Optimization Of β-mannanase Fermentation And Purification By RSM From Bacillus Licheniformis

Plackett-Burman design (PB) and Central composite design (CCD) in response surface methodology (RSM) were employed for optimizing fermentation and purification conditions ofβ-mannanase production by Bacillus licheniformis TJ-101. Flocculation, aqueous two-phase extraction (ATPE) and membrane separation were used to purify the rough zymotic fluid and also spray drying method was used to produce solidβ-mannanase preparation.A 6.6L auto-control fermenter BIOSTAT was used to studyβ-mannanase fermenting process from Bacillus licheniformis. Three significant key factors including rotate speed (r/min), air flow rate (L/min) and fermenting time (h) were investigated according to CCD. The optimal values of the variables were determined by RSM analysis and were listed as follows: 658.58r/min, 5.0L/min and 46.34h, respectively. The optimizedβ-mannanase activity of 440U/mL was obtained.With flocculant of cationic polyacrylamide (C-PAM) and anionic polyacrylamide (A-PAM) and aid-flocculant of polyaluminium chloride, flocculation of rough zymotic fluid, as an important method in the downstream bioprocess, was also designed using PB method and three key factors were screened to significantly effect the flocculating. And then CCD was used for further optimizing three key variables and RSM was applied to analyzing the experimental results. The optimal conditions, 240.55% (V) of water, 14.13% (V) of C-PAM and 16.97% (V) of A-PAM, were obtained. Under these conditions, the enzyme yield of 72.93% was obtained. The purified enzyme solution was converted into solid enzyme product by spray drying.To investigate the fraction behavior forβ-mannanase in ATPE, different combinations between polyethylene glycol (PEG) and salt (sulfate and phosphate) were tried and PEG1000/(NH4)2SO4 was the best one. After multi-goal optimization of RSM for three factors (PEG, (NH4)2SO4 and NaCl concentrations), different level enzyme extraction were obtained.Hollow fiber membrane was used to condense the ferment production and inlet amount, operating pressure and operating time were researched. The optimal conditions of 1.3L/h inlet and 0.04MPa operating pressure were found the best and from 20 minutes to 45 three level concentrate solutions can be collected.