重组菌产碱性果胶酯裂解酶酶学性质研究  

Properties of alkaline pectate lyase from recombinant strain E.coli JM109(pHsh PL)

作　　者：诸葛斌[1] 堵国成[1] 诸葛健[1] 陈坚[1] 

机构地区：[1]江南大学生物工程学院环境生物技术研究室,工业生物技术教育部重点实验室,无锡214122

出　　处：《微生物学报》2008年第1期121-125,共5页Acta Microbiologica Sinica

基　　金：国家"863计划"(2003AA322050)~~

摘　　要：利用盐析-透析-色谱流程建立快速高效纯化工程菌E.coliJM109(pHshPL)所产碱性果胶酯裂解酶(PL)的方法,纯化后酶达到电泳纯,比酶活为1079U/mg。重组菌所产PL酶促反应适宜的pH为9～10,适宜温度为50～66℃,与酶基因来源野生菌所产PL相比,重组菌所产PL适宜pH范围有所扩大,并保持了野生菌PL的热稳定性。通过金属离子种类、浓度及存在时间对PL酶活力影响考察发现:在考察的离子中除Mg2+对酶活有较好的促进作用外,其余对重组菌PL均有抑制作用,其中Fe2+对酶活力抑制作用最强。该酶的Km值为20.93mg/L,Vmax为105.3μmol/min,反应活化能Ea为21.74kJ/mol。对重组菌所产PL热稳定动力学进行分析,发现有底物情况下的失活常数kd(0.02min-1)小于无底物情况下的失活常数kd(0.0342min-1),说明当酶与底物结合形成复合物时对酶活具有保护作用。利用HPLC-ESI-MS对重组菌所产PL酶解产物进行测定发现,产物含有不饱和二聚半乳糖醛酸(m/z350.82)和不饱和三聚半乳糖醛酸(m/z527.04),同时测定结果中没有发现不饱和半乳糖醛酸单体(m/z175),可以初步推测重组菌PL不能以不饱和二聚半乳糖醛酸和不饱和三聚半乳糖醛酸为底物进一步裂解。Alkaline pectate lyase （PL） from recombinant strain E.coli JM109 （pHsh PL）was purified by a three-step process including （NH4）2SO4 precipitation followed by dialysis and chromatography. The purified enzyme ap- peared homologous on SDS-PAGE. The specific activity of the purified enzyme reached 1079 U/mg. The optimal pH and temperature were in the ranges of pH 9.0 to 10.0 and 50℃ to 66℃. The enzyme was preferable in optimal pH range in enzymatic retting of flax. Enzyme activity slightly increased in the presence of Mg2~ ion, whereas decreased in the presence of other ions, especially Fe2~. The Km of the purified enzyme for polygalacturonic acid was 20.93 mg/L, the Vm^x for polygalacturonic acid hydrolysis was 105.3 lmaol of unsaturated products per min and Ea was 21.74 kJ/mol. The results of the decay constant （kd） analysis on condition of PL bonding polygalactu- ronic acid （kd =0.02 min-1） and PL without polygalacturonic acid （kd =0.0342 min-l） showed the substrate was helpful to decrease thermal inactivation of PL. The products （unsaturated oligomers） from polygalacturonic acid degraded by PL were analyzed by electrospray ionization mass spectrometry（ESI-MS）. The following data were obtained： ESI-MS m/z, 350.82 （unsaturated bigalacturonic acid, uG2）, 527.04 （unsaturated trigalacturonic acid, uG3）. However, m/z 175 （unsaturated galacturonic acid, uG1） was not found. These results indicate that the final PGA degradation products was a mixture of unsaturated oligo-galacturonides including uG3 and uG2 except for uG1. It suggests that the recombinant PL cannot degrade uG3 and uG2.

关 键 词：碱性果胶酯裂解酶 重组大肠杆菌 纯化 酶学性质 

分 类 号：Q789[生物学—分子生物学]