Preliminary Studies On Cellulase Production By Aureobasidium Pullulans And Purification And Characterization Of CMCase

A total of 19 strains of marine yeasts which could produce cellulase were screened from the yeasts stored in the present laboratory, and strain 98 ,which was found to be able to produce a higher yield of cellulase, was selected for subsequent investigation. The marine yeast strain 98 was isolated from the surface seawater of Dongfeng saltern in Qingdao, China, and was identified to be Aureobasidium pullulans by 18S rDNA,, ITS gene sequences analysis and routine yeast identification methods. The maximum production of enzyme (CMCase 4.51U/mg, FPAase 4.75U/mg protein) was obtained in a medium containing 2.0 g of soluble starch, 0.5 g of peptone, 2.0 g of yeast extract, 100 mL of sea water, at an initial pH of 7.0 after fermentation at 28°C for 24 h. The TLC results indicate that the crude enzyme could hydrolyze CMC and filter paper, producing a large amount of monosaccharide and a trace amount of disaccharide. This is the first report that marine yeast can produce cellulase. The extracellular CMCase in the supernatant of the cell culture of the marine yeast Aureobasidium pullulans 98 was purified to homogeneity with a 1.98-fold increase in specific activity as compared to the concentrated supernatant by ammonium sulfate fractionation and gel filtration chromatography (SephadexTM G-75) and DEAE Sepharose Fast Flow . According to the data on SDS polyacrylamide gel electrophoresis show that the molecular weight of the purified enzyme was 67 kDa. The optimal pH and temperature of the purified enzyme were 5.6 and 40°C, respectively. The enzyme was activated by Na⁺,Mg²⁺,Ca²⁺,K⁺,Fe²⁺ and Cu²⁺. On the other hand, Fe³⁺, Ba²⁺,Zn²⁺ ,Mn²⁺ and Ag⁺ inhibited the enzyme. The enzyme was inhibited by PMSF ,SDS ,EDTA ,EGTA ,DTT and Iodoacetic acid. Km and Vmax values of the purified enzyme for CMC were 4.73 mg/mL and 0.566μmol/min/mg, respectively. Oligosaccharides with more than monosaccharides were detected in the hydrolysate of CMC, indicating that the purified enzyme was indeed endo-1, 4-β-D-glucanase.