Identification, Fermentation And Characterization Of A Strain YS1069 Producing Xylanase

Xylanases can effectively hydrolyze xylans into oligosaccharide. The xylanases are widely used in food industry, feed industry, paper and pulp industry, medicine industry and environmental protection industry. A strain producing Xylanase was isolated from Yellow Sea water(stored by enzyme engineering team). After purification culture, the strain was identified by physiological, biochemical and genetic characterization. In order to improve the yield of the xylanase, the fermentation conditions of strain YS1069 were optimized. Then the xylanase was preliminarily separated and prepared by ammonium sulfate precipitation and ultrafiltration. With the prepared enzyme as the research object, enzymatic properties and stability were studied. The purpose of the study was in order to lay a foundation for future research, development and application.1. Using the clarity circle method and shake flask fermentation, a marine bacterium YS1069 producing xylanase was screened. The production of xylanase activity was 710 U/m L. The strain is gram-positive bacteria which has spore. The bacterial colony is round, protuberance and milk white on the agar culture-medium. The surface of the bacterial colony is smooth, sticky. As the time increasing, the colony slightly shrivel bumps in the edge. The bacteria is short rod observed by the electronic microscope. The length is about 2 microns. The width is about 0.5 microns. The strains YS1069 is salt resistance, can grow well in 5% Na Cl and weak growth in 14% Na Cl. The growth p H ranges from 9.0 to 11.5, and can be well growth in 20 ℃ to 45 ℃ temperature. It was identified as Bacillus based on physiological, biochemical test and 16 S r DNA sequences analysis.2. The fermentation conditions of strain YS1069 were optimized in order to improve the yield of xylanase by one-single factor and response surface methodology. The single factor was used to screen these factors: the nitrogen source, carbon source, inorganic salt, inoculation volume, liquid volume, initial p H, fermentation temperature and fermentation time. The best result carried out of each single factor was as follows: bean cake powder 25 g/L, wheat bran 40 g/L, The optimum concentration of Na NO3, K2HPO4 and Mg SO4·7H2O was 0.9 g/L, 3 g/L and 0.6 g/L, the inoculums at 4%, 30 m L/250 m L(v/v) liquid volume, the Na2CO3 18 g/L, the culture temperature at 30 ℃ and the culture time at 96 h. Secondly, those main variables were valuated through the Plackett-Burman design method. Results showed Na2CO3, wheat bran and Mg SO4·7H2O that were the significantly affecting variables. Then, the central composite design and the analysis by Design-Expert 8.05 software were adopted to determine the optimal level of the three main factors. The most suitable variables were identified as follows: Na2CO3 21.86 g/L, wheat bran 51.41 g/L, Mg SO4·7H2O 0.59 g/L. The xylanase activity was predicted to be 4462.34 U/m L. The result of verification experiment under the optimum conditions showed that 4408.63 U/m L was the maximum activity. Comparison with that before optimization 738.21 U/m L, the productivity of alkaline xylanase increased 5 times. These results suggested that the predicted model was reliable and available for the optimization of xylanase fermentation conditions.3. Using ammonium sulfate precipitation and dialysis separate the enzyme. Under the condition of 25 ℃, adding ammonium sulfate to saturation 30% to remove miscellaneous protein. Continue adding ammonium sulfate in supernatant to 55% to precipitate target protein. The precipitation was dissolved by 50 mmol/L p H 8.0 phosphate buffer solution, then dialysed using 7 k Da dialysis bag. Using Ba Cl2 detected dialysis completely, then the dialysis solution was ultrafiltrated and concentrated by 10 k Da ultrafiltration membrane. Then we can get preliminarily separated and prepared enzyme.Enzymatic properties and stability were studied. The results showed that the optimal temperature of xylanase was 60 ℃ and the optimal p H of xylanase was 8.0. Under the condition of p H 8.0、55 ℃ heating 1 hour, the residual enzyme activity was 84.88% showing that xylanase has good stability. Ba2+、K+、Ca2+、Mg2+、Al3+、Na+ can promote the enzyme activity of xylanase. Li+、Co2+、Mn2+、Zn2+、Cu2+、Fe2+、Fe3+ can inhibit the enzyme activity of xylanase. Mn2+ and Li+ have stronger inhibitory effect on the enzyme activity of xylanase among these metal ions. The influence of additives on the enzyme activity of xylanase showed that nonionic surfactant Tween-60 and Triton X-100 have activation function. Tween-20, Tween-40, Tween-80 have slight inhibition. Anionic surfactant sodium dodecyl sulfate SDS, aprotic solvent DMSO and cationic surfactant cetyl trimethyl ammonium bromide CTAB have stronger inhibition. The properties and stability of xylanase shows that the enzyme has good application prospect.