【作者】 张根林；

【导师】 李春；

【作者基本信息】 石河子大学 ， 农产品加工及贮藏工程， 2007， 硕士

【摘要】 以自主筛选的产1,3-丙二醇菌株为基础,研究了甘油脱水酶的催化反应特性,通过克隆和表达甘油脱水酶与复活因子,定量研究了复活因子和其它因素与失活甘油脱水酶的复活关系。通过研究得到以下结果:从新疆特殊环境土壤中筛选到在厌氧条件下能够转化甘油生成1,3-丙二醇的菌株XJPDLi-2,经鉴定后命名为K. pneumoniae XJPD-Li(缩写XJPD-Li)。摇瓶发酵实验确定其最佳发酵条件为:温度40℃,pH8.0,硫酸铵6.0g·L-1,底物甘油20g·L-1和葡萄糖2.5 g·L-1。5L发酵罐批式发酵表明该菌在8h内可消耗20g·L-1甘油,1,3-丙二醇浓度达12.2 g·L-1,甘油的摩尔转化率达理论值0.75。补料批式发酵发现48h可消耗66.4g·L-1甘油合成38.1g·L-1的1,3-丙二醇,转化率达0.70,表明XJPD-Li菌是具高转化率的1,3-丙二醇生产菌株。以XJPD-Li菌甘油脱水酶为酶源研究了其酶促反应特性。结果表明该酶的表达合成与菌体生长呈现明显的耦合关系。催化反应的最适温度为40℃,且在40℃以下酶的稳定性较好。最适pH值为8.0,在6.0～8.0之间甘油脱水酶活性较高,稳定性也好。Na+、Mg2+、Mn2+和Fe2+对甘油脱水酶催化反应具有一定促进作用,而Ca2+、Co2+、Fe3+、Zn2+和Cu2+对其活性均有抑制作用。该酶分别以甘油和1,2-丙二醇为底物时最高反应浓度分别为0.15mol·L-1和0.3mol·L-1左右,动力学常数Km分别为5.57mmol·L–1和8.16 mmol·L–1。研究也表明该酶催化反应的活化能Ea为18.904kJ·mol– 1。利用PCR方法从XJPD-Li基因组DNA中分别克隆出甘油脱水酶基因dhaBCE和甘油脱水酶复活因子基因dhaFG片段。分别构建表达载体PET-28a(+)-dhaBCE和PET-28a(+)-dhaFG后转化E.coli BL21(DE3),在25℃、OD600为0.6时用0.4mMIPTG诱导后均成功表达出具有生物活性的目标蛋白,诱导5小时后重组菌中甘油脱水酶活力达299U·mg-1,约为原始菌的300倍。重组菌中复活因子可使甘油和氧失活的脱水酶活力均恢复90%以上。重组菌中甘油脱水酶在以甘油为底物催化反应时,3min酶活就损失约35%,1h后活性仅剩2.7%;通氧时其活性损失更快,3min损失48%,1h后仅剩1.5%。通过定量研究甘油失活脱水酶的复活效果确定其最佳的复活条件为:ATP 50mmol·L-1、Mg2+10mmol·L-1、辅酶B123μmol·L-1、甘油脱水酶与复活因子的质量比4:1,在此条件下甘油和氧失活脱水酶的活力可分别恢复98.9%和97.3%。各因子对甘油脱水酶的复活贡献程度研究表明,脱水酶与复活因子质量比和ATP对脱水酶有效复活的贡献程度最大,但四种因子均是甘油脱水酶复活的必需因子。复活动力学研究表明在ATP、Mg2+和辅酶B12存在情况下,复活因子均能快速复活甘油和氧失活的脱水酶,且反应前10min复活速率较大,甘油失活脱水酶更易复活,复活反应60min后丙醛的积累量是氧失活脱水酶复活后的1.5倍。通过基因工程手段获得甘油脱水酶并进行催化失活与复活关系研究,将为构建新的甘油脱水酶体外催化与复活体系提供依据,也为1,3-丙二醇的生物合成调控提供理论指导,对促进生物法合成1,3-丙二醇的工业化进程具有重要意义。更多还原

【Abstract】 To investigate the quantitative relation of glycerol dehydratase reactivation factor and other factors to inactived glycerol dehydratase in vitro, a new strain, XJPDLi-2 from Xinjiang was screened which could convert glycerol anaerobically to 1, 3-propanediol. The strain of XJPDLi-2 is a member of Klebsiella pneumoniae after identified, and named K. pneumoniae XJ-Li(XJPD-Li). The study of fermentation characteristic indicated that the optimal cultivation parameters for temperature and pH were determined as 40℃and 8.0, respectively; the optimized nitrogen source was 6.0g·L-1 of (NH4)2SO4, and the concentration of glycerol and glucose concentration were 20g·L-1 and 2.5g·L-1, respectively. Batch fermentation in 5L bioreactor was performed, and the results displayed that 20 g·L-1 of glycerol was consumed just in 8h with 12.2 g·L-1 of 1, 3-PD and the molar yield of 1,3-PD to glycerol 0.75 was achieved. Fed-batch fermentation also indicated a higher molar yield of 0.70, and 1,3-PD reached 38.1 g·L-1 by consumping 66.4 g·L-1 of glycerol after 48h. Thus, it was shown that this strain would be a excellent 1,3-PD producer.Research about the catalytic characteristic of glycerol dehydratase showed that the enzyme activity obviously goes simultaneously with the cell growth of XJPD-Li strain. The optimal reaction temperature and pH were 40℃and 8.0 respectively, and the enzyme was relatively stable under 40℃and pH 6.0~8.0. In all of the cation ions tested, Na+、Mg2+、Mn2+ and Fe2+ have activation effect, while Ca2+、Co2+、Fe3+、Zn2+ and Cu2+ have inhibition effect on the enzyme. The saturated concentration for catalyzing reaction of glycerol dehydratase was 0.15mol·L-1and 0.3mol·L-1, and Km was 5.57mmol·L–1 and 8.16 mmol·L–1 when glycerol and 1,2-propanediol were as substrate, respectively. The study also displayed that the activation energy of the catalyzing reaction of glycerol dehydratase was 18.904kJ·mol– 1.The genes of glycerol dehydratase(dhaBCE)and reactivation factor (dhaFG) were cloned by PCR from the genome DNA of XJPD-Li, respectively. The expression plasmids of PET-28a(+)-dhaBCE and PE-T28a(+)-dhaFG were conceived, and then were successfully transformed into E.coli BL21(DE3), respectively. The active proteins were induced after 5 hours by 0.4mMIPTG at 25℃while OD600 reached 0.6. The specific activity of glycerol dehydrates was 299 U·mg-1which was about 300 times compared with that of wild strain, and the reactivation factor could make the activity renew above 90% for glycerol and O2 -inactivated glycerol dehydratase.The studies of inactivation process of glycerol dehydratase displayed that the enzyme activity lossed 35% and 48% in 3 minutes, and only 2.7% and 1.5% after 1 hour when glycerol dehydratase reacted with glycerol and sparged with O2 at 40℃, respectively. The optimum reactivation conditions of inactivated glycerol dehydratase were ATP 50mmol·L-1、Mg2+10mmol·L-1、coenzyme B123μmol·L-1 and glycerol dehydratase/reactivation factor 4, and the activity of glycerol and O2 inactivated can be renewed 98.9% and 97.3% under these conditions, respectively. For the contributing extent to reactivate the inactivated glycerol dehydratase, glycerol dehydratase/reactivation factor and ATP are the most important to effective reactivation of inactivated glycerol dehydratase, but all factors are necessary to reactivation of inactivated glycerol dehydratase. The reactivation factor can reactivate effectively the glycerol and O2-inactivated glycerol dehydratase with ATP, Mg2 + and coenzyme B12. Compared with O2-inactivated glycerol dehydratase, glycerol -inactivated glycerol dehydratase can be reactivated more easily, and the concentration of propionaldelyde is 1.5 times after reactivation about 60 minutes.The inactivation and reactivation of glycerol dehydratase which was obtained by expressing in Ecoli BL21 were studied. This research will provide the guidance for the construction the newly catalyzing reaction and reactivation systems of glycerol dehydratase and also for the regulation of biosynthsis of 1,3-propanediol.更多还原