Introduction: Chitosan and chitosan oligosaccharides have aroused people's research interest in recent years because of their excellent biological activity. Chitosan can be obtained by further hydrolysis to obtain better water solubility, easier to use and better biologically active chitosan oligosaccharide. At present, the methods of chitin deacetyl are mainly enzymatic and chemical. The main ways to obtain chitosan oligosaccharides are enzymatic, chemical and physical. The shortcomings of these methods are low efficiency, pollution and extremely uncontrollable of the amount of product. In our study proposed a new method, that is, the use of E. coli cell surface display chitosan enzyme (CHI-1) to hydrolyze chitosan, the acquisition of chitosan oligosaccharides in the experimental process and after the experiment. We carried out a series of verification, to ensure that the experiment is successful, while ensuring that the chitosanase produced by the cell surface display technology is able to use, high efficiency and stability.
To verify that the newly synthesized genome contains the target gene (chitosanase gene).
In this experiment, the selected Bacillus cereus and Bacillus thuringiensis were used as chitosanase gene source strains. The gene sequences of the above genes were obtained from the NCBI database, and then the target gene was synthesized by direct DNA synthesis between the chitosanase gene and the N-terminal gene of ice crystal nuclear protein. PCR was used to determine whether a band was amplified.
Found the amplification of the band, that is, the new synthetic gene contains the target gene.
To verify the presence of the chitosanase gene in cultured Escherichia coli and to determine whether the chitosanase gene can be effectively expressed on the surface of Escherichia coli cells.
During the experiment of cell surface display technology, while cultivating DH5alpha, a small amount of BL21 is cultured simultaneously. Fluorescent proteins are inserted into the plasmids and examined under a microscope for luminescence. (Figure 1-A)
(1-A)
The fluorescence was observed successfully by microscope, which proved that the chitosanase gene was contained in E. coli, and the chitosanase gene could be expressed on the surface of E. coli cells.
Test the activity of chitosanase which is expressed by the cell surface display technique.
To determine the viability of the cell surface display technique, we must ensure that the expressed chitosanase is sufficiently active. we chose to use the DNS method to test the activity of the chitosanase. By using this method, we obtained the activity of chitosanase and compared the activity of chitosanase exhibited on the cell surface with that of crude chitosanase. The effects of temperature, PH value, and heavy metal ions on chitosanase activity were compared by this method. The relative enzyme activities of E. coli BL21-PET23b (+) -NICHI engineering bacteria suspension and cultured Bacillus thuringiensis supernatant were measured within 40 days. The results showed that E. coli BL21-PET23B (+) -NICHI recombinant could hydrolyses water-soluble chitosan effectively. Compared with water-soluble chitosan, the hydrolysis efficiency of colloidal chitosan was 76.3%. It was also verified that E. coli BL21-PET23B (+) control group could not degrade colloidal chitosan to produce reduced chitosan oligosaccharide. Furthermore, it determined that coli BL21-PET23B (+) -NICHI could not hydrolyze microcrystalline cellulose, xylan, carboxymethyl chitosan and carboxymethyl cellulose.
(2-A)
The relative hydrolysis efficiency of different substrates is hydrolyzed by E. coli BL21-pET23b(+) NICHI(2-A). Control presents the hydrolysis of water-soluble chitosan by E. coli BL21-pET23b(+).
The effects of different heavy metal ions on the enzyme activities of E. coli BL21-PET23B (+) -NICHI and CHI-1 crude enzyme solutions under the same heavy metal ion conditions（MnSO4•H2O、ZnSO4•7 H2O、 FeSO4•7 H2O、Mg SO4•7 H2O、CuSO4•5 H2O、FeCl3、CaCl2）were measured and compared. In addition, we found that the activity of E. coli BL21-PET23B (+) -NicHI was higher than that of crude enzyme solution.
(2-B)
Effect of several metal ions on activity of E. coli BL21-pET23b(+)-NICHI and crude CHI-1(2-B)
Under the same pH value conditions (3, 4, 5, 6, 7, 8, 9), E. coli BL21-pET23b(+)-NICHI showed higher enzymatic activity than crude enzyme solution. At the same time, it is concluded that the most suitable pH value for the performance enzyme and the crude enzyme solution is the same.
(2-C)
Finally, the enzymatic activity E. coli BL21-pET23b(+)-NICHI and CHI-1 crude enzyme solution at different temperatures were determined. Found at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, the enzyme activity of E. coli BL21-pET23b(+)-NICHI was higher than that of crude enzyme solution under the same temperature conditions. Moreover, the E. coli BL21-pET23b(+)-NICHI and crude enzyme solutions are measured at the same optimal temperature.
(2-D)
Effect of different temperatures and pH on the activity of E. coli BL21-pET23b(+)-NICHI and crude CHI-1(C, D)
By comparing the relative hydrolysis efficiency of enzymes and crude enzymes for the hydrolysis of different substrates, the effects of several metal ions, temperature, and pH values on enzyme activity. We found that the performance enzymes performed higher in the above tests. At the same time, the optimal pH value and temperature of the performance enzyme can be consistent with the crude enzyme. It was demonstrated that chitosanase expressed by the cell surface display technique was sufficiently active to be able to work.
Detect whether chitosanase expressed by cell surface technology is sufficiently stable and determine its hydrolysis capacity for chitosan.
We placed the crude chitosan solution and engineered bacteria at room temperature for 40 days, and took the PBS suspension and the crude chitosanase hydrolyzed substrate every 5 days respectively. As can be seen in Figure (3-A) at day 0, day 5, day 10, day 15, day 20, day 25, day 30, day 35, day 40 time points, engineering bacteria maintain 80% of the initial enzyme activity even after 40 days at room temperature, however, the crude chitosan is placed at room temperature for 40 days, nearly maintained 31% of the initial enzyme activity.
(3-A)
Relative enzyme activity of crude CHI-1 and E. coli BL21-pET23b(+)-NICHI in 40 days (3-A)
The amount of chitosan produced by engineering bacteria and crude chitosan enzymes and substrate hydrolysis was further compared. As shown in Figure (3-B), within 1-7 days, the hydrolysis capacity of engineering bacteria maintains a good stability and can interact with the substrate to hydrolyze the substrate, at day 7, its hydrolysis efficiency reached 41%, but the crude enzyme solution is almost inactivated after a day of interaction with the substrate, cannot continue to hydrolyze the substrate. At day 2, the crude enzyme solution is completely inactivated, and within 2 to 7 days, the preparation rate of chitosan oligosaccharides remains unchanged, only 9% eventually.
(3-B)
COS yield after the hydrolysis of chitosan (1.0 g/L) with crude CHI-1 and E. coli BL21-pET23b(+)-NICHI in 1-7 days (3-B)
Kinetics of the hydrolysis of chitosan (0.05−1.0 g/L) (3-C)
Through observations at room temperature for forty days, it is proved that cell surface display technology is an excellent technique that can guarantee and even improve the stability of chitosanase. At the same time, by comparing the hydrolysis efficiency of engineered bacteria and crude chitosanase, it is concluded that the hydrolysis capacity of engineered bacteria is much more stable than that of crude enzyme solution. It shows that the cell surface display technology can ensure and improve the hydrolysis ability of chitosanase for chitosan.