九孔鲍褐藻酸酶降解褐藻胶的反应条件与酶解产物的分析

Degradation effect factors and product analysis of alginate by Haliotis diversicoloe aquatilis lyase

以九孔鲍(Haliotis diversicoloeaquatilis)为原料制备一定纯度的褐藻酸酶,并对影响其降解褐藻胶的条件和产物的性质进行分析。该酶的最适温度和pH分别为45℃和7.0;与磷酸盐缓冲液相比,其在Tris-HCl缓冲液中与底物的亲和力相对较高。动力学曲线表明酶解反应主要发生在1 h之内,在2 h之内达到平衡,酶的剂量和底物的浓度对该平衡起到一定的影响作用。反应2 h之后,添加同数量的酶或者底物发现,产物的反馈抑制只是反应达到平衡的一个影响因素,而酶的变性失活也起到了重要的作用。金属离子对酶的活性具有明显的影响,Co2+、Mg2+对酶的活性具有较强的促进作用,而Cu2+、Ag+和Zn2+则具有较强的抑制作用。产物特性黏度的变化主要是在1 h内,2 h后基本不再改变。1HNMR图谱分析发现随着聚甘露糖片段,特别是M-M二聚体的降解,MG二聚体、GGM和MGM三聚体的量相应增多,表明该酶属于甘露糖裂解酶(EC 4.2.2.3)。

In this experiment, the effect factors on the degradation of alginate and the specificity of alginate lyase from Haliotis diversicolos aquatilis were studied. Temperature and pH strongly affected the enzyme activity, and the enzyme was more stable in an acidity pH at lower temperature than in an alkalinity pH at higher temperture. The optimal temperature and pH value of the lyase were 45 ℃ and 7.0, respectively. The Km and Vmax values of the lyase were 0. 211 mg/mL and 0. 209 UA/min in phosphate buffer, respectively; but they were 0.189 mg/mL and 0.258 UA/min in Tris-HCl buffer, respectively, Thus the enzyme is considerably more efficient in Tris-HCl buffer than in phosphate buffer. The progress curves indicated that an apparent endpoint level of maximal conversion was reached within 2 h. This level was not only affected by the initial substrate concentration, but also dependent on the enzyme dose. The activity was raised slowly when sodium alginate concentration reached 0.3 % and decreased with the increase of enzyme doses. Although the system have been reached the equilibrium level, but an addition of new substrate can lead immediately to an increased formation of product, The enzyme present cannot be considered inactivated or irreversibly inhibited. It should also be noted that the reaction induced by substrate addition at 2 h progressed at a much lower rate than the initial one and the reaction was too slow to reach a stable endpoint within the recorded period. This effect can only be explained by a reduced amount of enzyme accessible to the new sub- strate. A doubling of the enzyme concentration after 2 h could make curves approach those of the same total enzyme dose given initially. Thus, the apparent endpoint levels were unaffected by a multistep addition of enzyme as long as the final total dose was consistent. The reason may be that the product inhibited the en- zyme activity and the reduction of substrate concentration. The effect of the ion indicated that the activity of lyase was activated by CO^2＋ , Ca^2＋ , Al^3＋, Pb^2＋, Ba^2＋, Mg^2＋, NH4^＋ , K^＋, especially CO^2＋ and Mg^2＋ , which has increased by 62.7 %, 50.8 %, respectively. But Cu^2＋ , Ag^＋ and Zn^2＋ significantly inhibited the enzymatic activity（P 〈 0.01） ; the activity was inhibited by 76.3 %, 52.4 % and 49.8 %, respectively. The decrease of the viscosity with the time was due to the enzymatic degradation of alginate. It indicated that the change of[ η] isn＇t linear dependence; the viscosity of the solution almost reached an equilibrium level after 1 h. It may be explained by the strong, reversible product inhibition and the reduction of the substrate;on the other hand, the ratio of mannurate to guluronate in alginate also affected the mechanism of hydrolysis. 1H NMR monitoring of the degradation of a commercial alginate sample found that M block was attacked with the fraction of MM dimmers decreased while the fraction of MG dimmers, GGM and MGM trimmers increased after epimerization. It indicated that the alginate lyase from Haliotis diversicoloe aquatilis was a kind of mannuronate （EC 4.2.2.3）. But there is no obvious difference between algiante acted by the lyase for 1 h and alginate acted by the lyase for 4 h, it indicated that a majority of the polymer chains have alternated their structures during the first 1 hour. Compared with the degradation of alginate by enzyme, the GGM, MGM and GGG were first decreased and the GG and GM increased when the alginate was reduced by the acid （0.1 mol/L HCl）. [Journal of Fishery Sciences of China,2007,14（4）：659- 666]