Team:SZU-China/Project/AlkalineCellulase

BACKGROUND

      Cellulase refers to a group of enzymatic degradation of cellulose to form glucose. According to the optimum pH of valuecatalytic reaction, cellulase can be divided into acidic cellulase ( the optimal pH value is 3 ~ 5 ), neutral cellulase(the optimal pH value is 6 ~ 8) and alkaline cellulase (the optimal pH value is 8 ~ 10).

      Acidic cellulase has developed the earliest, applied widely at present. However, with the industrial application scope expanding, acidic cellulase revealed many problems. Such as in alkaline conditions, the enzyme activity is relatively low or no activity, poor stability, pH value in narrow scope, and under acidic conditions.Processing textile will produce the phenomenon of fading and so on, which greatly limits the application of cellulase in textile and detergent industry. Compared with the acidic cellulase, alkaline cellulase has to adapt to a wide range of pH value, good stability, and can withstand high temperature. As the advantages of detergent enzymes can make the clothes after being washed for many times feeling, keep bright appearance, not easy to cause textile fading.In the textile and detergent industry, alkaline cellulase has a wide application prospect.

       However, the current domestic research and application are more concentrated in the acidic cellulase.For the study of alkaline cellulase, because of many reasons of the enzyme low producing ability and the low specific activity, it has not yet reached the level of industrialization of production. Therefore,it is of great significance for seeking heat resistance, alkali resistance and specific activity of high quality alkaline cellulase. The SZU-iGEM team through the early study of alkali Bacillus III 23 screened from Inner Mongolia alkali Lake,which can produce enzyme up to 60 U /ml. On the basis of this, the SZU-iGEM team will transformed Escherichia Coli into a high yield, alkali resistance, can secrete alkaline cellulase super machine. This transformation will have a very important role in the sustainable development of resources and environmental protection.

1.Study on enzymatic properties of Alkaline cellulase

   1.1 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.

1.2 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 3). 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, alkaline cellulase is excellent in heat resistance of alkaline cellulase, application in production show a good application prospect.

Fig.2 Effect of Temperature on the stability

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

2. Expression of alkaline endoglucanase gene in Escherichia coli

      Study on enzymatic properties of Bacillus sp. alkaline cellulase bacteria endoglucanase the discovery of alkaline cellulase, the enzyme has to adapt to a wide range of pH, heat resistance, alkali resistance, enzymatic properties of metal ions and surfactants is excellent, especially suitable for the detergent and textile industries. However, Bacillus sp. alkaline cellulase bacteria enzyme production is not stable, failed to meet the requirements of industrial application. Therefore, we will alkaline endoglucanase alkaline cellulase gene Bacillus sp. alkaline cellulase (not including the signal peptide coding sequence) into Escherichia coli expression system and expressed. The experimental results show that, the method by adding surfactants to increase cell membrane permeability, can realize the expression of alkaline endoglucanase gene alkaline cellulase in Escherichia coli expression system of extracellular.

2.1Construction of recombinant Escherichia coli

2.1.1 Obtaining the target gene alkaline cellulase

      From the Bacillus sp. bacteria in 84 h medium, using the EZ-10 spin column Genomic DNA Minipreps Kit (For Beteria) kit to extract genomic total DNA, and alkaline cellulase gene was amplified by PCR method, as shown in figure 3. The expression vector pET-28a (+) of Escherichia coli can make the objective protein periplasmic expression, so in the PCR primers were designed according to the coding sequence of alkaline cellulase mature enzyme protein design. Can be seen from Figure 3, the PCR amplified product size was 1907bp, andalkaline cellulase gene size does not contain the signal peptide coding sequence match. The amplified product was connected to pMD18-T vector for sequence, showed that the amplified sequence is consistent with the corresponding sequence.

Fig.3 Alkaline cellulase mature enzyme sequence pcr

2.1.2 Construction of PET-28a (+) expression vector

      Escherichia coli expression vector pET-28a (+) containing Kan+ resistance marker, vector map and multiple cloning sites are shown in figure 4A and figure 4B.

      alkaline cellulase gene without signal peptide coding sequence by Hind I and EcoR I enzyme digestion and by the same enzyme digestion Escherichia coli expression vector pET-28a (+) connection, the recombinant plasmid pET-28a.

figure 4A. PET-28a (+) expression vector map

                                                                              figure4B. PET-28a (+) expression vector multiple cloning site

2.1.3Verification of recombinant gene

      Pick the single colonies , and then place in the containing kan resistance LB liquid culture medium, 220rpm, 37 degrees overnight culture.The next day extracting bacteria plasmid do PCR verification. The verification results shown in the figure 5, showed that the alkaline cellulase gene was successfully recombination.

The ups primer EcoRⅠ： 5’-GATgaattcGAAGGAAACACTCGTGAAGAC-3’

The down primer Hind Ⅲ： 5’-GTTaagcttTTATTTTTTCGTAGCCTCTTTC-3’

Fig.5 The PCR verification of CMCase gene after recombinant plasmid was transformed

2.2 E.coli BL21 Star (DE3) transformation and functional verification

2.2.1 Determine the activity of Alkaline cellulase by plate method

We transformed the recombinant plasmid pET-28a into E.EcolI BL21 Star(DE3), and cultured on the plate containing 25 g/ml kanamycin (Kan) resistance.

Fig.6 Halos produced by E.coli 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.

2.2.2 Determine the activity of Alkaline cellulase by DNS method

After transforming the recombinant plasmid pET-28a into E.EcolI BL21 Star,we placed it in 23℃, cultured for 48 hours and finally used the CMC-Na DNS method for detection of Alkaline cellulase activity.

To measure the ability of kil to exogenous protein secretion of Alkaline cellulase，we determine the activity of Alkaline cellulase by DNS colorimetric method.

As shown in the figure, the more deeper color represents the more amount of Alkaline cellulase secreting out of the Escherichia EcolI , which can express the secretion ability of kil is strong.

Fig.7 Using CMC-Na DNS method for detection of Alkaline Cellulase
Lane 1.The activity of Alkaline cellulase in cell supernatant of top10
Lane 2.The activity of Alkaline cellulase in cells of top10.
Lane 3.The activity of Alkaline cellulase in cell supernatant of BL21
Lane 4.The activity of Alkaline cellulase in cells of BL21
Lane 5.The activity of Alkaline cellulase in cell supernatant of DH5α
Lane 6.The activity of Alkaline cellulase in cells of DH5α

2.3The effect of Induction temperature on enzyme expression

The recombinant strain E.EcolI were inoculated into LB liquid medium for OD600 value bacteria liquid reaches 0.6,added at a final concentration 1mmol/L of inducer IPTG and placed in different temperature (23℃, 30℃, 37 ℃ ) induction training.Measured enzyme activity after disruption of the cells by timing sampling and repeating freeze-thaw method with liquid nitrogen.The results show in figure 7. From figure 7 visible, the enzyme activity of culturing at 23℃ and 30℃ were higher than the corresponding value at 37℃, and the induction time was reached the peak at 12h. At the same time, found that the enzyme activity decreased significantly at 37 ℃ after 9 hours. This is main that the enzyme expression speed is slow at low temperature, which is not easy to form inclusion bodies.At high temperature, the enzyme expression of the speed is too fast, the enzyme protein is not enough time to fold the mature and active enzyme protein , which is easy to form inactive inclusion body. Considering the induced temperature is too low will affect cell growth, therefore, the choice of induction temperature 23℃ to 30℃ is appropriate.

Fig.8 Effect of inducing temperature on the expression of endoglucanase

2.4 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.