Studies On The Protease From Bacillus Thuringiensis FS140

FS140 producing thermophilic proteinase was screened from 35 Bacillus thuringiensis(Bt)strains under temperature 55℃.Through single factor screen tests the results showed that the best carbon source and nitrogen source were glucose and the compounding of yeast powder and soya meal,respectively.Plackett-Burman design was used to select 3 important factors from 8 factors which affected prtotease fermentation.They were soya meal,yeast powder and glucose in culture medium. The optium components obtained by the response surface methodology(RSM)were soya meal 18%,yeast powder 0.36%and glucose 0.14%respectively.After the optimization,the yield of protease increased significantly and reached 837.71U/mL, of which only 1.34%differed from the prediction by RSM.The optimization of fermentation conditions showed that fermentation temperature was 31℃,the initial pH was 7.0,the loading volume of medium was 35mL culture in 250mL shake flasks, inoculation volume was 0.75mL cell suspension(cell concentration was 7×10⁶/mL) each shake.Under optimized fermentation conditions the protease production could reached 918.91U/mL.The studies of the metabolic characters of protease batch fermentation with Bt FS140 were conducted.Cell growth,protease production and sugar consumption throughout the protease fermentation were investigated.The kinetic models for cell growth,protease formation,sugar consumption were proposed according to the Logistic equation and Luedeking-Piret equation.The calculated results of models were compared satisfactorily with the experimental data.The model equations could really reflect the protease fermentation process and kinetic mechanism.Protease formation was partially associated with the growth of Bt FS140.FS140 protease fermentation data was analyzed according to saccharide metabolism.Glucose was a restrictive substrate for whole fermentation and the changes of pH in liquid could reflect glucose metabolism.Because pH was easy to be on-line control in automatic 7 liter fermentor,glucose fed-batch strategy under pH-control was adopted.The results showed that total fermentation sugar growed up from 5g/L to 7.86g/L,and protease production reached 1211U/mL from 1010U/mL.FS140 protease was purified by using ammonium sulfate precipitation and column chromatography on Sephadex G-25,DEAE Sepharose Fast Flow and Sephadex G-75.The Specific activity of the enzyme was 29900 U/mg.Purification times and recovery rate of enzyme were 12.8 and 28.6%respectively.The molecular weight of the purified enzyme was estimated to be 40.0 kDa by SDS-PAGE.The kinetic behavior of this enzyme was performed using casein as a substrate. The optimum temperature was at 55℃and the enzyme was heat-stable under 50℃after 30min.The optimum pH was determined to be at pH 7.0 and the enzyme was stable in the range 7-11.The enzyme activity was consistented with typical Michaelis-Menten kinetics for the hydrolysis of casein,bovine haemoglobin and bovine serum albumin.The K_m and V_m,values were determined to be 0.5,1.05, 1.28g/L and 333.3,500,312.5μg/L/min,respectively.The result of dynamics showed that the casein was the optimum substrate.The effects of metal ions on protease were also tested.The results showed that 5 mmol/L Na⁺,K⁺ and Li⁺ ions had no any effects on the enzyme activity.3 mmol/L Mg²⁺,Mn²⁺,Ca²⁺and Ba²⁺ions had few effects on the enzyme activity,while Cu²⁺, Hg²⁺,Cd²⁺,Zn²⁺and Al³⁺inhibited the enzyme,the residual activity were 32.4%, 35.5%,43.5%,67.2%and 58.5%of CK.Fe²⁺and Fe³⁺strongly activated the enzyme activity,the residual activity reached 140.5%and 119.4%of CK.Effects of different organic solvents on the activity of proteinase were investigated.The inhibitory increased gradually along with increase of organic solvents concentration.IC50 value of methanol,1.2-propaneediaol,alcohol and isopropyl were 15.5%,14.5%,8.5%and 6.8%,respectively.The protease was modified by 10 different modifications.The results indicated that the histidineresidues and tryptophane residues seemed to be essential to catalytic activity or located at the active site of protease.After treatment with metal chelating agent-ethylenediaminetetraacetic acid disodium(EDTA),the enzyme activity completely lost,which indicated that this protease was metal enzyme.