Screening Of Cellulase-producing Strains And Purification Of Cellulose From The Digestive Tract Of Grass Carp

Cellulose is the most abundant carbohydrate in the world, and it is the renewableresource that is available in large quantities. It is great realistic for human being to solvethe problems of environment pollution, food shortage and energy crisis by using cellulaseto transform cellulose into energy, chemical and food.A bacterial strain is isolated from the digestive tract of grass carp by using Congored as plate and various carbon sources. Research on cellulase producing conditions ofthe screened bacterial strains in liquid state fermentation is conducted. The optimalculture condition follows: 1% 1%(w/v) CMC-Na as carbon source; 1%(w/v) peptone asthe nitrogen source; inoculum's volume 5%; initial medium pH value 7.0; optimal culturetemperature 37℃; 24 hours in 180rpm/min.In the hereinabove cultural condition, the cultural mixture from the isolated strain isprocessed by smashing cell wall in an ultrasonic crashing instrument. Techniques such asmagnetic stirring and centrifugal filtration are applied to process fermentation liquor, andthen enzymatic activity of the cellulose is compared and examined, thus the enzymespecies generated from the strain is identified as extracellular enzyme. By means ofammonium sulphate fractional precipitation, Anion-exchange chromatography onDEAE-Sephadex A-50, and Gel-filtration chromatography on Sephadex G-75, thecellulose was separated and purified, and the purified cellulose is resolved into at least 3subunits by sodium dodecyl sulphate-polyacrylamide gel electrophresis.The biocatalyzed characters of the purified cellulose are researched when sodiumcellulose glycolate (CMC-Na) is as the substrate. The most appropriate catalyzedtemperature for the cellulose is 55℃. When the temperature stability range of thecellulose is 45℃-65℃, the enzyme has optimal stability to the changing temperature,and it even retains 30% activity at 80℃in 60min. The enzyme vitality is comparativelystable as its pH value is 4.0-7.0, and the most appropriate pH for cellulose is 7.0. Mn²⁺,Mg²⁺ can activate the enzyme vitality of the cellulose, while Fe²⁺, Ca²⁺, Zn²⁺ canobviously inhibit the vitality and Cu²⁺,Co²⁺ can have the toxic action on the cellulase,The Km value based on sodium cellulose glycolate (CMC-Na) substrate is 5.3×10^-3g/ml.