Study On The Fermentation And Purification Of Dextranase From Marine Microorganism

Dextranase (α-D-1,6-Glucan-6-D-Gluca-nohydrolase, EC3.2.1.11), can hydrolyze α-1,6 glucosidic dextran bonds specially and has been applied extensively in the field of sugar industry, the production of low molecular dextran and caries prevention. In this study, the fermentation conditions, the stability, the purification and characteristics of dextranase from Arthrobacter oxydans KQ11 were explored. Initially,1. The fermentation conditions of Arthrobacter oxydans KQ11 stored in lab were optimized through single-factor experiment, orthogonal experiment, response surface methodology and fitting amplification. The best carbon source and nitrogen source were wheat bran, yeast extract, tapioca, peptone and soya bean meal respecially with single-factor experiment. With the orthogonal experiment and response surface methodology, the optimal medium compositions and conditions to achieve the maximum dextranase production were determined (g/L):yeast extract,5; wheat bran,10; NaCl,4; MgSO4,0.4; dextran,7.2; soybean meal,7.57; tapioca,7.43 and the initial pH was 6.99. The highest dextranase activity of 500 L was observed at 32 h, which enhanced the dextranase from 11.424 U/mL to 28.128 U/mL.2. The preparation of dextranase from Arthrobacter oxydans KQ11 was studied. The fermentation liquor producted with the best fermentation conditions optimized was disposed through flocculation, centrifugation, ultrafiltration and lyophilization. Then the stability of dextranase was studied with the power observed from lyophilization. The best enzymatic protective reagents were glycerol (16%), sodium acetate (18%), sodium citrate (20%), acetate (0.05%), D-sodium isoascorbiate (0.03%) and potassium sorbate (0.05%) with single-factor experiment and orthogonal experiment. Subsequent validation experiment showed that dextranase with enzymatic protective reagents maintained 70.8% and 28.96% activities at the 13th week at 25 and 37℃, respectively.3. The purification and characteristics of dextranase from Arthrobacter oxydans KQ11 were studied. The crude dextranase was purified by a combination of ammonium sulfate fractionation and ion-exchange chromatography, and then the enzyme was characterized. The enzyme was 66.2 kDa with an optimal temperature of 50℃ and a pH of 7. The enzyme had greater than 60% activity at 60℃ for 1 h. Moreover,10 mM Co2+enhanced dextranase activity (196%), whereas Ni2+and Fe3+negatively affected activity.0.02%xylitol and 1% alcohol enhanced activity (132.25%and 110.37%, respectively) whereas 0.05%SDS inhibited activity (14.07%). Through the hydrolysis experiments of substrate, the enzyme hydrolyzed a-1,6 bonds rather than a-1,4 bonds. Through the thin-layer chromatography experiment, the enzyme hydrolyzed dextran into maltotriose and isomaltotriose, so the enzyme was belong to endo-dextranase.