Clone And Fuctional Analysis Of The Multi-fuction Cellulase Gene Egx From Mollusc, Ampullaria Crosean

The target gene is cDNA of egx from mullusc, Ampullaria crossean (Gene Bank Accession NO. AY285999). The EGX is a multi-fuctional celllulase with the activities of exo-β-1,4-glucanase (E.C.3.2.9.11), endo-p-1,4-glucanase (E.C.3.2.1.4) and endo-β-1,4-xylanase(E.C.3.2.1.8). Accoding to the full sequence of egx cDNA, a series of primers (5-1, 5-2, 5-3, 5-4, 5-5, 3-1, 3-2) were designed to amplify different fragment of egx. Seven deletions in different length of 1132bp, 1089bp, 1056bp, 891bp, 828bp, 1044bp, 921bp were obtained respectively. These DNA fragments were inserted into expression vector pPIC9K and expressed in Pichia pastoris GS115 strain. Seven truncated proteins were obtained which named 5-1 (lacking the structure ofαhelix of N terminal),5-2 (lacking the structure ofαhelix-βstrand of N terminal), 5-3 (lacking the structure ofαhelix-βstrand-αhelix of N termial), 5-4 (lacking the structure of a helix-βstrand-αhelix-βstrand of N terminal), 5-5 (lacking the structure ofαhelix-βstrand-αhelix-βstrand-αhelix of N terminal), 3-1 (lacking the structure ofβstrand-βstrand-βstrand-βstrand of C terminal) and 3-2 (lacking the structure ofβstrand-βstrand-βstrand-βstrand-αhelix-a helix of C terminal).The activity assays of these seven truncated proteins were performed. The result indicated that the endo-p-l,4-glucanase, exo-β-1,4-glucanase and endo-β-1,4-xylanase activity of 5-1 were similar to that of the full length EGX protein. The endo-P-1,4-xylanase activity of 5-2 was similar to that of the full length EGX protein while the endo-β-l,4-glucanase and exo-β-l,4-glucanase decreased sharply. The endo-β-1,4-glucanase, exo-β-1,4-glucanase and endo-β-1,4-xylanase activity of 5-3, 5-4, 5-5, 3-1, 3-2 decreased greatly.These results suggested that the sequence between the firstβstrand of N terminal and C terminal was important to the activities of the endo-p-l,4-glucanase and exo-p-l,4-glucanase. The sequence between the second a helix of N terminal and C terminal was important also.Three EST (Expressed Sequence Tags) were obtained. According to blast them on NCBI (The National Center for Biotechnology Information), three domains were found which were vWFA_subfaminly_ECM, Peptidase_M41, FN3.