Part:BBa_K5002002
Cellulose degrading enzyme,cep94A+cex+cenA
Considering the fact that the discharge of printing and dyeing wastewater has a very bad impact on the environment, we wanted to reduce the content of harmful substances in printing and dyeing wastewater to the standard of discharge. Therefore, we decided construct a cellulose synthesis operon and a group of plant cellulose discomposing genes. They are derived from artificial synthesis, one can synthesize bacterial cellulose, and another can discompose the plant cellulose into glucose.
The cellulose production operon in Acetobacter xylinum consists of a 10 kb genomic region composed of four major elements: acsAB, known as cellulose synthase, comprises two domains: acsA, which is the catalytic domain, and acsB, which is the cyclic-di-GMP binding domain. This modular enzyme requires a second messenger, cyclic-di-GMP, for efficient catalysis. acsC encodes for an embedded outer membrane protein that facilitates the transport of oligomers across the cell membrane, allowing the polymer to be extruded into the external environment. acsD is considered a non-essential element, even though it plays a crucial role in controlling cellulose crystallinity and is necessary for achieving maximum cellulose production rates (Lee et al., 2014).
Usage and Biology
We synthesized cex and cenA genes and inserted them into pET23b vector. The constructed plasmid was then transferred into Rosetta E. coli for exoglucanase production, which is used for plant cellulose decomposition.Similarly, we synthesized the cep94A gene and inserted it into pET23b vector. The constructed plasmid was also introduced into Rosetta E. coli to produce cellobiose-phosphorylase for plant cellulose degradation.
Figure 1 Design of the cenA and cex.
Figure 2 Design of the cep94A.
Characterization
To investigate whether the combination of crude enzyme extracts from cex, cenA, and cep94A could enhance glucose production, we did enzymatic hydrolysis experiment. We first prepared crude enzyme extracts of cex, cenA, and cep94A. The reaction conducted in a 100 μL reaction mixture containing 1% (w/v) carboxymethyl cellulose (CMC), 50 mM citrate buffer (pH 4.8), and 10 μL of crude enzyme extract. We have set four experimental group (cex alone, cenA alone, cep94A alone or a combination of Cex, CenA, and Cep94A) and one control without enzymes. After incubating the mixture at 50°C for 30 minutes, we used DNS method to quantify the glucose production. We expressed enzyme activity as the amount of glucose produced per minute. Lastly, we used a standard curve of glucose to calculate the reducing sugars concentration in the reaction mixtures.
Figure 3 Gel electrophoresis of cex,cenA and cep94A .
Figure 4 （A） Synergistic action of cellulolytic enzymes (B) Plasmind map.
As the column diagram shown in figure 4, the combination of Cex, CenA, and Cep94A group have produced the largest amount of glucose. Quantitatively, it produced 6.20 U/mg per minute. Due to the striking contrast with other experimental groups and control group, we can conclude that the combination of crude enzyme extracts from cex, cenA, and cep94A could enhance glucose production.
Potential application directions
This experiment demonstrates that the co-expression of cep94A with cex and cenA genes can enhance glucose production. In the future, it could make paper decomposition more efficient and provide technical support for the development of paper waste treatment products, showing promising prospects for development.
References
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.Lee, Koon‐Yang, et al. "More than meets the eye in bacterial cellulose: biosynthesis, bioprocessing, and applications in advanced fiber composites." Macromolecular bioscience 14.1 (2014): 10-32.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 2966
Illegal NotI site found at 3943
Illegal NotI site found at 5087 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 768
Illegal BglII site found at 5000
Illegal BamHI site found at 4136
Illegal XhoI site found at 4498
Illegal XhoI site found at 4747 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 846
Illegal NgoMIV site found at 1942
Illegal NgoMIV site found at 2599
Illegal NgoMIV site found at 2972
Illegal NgoMIV site found at 3474
Illegal NgoMIV site found at 4278
Illegal NgoMIV site found at 5203 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 108
Illegal BsaI site found at 4182
Illegal BsaI.rc site found at 3019
Illegal SapI.rc site found at 3102
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