Study On Application Of Cold-active Cellulase From Antarctic To Simultaneous Saccharification And Fermentation (SSF)

This study isolates and selectes an Antarctic fungus—QP7, which can secrete high activity cold-active cellulose. And then we research for its application to SSF.Samples of soil collected during the 24^th Antarctic scientific expedition were primary screened with CMCNa Congo red medium, and rescreened with liquid fermentation medium, one strains of higher cellulase production—QP7 were selected. By morphology and microscopic observationand, physiological characteristic mensurated, we can identify that the strain belongs to Verticillium longisporum.The research showed that the optimum pH and temperature of cellulase produced by QP7 was 5 and 38℃, which was close to the suitable growth pH and temperature of yeast, and adapted to the weak acidic lignocelluloses after acid treatment. In addition, it has good thermal and pH stability. It can reserve for a few days if stored in a 4℃, dark area. It can keep a high activity with the presence of a certain concentration of ethanol. So the cellulase produced by QP7 has better adaptability to SSF.Then we found that the enzymatic activity could be increased by Ca²⁺ and K⁺, this can be applied to the production.The optimum conditions of cellulase production were confirmed as follows:The carbon source is 3% bran, nitrogen source is 0.5% (NH₄)₂SO₄. There are also 0.02% MgSO₄, 0.1% sorbitol, 0.1% lactose, 0.1% maltose, 0.1% Tween80, and 0.01mol/L Ca²⁺, 0.01mol/L K⁺. The initial pH of culture medium was 5.5. inoculating volume was 5 %. After culture at 10℃for 96 h, it could reach the highest activity.We also found two details that can improve the activity: The activity may increase more than 20% if the medium was placed overnight before sterilization; and the seed should be put at refrigerator for a few days before inoculate. At the UV mutagenesis box with 254 nm, 11W×3, after UV irradiation for 5min, a high cellulase yield strain was obtained—QP7-1, and its production property was stable.The activity of which was increased 24%. Another stable mutants was achieved by N⁺ ion implantation, its activity was increased 5%.A preliminary SSF was carried on with the cold-active cellulase and halophyte suaeda. 10 g raw material can produce 0.4 g ethanol.In summary, this cold–active cellilase produced by QP7 can solve the problem of the large gap between the optimum temperature of common cellulase(45-65℃)and the optimum growth temperature of yeast, and reduce costs. It has great potential to SSF.