Study Of Key Enzymes In Bacillus TSH1

Recent years,energy crisis and environmental problems has become an urgent global issue,it improve the development of new renewable energy sources which has great potential to replace traditional fossil resources.As can be mixed with gasoline and used for clean energy,has higher energy density,less corrosive than ethanol,butanol has become hot for scholars from various countries.Currently,selection of Clostridium acetobutylicum strain and improvements of fermentation process has made great progress.Butanol-forming strains are commonly obligative anaerobic,which has very strict requirements about equipment.Bacillus TSH1(B.TSH1)is a new butanol-producing microorganism isolated in our laboratory.B.TSH1 can grow and ferment under facultative anaerobic conditions,while sharing similar fermentation pathways and products with Clostridium acetobutylicum.To illustrate the relationship between the products and the enzyme activity in B.TSH1,improve the butonal.concentration and yield,key butanol-and ethanol-forming enzymes were assayed.Under different aeration conditions,fermentation results of B.TSH1 were contrast.During acid-produce period,glucose is mainly used for the generation of cell growth and acid production,in solvent-produce period,glucose is mainly used for the generation of solvent,at the same stage,acetate and butyrate were re-used.VWhlie initial glucose concentration is 36.33 g/L,solvent is generated in 12 h,fermentation terminated at 36 h,ratio of acetone,butanol and ethanol is A:B:E= 2:7:1.Final concentration of Butanol is 7.13 g/L,yield is 19.6%,accounting for 70%of the total ABE.Secondly,key butanol-and ethanol-forming enzymes were assayed,the activity of alcohol dehydrogenase reached a maximum of 0.054 U/mg at 12 h;the enzymes activity of butyraldehyde dehydrogenase and acetyl coenzyme A reached a maximum of 0.01 U/mg and 11900 U/mg both at 21 h;butanol dehydrogenase reached a maximum of 0.016 U/mg at 15 h;the enzymes activity of acetoacetate decarboxylase reached a maximum of 7.46 U/mg at the end of the fermentation.Compared with fermentation in ventilation(0.05 vvm),key enzymes activity are lower than in aerobic conditions,and peaked earlier(acetoacetate decarboxylase opposite to be).In semi-quantitative conditions,key enzymes in different fermentation period of B.TSH1 were researched at the RNA level.Enzymes such as ct,hbd and crt both has a constant expression in whole fermentation;expression of adc is very weak in the acid-producing period,while had little improve in solvent-producing period;ak participate in the metabolic pathway early in the acid-producing period;between the metabolism from butyrale coenzyme A to butyrate,bk played a major role;the three kinds of adh,bdh and two kinds of thl all expressed,among them,adh1,bdh had a relatively more and substantially stable expression as well as thlp,and played a relatively major role in the entire fermentation process.When butanol concentration reached more than 4 g/L,and production rate began to increase,the heat shock protein inhibitors became to play.Finally,analysed the proteomic sample in different fermentation stage,in acid-producing period and solvent-producing period,24 point of protein concentration differences in more than three times,in which 20 raised in acid-producing period,4 raised in solvent-producing period.