绿色木霉产内切壳聚糖酶的鉴定及酶学性质的研究

Purification and properties of endo-chitosanase secreted by Trichoderma viride

以实验室前期从Trichoderma viride XW01微晶纤维素诱导的发酵液中分离纯化的一种新壳聚糖酶组分Csn为对象,采用SDS-PAGE分析、不同反应时间水解产物的粘度测定和薄层层析(TLC)分析、还原糖含量的测定等手段对壳聚糖酶Csn进行了初步鉴定及酶学性质研究,结果显示:Csn的分子量为45ku,最适作用温度和最适pH分别为60℃和pH5.0,在低于50℃、pH4.8～7.5范围内非常稳定;脱乙酰度DD对Csn的催化速率影响显著,其对83%DD的壳聚糖的催化活力最高;金属离子Mn2+、Mg2+、Ca2+、Zn2+对该酶有明显的促进作用,而Fe3+、Cu2+和Hg2+则对该酶有强烈的抑制作用;该酶的米氏常数为1.10mg/mL,最大反应速度为7.15μmol/mL·min;该酶以内切方式作用于壳聚糖,主要水解GlcNAc-GlcN和GlcN-GlcN之间的糖苷键,水解产物以四糖以上的壳寡糖为主。

In our previous study,a novel chitosanase Csn was purified from the fermented solution of Trichoderma viride WX01 with the induction of microcrystalline cellulose. The characteristics of the Csn toward chitosan were studied in detail. SDS-PAGE analysis showed that the enzyme Csn was a mono subunit and the molecular weight was estimated as 45ku. Csn was stable below 50℃ and within the pH range from 4. 8～7. 5. The optimal temperature for the enzyme was 60℃ and optimal pH was 5. 0. Mn2＋,Mg2＋,Ca2＋,Zn2＋ enhanced the enzyme activity,whereas Fe3＋,Cu2＋ and Hg2＋ inhibited the enzyme activity significantly. Kinetic parameter Km was 1. 10mg/mL,Vmax was 7. 15μmol/mL·min. Substrate specificity study showed that the effect of deacetylation of chitosan on the activity was evident. Csn exhibited the highest activity toward 83%DD chitosan. The hydrolysates of Csn toward 83%DD chitosan were analysis by TLC and viscosity changing. The results indicated that Csn could split GlcNAc-GlcN as well as GlcN-GlcN,with the main product of hydrolysis showing the degrees of polymerization more than four.