Studying On Producting Of L-Arabia Glucose Isomerase And D Arabla Glucose Isomerase And D-tagatose

D-tagatose is a sweetener with the characteristics of low energy to be using in the fields of conquering of blood sugar, Improving of intestinal flora, Inhibing of intestinal pathogenic bacteria, resistancing of caries and reducing of obesity, and so on, Now, the product of D-tagatose is mainly come from artificial synthesis. But it’s so complicated of the production process that the price of the D-tagatose product is higher. L-Arabia glucose isomerase behaves the function of changing D-galactose as D-tagatose. Here, we studying on producting of L-Arabia glucose isomerase an changing D-tagatose as D-tagatose.Here a L-Arabia glucose isomerase (L-ai) gene was amplified by PCR amplification from E. coli. DH5a. For studying expression of recombinant bslac2protein in Pichia pastoris, the gene of L-ai was cloned to the MCS of pGAP9K and the pGAP9K-L-ai were transformed into the genome of Pichia pastoris GS115by electroporation. The recombinants containing of this gene were proved by PCR with gene specific primers and the recombinants of higher expressiuon of L-ai protein was selected as engineering strain of GS115(pGAP9K-L-ai).Studying on fermentation conditions for the GS115(pGAP9K-L-ai), the yield from solid state fermentation in shaking flask slightly higher than the liquid fermentation in shaking flask。 Investigating of expanding production of the recombinant enzyme by solid state fermentation. While increasing the medium till100times to fermentate the strain in homemade iron suitcase for3days, the aim protein expression is slightly less than the fermentation in shaking flask but no significant difference. SP-Sepharose FF ion-exchange chromatography was used to purify of the recombinant L-AI protein. The results from testing of the L-AI protein activity indicated its best L-ai enzyme activity is at pH5.8and55℃.27110U/L fermentation broth of L-AI was secreted to the fermentation by the GS115(pGAP9K-L-ai). The fermentation broth or the purified L-AI protein are all can react with D-galactose to produce of D-tagatose. Purification of D-tagatose was progressed by Ca2+separation of resin under65℃then the obtained D-tagatose containing solution was went thorought anion and cation column to remove colour an salt. The result from infrared spectroscopy indicated that the obtained product from the purification shown the same spectrum as the staindard D-tagatose.The fermentation residue was used to produce of biogas. In a4days fermentation,5200mL biogas was obtained from each kilogram of fermentation residue and also the number of the GS115(pGAP9K-L-ai) is significant reduction in the anaerobic environment.Our works of cloning L-Arabia glucose isomerase from E. coli DH5a, expressing this gene in Pichia pastoris and studying the processes of fermentation engineering strain, purification foreign protein, producing of D-tagatose and recycling by-product had established foundation on scale production of D-tagatose by using the L-Arabia glucose isomerase from E. coli DH5a to catalyze of D-galactose.