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/

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 3.906 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