The Characterization Of Cellulase And Isolation Of Endo-1,4-β-glucanase From Omphis Fuscidentalis

Cellulose is the most abundant renewable resource in nature. There are billions of productsby photosynthesis every year which cellulose taking almost half ones, but only a small partare utilized. It has great realistic significance for solving energy crisis, by utilizing andtransforming cellulose to available energy for human beings. Specifically, it first degradesthe cellulose to monose by cellulase, then transforms it to green resourse-cellulosic alcohol.Since the product of enzymolysis is easily recycled and utilized, most importantly lowerpollution, the enzymolysis of cellulose becomes an idea and recognized transformingmethod. However, the high cost of cellulase can’t be avoided if it plans to massive producein the future. Therefore, it is a critical factor to obtain high activity and high stabilitycellulase during the production of cellulosic ethanol. In order to find this kind of cellulase,scientists have conducted a large number of selections from different species, and deepstudied the activity and property of the obtained ones. Moreover, it is an important researchfield in exploiting the cellulase resource to obtain high activity and special propertycellulase, which can also provide more information for analyze and remould of proteinstructure of cellulase. Thanks to the speciality and high activity, animal cellulase is a natureresource with excellent property. Hence, we select Omphis fuscidentalis as research objectto extract and purify its cellulase and then analyze the properties of this cellulase.The O. fuscidentalis larvaes live in the body of Gigantochloa levis, and feed on thecellulose-rich materials. It can hydrolyze cellulose to glucose as energy resource throughits cellulose, which might be different from grazing animals because they depend ondifferent digestive environment. Through obtaining and analyzing this kind of animal’scellulase, we look forward to find some cellulase with new characteristics. It hopes toprovide new gene resource by utilizing biotechnology method to obtain high efficientcellulase.O. fuscidentalis are used as the experimental materials in this research. Crude enzymewas obtained after homogenization and extraction by citric acid buffer solution. Furtherprotein precipitation was prepared to test the activity of cellulase. The activity and theoptimum environmental conditions of the O. fuscidentalis cellulase were tested withdifferent enzyme concentrations by DNS reaction. Besides, the effects of different mentalions on the cellulase were investigated. The results showed that filter paper units activitywas FPU=0.5438U/ml, activity test about substrate for the microcrystalline cellulose activity was CB=0.4296U/ml, sodium carboxymethyl cellulose activity was CMC=0.3657U/ml, salicin activity was SA=0.5285U/ml.We also found the cellulase system of O. fuscidentalis adapt to the neutral to slightlyalkaline pH environment, high temperature and short reaction time and other properties,which provide a reference for selection of good cellulase system. Our results showed mostof the metal ions inhibited cellulase activity of O. fuscidentalis, and high concentrations ofmetal ions expressed stronger inhibition than low concentrations, such as Na⁺、K⁺、Ca²⁺、Mg²⁺、Cu²⁺、Fe³⁺、Zn²⁺inhibited the FPU and SA, while improved the CB and the CMC atlow concentrations and inhibited these reactions at high concentrations. The furtherresearch about effects of Ag⁺、Mn²⁺、and Fe²⁺ cellulase is necessary because of thedisturbance of these ions to DNS reaction.The crude enzyme of O. fuscidentalis was processed by the Folin protein precipitationafter ion exchange chromatography, which can remove the impact of the reducingsubstance on testing the enzyme activity in the crude enzyme. We successfully purified theendo-1,4-β-glucanase from O. fuscidentalis by the improved ion exchange chromatographyand gel filtration chromatography. This endoglucanase avtivity of O. fuscidentalis was13.23U/mg, the molecular weight was55.2kDa, the purify multiple was4.35andrecovery rate was19.4%. The purified cellulase can be sequenced within the N-terminalsequencing of protein, and the primers can be designed to get its coding sequence, whichcan provide the basic research data for cloning and expression of cellulase.