The Isolation Of β-mannanase From Snail Liver And The Amplification Partial Region Of The Gene

To select healthy large snail, diluted liver tissue of the snail with pH value of 7, 0.02M Na2HPO4-NaH2PO4 buffer diluted concentration of 1:4, prepared crude enzyme solution, ammonium sulfate fractionation and polyacrylamide gel electrophoresis purification by two times, detected ?-mannanase by direct and indirect ELISA assay, gained products of enzyme with homogeneous purity, identified the enzyme's molecular by SDS-PAGE ,the result of determination of molecular shows it weight 37KD,with specific activity 975.61U/mg, purifyication times 5.05,and recovery rate 24.84%.β-manna- nase with homogeneous purity by SDS-PAGE, trarsmembrane to semi-dry membrane, determination the sequence of the enzyme by N-terminal 10 amino acid. According to available amino acid sequences, combined with online software Primer5 to design degenerate primers A and B, meanwhile extract snail total RNA of digestive gland, processing RT-PCR, amplified partialβ-mannanase gene fragment with 400bp by application of degenerate primers A and B .By gene sequence and protein sequence alignment of mollusks(Snail,Biomphalaria glabrata,Haliotis discus hannai,Mytilus edulis),found that homology of snail and mytilus edulis is more higher.