乳酸片球菌AS1.2696来源的α-L-鼠李糖苷酶酶学性质分析
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摘要
克隆表达乳酸片球菌AS1.2696菌株中的α-L-鼠李糖苷酶基因,研究重组酶的酶学性质。分析乳酸片球菌AS1.2696菌株的全基因组序列,聚合酶链式反应扩增目的基因,以p SE380为表达载体构建重组质粒p SE-prha2和p SE-prha3,在大肠杆菌Escherichia coli XL-blue进行表达,使用镍亲和层析纯化重组蛋白,研究重组酶PRHA2、PRHA3的酶学性质。结果表明,重组酶PRHA2的最适pH值和温度分别为5.0和60℃,Km值为(3.039±0.581)mmol/L,Vm a x值为(2.032±0.186)μmol/(min·mg);PRHA3的最适p H值和温度分别为5.5和45℃,Km值为(2.797±0.132)mmol/L,Vmax值为(113.35±1.485)μmol/(min·mg)。PRHA2和PRHA3能够水解人工底物对硝基苯基-α-L-吡喃鼠李糖苷(p-nitrophenyl-α-L-rhamnopyranoside,p NPR)以及α-1,6键的橙皮苷、芦丁;PRHA2对4-硝基苯基-β-D-阿拉伯糖苷(4-nitrophenyl-β-L-arabinoside,p NPA)有一定的水解活性;PRHA3对淫羊藿苷、淫羊藿次苷I、朝藿定A、朝藿定B、朝藿定C这几种物质C-7位的葡萄糖糖苷键有较弱的水解作用。此外,PRHA3能够水解朝藿定C的C-3位以α-Rha(2→1)α-Rha连接的外侧的鼠李糖苷键,在食品及医疗方面具有一定的应用价值。
The aim of this study was to evaluate the enzymatic properties of recombinant α-L-rhamnosidases PRHA2 and PRHA3 from Pediococcus acidilactici AS1.2696. The whole genome sequence of P. acidilactici AS1.2696 was analyzed and the two genes encoding α-L-rhamnosidase were amplified by PCR and ligated into expression vector pSE,yielding recombinant plasmids pSE-prha2 and pSE-prha3. The recombinant plasmids were separately transformed and expressed in E.coli XL-blue. The recombinant proteins PRHA2 and PRHA3 were purified by Ni-NTA affinity column chromatography and their enzymatic characteristics were investigated in detail. The results showed the optimal pH and temperature of PRHA2 were 5.0 and 60 ℃, respectively. Its Km and Vmax values were(3.039 ± 0.581) mmol/L and(2.032 ±0.186) μmol/(min·mg), respectively. The optimal pH and temperature of PRHA3 were 5.5 and 45 ℃, and its Km and Vmax values were(2.797 ± 0.132) mmol/L and(113.35 ± 1.485) μmol/(min·mg), respectively. PRHA2 and PRHA3 could not only hydrolyze the artificial substrate p-nitrophenyl-α-L-rhamnopyranoside(p NPR) but could also hydrolyze the α-1,6 glucosidic linkages of the natural substrates hesperidin and rutin. PRHA2 had hydrolytic activity on 4-nitrophenyl-β-L-arabinoside(p NPA). PRHA3 weakly hydrolyzed the glucosidic linkages at the C-7 position of icariin, icariside I, epimedin A, epimedin B and epimedin C. In addition, PRHA3 could hydrolyze the outer rhamnosidic linkage to the C-3 position of epimedin C through α-Rha(2→1)α-Rha, leading to potential applications in the food and medicinal fields.
The aim of this study was to evaluate the enzymatic properties of recombinant α-L-rhamnosidases PRHA2 and PRHA3 from Pediococcus acidilactici AS1.2696. The whole genome sequence of P. acidilactici AS1.2696 was analyzed and the two genes encoding α-L-rhamnosidase were amplified by PCR and ligated into expression vector pSE,yielding recombinant plasmids pSE-prha2 and pSE-prha3. The recombinant plasmids were separately transformed and expressed in E.coli XL-blue. The recombinant proteins PRHA2 and PRHA3 were purified by Ni-NTA affinity column chromatography and their enzymatic characteristics were investigated in detail. The results showed the optimal pH and temperature of PRHA2 were 5.0 and 60 ℃, respectively. Its Km and Vmax values were(3.039 ± 0.581) mmol/L and(2.032 ±0.186) μmol/(min·mg), respectively. The optimal pH and temperature of PRHA3 were 5.5 and 45 ℃, and its Km and Vmax values were(2.797 ± 0.132) mmol/L and(113.35 ± 1.485) μmol/(min·mg), respectively. PRHA2 and PRHA3 could not only hydrolyze the artificial substrate p-nitrophenyl-α-L-rhamnopyranoside(p NPR) but could also hydrolyze the α-1,6 glucosidic linkages of the natural substrates hesperidin and rutin. PRHA2 had hydrolytic activity on 4-nitrophenyl-β-L-arabinoside(p NPA). PRHA3 weakly hydrolyzed the glucosidic linkages at the C-7 position of icariin, icariside I, epimedin A, epimedin B and epimedin C. In addition, PRHA3 could hydrolyze the outer rhamnosidic linkage to the C-3 position of epimedin C through α-Rha(2→1)α-Rha, leading to potential applications in the food and medicinal fields.
引文
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[31]LIU T Q,YU H S,ZHANG C Z,et al.Aspergillus niger DLFCC-90rhamnoside hydrolase,a new type of flavonoid glycoside hydrolase[J].Applied and Environmental Microbiology,2012,78(13):4752-4754.DOI:10.1128/AEM.00054-12.
[32]TAMAYO-RAMOS J A,FLIPPHI M,PARDO E,et al.L-Rhamnose induction of Aspergillus nidulansα-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake[J].Microbial Cell Factories,2012,11(26):1-17.
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[35]TURNER P,SVENSSON D,ADLERCREUTZ P,et al.A novel variant of Thermotoga neapolitanaβ-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation[J].Journal of Biotechnology,2007,130:67-74.DOI:10.1016/j.jbiotec.2007.02.016.
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[2]YADAV V,YADAV P K,YADAV S,et al.α-L-Rhamnosidase:a review[J].Process Biochemistry,2010,45(8):1226-1235.DOI:10.1016/j.procbio.2010.05.025.
[3]VILA-REAL H,ALFAIA A J,ROSA J N,et al.α-Rhamnosidase andβ-glucosidase expressed by naringinase immobilized on new ionic liquid sol-gel matrices:activity and stability studies[J].Journal of Biotechnology,2011,152:147-158.DOI:10.1016/j.jbiotec.2010.08.005.
[4]IZZO V,TEDESCO P,NOTOMISTA E,et al.α-Rhamnosidase activity in the marine isolate Novosphingobium sp.PP1Y and its use in the bioconversion of flavonoids[J].Journal of Molecular Catalysis B:Enzymatic,2014,105:95-103.DOI:10.1016/j.molcatb.2014.04.002.
[5]LIU Q,LU L L,XIAO M.Cell surface engineering ofα-Lrhamnosidase for naringin hydrolysis[J].Bioresource Technology,2012,123:144-149.DOI:10.1016/j.biortech.2012.05.083.
[6]CELIZ G,RODRIGUEZ J,SORIA F,et al.Synthesis of hesperetin7-O-glucoside from flavonoids extracted from citrus waste using both free and immobilizedα-L-rhamnosidases[J].Biocatalysis and Agricultural Biotechnology,2015,4(3):335-341.DOI:10.1016/j.bcab.2015.06.005.
[7]WEIGNEROVA L,MARHOL P,GERSTORFEROVA D,et al.Preparatory production of quercetin-3-β-D-glucopyranoside using alkali-tolerant thermostableα-L-rhamnosidase from Aspergillus terreus[J].Bioresource Technology,2012,115:222-227.DOI:10.1016/j.biortech.2011.08.029.
[8]张霞,李利君,倪辉,等.微生物来源α-L-鼠李糖苷酶的分子和结构生物学研究进展[J].生命科学研究,2015,19(1):68-74.DOI:10.16605/j.cnki.1007-7847.2015.01.008.
[9]LISE D F,MENSITIERI F,TARALLO V,et al.RHA-P:Isolation,expression and characterization of a bacterialα-L-rhamnosidase from Novosphingobium sp.PP1Y[J].Journal of Molecular Catalysis B:Enzymatic,2016,134:136-147.DOI:10.1016/j.molcatb.2016.10.002.
[10]巩建业,吴喆瑜,李利君,等.GH78家族真菌-L-鼠李糖苷酶分子系统进化关系分析[J].现代食品科技,2017,33(10):13-20.DOI:10.13982/j.mfst.1673-9078.2017.10.003.
[11]O’NEILL E C,STEVENSON C E M,PATERSON M J,et al.Crystal structure of a novel two domain GH78 familyα-rhamnosidase from Klebsiella oxytoca with rhamnose bound[J].Proteins,2015,83(9):1742-1749.DOI:10.1002/prot.24807.
[12]CUI Z L,MARUYAMA Y,MIKAMI B,et al.Crystal structure of glycoside hydrolase family 78α-L-rhamnosidase from Bacillus sp.GL1[J].Journal of Molecular Biology,2007,374(2):384-398.DOI:10.1016/j.jmb.2007.09.003.
[13]GRANDITS M,MICHLMAYR H,SYGMUND C,et al.Calculation of substrate binding affinities for a bacterial GH78 rhamnosidase through molecular dynamics simulations[J].Journal of Molecular Catalysis B:Enzymatic,2013,92(100):34-43.DOI:10.1016/j.molcatb.2013.03.012.
[14]金城.黑曲霉α-鼠李糖苷酶[J].微生物学通报,2011,38(3):446.DOI:10.13344/j.microbiol.china.2011.03.019.
[15]BIRGISSON H,HREGGVIDSSON G O,FRIDJONSSON O H,et al.Two new thermostableα-L-rhamnosidases from a novel thermophilic bacterium[J].Enzyme and Microbial Technology,2004,34(6):561-571.DOI:10.1016/j.enzmictec.2003.12.012.
[16]ISHIKAWA M,SHIONO Y,KOSEKI T.Biochemical characterization of Aspergillus oryzae recombinantα-L-rhamnosidase expressed in Pichia pastoris[J].Journal of Bioscience and Bioengineering,2017,124(6):630-634.DOI:10.1016/j.jbiosc.2017.07.007.
[17]GERSTORFEROVáD,FLIEDROVáB,HALADA P,et al.Recombinantα-L-rhamnosidase from Aspergillus terreus in selective trimming of rutin[J].Process Biochemistry,2012,47:828-835.DOI:10.1016/j.procbio.2012.02.014.
[18]LI L J,YU Y,ZHANG X,et al.Expression and biochemical characterization of recombinantα-L-rhamnosidase r-Rha1from Aspergillus niger JMU-TS528[J].International Journal of Biological Macromolecules,2016,85:391-399.DOI:10.1016/j.ijbiomac.2015.12.093.
[19]QIAN S,WANG H Y,ZHANG C Z,et al.Isolation and characterization of dioscin-α-L-rhamnosidase from bovine liver[J].Journal of Molecular Catalysis B:Enzymatic,2013,97:31-35.DOI:10.1016/j.molcatb.2013.07.007.
[20]赵力斌.固定化柚苷酶及其对桔子汁脱苦的研究[J].食品与发酵工业,1987(1):25-34.DOI:10.13995/j.cnki.11-1802/ts.1987.01.003.
[21]LI L J,WU Z Y,YU Y,et al.Development and characterization of anα-L-rhamnosidase mutant with improved thermostability and a higher efficiency for debittering orange juice[J].Food Chemistry,2018,245:1070-1078.DOI:10.1016/j.foodchem.2017.11.064.
[22]陈俊,李利君,倪辉,等.重组α-L-鼠李糖苷酶制备普鲁宁[J].中国现代应用药学,2016,33(8):1025-1030.DOI:10.13748/j.cnki.issn1007-7693.2016.08.015.
[23]胡群芳,李利君,陈艳红,等.从黑曲霉固态发酵产物中纯化α-L-鼠李糖苷酶及酶法制备普鲁宁[J].现代食品科技,2015,31(1):107.DOI:10.13982/j.mfst.1673-9078.2015.1.020.
[24]VILA-REAL H,ALFAIA A J,PHILLIPS R S,et al.Pressureenhanced activity and stability ofα-L-rhamnosidase andβ-Dglucosidase activities expressed by naringinase[J].Journal of Molecular Catalysis B:Enzymatic,2010,65(1/2/3/4):102-109.DOI:10.1016/j.molcatb.2010.01.022.
[25]王侃,鱼红闪,金凤燮.芦丁-α-鼠李糖苷酶分离提纯及其酶性质[J].大连轻工业学院学报,2004,23(1):30-33.
[26]韩冰,付绍平,金凤燮,等.两种菌产两种不同天然苷类-α-鼠李糖苷酶的研究[J].大连工业大学学报,2008,27(2):105-109.
[27]YADAV S,YADAV S,YADAV K D S.α-L-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224[J].Bioorganic Chemistry,2017,70:222-228.DOI:10.1016/j.bioorg.2017.01.002.
[28]南敏伦,李世财,赵昱玮,等.淫羊藿苷元制备方法及药理活性研究进展[J].中国实验方剂学杂志,2015,21(7):227-231.DOI:10.13422/j.cnki.syfjx.2015070227.
[29]XIA Q,XU D J,HUANG Z G,et al.Preparation of icariside II from icariin by enzymatic hydrolysis method[J].Fitoterapia,2010,81(5):437-442.DOI:10.1016/j.fitote.2009.12.00.
[30]MANZANARES P,OREJAS M,GIL J V,et al.Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene,encoding anα-L-rhamnosidase of enological interest[J].Applied and Environmental Microbiology,2003,69(12):7558-7562.DOI:10.1128/AEM.69.12.7558-7562.2003.
[31]LIU T Q,YU H S,ZHANG C Z,et al.Aspergillus niger DLFCC-90rhamnoside hydrolase,a new type of flavonoid glycoside hydrolase[J].Applied and Environmental Microbiology,2012,78(13):4752-4754.DOI:10.1128/AEM.00054-12.
[32]TAMAYO-RAMOS J A,FLIPPHI M,PARDO E,et al.L-Rhamnose induction of Aspergillus nidulansα-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake[J].Microbial Cell Factories,2012,11(26):1-17.
[33]LI X,LIU X H,YIN Z H,et al.Site-directed mutagenesis ofα-Lrhamnosidase from Alternaria sp.L1 to enhance synthesis yield of reverse hydrolysis based on rational design[J].Applied Microbiology and Biotechnology,2016,100(24):10385-10394.DOI:10.1007/s00253-016-7676-4.
[34]李彬春,张甜,吉亚茹.α-L-鼠李糖苷酶以催化活性包涵体形式异源表达及其酶学性质[J].食品科学,2018,39(14):79-84.DOI:10.7506/spkx1002-6630-201814012.
[35]TURNER P,SVENSSON D,ADLERCREUTZ P,et al.A novel variant of Thermotoga neapolitanaβ-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation[J].Journal of Biotechnology,2007,130:67-74.DOI:10.1016/j.jbiotec.2007.02.016.
[36]张季林,杨硕,徐彭.淫羊藿活性成分抗肿瘤作用的研究进展[J].实用中西结合临床,2017,17(5):163-165.DOI:10.13638/j.issn.1671-4040.2017.05.106.