Purification And Stabilization Of β-mannanase From Bacillus

β-Mannanase is an important hemicellulase, which is widely used and usually produced by microbial fermentation method. In this thesis, firstly, the fermentation medium was simplified and optimized, and the purified enzyme was obtained by separating and purifying. Then, the circular dichroism and the prediction of protein secondary structure were employed in investigating the structures and stabilization ofβ-mannanase. Finally, the stability of spray-drying product was improved by adding stabilizer and optimizing operating parameters. The main results were shown as following:The nitrogen sources, carbon sources and inorganic salts of the fermentation medium were screened and optimized, the simplified fermentation medium was determined. The unit enzyme activity cost of final fermentative solution deceased about 63% comparing with that before optimization, which suggested that the benefit was largely increased. Meanwhile, the pigment and impurity contents of the fermentative solution were obviously lower than that before optimization, which is beneficial to the subsequent enzyme refining.Theβ-mannanase of electrophoretic purity was prepared from crude enzyme by ammonium sulfate precipitation, UF, DEAE-52 sepharose, SephadexG-100, and hydroxyapatite chromatography. The molecular weight ofβ-mannanase estimated through GEC is consistent with that through SDS-PAGE, which is about 39kDa.Four sequence analysis methods-APSSP2, SSPRO4, PSIPRED and PHD were used to predict the secondary structure and some properties ofβ-mannanase based on its amino acid sequence. The results indicated the pI ofβ-mannanase was 5.505 and the molecular weight was 38005.4. The hydrophobicity curve and titration curve were also predicted.Through investigating changes in secondary structure and enzyme activity ofβ-mannanase at different temperature and pH by far UV circular dichroism, the effects of secondary structure on enzyme stabilization were preliminarily studied. The secondary structure relative contents ofβ-mannanase were calculated by the optimal CDSSTR method asα-helix 42%,β-sheet 13%, turns 17%, and random coil 28%. And the tertiary structure class ofβ-mannanase was also determined asα+β. The effects of four additives (NaCl, sucrose, amylum, and dextrin) on the stability ofβ-mannanase during spray drying were discussed. The experimental results of the above additives with various concentrations were shown that the addition of sucrose and dextrin might largely enhance the thermal stability of enzyme during spray drying, and the maximum activity of beta-mannanase was increased about 50 % when dextrin was 50 mg/ml. Furthermore, through RSM, the optimal conditions of spray drying were calculated as follows: feed flow rate of 4.3 mL/min, heated air flow rate of 31.3 L/h, and inlet temperature of 177 oC. Under these conditions,β-mannanase activity of 3715.9 U/g was experimentally obtained, which increased about 21.9 % comparing with that before optimization.