A Comparative Study Of Characterization Of Four Endogenous Cellulases From Longicorn Beetles

In nature, cellulose is the most abundant and the most widely distributed polysaccharide, which constructed from repeating glucose units linked by ?-1, 4-glucosidic bond. It is also the most abundant biomass resource, and the bioenergy from cellulosic materials is great significance for the sustainable development of human beings. Cellulases are multicomponent complexes which are composed of endoglucanases?EC 3.2.1.4?, exoglucanases?EC 3.2.1.91?, and cellobiases?EC3.2.1.21?. It is a common enzyme found in bacteria, actinomycetes, fungi, mollusks, insects, higher plants, and so on. After constant research and exploiture, the application of cellulase is being further extended to many industries, including textile, brewing, feed, food, household chemicals, Chinese medicine extraction and other fields.Longicorn beetles as a kind of wood-boring insect pests, which caused economic losses to forestry industry. To find its cause, there are some endogenous cellulase systems in the body of longicorn beetles. The study on the characterization of these enzymes is expected to provide basic theories to control longicorn beetles using cellulase inhibitors. In this study, we cloned, expressed, purified and analyzed the characterization of novel cellulase genes successfully from Batocera horsfieldi, Psacothea hilaris, Anoplophora malasiaca, respectively. At the same time, we separated Endoglucanase in the midgut from Psacothea hilaris by Native-PAGE and identified endoglucanase species with LC-MS/MS, then determined enzyme activity of endoglucanase. Main results for this study are as follows:1. Molecular cloning, expression, and characterizations of novel endogenous cellulases from three kinds of longicorn beetles.?1? With 5' and 3'-RACE method, we obtained four complete cellulase cDNA named Am-EGase ??Bh-EGase ??Bh-EGase I?Ph-EGase I from B. horsfieldi, P. hilaris, A. malasiaca, respectively. By the homologous comparison, we found Am-EGase ? and Bh-EGase ? belong to the family of glycoside hydrolase 5?GHF 5? 5, Bh-EGase I and Ph- EGase I belong to the family of glycoside hydrolase 45?GHF 45?.?2? From the results of bioinformatics analysis, we can know that the physicochemical properties of Bh-EGase ? and Am-EGase ? are similar, moreover, the biochemical characterications of Bh-EGase I are same as Ph-EGase I. However, there are differences between Bh-EGase I and other three kinds of cellulase. Am-EGase ?, Bh-EGase ? and Ph-EGase I are all hydrophobic proteins, while I Bh-EGase is a hydrophilic protein. Bh-EGase ?, Am-EGase ? and Bh-EGase I contain different numbers and sites of the potential N- glycosylation, but Ph-EGase I has no potential N- glycosylation sites.?3? By Bac-to-Bac baculovirus expression system, we have successfully expressed recombinant protein in the fifth larvae of silkworm. Under native conditions, we purified the recombinant protein and obtained biological activity protein. We analyzed the glycosylation of cellulase with Endo H. The results showed that the molecular of cellulase was decreased, but the activity of cellulase was not lost. So we think the N- glycosylation modification is not the decisive factor of activity of cellulase.?4? We used TLC analysis to clarify how the purified Ph-EGase I and Bh-EGase ? degarade cellulose derivatives. Ph-EGase I can convert CMC and cellotriose into cellobiose and cellotriose but not into glucose, Bh-EGase ? can convert CMC, cellotriose and cellobiose into glucose. The results not only proved the Ph-EGase I and Bh-EGase ? belong to endoglucanase, but also showed the Bh-EGase ? may have properties of ?- glucosidase.?5? Characterization of the expression product showed that the the optimum reaction optimal pH value was 4.0 and optimal temperaturewas at 50 °C of Am-EGase ? and Ph-EGase I, however, Bh-EGase ? and Bh-EGase I were different. Bh-EGase ? showed the maximum enzyme activity occurred at pH 5.0, 40°C. Bh-EGase I showed the highest enzyme activity at pH 4.0, 40°C. ions can affect enzymatic activity of cellulase. In general, tha activity of the four kind of cellulase was enhanced in the presence of metal cations like Ga²⁺, Mg²⁺ and Co+, but was insensitive to Cu²⁺.2. Identification and determination of enzyme activity of endoglucanase in the midgut of Psacothea hilaris?1? According to technology of Native-PAGE and LC-MS/MS, there were four kinds of endoglucanases in the midgut of Psacothea hilaris, which belong to GHF 5. In addition, we also found two endoglucanases bands, whose molecular weight below 15 kDa. These results showed that complexity of cellulase system in the midgut of longicorn beetles.?2? Endoglucanases exhibited maximum activity at pH 6 and 50 °C. It showed that endoglucanases have a wide range of adaptability. Differnent metal ions have a different effect on enzymatic activity. Co²⁺and low concentration of Mg²⁺ have a strong positive effect on the anzyme, but Cu²⁺ and high concentration of Mg²⁺ have a negative effect on the enzyme.