Studies On Screening Of Strains Producing Thermophilic Proteinases From Bacillus Thuringiensis, Fermentation Optimization And Characteristics Of Enzyme

FZ62 producing thermophilic proteinase was screened from 34 Bacillus thuringiensis strains.First, the factors which affected proteinase fermentation were screened out by single factor screening tests.The result showed that the best Carbon and Nitrogen source were glucose and yeast powder respectively. Second, Plackett-Burman design was used to evaluate eight factors which affect fermentation. Three prominent factors were yeast powder, glucose and initial pH in the culture medium. Third, the optimal levels of these factors were determined by Response Surface Methodology (RSM). Studies showed that optimal fermentation medium components were yeast powder 20.4 g/L, glucose 1 g/L, KH₂PO₄ 2.5 g/L, MgSO₄·7H₂O 0.35 g/L, CaCO₃ 0.5 g/L, initial pH 7.07. The yield of thermophilic proteinase by FZ62 increased from 263.43 U/mL to 1112.39 U/mL.There was only +5.66% differences from the forecast by RSM. The last,the optimal conditions were 35mL culture in 250mL shake flasks, amount of inoculation 0.75mL, fermentation temperature 31℃, culturing duration 20h by optimization, and the activity of enzyme reached 1288 U/mL.Preliminary study of the metabolic characters of thermophilic proteinase batch fermentation with FZ62 was carried out.The kinetic models for cell growth; enzyme production and substrate consumption were proposed basing on Logistic equation and Luedeking-piret eqution. Enzyme formation was growth-associated with cell growth in all batch cultures, the calculated results of models were compared satisfactorily with the experimental data.Thermophilic proteinase was purified by using ammonium sulfate precipitation, column chromatography on DEAE Sepharose Fast Flow and Sephadex G-75. The Specific activity of the enzyme was 802.40 U/mg. Purification times and recovery rate of enzyme were 10.49 and 38.38 % respectively.The molecular weight of the purified enzyme was estimated to be 38.91 kDa by SDS-PAGE.The kinetic behavior of this enzyme was performed using casein as substrate. The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of casein and the K_m and V_m values were determined to be 4.8g/L and 322.58U/mL. The optimum temperature was 55℃, and enzyme was stable below 60℃.The optimum pH was determined to be at pH 7.0 and showed to be stable in the range 7～11.The effects of metal ions on the enzyme activity of thermophilic proteinase were studied. The salt concent ration was 2mmol/L, K⁺ and Mg²⁺ strongly activated the enzyme activity, but Cu²⁺,Mn²⁺,Zn²⁺ had inhibitory effect.Effects of different organic solvents on the activity of thermophilic proteinase were investigated. The inhibitory was increased gradually along with increase of organic solvents concentration.