重组木聚糖酶生产低聚木糖的实验研究

31220114ChemistryandIndustryofForestProductsVo.l31No.2Apr.2011:2010-09-25:(31070513);(973)(2007CB707801);(10KJA22019):(1972-),,,,,,;Emai:[e-mail]欧阳嘉,刘明,李鑫,陈牧,连之娜(南京林业大学化学工程学院,江苏南京210037):研究了重组木聚糖酶C0602生产低聚木糖的制备工艺,考察了不同类型底物底物浓度和酶用量对酶解效率和低聚木糖产率的影响实验结果表明,低聚木糖生产原料以抽提木聚糖为宜桦木木聚糖质量浓度30g/L,重组木聚糖酶用量20IU/g条件下水解4h,酶解率可达56.05%,低聚木糖产率(C2~C6)为29.76%,产品平均聚合度为2.76724.5g/L碱抽提玉米芯木聚糖在酶用量80U/g条件下水解48h,酶解率68.33%,低聚木糖产率为14.88%产品不含木糖,平均聚合度为3.45利用重组木聚糖酶生产低聚木糖可以大幅度降低产品中的木糖含量,显著提高低聚木糖产品质量:木聚糖酶;低聚木糖;酶水解;聚合度:TQ351:A:0253-2417(2011)02-0037-06ExperimentalInvestigationofXylooligosaccharidesProductionbyRecombinantXylanaseOUYANGJia,LIUMing,LIXin,CHENMu,LIANZhina(TheCollegeofChemicalEngineering,NanjingForestryUniversity,Nanjing210037,China)Abstract:Xylooligosaccharides(XOs)productionusingtherecombinantxylanaseC0602wasstudied.Effectsofdifferentsubstrates,substrateconcentrationandenzymedosageontheenzymatichydrolysisandxylooligosaccharideyieldwereinvestigated.Theresultsshowedthattheextractedxylanwasthesuitablesubstrateforxylooligosaccharidesproduction.When30g/Lbirchwoodxylanwastreatedwiththedosageof20U/gxylanaseforhydrolysis4h,thehydrolysisyieldwas56.05%andthexylooligosaccharideyield(C2-C6)was29.76%.Theaveragedegreeofpolymerization(DP)was2.767.When24.5g/Lxylanextractedfromcorncobwashydrolyzedby80U/gxylanasefor48h,thehydrolysisyieldreached68.33%andthexylooligosaccharideyieldwas14.88%.Correspondingly,theproductdidnotcontainxyloseanditsDPwas3.45.Theresultsshowedthatxylooligosaccharideproductionusingtherecombinantxylanasecouldgreatlydecreasethecontentofxyloseandsignificantlyimprovethequalityofxylooligosaccharides.Keywords:xylanase;xylooligosaccharide;enzymatichydrolysis;degreeofpolymerization,,2~8-1,4-[1],,,[2-3][4-5],,,,[6-8],[9]3831[10],,,11.1C0602,1400IU/mL,Megazyme,Wicklow,Ireland:,55.94%,16.33%;2009,0.4~0.8mm,34.88%,21.19%,21.35%;,55.90%,25.39%,10.53%;Sigma1.20.9g50mL,1mol/L1mL,30mL,,50170r/min3000r/min10min,r=(C0.88)/G100%DP=C/B100%y=(D2+D3+D4+D5+D6)/G100%:C,g/L;0.88;G,g/L;B,g/L;D2,D3,D4,D5D6,,,,g/Lr,%;DP,%;y,%1.3NREL[11],DNS[12];HPLC,HPLCHPLCBio-RadAminexHPX-87H(300mm7.8mm),5mmol/L,0.6mL/min,55,(Megazyme,)DIONEXICS3000,CarbonPacPA200,100mmol/LNaOH500mmol/LNaOAc,0.3mL/min,3022.1,[13-15],30g/L,40IU/g(,),pH4.850,148h,,69.42%,,63.72%10%,,,2,:39,,2~3,,,;,1Table1Comparisonoftheenzymatichydrolysiswithdifferentsubstrates1)substrate/(gL-1)xyloseDP/%enzymaticyieldbirchwoodxylan0.2332.46169.42beechwoodxylan0.2362.08662.08straw0.74810.0638.10alkaliextractedcornstalkresidue1.4971.40767.55alkaliextractedxylan*04.08963.721)*:24.5g/Lalkaliextractedxylanmassconcentrationis24.5g/L2.25030g/L4h,(C2~C6,)212:5IU/g20IU/g,43.85%57.95%,,,40IU/g,2.346,,,,,,2Table2Effectsofenzymedosageontheenzymatichydrolysisofbirchwoodxylan/(IUg-1)enzymedosage/(gL-1)xyloseDP/%enzymaticyield50.0743.74643.85100.1123.16656.41200.1342.86057.95400.1982.34654.211:5~40IU/g26.60%~32.05%,,,20IU/g,,20IU/g,20IU/g,32,,30g/L,56.05%,2.767;20g/L,2.712,,,30g/L,29.76%,,,,30g/L,4031169.29,,1.88g/L,21.35%,60.67%,17.98%3Table3Effectsofsubstratemassconcentrationontheenzymatichydrolysisofbirchwoodxylan/(gL-1)substratemassconcentration/(gL-1)xyloseDP/%enzymaticyield20.0139.1513.1950.0084.3076.70100.0773.52012.85200.0742.71229.22300.1272.76756.051Fig.1Effectsofenzymedosageonxylooligosaccharideconcentrationsandyields2Fig.2Effectsofsubstratemassconcentrationonxylooligosaccharideconcentrationsandyields2.3,[16]24.5g/L50pH4.848h,34h,,,,80IU/g48h,,5.512g/L20IU/g,4h,52.81%;,6.04,,48h44Table4Effectsofenzymedosageontheenzymatichydrolysisofalkaliextractedcorncobxylan1)/(IUg-1)enzymedosage/(gL-1)xyloseDP/%enzymaticyield/%xylooligosaccharidesyield20*6.0452.81200.064.6959.797.10400.004.0963.729.26800.003.4568.3314.881)*:20IU/g4hdataat20IU/genzymeafter4hhydrolysis2,:414:,20IU/g80IU/g,,59.79%68.33%,,14.88%,,40IU/g,4.09(4),,,80IU/g18.82%,47.92%,20.87%;,1.749g/L3Fig.3Timecourseoftheenzymatichydrolysisofalkaliextractedcorncobxylanusingtherecombinantxylanase4Fig.4Effectsofsubstrateconcentrationontheaveragedegreeofpolymerizationofxylooligosaccharides33.1C0602,,3.2,50pH4.8,30g/L,20IU/g4h,56.05%,29.76%2.767,169.29,21.35%,60.67%,17.98%3.324.5g/L50pH4.880IU/g48h,68.33%,14.88%,3.45,18.82%,47.92%,20.87%:[1],,,.[J].,2006,9:14-15.[2],,.[J].,2009,2:56-57.[3],,.[J].:,2010,34(1):5-8.[4],,,.ACCC30566[J].,2009,37(12):126-129.[5]MUSSATTOSI,MANCILHAIM.Nondigestibleoligosaccharides:Areview[J].CarbohydratePolymers,2007,68:587-597.[6],,.[J].,2008,38(1):53-59.[7],,.[J].,2006,26(1):124-126.4231[8],.[J].,2005,25():77-81.[9]MUKUNDGA,KULBHUSHANBB,DIGAMBARVG.BiochemicalcharacterizationoftwoxylanasesfromyeastPseudozymahubeiensisproducingonlyxylooligosaccharides[J].BioresourceTechnology,2009,100:6488-6495.[10]OUYANGJ,WANGS,WANHY,eta.lProductionofaTrichodermareeseiQM9414xylanaseinPichiapastorisanditsapplicationinbiobleachingofwheatstrawpulp[J].WorldJournalofMicrobiologyandBiotechnology,2010,27(4):751-758.[11]SLUITERA,HAMESB,RUIAR,eta.lLaboratoryanalyticalprocedure[S].NRELBiomassProgram,2004.[12]MILLERGL.Useofdinitrosalicylicacidreag