Figure 1.  Illustration of Waste Paper Degradation System.

Figure 2.  Gel electrophoresis of cex,cenA and cep94A . 

Figure 3.   Experimental Results of Bacterial Cellulose Production System.(a, testing of Exoglucanase Cex.b,testing of Endoglucanase CenA.c,d,testing of Cellulose Disaccharide Phosphorylase Cep94A.e,synergistic action of cellulose-degrading enzymes )
All experiments were repeated three times. Data are expressed as mean ± standard deviation. Differences were analyzed using t test, P values < 0.05 was considered statisticall

As shown in Figures A, B, and C, three plasmids, pET23b-cex, pET23b-cenA, and pT7-cep94A, were constructed and transformed into Escherichia coli. The corresponding enzyme levels were tested, and it was observed that compared to the control group, there was a significant increase in enzyme levels.As shown in the figure 3D, E. coli Rosetta carrying an empty vector, the control group had enzymatic activities of 13.83 U/mg, and the genetic engineered E.coli Rosetta, whose plasmid is recombinant with cep94A gene had enzymatic activities of 255.20 U/mg. The results demonstrate that the enzyme activity of cellulose diphosphorylation enzyme expressed by the cep94A gene is significantly enhanced.Under the same condition, we set up several group to let the engineered E.coli to express the enzyme under different temperature. Quantitatively, our results in  figure 3Eshow that At 50℃, the cellobiose phosphorylase of engineered bacteria is about 255.2 U/mg. By changing the ambient temperature, it is shown that 37℃ is the best reaction temperature, which has the cellobiose phosphorylase about 446.99 U/mg. The results indicate that the enzyme activity of the engineered bacteria's cellulose diphosphorylation enzyme is approximately 255.2 U/mg at 50℃. By altering the environmental temperature, it was found that 37℃ is the optimal reaction temperature for the enzyme.

We expressed the bacterial cellulose synthase with E.coli Rosetta. We broke them with the ultrasonic wave. Then, we added NaOH into the to dissolves the non-cellulosic components and precipitates the cellulose. After removing the remaining NaOH, we weighed the dried cellulose to determine the cellulose content.

Figure 4.  Design of the acsAB.

Figure 5.  Gel electrophoresis of acsAB . 

Figure 6. Cellulose synthesis capacity test of AcsAB.
As the picture shown, in bacterial precipitation samples, engineered E. coli expressing AcsAB produced about 1.71g/L bacterial cellulose in LB medium. But in the culture medium samples, there was almost no detectable presence of cellulose. The results indicate that our acsAB gene's ability to express bacterial cellulose synthase is significantly improved.

1. Sekar, Ramanan, Hyun-Dong Shin, and Rachel Chen. "Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism." Applied and environmental microbiology 78.5 (2012): 1611-1614.
2. Lakhundi, Sahreena Saleem. "Synthetic biology approach to cellulose degradation." (2012).
3. Wood, Thomas M., and K. Mahalingeshwara Bhat. "Methods for measuring cellulase activities." Methods in enzymology. Vol. 160. Academic Press, 1988. 87-112.