Project long-term planning

Expression and Reaction of endocellulase & exocellulase

Genes of endocellulase and exocellulase expressed conducted by λ promoter at first.Without inhibition for λ promoter,genes of endocellulase and exocellulase transcribed and translated as E.coli strain grew.So we aimed to incubate E.coli at liquid medium and predicted the concentration of bacteria and activity of enzymes with Logistic equation and Luedeking-Piret equation.As a result,we assumed the relationship between concentration of E.coli and enzymes was semi-relative and predicted how much we could get enzymes.

Then,with results of activity of enzymes and concentration of cellulose,we had used Langmuir adsorption equation and modeled Michaelis-Menten equation to predict concentration of cellobiose.Langmuir adsorption equation was commonly used for enzyme adsorption to solid substrate.Especially,for cellulose,just when enzymes adsorbed long chains of cellulose they could work.Michaelis-Menten equation was a classical equation for enzyme reaction,but because of inactivity in process of enzyme adsorption,it was modified to match the actual enzyme reaction rate.To calculate the concentration of cellobiose,we applied the empirical second exponential decay equation which could predict the maximum concentration of cellobiose under different concentrations of cellulose.We found the most suitable concentration of cellulose was 20g/L and time of fermentation was 24hours

Regulation model

For the former was hydrolysis reaction and the latter was synthesis reaction,we considered the difference of the enzymes under different pH and temperature to avoid the conflict which cellulase catalyzed the bacteria cellulose.So we comprehensively considered the effect of pH and temperature to cellulase and bacterial cellulose synthase to ensure suitable fermentation condition.The best condition for bacterial cellulose synthase was that pH is 6.0 and temperature was 28℃,but for E.coli growth and cellulase synthesis,we set 37℃ and pH 7.0.After changing pH and temperature,the enzyme activity of cellulase was down to 15% and 10% of original activity of cenA and cex.

When Lac promoter worked,the gene of CI expressed CI protein as inhibitor for λ promoter.So we aimed to know about effect of the concentration of IPTG to λ promoter.To realize the close of λ promoter and latter enzymes expression,we had to clear what concentration was suitable for our experiment.

Reaction of Cellobiose Phosphorylase and Bacterial Cellulose Synthesis

When cellobiose in fermentation liquor entered E.coli,with expression of Cellobiose Phosphorylase and Bacterial Cellulose Synthase,the strain began to produce bacterial cellulose utilizing cellobiose.So,we analyzed the metabolic pathways from glucose to UDPG and from UDPG to bacteria cellulose,predicting the varieties of the concentration of cellobiose and bacteria cellulose.

Bio-manufacturing by fermentation

how we make paper transformer work and maximize our profit by recycling and reuse waste paper,so we build a factory to produce bacteria cellulose according to recent statue of waste paper utilization.

By investigation, we find pretreated waste-paper pulp contains 65% cellulose and its pH is 2~3 and after adjustment,it can act as substrate for fermentation.So we design a set of 500L fermentor as a factory model to produce bacteria cellulose.We use waste-paper pulp and industrial molasses as substrate for E.coli to produce and in the early fermentation we use airlift fermentor to keep strain grow and maintain suitable level of cellulase concentration for catalyzing cellulose to cellobiose.When the concentration of cellobiose does not change,we change fermentation liquor condition and control the temperature and pH to open later-stage fermentation switch by adding lactose as a inducer to synthesize BC.

But how to ensure the concentration of cellulose and get maxium quality of cellobiose,we build logistic model for strain growth,cellulase enzymatic reaction equation model and the mathematical models for transformation from cellulose to cellobiose.Finally,we consider the concentration of cellulose is 20g/L and can get 6g/L cellobiose after 24-hr fermentation under 37℃ in the first fermentation.Then,we analyze the metabolic pathway of UDPG which transfers about 5% cellobiose and other sugar to BC and will get 1.3g/L cellobiose in the end.To avoid to destroy the length and diameter of BC,we decline the ventilation in the airlift fermentor for latter process.

We analyze the expense of all materials and items to evaluate economic benefits,if we build a factory and have such a produce process in 200 m3 fermentor ,we can make a profit 8062780 dollars per year,decrease waste water 412200 ton ,reduce waste paper pulp 2061 ton,so environmentally friendly and economical!