Research For β-mannanase-producing Endophytic Bacteria: Mutation Breeding And Enzymatic Characterization

β-mannanase (1,4-β-D-mannan mannanosidase, EC3.2.1.78) is an endohydrolase, which randomlyhydrolyzes β-1,4-linkages of mannan-based polysaccharides, hydrolyzing mannan into mannoseoligosaccharide or monosaccharide. Over the past decade, endo-b-1,4-mannanases have attractedworldwide researchers and played an important role in paper and pulp industry, food and feed technology,spinning, bleaching, coffee extraction, oil drilling and detergent industry. Despite having high practicalpotentialities, however, in china, the use of mannanase is still limited due to low yields and high-productioncosts,the study of β-mannanase is still at the laboratory stage, so the work of breeding high-yieldβ-mannanase strains is the direction of our future research.Compared the mutagenic effects of the ultraviolet ray, diethyl sulfate's single factor andultraviolet-visible, ultraviolet-diethyl sulfate compound mutagenesis. By observing the clear circles onselective plates and rescreening with rescreening medium, the results of enzyme activity show thatcompound mutation is better. Using20W UV lamp irradiation for6min in the distance of30cm, and then1.6%diethyl sulfate treat40min. A mutat strain, named Bacillus subtilis UD-20with high-levelβ-mannanase production was obtained after ultraviolet-diethyl sulfate compound mutagenesis fromBacillus subtilis HD-1which is an endophytic bacteria screened from soybean seeds, the β-mannanaseactivity of Bacillus subtilis UD-20increased63.9%compared with that of the original strain Bacillussubtilis HD-1(54.6U/ml) and was stable during the generations.The optimum of fermentation conditions was preliminary determined as follows: konjacglucomannan (KGM)30g/L, peptone3g/L, NH4NO33g/L, CaCl23.0mmol/L, MgCl22.0mmol/L,BaCl20.2mmol/L, Tween801.0g/L, glycine0.2g/L (which was added into the fermentation mediumafter10h of the fermentation beginning) pH7.0, agitation speed180r/min, inoculation volume of2%incubation at37℃for72h, the highest β-mannanase activity was213.6U/ml. In addition, thefed-batch fermentation activity is275.1U/ml,34%higher than not feeding, and5.0-fold of Bacillussubtilis HD-1, Static culture β-mannanase activity increased by24.5%than the conventionalfermentation.The enzymatic properties of β-mannanase analysis shows that, The optimal temperature and pH for β-mannanase activity was50℃and7.0, and the enzyme was stable up to50℃and demonstratedbroad pH stability within a pH range of5.0-8.0; The β-mannanase activity was strongly inhibited by2.0mmol/L Mn2+(55.6%), but was activated by2.0mmol/L Ca2+,2.0mmol/L Mg2+,2.0mmol/L Fe2+,2.0mmol/L Co2+,0.2mmol/L K+,0.2mmol/L Ca2+,0.2mmol/L Cu2+and0.2mmol/L Ni2+, was stronglyactivated by2.0mmol/L Fe2+(113.2%),2.0mmol/L Co2+(131.7%).The β-mannanase stability wasactivated by2.0mmol/L K+,2.0mmol/L Ca2+,0.2mmol/L Ni2+, and was strongly activated by2.0mmol/L Co2+(148%). But the β-mannanase stability was strongly inhibited by2.0mmol/L Mn2+(59.4%).The Michaelis-Menten constants and substrate specificity were determined for locust bean gum,glucomannan and guar gum, The Kmvalues were10.9mg/ml,15.6mg/ml,33.4mg/ml, and the Vmaxvalues were9960.2U/ml,526.3U/ml,769.2U/ml. The β-mannanase showed high activity(6116.6U/ml)on glucomannan, which was the best substrate,22.2-fold of locust bean gum and28.2-fold of guar gum.The most product of locust bean gum by β-mannanase hydrolyzing was mannose oligosaccharide. Theβ-mannanase of from Bacillus subtilis strain UD-20can be useful in several processes in the food, feed,as well as in the health care product industries.