| β-mannanase (EC3.2.1.78), a kind of endo-hydrolases, belonging to hemicellulases could catalyze the hydrolysis of theβ-1,4 glycosidic linkages within the main chain ofβ-1,4-mannans including galactomannan, glucomannan, galactoglucomannan and mannan, yielding monosaccharides and oligosaccharides as products. With exploitation of the resources of hemicellulose and discovery of pharmaceutical value in mannan oligosaccharides, the study ofβ-mannanase entered a new high tide. They had been found applications in food, feed, pharmaceutical, biotechnology, paper and pulp industry and so on.A.tabescens EJLY2098 is a fungus of Tficholomataceae, it parasitized on the stem or root of trees, and can cause many tree's decayed diseases. Through the searching, the author didn't find any report aboutβ-mannanase of A.tabescens EJLY2098 from other laboratories or research institutes except ours. Our previous research had indicated that A.tabescens EJLY2098 could pruduce two active ingredient ofβ-mannanase:β-mannanaseP1 andβ-mannanaseP2(79K).β-mannanaseP2(79K) had been purified and characterized and the gene cloning and expression ofβ-mannanase(46K) had also been finished.The purpose of this article were: 1.Purification ofβ-mannanase46K from A. tabescens EJLY2098 by column chromatography and characterization ofβ-mannanase46K; 2.β-mannanase(P2)79K gene cloning from A. tabescens EJLY2098 by SMART-RACE, initiating with degenerate prime design based on N-termianl sequence of the protein.First, konjac was used to induce A.tabescens EJLY2098 to produceβ-mannanase, then three different purification protocols were used to purifyβ-mannanase: 1) Sephadex G-25,DEAE ion exchange chromatography and hydrophobic interaction chromatography(HIC); 2) SephadexG-25,DEAE ion exchange chromatography and SephadexG-75; 3) DEAE ion exchange chromatography. And experiments showed thatβ-mannanaseP1(also namedβ-mannanase47K) could be purified using the third protocol. The multiple of this purification was 12.07 with recovery rate of 3.2％; SDS-PAGE showed that the molecular weight ofβ-mannanase47K was 47KD approximately; The isoelectric point ofβ-mannanase 47K was estimated to be 5.6-5.7; the result of HPTLC revealed that this 47K enzyme was endo-β -mannanase; Analyses of zymological characterization showed the optimum activity of the enzyme was obtained under 50℃and pH5.0-6.5. The stable pH and temperature of the enzyme was pH5.0-6.5 and 30～60℃, respectively; the activity ofβ-mannanase were enhanced by Na+andBa2+. Compared with recombinationβ-mannanase P1(β-mannanase 47K): the natural and recombinant enzmye had similar optimum activity pH and thermal stability. The optimum activity temperature of the natural enzyme was lower than that of the recombinant enzyme which had a wider stable pH of pH4.0～5.5.In the article, attempts was done to cloneβ-mannanase79K(P2) gene. First of all, molecular weight and activity ofβ-mannanase79K(P2) were anamyzed by Native-PAGE, and the active zone, which MW is 79KD was chosen for sequencing to determine N-terminal sequences. Nine amino acids: AFLNVDLDF was got. The upstream primer was designed based on this sequence, and then PCR was done using this upstream primer and three different downstream primers (1) OligodT20, (2) downstream primer---M13PrimerM4 from LA kit, (3) downstream primer based on homology from NCBI Protein Data Bank) to cloneβ-mannanase79K(P2) gene. Eventually, a 380bp cDNA fragment was amplified from A. tabescens EJLY2098 using (3) downstream primer. The homologous analysis (blastx) showed that the sequence was not similar to that ofβ-mannanase from other fungi, indicatedβ-mannanase79K(P2) gene was not cloned successfully.In short,β-mannanase47K was purified from A. tabescens EJLY2098 bacterium supematant, and its enzymology characterization was well analyzed. Primary research of cloning unknown gene from protein sequence using different primers was carried out. This work played a partial important role in fully understanding ofβ-mannanase from A. tabescens EJLY2098.
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