Partitioning and purification of extracellular beta-1,3-1,4-glucanase in aqueous two-phase systems.

The partition behaviors of beta-1,3-1,4-glucanase, alpha-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system (polyethylene glycol (PEG)/MgSO(4)) was examined with regard to the effects of PEG molecular weight (MW) and concentration, MgSO(4) concentration, pH and NaCl concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of beta-glucanase with the PEG/MgSO(4) system. MgSO(4) concentration influenced the partition and extraction of beta-glucanase significantly. pH had little effect on beta-glucanase or proteases partition but affected alpha-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of beta-glucanase but had very significant effects on the partitioning of alpha-amylase and on the neutral proteases. The partition behaviors of beta-glucanase, alpha-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying beta-glucanase was developed, which achieved beta-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.

Medium optimization for the production of thermal stable beta-glucanase by Bacillus subtilis ZJF-1A5 using response surface methodology.

Polysaccharides, such as barley flour, dextrin and soluble starch, were better carbon sources than monosaccharides and disaccharides, such as glucose and maltose, for cell growth of Bacillus subtilis ZJF-1A5 and beta-glucanase production. beta-Glucanase produced by B. subtilis ZJF-1A5 was associated partially with cell growth and increased significantly when cells entered stationary phase; yeast extract was the best nitrogen source, followed by soybean flour. All inorganic nitrogen sources chosen in the experiments were not favorable for cell growth and enzyme production. A fractional factorial design (2(6-2)) was applied to elucidate medium components that significantly affect beta-glucanase production. The concentration of barley flour, corn flour and soybean flour in medium were significant factors. The steepest ascent method was used to locate the optimal domain and a central composite design was used to estimate the quadratic response surface from which the factor levels for maximum production of beta-glucanase were determined. The composition of fermentation medium optimized with response surface methodology was (g/l): barley flour, 63.5; corn flour, 44.8; KH2PO4, 1.0; MgSO4 x 7H2O, 0.1; CaCl2, 0.1. beta-Glucanase activity was 251 U/ml at 48 h using optimized medium, 1.4 times higher than that in original medium.

Optimization of cultural conditions for thermostable beta-1,3-1,4-glucanase production by Bacillus subtilis ZJF-1A5.

The optimization of cultural conditions for Beta-glucanase production by Bacillus subtilis ZJF-1A5 was investigated in flask trials. Temperature had great effect on Beta-glucanase production which maximized at optimal temperature of 37 degrees C and decreased significantly when temperature was over 37 degrees C. Charge quantity affected Beta-glucanase production significantly. Adding oxygen vector N-dodecane or acetic ether benefited Beta-glucanase production, but it depended on the concentration and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting Beta-glucanase production and the cultivation time span to reach the highest Beta-glucanase activity. The optimal cultural conditions for Beta-glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 210 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37 degrees C for 50 h. Repeated experimental results accorded with those predicted by a second-order polynomial model. The amount of Beta-glucanase, Alpha-amylase and neutral protease produced by B subtilis ZJF-1A5 was associated partially with cell growth. Those three enzymes' activities increased following the cell growth and increased significantly when cells entered the stationary phase.