cep94A

This is the so called cellobiose phosphorylase (Cep94A), which phosphorylates the cellobiose β-linkage resulting in its breakdown to D-glucose and α-D-glucose-1-phosphate. It is used in the following biobrick BBa_K2449004 For more info visit https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294459/

Squirrel-Beijing-I 2023

Description

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 one can discompose the plant cellulose into glucose.

Usage and Biology

We synthesized the cep94A gene and inserted it into the pET23b vector. The recombinant plasmid was then introduced into Escherichia coli Rosetta for the production of cellobiose-phosphorylase for plant cellulose decomposition.

Figure 1 Design of the cep94A.

Characterization

We expressed the cellobiose-phosphorylase with the E.coli Rosetta. To determine the activity of cellobiose phosphorylase, we measured the production of the glucose. We constructed a 100 μL reaction system that include 1 mM cellobiose, 10 μL enzyme solution, and 20 mM citrate buffer (pH = 4.8). We used the GOD-POD assay kit (Yuanye, China) to measure the glucose content in this system. We defined enzymatic activity as the micromoles of glucose produced per minute, and E. coli Rosetta carrying an empty vector was used as a control. We used a glucose standard curve to calculate the amount of glucose produced in the reaction based on the absorbance values.

Figure 2 Gel electrophoresis of cep94A.

Figure 3 Experimental Results on cep94A.

As shown in the figure 3A, 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 3B show 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.

Potential application directions

This experiment demonstrates that artificially synthesized cep94A can express cellulose phosphorylase, which could be applied in waste paper decomposition to provide a food source for engineering bacteria, solving environmental problems, and having promising development prospects.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 768
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 846
Illegal NgoMIV site found at 1942
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 108

ECUST_China 2019 characterization

Usage and Biology

Cellobiose phosphorylase is an enzyme that catalyzes the chemical reaction cellobiose + phosphatealpha-D-glucose 1-phosphate + D-glucose. In ECUST_China 2019 characterization, we obtained the sequence of cellobiose phosphorylase from Part:BBa_K2449003. Unfortunately, this part was not contained in iGEM distribution kit, so we synthesized this gene (cep 94A) from Genewiz.

Characterization

In order to characterize the function of Cep94A specifically, we cloned the cep94A into p15a. Two aspects of characterization were condidered: the expression of Cep94A protein and the enzyme property.

Cep94A and the negative control (p15a) grew in 5mL LB (containing 0.1% Chl) at 37℃ for 12 hr and then was transferred to the 100 mL LB for enlarge culture. After almost 16 hours in 100mL LB, the Cep94A and p15a was collected to perform the SDS-PAGE. The result showed that Cep94A was actually expressed (about 91kDa) after induction.

Figure 1. SDS-PAGE results of Cep94A (about 91kDa)

After verifying the expression of Cep94A, we decided to measure the Cep94A enzyme activity and evaluate the enzyme property to perform the quantitative experimental characterization. After induction for 16 hours, the cells were collected and resuspended in 10 ml of PBS buffer, afterwards sonicated to obtain 10 ml supernatant. The enzyme activity of Cep94A was measured by the DNS method: 2 μL cellobiose +1 μL enzyme solution + 17 μL citrate buffer (pH =4.8) was reacted at 50 ° C for 30 min. Then 30 μL DNS reagent was added, boiling for 10 min. The reacton mixture was diluted 5 times and transferred to 96-well microplate to measure the OD540 with a microplate reader.

Figure 2. The DNS assay of Cep94A

Enzyme activity = moles of substrate converted per unit time. So we could measure the activity of Cep 94A by monitoring the rate at which a product (glucose) formed. Firstly, we drew the glucose standard curve: y = 0.6746x - 0.0491，R² = 0.9775 (y represents OD540, x represents glucose concentration), then the enzyme activity was calculated based on the glucose standard curve. Pluging OD540=0.093 into y, we worked out that x (glucose concentration) = 0.211mg/ml. According to the formula: Cep94A enzyme activity (U/mL) =enzyme dilution factor×moles of glucose /reaction time, we calculated that the enzyme activity of Cep94A was 0.781 U/ml.

Figure 3. Glucose standard curve

In addition, we also evaluated the Cep94A enzyme property at different temperatures and pHs.

Table 1. The absorbance(540nm) at different temperatures and pHs

temperatures	19℃	28℃	37℃	46℃
pH 6.0	0.351	0.473	0.483	0.475
pH 7.0	0.452	0.583	0.456	0.471

Figure 4. The enzyme activity at different temperatures and pHs

Reference

[1]Sekar R, Shin HD, Chen R. Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism. Appl Environ Microbiol. 2012;78(5):1611–1614. doi:10.1128/AEM.06693-11