绿色木霉非专一性产双功能酶的鉴定

目的]对绿色木霉诱导产生的具有壳聚糖和纤维素降解活性的双功能酶组分CCBEⅡ进行酶学性质研究和酶类鉴定。方法]采用3,5-二硝基水杨酸(DNS)法测定CCBEⅡ的壳聚糖酶和纤维素酶活力,对酶的温度、pH值稳定性、底物特异性、金属离子影响等酶学性质进行研究。结果]双功能酶CCBEⅡ作用壳聚糖和羧甲基纤维素钠(CMC)的最适温度均为60℃,最适作用pH值分别为5.2和4.2。金属离子Mn2+、Mg2+对酶的2种活性都有激活作用,Fe3+、Cu2+、Ag+和Hg2+则能强烈抑制2种酶活。CCBEⅡ对CMC和壳聚糖(84%脱乙酰度)显示出相当的降解活力,而对对硝基苯-β-D-吡喃葡萄糖苷无降解活性;以二糖方式切割CMC,而以单糖形式切割壳聚糖,可以水解GlcN-GlcN和GlcNAc-GlcN键或GlcN-GlcNAc键。经鉴定,CCBEⅡ与糖苷水解酶7族的绿色木霉所产的CBHⅠ(gi|295937)序列具有很高的同源性,二级结构以β-折叠为主。结论]双功能酶CCBEⅡ与从商品纤维素酶制剂中纯化的双功能酶性质基本一致,两者为同一蛋白;经鉴定实际为具有外切壳聚糖酶活性的CBHⅠ,属于糖苷水解酶7族。

Identification of Non-specific Trichoderma viride Producing Bifunctional Enzyme

Objective] The property of the bifunctional enzyme CCBE Ⅱ,introduced by Trichoderma viride,with the degradation activity of chitosan and cellulose was researched and its type was identified.Method] With the method of dinitrosalicylic acid(DNS) the enzyme activity of chitosan and cellulase was tested and the effect of the temperature of enzyme,the stability of pH value,the specificity of substrate,the metal ion and others on enzyme property was studied.Results] The optimum temperature of the bifunctional enzyme CCBE Ⅱ for chitosan and carboxyn methyl cellulose sodium(CMC) was 60 ℃,the optimum pH value of the bifunctional enzyme CCBE Ⅱ for chitosan and CMC was 5.2 and 4.2,respectively,the metal ion: Mn2+,Mg2+,had the activation to the activity of both enzymes and Fe3+,Cu2+,Ag+ and Hg2+,strongly inhibition.The CCBE Ⅱ was with considerable degradation energy to the CMC and chitosan(84% deacetylated) but no degradation activity to p-nitrophenyl-β-D-glucopyranoside.The bond of GlcN-GlcN,GlcNAc-GlcN or the bond of GlcN-GlcNAc could be hydrolyzed while the CMC was cut with disaccharide form and the chitosan was cut with single sugar.The identification indicated that there was high homology between CCBE Ⅱ and the sequence of CBH Ⅰ(gi | 295937) produced by Trichoderma viride in glycoside hydrolase family 7 and the β-sheet was main form in its secondary structure.Conclusion] The property of the bifunctional enzyme CCBE Ⅱ and the bifunctional enzyme purified from the commercial cellulase preparation was basically with the same nature and both of them was consisted of by same protein.And the identification indicated that it was the CBH Ⅰactually having the activity of exonuclease chitosan,belonging to glycoside hydrolase family 7.