Apple Snails Multifunctional Cellulose Enzyme Egxa Its Domain In The Reorganization Of The Insect Cell Tn5 Analysis, Expression And Characterization

Cellululose is the major component of plant biomass resulting from the photosynthesis of life on earth. And cellulase is a kind of enzyme that plays a great role in the conversion process from cellulose into the glucose. Thus it would be of giant value for both academic researches and industry applications.The cellulase designated as EGX was purified from gastric juice of Ampullaria crossean. The molecular mass of EGX was 41.5kDa. According to the central amino acid sequence of EGX, a DNA sequence was cloned from Ampullaria crossean cDNA expressional library. And the 3'end was totally same to the EGX open read frame. Thus we named the DNA sequence as egxa, and the protein expressed as EGXA. It was needed to functionally express egxa to obtain recombinant protein which possesses similar enzymatic properties with animal cellulase.Insect cell baculovirus expression system offers many advantages over other expression system. Such as safety, easy of scale up, high level of recombinant gene expression, accuracy and the use of cell lines ideal for suspension culture, especially insect cells have similar post-translated modification system to Ampullaria crossean.We expressed egxa in the insect cell Tn5(Trichoplusia ni.) using baculovirus vectors system. And we found that recombinant EGXA showed hydrolytic activities towards p-nitrophenyl -β-D-cellobioside (pNPC), 2-hydroxythel-cellulose, hydroxythel-cellulose, sigmacell, carboxylmethyl cellulose sodium salt (CMC-Na) and xylan from oat spelt, which implied recombinant EGXA was an multi-functional enzyme with the activities of exo-β-1,4-glucanase, endo-β-1,4-glucanase and endo-β-1,4-xylanase. The hydrolytic activities of the enzyme towards soluble pNPC, xylan from oat spelt and CMC-Na reached their maximum at 48℃, 45℃and 40℃respectivily, and the optimum pH were about pH5.8, pH 5.5 and pH 5.2. In addition, the purified recombinant EGXA was glycosylated by the insect cells.Additionally, CBM-A, the recombinant cellulose-binding module showed ability of binding to sigmacell, while the recombinant catalysitic domain CD-A lost the enzymatic activities, which implied the binding domain played a vital role in the hydrolyzing course of cellulose.In summary, recombinant EGXA showed similar hydrolytic activities to wild EGX. So it can be used as an animal cellulase in both academic researches and industrial applications. And the catalystic domain could not work without its cellulose-binding module.