Construction Of A High-effective Expression System In Bacillus Subtilis Using Xylanase Gene As Mark

To date, various genes have been identified and cloned using molecular biology. Their expression and the production of proteins of interest are highly important for both basic research and practical applications. Thus, the demand for foreign gene expression systems is rapidly increasing. Production of heterologous proteins at high levels by bacteria is commonly achieved using Escherichia coli as the host. However, the E. coli expression system still has disadvantages. For example, it is a pathogenic bacterium and has endotoxins (lipopolysaccharide); it secretes protein into the periplasm and often into inclusion bodies. Bacillus subtilis is an attractive host for the production of heterologous secretory proteins. Many genes has succeed expressed in Bacillus subtilis, and the plasmid vectors for Bacillus subtilis are now taken from other genus bacterium, such as Staphylococcus aureus or lactococcus lactis and so on. As it is clear to known that these plasmids are far from optimal for Bacillus subtilis, because of low efficiencies of cloning and high levels of replicating instability. So the integration vector is developed, integrate foreign gene into the genome of Bacillus subtilis is an effective strategy to avoid genetic instability. However, an efficient expression system is not limited to secreting protein, keeping the ability of genetic and replication, it also has high expressive element (such as strong promoter).This reseach used xylanase gene as object, constructed a stable, high-effective expression system in Bacillus subtilis. The complete and promoterless xynA gene was amplified from Bacillus sp. by PCR, the latter was connected with the strong promoter of Bacillus thuringiensis, namely S-layer protein promoter, to constructed recombinant S-xynA gene. The xynA gene and recombinant S-xynA gene were respectively inserted into integration vector pDG1730, transferred Bacillus subtilis B47, which producing highα-amylase.Experiment results showed that the production can secrete out of cell and had normal bioactivity. The xylanase activities of two recombinant strains were both increased. The activity of xylanase expressed with native promoter was 50.1 IU/mL, and the activity of xylanase expressed with S-layer promoter was 84.9 IU/mL, which was 1.69 times of that with native promoter. The molecule weight of xynA was determined as about 26kD. The analysis of medium showed that the activity was increased, when fecula was added to the medium. And the activity was increased, when lactose was added to the medium.The biochemical properties of the xylanases produced by combinant strain showed that it had an optimum temperature of 50℃and an optimum pH of 6.0. The xylanase had good activity at temperature ranged from 40℃to 60℃and at pH ranged from 5.0 to 8.0. The xylanase had good temperature stability. So the xylanase had a wide foreground in exploitation and utilization of animal feedstuf resources.