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β-Mannanase is an endo-wise glycosyl hydrolase catalyzes random hydrolysis ofβ-1,4-mannosidic linkages in the main chain of mannan and it is a kind of hemicellulases. Mannanase existed widely in animals, plants and microorganism. Mannanase has wide commercial applications in industry processes such as foodstuff, pharmaceutical, feed, paper and pulp industry. Konjac oligosaccharide has a variety of physiological functions, the mannanase hydrolysis konjac could be used to produce purity oligosaccharide which has a bright future.36β-mannanase producing strains were isolated from soil and factory waste liquid. One strain named CYS4 was obtained after screening by determining mannanase activity in DNS method, its enzyme activity was 5.14 U/mL. The research also found that the CYS4 strain is a exocelluarβ-mannanase producing strain.16S rDNA sequence of CYS4 strain was determined, comparative 16S rDNA sequence analyses revealed that CYS4 strain was most related to Bacillus subtilis（Identities>99 % ）, the similarity of Bacillus subtilis CD-23 was 100 % . According to cultivation characteristics, morphological characteristics, physiological and biochemical properties, CYS4 strain was preliminarily identified as Bacillus subtilis.The fermentation conditions of CYS4 strain were optimized by the single factor tests and orthogonal tests, the results showed that the compositions of fermentation medium were: 5%konjac powder, 2%yeast extract, 0.6%KH2PO4, 0.03%MgSO4, pH7.5. The optimal cultivation temperature was 40℃, and inoculum volume was 7%. 48h’s cultivation under these condition, the activity ofβ-mananase can reach 20.67U/mL,which was 4.02 times than before.Enzymatic characteristics analysis showed that the optimum pH for the enzyme was 7.0, and the enzyme could maintain well stability between pH5.0 and pH11.0, the enzyme activity was still higher than 80% after 2 hours’treatment at 37℃, and the optimum tempemture was 60℃, it was stable below 40℃, the enzyme activity was still more than 70% after 3 hours. The enzyme activity was activated by metallic ions such as Na+、Fe2+、Mg2+, while was inhibited by Hg+.
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