用于高果糖浆制备的宛氏拟青霉嗜热嗜酸菊粉酶酶学特性分析
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摘要
为实现酶法水解菊糖制备高果糖浆,从宛氏拟青霉(Paecilomyces variotii)XS27发酵液中分离纯化菊粉酶,并对其酶学特性进行研究。发酵液经过硫酸铵盐析、透析、DEAE-Sepharose Fast Flow层析、Sephacry S-100分子筛过滤层析,得到电泳纯的菊粉酶,比活力327.4 U/mg,纯化倍数37.85。十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得菊粉酶为单一亚基的酶蛋白,分子质量62.0 kDa。菊粉酶能在较宽的pH值范围(3.5～6.5)内保持高活性,最适作用pH 4.0。在温度40～65℃之间,酶活力较高,最适作用温度为60℃。薄层色谱分析显示菊粉酶水解菊糖最终产物为果糖。以菊糖为底物,酶的K_m和V_(max)分别为5.93μmol/L和75.18μmol/(L·min)。Mg2+、Mn2+、Ca2+对酶有显著激活作用,Ba2+、Ni2+和Hg2+对酶有一定抑制作用。β-巯基乙醇、二硫苏糖醇和乙二胺四乙酸对酶有抑制作用,表面活性剂(十二烷基硫酸钠、Tween 80和Trition X100)以及乙醇对酶活力没有影响。从宛氏拟青霉XS27发酵液中分离纯化的菊粉酶在强酸高热的环境下具有强活性和稳定性,对表面活性剂乙醇有高耐受性,适合于果葡糖浆的工业化生产。
Electrophoretically pure inulinase for use in enzymatic production of high fructose corn syrup was isolated from the fermentation broth of Paecilomyces variotii XS27 by consecutive ammonium sulfate precipitation, dialysis, DEAESepharose Fast Flow chromatography and Sephacry S-100 gel ?ltration chromatography. The enzyme was puri?ed 37.85 folds with a speci?c activity. Its relative molecular mass was estimated to be 62.0 kDa by SDS-PAGE, consisting of a single subunit. This inulinase was active over a broad pH range of 3.5–6.5 and the optimum pH was 4.0; it had a high activity at temperatures ranging from 40 ℃ to 65 ℃, and the optimum temperature was 60 ℃. Thin-layer chromatography(TLC)analysis showed that the ?nal hydrolysis product of inulin by this enzyme was fructose. When inulin was used as a substrate,the Km and Vmax values were 5.93 μmol/L and 75.18 μmol/(L·min), respectively. The inulinase could be activated by Mg~(2+),Mn~(2+) and Ca~(2+), but inhibited by Ba~(2+), Ni~(2+) and Hg~(2+). The inulinase activity was inhibited by β-mercaptoethanol, DTT and EDTA but was not affected by surfactants(SDS, Tween 80 and Trition X100) or the organic solvent ethanol. In summary,the strong activity and stability of this inulinase against strong acid and high temperature as well as ethanol makes it suitable for the production of fructose and fructooligosaccharides.
Electrophoretically pure inulinase for use in enzymatic production of high fructose corn syrup was isolated from the fermentation broth of Paecilomyces variotii XS27 by consecutive ammonium sulfate precipitation, dialysis, DEAESepharose Fast Flow chromatography and Sephacry S-100 gel ?ltration chromatography. The enzyme was puri?ed 37.85 folds with a speci?c activity. Its relative molecular mass was estimated to be 62.0 kDa by SDS-PAGE, consisting of a single subunit. This inulinase was active over a broad pH range of 3.5–6.5 and the optimum pH was 4.0; it had a high activity at temperatures ranging from 40 ℃ to 65 ℃, and the optimum temperature was 60 ℃. Thin-layer chromatography(TLC)analysis showed that the ?nal hydrolysis product of inulin by this enzyme was fructose. When inulin was used as a substrate,the Km and Vmax values were 5.93 μmol/L and 75.18 μmol/(L·min), respectively. The inulinase could be activated by Mg~(2+),Mn~(2+) and Ca~(2+), but inhibited by Ba~(2+), Ni~(2+) and Hg~(2+). The inulinase activity was inhibited by β-mercaptoethanol, DTT and EDTA but was not affected by surfactants(SDS, Tween 80 and Trition X100) or the organic solvent ethanol. In summary,the strong activity and stability of this inulinase against strong acid and high temperature as well as ethanol makes it suitable for the production of fructose and fructooligosaccharides.
引文
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[19]LI Y,LIU G,WANG K,et al.Overexpression of the endoinulinase gene from Arthrobacter sp.S37 in Yarrowia lipolytica and characterization of the recombinant endo-inulinase[J].Journal of Molecular Catalysis B:Enzymatic,2012,74(1):109-115.DOI:10.1016/j.molcatb.2011.09.006.
[20]ZHANG S,YANG F,WANG Q,et al.High-level secretory expression and characterization of the recombinant Kluyveromyces marxianus inulinase[J].Process Biochemistry,2012,47(1):151-155.DOI:10.1016/j.procbio.2011.10.002.
[21]ZHANG T,GONG F,PENG Y,et al.Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase[J].Process Biochemistry,2009,44(12):1335-1339.DOI:10.1016/j.procbio.2009.07.008
[22]SILVA M F,RIGO D,MOSSI V,et al.Evaluation of enzymatic activity of commercial inulinase from Aspergillus niger immobilized in polyurethane foam[J].Food and Bioproducts Processing,2013,91(1):54-59.DOI:10.1016/j.fbp.2012.08.003.
[23]LIU X D,XU Y.A novel raw starch digestingα-amylase from a newly isolated Bacillus sp.YX-1:purification and characterization[J].Bioresource Technology,2008,99(10):4315-4320.DOI:10.1016/j.biortech.2007.08.040.
[24]MOHAMED S A,SALAH H A,MOHARAM M E,et al.Characterization of two thermostable inulinases from Rhizopus oligosporus NRRL 2710[J].Journal of Genetic Engineering and Biotechnology,2015,13(1):65-69.DOI:10.1016/j.jgeb.2014.12.001.
[25]MUGHAL M S,ALI S,ASHIQ M,et al.Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design[J].Bioresource Technology,2009,100(14):3657-3662.DOI:10.1016/j.biortech.2009.01.051.
[26]GILL P K,MANHAS R K,SINGH J,et al.Purification and characterization of an exoinulinase from Aspergillus fumigatus[J].Applied Biochemistry and Biotechnology,2004,117(1):19-32.
[27]TRIVEDI S,DIVECHA J,SHAH A.Optimization of inulinase production by a newly isolated Aspergillus tubingensis CR16 using low cost substrates[J].Carbohydrate Polymers,2012,90(1):483-490.DOI:10.1016/j.carbpol.2012.05.068.
[28]KARIMI M,HABIBI-REZAEI M,REZAEI K,et al.Immobilization of inulinase from Aspergillus niger on octadecyl substituted nanoporous silica:inulin hydrolysis in a continuous mode operation[J].Biocatalysis and Agricultural Biotechnology,2016,7:174-180.DOI:10.1016/j.bcab.2016.06.001.
[29]OHTA K,SUETSUGU N,NAKAMURA T.Purification and properties of an extracellular inulinase from Rhizopus sp.strain TN-96[J].Journal of Bioscience and Bioengineering,2002,94(1):78-80.DOI:10.1016/S1389-1723(02)80120-7.
[30]CHEN X M,XU X M,JIN Z Y,et al.Expression of an exoinulinase gene from Aspergillus ficuum in Escherichia coli and its characterization[J].Carbohydrate Polymers,2013,92(2):1984-1990.DOI:10.1016/j.carbpol.2012.11.087.
[31]MORIYAMA S,AKIMOTO H,SUETSUGU N,et al.Purification and properties of an extracellular exoinulinase from Penicillium sp.strain TN-88 and sequence analysis of the encoding gene[J].Bioscience Biotechnology and Biochemistry,2002,66(9):1887-1896.
[2]ROCHA J R,CATANA R,FERREIRA B S,et al.Design and characterisation of an enzyme system for inulin hydrolysis[J].Food Chemistry,2006,95(1):77-82.DOI:10.1016/j.foodchem.2004.12.020.
[3]VANDAMME E J,DERYCKE D G.Microbial inulinases:fermentation process,properties,and applications[M]//Advances in applied microbiology.Academic Press,1983:139-176.DOI:10.1016/S0065-2164(08)70356-3.
[4]ZHOU J,LU Q,PENG M,et al.Cold-active and NaCl-tolerant exoinulinase from a cold-adapted Arthrobacter sp.MN8 and its potential for use in the production of fructose at low temperatures[J].Journal of Bioscience and Bioengineering,2015,119(3):267-274.DOI:10.1016/j.jbiosc.2014.08.003.
[5]KULMINSKAYA A A,ARAND M,ENEYSKAYA E V,et al.Biochemical characterization of Aspergillus awamori exoinulinase:substrate binding characteristics and regioselectivity of hydrolysis[J].Biochimica et Biophysica Acta(BBA)-Proteins and Proteomics,2003,1650(1):22-29.DOI:10.1016/S1570-9639(03)00187-0.
[6]GILL P K,MANHAS R K,SINGH P.Hydrolysis of inulin by immobilized thermostable extracellular exoinulinase from Aspergillus fumigatus[J].Journal of Food Engineering,2006,76(3):369-375.DOI:10.1016/j.jfoodeng.2005.05.052.
[7]SINGH R S,SINGH R P.18-Inulinases[M]//Current developments in biotechnology and bioengineering.Elsevier,2017:423-446.DOI:10.1016/B978-0-444-63662-1.00018-X.
[8]ZHOU J,GAO Y,ZHANG R,et al.A novel low-temperature-active exo-inulinase identified based on molecular-activity strategy from Sphingobacterium sp.GN25 isolated from feces of Grus nigricollis[J].Process Biochemistry,2014,49(10):1656-1663.DOI:10.1016/j.procbio.2014.06.013.
[9]李娟,曹泽虹,李超,等.响应面法优化黑曲霉深层发酵产内切型菊粉酶工艺[J].食品科学,2014,35(9):207-212.DOI:10.7506/spkx1002-6630-201409041.
[10]TRIVEDI S,DIVECHA J,SHAH A.Optimization of inulinase production by a newly isolated Aspergillus tubingensis CR16 using low cost substrates[J].Carbohydrate Polymers,2012,90(1):483-490.DOI:10.1016/j.carbpol.2012.05.068.
[11]ABD EL ATY A A,WEHAIDY H R,MOSTAFA F A.Optimization of inulinase production from low cost substrates using PlackettBurman and Taguchi methods[J].Carbohydrate Polymers,2014,102:261-268.
[12]SHARMA A D,GILL P K.Purification and characterization of heat-stable exo-inulinase from Streptomyces sp.[J].Journal of Food Engineering,2007,79(4):1172-1178.DOI:10.1016/j.jfoodeng.2006.04.008.
[13]ETTALIBI M,BARATTI J C.Sucrose hydrolysis by thermostable immobilized inulinases from Aspergillus ficuum[J].Enzyme and Microbial Technology,2001,28(7/8):596-601.DOI:10.1016/S0141-0229(00)00342-2.
[14]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72(1):248-254.DOI:10.1016/0003-2697(76)90527-3.
[15]SINGH R S,CHAUHAN K.Inulinase production from a new inulinase producer,Penicillium oxalicum BGPUP-4[J].Biocatalysis and Agricultural Biotechnology,2017,9:1-10.DOI:10.1016/j.bcab.2016.10.012.
[16]CHESINI M,WAGNER E,BARUQUE D J,et al.High level production of a recombinant acid stable exoinulinase from Aspergillus kawachii[J].Protein Expression and Purification,2018,147:29-37.DOI:10.1016/j.pep.2018.02.007.
[17]SHENG J,CHI Z,LI J,et al.Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase[J].Process Biochemistry,2007,42(5):805-811.DOI:10.1016/j.procbio.2007.01.016.
[18]ZHANG L,ZHAO C,ZHU D,et al.Purification and characterization of inulinase from Aspergillus niger AF10 expressed in Pichia pastoris[J].Protein Expression and Purification,2004,35(2):272-275.DOI:10.1016/j.pep.2004.02.015.
[19]LI Y,LIU G,WANG K,et al.Overexpression of the endoinulinase gene from Arthrobacter sp.S37 in Yarrowia lipolytica and characterization of the recombinant endo-inulinase[J].Journal of Molecular Catalysis B:Enzymatic,2012,74(1):109-115.DOI:10.1016/j.molcatb.2011.09.006.
[20]ZHANG S,YANG F,WANG Q,et al.High-level secretory expression and characterization of the recombinant Kluyveromyces marxianus inulinase[J].Process Biochemistry,2012,47(1):151-155.DOI:10.1016/j.procbio.2011.10.002.
[21]ZHANG T,GONG F,PENG Y,et al.Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase[J].Process Biochemistry,2009,44(12):1335-1339.DOI:10.1016/j.procbio.2009.07.008
[22]SILVA M F,RIGO D,MOSSI V,et al.Evaluation of enzymatic activity of commercial inulinase from Aspergillus niger immobilized in polyurethane foam[J].Food and Bioproducts Processing,2013,91(1):54-59.DOI:10.1016/j.fbp.2012.08.003.
[23]LIU X D,XU Y.A novel raw starch digestingα-amylase from a newly isolated Bacillus sp.YX-1:purification and characterization[J].Bioresource Technology,2008,99(10):4315-4320.DOI:10.1016/j.biortech.2007.08.040.
[24]MOHAMED S A,SALAH H A,MOHARAM M E,et al.Characterization of two thermostable inulinases from Rhizopus oligosporus NRRL 2710[J].Journal of Genetic Engineering and Biotechnology,2015,13(1):65-69.DOI:10.1016/j.jgeb.2014.12.001.
[25]MUGHAL M S,ALI S,ASHIQ M,et al.Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design[J].Bioresource Technology,2009,100(14):3657-3662.DOI:10.1016/j.biortech.2009.01.051.
[26]GILL P K,MANHAS R K,SINGH J,et al.Purification and characterization of an exoinulinase from Aspergillus fumigatus[J].Applied Biochemistry and Biotechnology,2004,117(1):19-32.
[27]TRIVEDI S,DIVECHA J,SHAH A.Optimization of inulinase production by a newly isolated Aspergillus tubingensis CR16 using low cost substrates[J].Carbohydrate Polymers,2012,90(1):483-490.DOI:10.1016/j.carbpol.2012.05.068.
[28]KARIMI M,HABIBI-REZAEI M,REZAEI K,et al.Immobilization of inulinase from Aspergillus niger on octadecyl substituted nanoporous silica:inulin hydrolysis in a continuous mode operation[J].Biocatalysis and Agricultural Biotechnology,2016,7:174-180.DOI:10.1016/j.bcab.2016.06.001.
[29]OHTA K,SUETSUGU N,NAKAMURA T.Purification and properties of an extracellular inulinase from Rhizopus sp.strain TN-96[J].Journal of Bioscience and Bioengineering,2002,94(1):78-80.DOI:10.1016/S1389-1723(02)80120-7.
[30]CHEN X M,XU X M,JIN Z Y,et al.Expression of an exoinulinase gene from Aspergillus ficuum in Escherichia coli and its characterization[J].Carbohydrate Polymers,2013,92(2):1984-1990.DOI:10.1016/j.carbpol.2012.11.087.
[31]MORIYAMA S,AKIMOTO H,SUETSUGU N,et al.Purification and properties of an extracellular exoinulinase from Penicillium sp.strain TN-88 and sequence analysis of the encoding gene[J].Bioscience Biotechnology and Biochemistry,2002,66(9):1887-1896.