| β-Mannanases are the second most important enzymes for the hydrolysis of hemicelluloses, which could cleaves the β-1,4-glycosidic linkages within the backbone of mannan, glucomannan, galactomannan and galactoglucomannan, yielding mannooligosaccharides. The β-mannanase has widespread applications in various processes including the feed additive, food, as well as in the pulp and textile industries. In this paper, Konjac powder was used as the only carbon source to screen the strain which can produce β-mannanases efficiently. Then, the optimization of fermentation process, purification and characterization of the enzyme, and application in paper industry had also been researched in the paper.The major researching contents are as follows:(1) A strain producing β-mannanase, named as MY271, was isolated from soil by conga red staining. Its initial β-mannanase activity was 2.87 U/mL. The strain was identified as Enterobacter ludwigii through colonial morphology, physiological and biochemical characteristics combined with phylogenetic analysis.(2) The β-mannanase fermentation conditions of MY271 were optimized by single factor experiments. The optimal fermentation conditions were as follows: fermentation temperature 31 °C, fermentation time 48 h, initial pH 7.0, inoculation amount 9%, loaded liquid volum 50 mL/250 mL. Then, the fermentation medium components were optimized by Plackett-Burman experiments, the steepest ascent experiments and response surface methodology. The optimal fermentation components(g/L) were as follows: konjac powder 14, peptone 18, Na2HPO4 0.7, KH2PO4 0.5, ZnSO4 0.15 and MgSO4 0.01. The enzyme activity in vertification test reached 62.76 U/mL, which consistents with the model predicted value.(3) The MY271 strain was mutated by ultraviolet(UV) radiation and DES. A mutant M108 was obtained. The β-mannanase activity of M108 was 83.68 U/mL, which was 33.33% higher than MY271. By genetic stability test, M108 strain manifested a good genetic stability.Using single factor experiments, the optimal fermentation conditions of M108 were as follows: fermentation temperature 31 °C, fermentation time 24 h, initial pH 7.0, inoculation amount 7%, loaded liquid volume 25 mL/250 mL. Then, the fermentation medium components were optimized by Plackett-Burman experiments, the steepest ascent experiments and response surface methodology. The optimal fermentation medium components(g/L) were as follows: konjac powder 13.6, peptone 26, NaH2PO4 1.0, KH2PO4 0.6, MnSO4 0.1, ZnSO4 0.23 and MgSO4 0.015. The enzyme activity in vertification test reached 174.57 U/mL, which consistents with the model predicted value.(4) A high active β-mannanase was purified by ammonium sulfate fractionation, Sephadex G-25, DEAE anion-exchange and Sephadex G-100 chromatography. The purified enzyme was demonstrated a single protein and its molecular mass was caculated 43.16 kDa by SDS-PAGE.The optimal pH and temperature of the purified enzyme was 7.0 and 55°C. The β-mannanase had high active over a broad pH range. In addition, the purified enzyme was highly activated by several metal ions and chemical reagents such as Ca2+, Mg2+, Ba2+, Co2+, l-cysteine, GSH, urea, tween-80 and β-mercaptoethanol. Whereas, the enzyme was strongly inhibited by Hg2+, Cu2+, SDS, PMSF and NBS. EDTA and 1,10-phenanthroline had no effect on its activity. The β-mannanase has higher activity towards konjac glucomannan. As an endo β-mannanase, it can hydrolyze konjac glucomannan into a mixture of oligosaccharides. Moreover, the enzyme seemed to preferably hydrolyze mannooligosaccharides of DP>3. When the ratio of xylanase to β-mannanase was 1:2, the brightness of the paper increased significantly, but the tensile strength of the paper was not remarkably changed. |
PDF Full Text Request