The Study On The Multi-functional Cellulase EGX From Mollusca, Ampullaria Crossean

In these years, the field of animal cellulases has attracted much sight. A cellulase system with high specific activity from Ampullaria crossean, was screen out using the substrate of dehydrated straw without any chemical pretreatment. In the paper, we have studied the cellulase system of mollusca, Ampullaria crossean. The stomach juice of Ampullaria crossean was isolated to purify cellulases. A cellulase designated as EGX was purified to homogenity by 50% ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column and phenyl-Sepharose CL-4B column chromatography. The molecular mass of EGX is 41.5kDa. The pI of the enzyme is pI 7.35. EGX shows hydrolytic activities towards p-nitrophenyl β-D-cellobioside (pNPC), microcrystalline cellulase Sigmacell, carboxylmethyl cellulose (CMC-Na), xylan from oat spelt and the soluble xylan prepared from the birchwood xylan with the specific activities of 12.5, 37.5, 40.3,196 and 275U/mg, respectively, which imply EGX is an multi-functional enzyme with the activities of exo-β-1,4-glucanase, endo-β-1,4-glucanase and endo-β-1,4-xylanase. In addition, the purified EGX could hydrolyse the dehydrated straw. The hydrolytic activities of the enzyme towards soluble xylan, pNPC and CMC-Na reach their maximum at 53℃, 55℃ and 50℃, respectively, and the most favor pH range are pH 4.8-6.0, pH 4.8-5.6 and pH 4.4-4.8. In the presence of 0.2mM and 100mM NaCl, 0~100mM of cellobioside have no detectable inhibition effect on the pNPC hydrolytic activity. The free –SH of EGX are inside of the protein. EGX does not have disulfate bond between peptides. The effect of the monovalent anions, such as F-, Cl-, Br-, I- and NO3-, on the activity of EGX was studied. The five monovalent anions are necessary for the pNPC hydrolytic activity (exo-β-1,4-glucanase activity) and have activation effect. I- concentration of higher than 5mM would inhibit the pNPC hydrolytic activity. Cl- and Br- have activation effect on the CMC-Na hydrolytic activity (endo-β-1,4-glucanase activity) and are necessary. On the xylan hydrolytic activity (endo-β-1,4-xylanase activity), the five monovalent anions have no effect. Cl-, Br- and NO3- reduce the Km of EGX towards pNPC, but do not alert Vmax. In the presence of Cl-, the fluorescence spectrum and circular dichroism spectrum of EGX do not change but the thermostability of the enzyme improves.The 10 amino acids sequences of the N-terminal and the internal of EGX were determined to isolate the cDNA of EGX by PT-PCR. The cDNA is length of 1411bp, including 1185bp of opening reading frame (ORF), encoding 395 amino acids. There is a sequence "AATAAA" in 17bp downstream of TAG, which might be a polyA tailing signal. As deduced from cDNA ORF sequence, EGX contains 395 amino acids without typical signal peptide. It is indicated by amino acid sequence comparison that EGX should be classified to glycosyl hydrolase family 10 (GHF10). The cDNA was expressed in E. coli, and the product was inclusion body. EGX was successfully expressed in yeast Pichia pastoris. The recobinant EGX with the acitivities of exo-β-1,4-glucanase, endo-β-1,4-glucanase and endo-β-1,4- xylanase, which was similar with the EGX purified from the stomach juice of the mollusca, was secreted to the culture medium and hyperglycosylated.To prove the endogenous origin of EGX, the amplification of the EGX gene on the genomic DNA prepared from ovary of the mollusca confirms the endogenous origin of the gene. The genomic fragment is 4892bp and contains 9 exons and 8 introns. The 8 introns locat at 74bp, 122bp, 280bp, 336bp, 473bp, 646bp, 753bp and 972bp of opening reading frame, respectively. It proves that the multifunctional cellulase is secreted by the mollusca, Ampullaria crossean, itself.The cloning of EGX gene proves cellulase system in animal also includes exo-β-1,4-glucanase, which is similar to cellulase system in microorganisms. It also indicates the existence of endogenous xylanase in Ampullaria crossean, which was the firstly discovere...