2 611.40 U/mg,是原始酶的1.26倍。这可能是由于该位点的改变减小了结合口袋的外表面积、体积和深度,增强了酶与底物结合,从而增加了酶活力。M46A突变株具有工业生产低聚果糖的应用前景,也为定点突变技术改造酶活性提供了新思路。
Inulooligosaccharides (IOS) is a kind of prebiotics which favored human health. Endo-inulinase can effectively hydrolyze inulin into IOS. In previous work, the endo-inulinase gene inu3B from Lipomyces starkeyi NRRL Y-11557 was cloned and expressed in Escherichia coli BL21 (DE3). To further improve the activity of INU3B, homology modeling and molecular docking was used. The selected amino acids were mutated into alanine, respectively. The specific activity of mutant M46A was 2 611.40 U/mg and 0.26 times higher than that of wild-type. The enhancement of catalytic activity might by the changes of shape, volume and the depth of the substrate binding pocket. M46A showed great potential to apply in IOS production industry and might provide guidance for engineering of industrial enzymes in the future.
[1] SANGEETHA P T,RAMESH M N,PRAPULLA S G.Recent trends in the microbial production,analysis and application of fructooligosaccharides[J].Trends Food Sci Technol,2005,16(10):442-457.
[2] O'CALLAGHAN A,VAN SINDEREN D.Bifidobacteria and their role as members of the human gut microbiota[J].Front Microbiol,2016,7:925.
[3] AL-SHERAJI S H,ISMAIL A,MANAP M Y,et al.Prebiotics as functional foods:A review[J].J Funct Food,2013,5(4):1 542-1 553.
[4] SCHMIDT K,COWEN P J,HARMER C J,et al.Prebiotic intake reduces the waking cortisol response and alters emotional bias in healthy volunteers[J].Psychopharmacology (Berlin),2015,232(10):1 793-1 801.
[5] KANGO N,JAIN SC.Production and properties of microbial inulinases:Recent advances[J].Food Biotechnol,2011,25(3):165-212.
[6] GANAIE M A,LATEEF A,GUPTA U S.Enzymatic trends of fructooligosaccharides production by microorganisms[J].Appl Biochem Biotechnol,2014,172(4):2 143-2 159.
[7] LIU G L,CHI Z,CHI Z M.Molecular characterization and expression of microbial inulinase genes[J].Crit Rev Microbiol,2013,39(2):152-165.
[8] CHEN M,LEI X,CHEN C Z,et al.Cloning,overexpression,and characterization of a highly active endoinulinase gene from Aspergillus fumigatus CL1 for production of inulo-oligosaccharides[J].Appl Biochem Biotechnol,2015,175(2):1 153-1 167.
[9] WATERHOUSE A,BERTONI M,BIENERT S,et al.SWISS-MODEL:Homology modelling of protein structures and complexes[J].Nucleic Acids Res,2018,46(W1):W296-W303.
[10] 曲戈,赵晶,郑平,等.定向进化技术的最新进展[J].生物工程学报,2018,34(1):1-11.
[11] POUYEZ J,MAYARD A,VANDAMME A M,et al.First crystal structure of an endo-inulinase,INU2,from Aspergillus ficuum:Discovery of an extra-pocket in the catalytic domain responsible for its endo-activity[J].Biochimie,2012,94(11):2 423-2 430.
[12] BAO M,NIU C T,XU X,et al.Identification,soluble expression,and characterization of a novel endo-inulinase from Lipomyces starkeyi NRRL Y-11557[J].Int J Biol Macromol,2019,137:537-544.
[13] TROTT O,OLSON A J.AutoDock Vina:Improving the speed and accuracy of docking with a new scoring function,efficient optimization,and multithreading[J].Journal of Computational Chemistry,2010,31(2):455-461.
[14] MILLER G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Anal Biochem,1959,31(3):426-428.
[15] BRADFORD M M.A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Anal Biochem,1976,72(s 1-2):248-254.
[16] LAMMENS W,LE ROY K,SCHROEVEN L,et al.Structural insights into glycoside hydrolase family 32 and 68 enzymes:Functional implications[J].Journal of Experimental Botany,2009,60(3):727-740.
[17] KIM K Y,NASCIMENTO A S,GOLUBEV A M,et al.Catalytic mechanism of inulinase from Arthrobacter sp.S37[J].Biochemical and Biophysical Research Communications,2008,371(4):600-605.
[18] NI H,ZENG S Q,QIN X,et al.Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic,synergistic lepidopteran-larvicidal and nematicidal activities[J].Int J Biol Sci,2015,11(3):304-315.
[19] GLIBOWSKI P,PIKUS S.Amorphous and crystal inulin behavior in a water environment[J].Carbohydr Polym,2011,83(2):635-639.
[20] 马俊,安启坤,唐文竹.菊粉外切酶的异源表达、纯化及酶学性质[J].食品与发酵工业,2019,45(4):25-30.
[21] MIAO R,XIE H,HO F M,et al.Protein engineering of α-ketoisovalerate decarboxylase for improved isobutanol production in Synechocystis PCC 6803[J].Metab Eng,2018,47:42-48.
[22] YIN S Y,DING F,DOKHOLYAN N V.Eris:An automated estimator of protein stability[J].Nat Methods,2007,4(6):466-467.
