| As a broad-spectrum fungicide,chlorothalonil(2,4,5,6-tetrachloroisophthalonitrile,TPN)is widely used over the last 30 years and has become one of the most widely used agricultural fungicides in the world.TPN is very stable in the environment,and it has a long half-life and a significant cumulative toxicity.Thus,owing to its extensive use and difficulty in degradation,TPN could be easily detected from greenhouse gases,vegetables and fruits,which adversely affected human health and also caused food safety issues.TPN can be degraded by biological,photochemical,ozonic and water treatments.Among all these methods,biodegradation is considered to be an effective and reliable one without secondary pollution.In our previous study,a novel TPN hydrolytic dehalogenase(Chd)from Pseudomonas sp.CTN-3 has been identified in our lab.However,Chd is an intracellular enzyme,which makes difficulty in the following massive recovery and purification of the enzyme product.Thus,extracellular production of Chd using recombinant strain is an effective solution.It is a promising way to use Chd to remove TPN residues from soil,water,vegetables and fruits because the metabolite 4-hydroxy-trichloroisophthalonitrile(4-TPN-OH)is less toxic and more soluble in waterBacillus species have been among the most important industrial enzyme producers for their variety advantages,including the capacity of producing and secreting large quantities of extracellular enzymes and the microbial safety.Bacillus subtilis WB800 is a strain deficient in eight proteases and therefore can serve as an excellent host for the expression and secretion of foreign proteins.In this study,the gene chd was cloned into the vector pP43NMK under the control of the P43 promoter and the nprB signal peptide-encoding sequence in the protease-deficient Bacillus subtilis WB800.Therefore,engineering Bacillus subtilis for extracellular expression of Chd could be used to solve the problems of subsequent separation and purification.Moreover,the optimization strategy for submerged fermentation(SmF)and solid state fermentation(SSF)were also used to increase the yield of enzyme production after genetic modification.A 2.13 fold of initial enzyme activity,i.e.14.50 U/l broth was achieved by optimization of Chd production in submerged culture using the combined methodologies of Plackett-Burman and Box-Behnken designs compared to the original non-optimized conditions.The optimal conditions fermented by B.subtilis WB800(pP43Chd)were as follows:pH7.0,2.5%tryptone,3.12%yeast extract,5%glucose,0.3%K2HPO4,0.3%NaCl,0.3%MgSO4,and an inoculum amount of 10%at 37℃,180 rpm,24 h,meanwhile after 12 hours of fermentation 20 mg/1 of TNP was added to the medium.Optimization of important physical parameters for Chd production by solid state fermentation offered enhanced activity from 22.42 U/kg to 85.60 U/kg substrate,i.e.3.82 fold of the initial enzyme activity.The optimal conditions fermented by B.subtilis WB800(pP43Chd)were as follows;2 g bran,1g rice hull and 2 g wheat straw powder placed in a 200 ml tissue culture bottle,1 ml salt solution(K2HPO4 0.4 g/1 and MgSO4 0.2 g/1),initial water content 60%,20%inoculum level,with the important factors of stacking height 3.3 cm,particle mesh 5,stacking density 0.047 g dry substrate weight/cm3,porosity 84.86%,incubated at 37℃ for 36 h.Chd is proved to have great potential in the cleaning up fruits and vegetables which are contaminated with TPN.TPN degradation assay was performed to testify the degradation ability of TPN on vegetables by the crude enzyme Chd.The concentration of TPN was gradually reduced due to the degradation of Chd.After enzymatic reaction carried out for 120 min,almost all the TPN residues in tomatoes which contained 25 mg/kg TPN were degraded,about 82%TPN could be degraded in asparagus lettuce which contained 45 mg/kg TPN,in lettuce which contained 29 mg/kg TPN,the TPN degradation rate could reach about 67%and the TPN degradation rate was between 40%-55%in the other three kinds of vegetables.No significant decrease in the concentration of TPN in the control test.At the same time,TPN eluted from vegetables could be degraded to<1 mg/l by the crude enzyme Chd after enzymatic reaction carried out for 3 h.The enzymatic reaction after 2 hours,residual Chd activity was maintained between 76%-86%of the initial Chd activity.Therefore,the decrease of TPN was resulted from the existence of crude enzyme Chd,showing that Chd has great potentials in removing TPN residues in vegetables. |
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