Fermentation, Purification and Characterization of Cellobiase from Marine Aspergillus Sp.
|Keywords||cellulose cellobiase marine Aspergillus sp. fermentation optimization purification characterization salt-tolerant thermostability|
There are three types of enzymes involved in the process of cellulose hydrolysis: endoglucanase, exoglucanase and β-glucosidase (which is also named cellobiase). Cellobiase plays a very important role in hydrolysis. It could hydrolyze cellobiose to glucose in order to eliminate cellobiose inhibition. So the step performed by cellobiase is the rate-limiting step for the whole cellulose hydrolysis process.The Aspergillus sp. isolated from the marine sediment was used to produce cellobiase and endoglucanase by solid state fermentation. And the optimal culture medium and condition of fermentation were studied well. The results indicated that the best carbon source were hyacinth and bran (hyacinth:bran=1:1) and the best nitrogen source was urea. The enzymes activities reached the highest in condition of that the concentration of urea is0.5%, the initial moisture is55%, the initial pH value is5.0, the fermentation temperature is34℃and the fermentation time is9days. And under the optimal circumstances the maximal cellobiase and endoglucanase activities reached79.78U and293.95U, respectively.The crude enzymes were pre-separated by ammonium sulfate precipitation in the concentration of50%～70%and ultra-filtration with30kDa filter membrance. The purified cellobiase was obtained by Q-Sepharose FF anion exchange chromatograph and Q-Sepharose HP anion exchange chromatograph. The cellobiase total activities were reduced from1768.37U to187.65U as well as the total proteins were reduced from161.04mg to1.43mg. However, the specific activities of cellobiase increased to131.21U·mg-1while the crude enzyme specific activity is10.98U·mg-1.Overall levels of11.93-fold purification of cellobiase was observed. The purified enzyme showed a single protein band on SDS-PAGE with an estimated molecular mass of101kDa.The optimal reaction temperature and pH of the cellobiase were65℃and4.5. The optimal activity occurred at40g/L of NaCl, and the activities increased by23%under this circumstance. The cellobiase activity was higher over the range of20g/L-150g/L NaCl than that in NaCl-free. So it was a typical salt-tolerant enzyme. Moreover the thermostability of the cellobiase was remarkable improved in high salinity solution and its half-life time increased by2-3folds of that in NaCl-free solution. In the condition of40g/L,60g/L and80g/L NaCl, the half-life time were respectively improved to144min,157min and203min when the temperature is65℃, while the contrast is51min. Both the activity and thermostability of the cellobiase from the marine Aspergillus sp. were better than those from other terrestrial fungus. The purified cellobiase was thermostable and salt-tolerant and showed great potential in industry.