白地霉红枣果胶酶的分离纯化及酶学性质的研究

Purification and enzymatic properties of jujube pectinase from Galactomyces candidum

为了得到专用于红枣浓缩汁的果胶酶制剂,通过硫酸铵盐析、Sephadex G-75凝胶过滤层析、DEAE-Sephadex A-50阴离子交换层析技术对白地霉发酵产得果胶酶粗酶液分离纯化,得到电泳纯的果胶酶,并研究了其相关的酶学性质。结果表明:发酵上清液经三步分离纯化后比活力达到8036.34 U/mg,纯化倍数为3.12,回收率37.22%,SDS-PAGE电泳检测分子质量为59.62 ku。最适的作用温度为50℃,p H为6。在低于50℃保温2 h酶活剩余80%以上,60℃保温1 h酶活损失60%;p H稳定范围较窄,在p H(5~7)范围内稳定性较好。Ca^(2+)、Mg^(2+)、K^+对果胶酶有激活效应,尤以Ca^(2+)(180.29%)最为突出,Mn^(2+)、Ag^+、Fe^(3+)对果胶酶有抑制作用,其中Ag^+(44.52%)抑制效果显著。通过Lineweave-Burk作图法得到K_m=8.75 mg/m L,V_(max)=60.24μg/min·m L,表明果胶酶对底物具有较强的亲和力。

In order to get the pectinase, that be exclusively used in jujube concentrated juice, the fermented pectinase from Galactomyces candidum were purified by ammonium sulfate, Sephadex G-75 gel filtration chromatography, DEAE-Sephadex A-50 anion exchange chromatography, electrophoresis purity pectase was obtained, and studied the enzymatic properties of pectinase. The result showed that： the specific activity reached 8036.34 U/mg by three steps of purify, the pectase was purified 3.12 folds, the rate of recovery was 37.22%, SDS-PAGE analysis showed the molecular weight of the enzyme was estimated to be 59.62 ku. The optimum reaction temperature of pectase was 50 ℃, pH 6. 80% pectase activity were persisted when incubated two hours under 50℃, 60% pectase activity were lost when incubated one hour in 60℃. It had narrow range of pH stability, in the range of pH 5-7 existed better stability. Ca2＋, Mg2＋, K＋ had activation effect on pectinase, especially of Ca2＋（180.29%）, Mn2＋, Ag＋, Fe3＋ had inhibitory effect on pectinase, Ag＋ （44.52%） had significant inhibitory effect. Through Lineweave-Burk method, we knew that Km=8.75 mg/ mL, Vmax=60.24μg/min.mL, the result showed that the pectinase had better affinity to the substrate.