脂肪酶产生菌发酵条件优化

绵阳师范学院本科生毕业论文（设计）题目脂肪酶产生菌M-6-2发酵条件的优化专业生物技术院部生命科学与技术学院学号0811420218姓名杜长蔓指导教师李俊刚答辩时间2012年5月论文工作时间：2011年7月至2012年5月脂肪酶产生菌M-6-2发酵条件的优化学生：杜长蔓指导老师：李俊刚摘要：本文对绵阳师范学院微生物实验室筛选和鉴定的产脂肪酶细菌M-6-2的生长动力学和产酶动力学进行了研究；通过单因素实验和正交试验，对脂肪酶产生菌M-6-2摇床发酵产脂肪酶的培养基组成和培养条件进行优化，得出较佳的产酶培养基组成配方为：1.5%淀粉+0.5%酵母膏为碳源、4.5%豆饼粉+1.5%硝酸铵为最佳的氮源、0.05%磷酸氢二钠和0.15%硫酸镁；最优的发酵条件为：初始pH7.5，接种量1.5%，装液量20ml/250ml，发酵温度35℃，在转速180r/min下，培养16h，经过优化后发酵液脂肪酶酶活力最高可达到15.60U/ml,较优化前提高了49.57%。脂肪酶产生菌M-6-2与国内文献报道的产脂肪酶细菌相比产酶活力高。对该菌株发酵条件进行优化后，为生产性试验打下了基础。关键词：脂肪酶产生菌M-6-2；脂肪酶；发酵条件；优化；正交试验；LipasetoproducebacteriaM-6-2OptimizationoffermentationconditionsUndergraduate:DuChangmanSupervisor:LiJunGangAbstract:Inthispaper,LaboratoryscreeningandidentificationoflipaseproductionbybacteriaintheM-6-2growthkineticsandenzymeproductionkineticswerestudied;throughsinglefactorexperimentsandorthogonaltest,thelipasetoproducebacteriaM-6-2shakerfermentationlipasemediumcompositionandcultureconditionswereoptimizedtocometoabetterenzymeproductionmediumcompositionformula:1.5%starchand0.5%yeastextractascarbonsource,4.5%ofthesoybeanpowderand1.5%ammoniumnitrateforthebestsourceofnitrogen,0.05%disodiumhydrogenphosphateand0.15%magnesiumsulfate.Optimalfermentationconditionswere:initialpH7.5,1.5%oftheinoculumsize,liquidvolume20ml/250ml,fermentationtemperature35°C,inthespeed180r/minnext,cultured16hAfteroptimizationofthefermentationbrothlipaseactivitycanreach49.57%to15.60U/ml,comparedtobeforeoptimization.LipasetoproducebacteriaM-6-2andreportedinChinaintheproductionoflipasebacteriacomparedtothehighactivityofenzymeproduction.Ofthestrainfermentationconditionsoptimized,laidthefoundationfortheproductionoftest.Keywords:LipaseproducingstrainM-6-2；lipase；fermentationconditions；optimization；orthogonaltest目录1前言............................................................................11.1脂肪酶的概况..................................................................11.2国内外脂肪酶的研究概况........................................................21.3本研究的目的及意义............................................................22实验材料和器具..................................................................32.1实验菌种......................................................................32.2主要实验仪器..................................................................32.3实验试剂......................................................................32.4培养基........................................................................43实验流程.......................................................................44.实验方法.......................................................................44.1生长曲线的测定...............................................................44.2产酶曲线的测定...............................................................54.3脂肪酶产生菌M-6-2菌体生长的最佳条件的确定....................................54.3.1PH对M-6-2菌体生长的影响..................................................54.3.2温度对M-6-2菌体生长的影响..................................................54.3.3装液量对M-6-2菌体生长的影响................................................64.3.4摇床转速对M-6-2菌体生长的影响..............................................64.4脂肪酶培养基的优化............................................................74.4.1脂肪酶培养基单因素条件的优化...............................................74.4.2产脂肪酶培养基的正交试验...................................................74.5脂肪酶发酵条件优化............................................................84.5.1发酵条件单因素条件优化.....................................................84.5.2产脂肪酶发酵条件的正交试验.................................................95分析方法........................................................................95.1脂肪酶酶活测定与计算方法......................................................95.2酶活测定的原理...............................................................95.3酶活的定义...................................................................95.4酶活的测定...................................................................95.5酶活的计算方法：..............................................................95.6生物量的测定................................................................106结果与讨论...................................................................106.1生长曲线的测定结果...........................................................106.2产酶曲线的测定结果...........................................................116.3菌体的生长和产酶动力学关系...................................................116.4脂肪酶产生菌M-6-2菌体生长最佳条件的确定结果.................................116.4.1PH对M-6-2菌体生长的影响.................................................116.4.2温度对M-6-2菌体生长的影响.................................................126.4.3装液量对M-6-2菌体生长的影响..............................................136.4.4摇床转速对M-6-2菌体生长的影响............................................136.5脂肪酶培养基的优化..........................................................146.5.1单因素实验................................................................146.5.2正交试验...................................................................176.6脂肪酶产生菌M-6-2发酵条件的优化............................................196.6.1单因素实验.................................................................196.5.2