Reuse, Immobilization Of β-glucosidase And Its Application In Hydrolysis

Bio-ethanol is one of the most focuses on alternative-enenrgy research for the harms of large scale use of fossil energy. The enzyme is a restrictive factor for bio-ethanol application in industry. This paper studied the reuse and immobilization ofβ-glucosidase combined with appiliction in hydrolysis. It shows as follows:Reuse ofβ-glucosidase in low temperature by organic solvent is available. In the studies, it is found that Ethylacetat is the most effective co-precipitant of Butanol, Waschmittel and Ethylacetat. 20ml Ethanol with 4.45ml Ethylacetat or 25ml Ethanol with 2.10ml Ethylacetat is the best mixture in 20ml enzymatic hydrolysis system. It could get 90% enzymatic activity after 30 times repeat. But the latter compose has a better stability. Until 50 times repeat, coefficient of recovery decreased to less than 80%. This is a new method to reuse protein by organic solvent and enrich the way to reuse protein by organic solvent.Immobilization ofβ-glucosidase by ceramisite agitator is a new style: first crosslinked by cross linker , and then freeze dried and the last using organic acid adhesive which dissolved in acetone to immobilize theβ-glucosidase. The immobilizedβ-glucosidase made by this method has better stability than that without cross linker or acrylic. The optimum coupling conditions: cross linker concentration, coupling time, temperature, speed of shakes, pH, amount of adhesive A and initialβ-glucosidase amount per grams of carrier were determined as, 1%, 2h, 25℃, 100r/min, 4.8, 0.1 and 15IU/g respectively. By the analysis of immobilizedβ-glucosidase protein, the immobilized enzyme has 29.4% of initial proteins.The optimal pH value and temperature of immobilizedβ-glucosidase is 4.5 and 65℃respectively which is the same as freeβ-glucosidase. Compared to freeβ-glucosidase, immobilizedβ-glucosidase has higher stability both of pH and thermal: Immobilizedβ-glucosidase is stable from 3.0 to 10.0 while freeβ-glucosidase is from 3.0 to 6.0; From 30℃to 70℃, Immobilizedβ-glucosidase is stable while freeβ-glucosidase is stable from 30℃~60℃. The Km value of immobilized and freeβ-glucosidase is 16.6mmol/l and 0.89mmmol/l respectively which means freeβ-glucosidase has a higher substrate affinity than immobilizedβ-glucosidase. And it shows as protein analysis: the immobilizedβ-glucosidase contains about 30% of primary protein amount, but the activity of immobilizedβ-glucosidase shows much lower than theory value.Immobillizedβ-glucosidase was packed as a column to continuously hydrolyze the mixture of 30g/l glucose and 30g/l cellulose. The mixture was hydrolyzed at 50℃, pH 4.8, 5r/min lasting 31 days. The yield was above 90% from the beginning to the last and the average amount of glucose was 59.25g/l, cellulose 2.78g/l. It means the immobilized enzyme had a good stability In this way, there is a tempting application foreground in immobilization of enzyme.