The Use Of Fusion Tags, Purification And Expression Of ¦Â-mannanase

Mannanase is a very important enzyme for hemicellulose degradation,especially in biological-degumming field. In this research, the genome DNA of Bacillus licheniformis HDYM-04, the mannanase-producing strain, was used as the template of PCR, after the gain of mannanase gene by PCR, the fragment of mannanase gene was inserted into the fusion expression vector pET28a, and the recombinant DNA was transferred into Escherichia coli BL21(DE3) , and then the enzymological properties of the fusion enzyme was analyzed.The length of mannanase gene, named as man3, gained by PCR was 1282bp. The sequence of man3 was analyzed by bioinformatics methods. The results showed that the enzyme was homologous with Bacillus sp., acidic, MW 38kD, without signal peptide and transmembrane region, two structural region of MANB and Glycosyl hydrolase. The original enzyme activity of Escherichia coli BL21(DE3) recombinant train HDEM8 of mannanase gene was determined as 38.60U/mL, and after optimized by orthogonal experiment, it was 45.57U/mL with the induced condition IPTG 1.0mmol/L, 30℃, 7h. After the purification by His·Bind Columns, the results of enzymological analysis showed that the optimum temperature and pH was 55℃and 5.5, the relevant activity at the range 50℃-60℃and 4.5-6.0, temperature stability was 30℃-60℃and pH stability was 3.0-6.0.The gain and analysis of fusion expressed mannanase made a foundation for using HDYM04 and it's mannanase in the biological-degumming and the other applications. In this research, the system of an allogenetic gene fusion expressed in Escherichia coli BL21(DE3) was established for our laboratory.