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
•Fig.1 shows the most suitable III-3-A enzyme pH is around 9, as a typical alkaline cellulase.For study on the stability of III-3-A 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 III -3-A enzyme
he 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
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 III-3-A 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, III-3-A is excellent in heat resistance of alkaline cellulase, application in production show a good application prospect.
E.coli BL21 Star (DE3) transformation and functional verification
We transformed the recombinant plasmid pET-28a-III-3-a into E.coli BL21 Star(DE3), and cultured on the plate containing 25 g/mL kanamycin (Kan) resistance.
Fig.3 Halos produced by E.coli Ⅲ-3-A on CMC-containing solid plate
Results show that alkaline cellulase Ⅲ-3-A can form a large transparent circle in the containing CMC-Na plate by 3 days of culture. Alkaline cellulase Ⅲ-3-A has the property of high enzyme activity and stability.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Unknown
- 1000COMPATIBLE WITH RFC[1000]
| n/a | Alkaline Cellulase |