Research For Screening Of Endophytes Producing β-Mannanase And Its Enzymatic Properties

β-mannanase, a kind of endohydrolases, belongs to the hemicellulases. At present, there are many reports about screening ofβ-mannanase strains at home or abroad, most of their strains were isolated from soil or water body. The endophyte has been concerned widely as a new microbial resources since Vogl isolated the first endophtyes strain from ryegrass seeds in 1898. Most of researches about endophtyes focused on the screening of antagonistic strains in the early years. In recent years, the researches focused on the isolation of those producing bioactive substances. However, the studies about screening the endophtyes producing enzyme have seldom been done at home or abroad. In this study, we try to screen strains which productβ-mannanase from endophtyes.In this paper, forty-six endophtyes strains were isolated from plant tissue which rich in mannan by enrichment culture, and seven endophytes strains producingβ-mannanase were screened using Congo red dye method. By the shaking-flask culture, HD-1 and CH-3with the higher enzyme activity of 54.59 U/mL and 42 U/mL was obtained. The two strains were worth further studying.The 16S rDNA of CH-3 were amplified by PCR and cloned into vector, and then sequenced. The nucleotide sequences were analyzed by performing BLAST search. It was most closely related to Paenibacillus polymyxa strain YRL13 (97% sequence similarity). CH-3 was identified tentatively as Paenibacillus sp.coupled with classic morphological, physiological and biochemical characteristics. Its genebank accession number is HQ329105. The 16S rDNA of HD-1 were amplified by PCR and cloned into vector, and then sequenced. The nucleotide sequences were analyzed by performing BLAST search. It was most closely related to Bacillus subtilis (99% sequence similarity). HD-1was identified as Bacillus sp. coupled with classic morphological, physiological and biochemical characteristics. Its genebank accession number is HQ329104.Enzymic properties of HD-1revealed that the optimal temprature and pH for producing enzyme were 30°C?50°C and 7.0, respectively; the enzyme activity still remained 68 % when the enzyme was treated at 50°C for 2 h; and more than 64 % enzyme activity was remained after 1 h treatment at pH 5.0?pH 9. 0. In addition, the enzyme activity can be activated by Zn²⁺,Co²⁺,Ba²⁺ and K+, and Ca²⁺ has the most significant effect on the enzyme activity, and 31% of activity was improved when the ion appeared in the enzyme solution. However, Mn²⁺ and EDTA can inhibit its activity. Enzymic properties of CH-3 revealed that the optimal temprature and pH for producing enzyme were 40°C and 5.0-8.0, respectively; the enzyme activity still remained 63 % when the enzyme was treated at 50°C for 2 h; and more than 66 % enzyme activity was remained after 1 h treatment at pH 5.0?pH 10. 0. In addition, the enzyme can be activated by Fe²⁺,Co²⁺,Ca²⁺, and Co²⁺ has the most significant effect on the enzyme activity, and 23% of activity was improved when the ion appeared in the enzyme solution. However, Mn²⁺,Cu²⁺ and EDTA can inhibit its activity.The effects of fermentation temperature and initial medium pH on enzyme production of HD-1 were investigated by single factor experiments. Results revealed that the optimal conditions of them were 35℃and pH 7.0. By orthogonal array design experiments, the optimal conditions of rotation speed, inoculum size and fermentation time were 200r/min, 5% and 72h, respectively. The enzyme production of HD-1 was investigated based on the type and concertration of carbon, nitrogen source and the ratio of carbon source to nitrogen source by single factor experiments. Results revealed that the optimal conditions of them were 3.0 % of konjak power and 0.375 % of (NH4)2SO4. The optimal types and concentrations of ions were FeSO4 0.001%, NaCl 0.5%, MgSO4 0.01% by orthogonal array design experiments. The average enzyme activity achieved 98.3 U/mL by optimizing medium and culture conditions. It is about 1.8 times the initialβ-mannanase activity .The strain HD-1fermentation was carried out in the 50L fermentor under the optimized medium and culture conditions. The sample was detected their enzyme activity every four hours after eight-hour cultivation. Results revealed that the enzyme activity began to increase rapidly since 56 h, and achieved its highest peak at 64 h. The maximum enzyme activity was 193.12 U/mL which is 3.6 times the initialβ-mannanase activity and 2 times the shaking-flask's .That proved the enzyme-production abilty can be improved by fermentating, which implied the industrialization of this strain.