Part:BBa_K1350003

Alkaline Cellulase

Alkaline cellulase gene from bacillus subtilis saline lake monoclonal, it can survive in high alkalinity and high heat environment, and keep good enzyme activity.Therefore, in the industrial production, such as in the production of paper making, washing and so on, the alkaline cellulose enzyme can have higher, more extensive application range, and can greatly reduce the energy consumption and production cost.

The optimum reaction pH and pH stability

Take proper amount of the pure enzyme, adding 1% CMC-Na solution of different pH value, according to the conventional method of measuring the enzyme activity, as shown in figure 1.

Fig.1 Effect of pH on the activity and stability ■ optimum pH value; ◇ pH value stability

•Fig.1 shows the most suitable Alkaline cellulase pH is around 9, as a typical alkaline cellulase.For study on the stability of Alkaline cellulase pH, respectively save the pure enzyme solution under different predetermined pH for 30min, then according to the conventional method determine the enzyme activity under pH9.The results showed that, the enzyme in the pH 6-11 range,the enzyme activity can be maintained above 80%.Visible on the alkaline cellulase in weak acidic and alkaline conditions with strong stability.

Research on temperature stability of Alkaline cellulase

The pure enzyme liquid are respectively arranged in the containing 5mM CaCl2 and CaCl2 free system, were placed in different temperature conditions (3℃-80℃) insulation 10min, then determined the enzyme activity of 40℃ to the reaction of 20min (Figure 2).

Fig.2 Effect of Temperature on the stability

■ without CaCl2; ◇ containing 5mM CaCl2. 30 ℃ enzyme activity was 100%

The results showed that, the enzyme 50℃ has better stability, temperature more than 50 ℃ is rapidly inactivated. While the addition of Ca2+ can significantly improve the thermal stability of Alkaline cellulase at 50℃-80℃, which is at 55℃ still maintained a high stability and there are still a small number of enzyme activity at 70℃. This indicates that the enzyme protein,the enzyme is excellent in heat resistance of alkaline cellulase, application in production show a good application prospect.

E.coli BL21 Star transformation and functional verification

We transformed the recombinant plasmid pET-28a into E.coli BL21 Star, and cultured on the plate containing 25 g/mL kanamycin (Kan) resistance.

Fig.3 Halos produced by E.coli BL21 on CMC-containing solid plate

Results show that alkaline cellulase can form a large transparent circle in the containing CMC-Na plate by 3 days of culture. Alkaline cellulase has the property of high enzyme activity and stability.

Detect deinking performance of Alkaline cellulase

We put the A4 small paper written words in the control bacteria liquid and the fermentation liquid with Alkaline cellulase, to judge by doing so if there is obvious effect of Alkaline cellulase to deink, so it can be applied to actual production.

In contrast to the picture in the control bacteria liquid, the picture in fermentation liquid with Alkaline cellulase has a strong degradation.So the pictures tell us Alkaline cellulase studied by SZU-iGEM has a good deinking ability,and it will have a good application prospect.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]