Study On Screening,Identification,Endoglucanases Of A Cellulase-producing Strain And Its Characteristics Of Alcohol-tolerance

Alcohol resistant cellulase producing strains were screed from different material in this paper, firstly by methods of ethanol-CMC-Na agar plate, secondly followed by affination of shake flask fermentation.We got a relatively stable cellulose activities producing strain Lys-1249.The strain Lys-1249 were identified as Bacillus subtilis by physiological and biochemical assay, morphological observation and homology analysis of sequence of 16S rDNA.For further studies, many more determinations were porformed including orthogonal optimization of enzyme producing conditions, alcohol tolerance degree analysis of strain and Its cellulase, identification the composition of endoglucanases system, peptide mass fingerprint identification of cellulase fragment by trypsin digestion through mascot searching from Swissprot database, MS/MS Ions searching for secondly mass spectrometry matching for the fragments. Finally the cellulase system information related to the strain were compared and analyzed with Uniprot protein database. The main conclusions were drawn in this paper as follows:1, The conditions of preparation enzyme were optimized and indicated that the ethanol tolerance of cellulase activity reached 158.54 U/mL when the strain Lys-1249 was cultured in medium of wheat bran in 2.0%, peptone 0.6%, K2HPO4 0.1%, MgSO4,7H2O 0.05%, FeSO4 7H2O 0.02%, MnSO40.01% initial pH 7.0, at 37℃ for 48h. The strain Lys-1249 grew well in the medium containing ethanol 6.0%. Also the cellulase activity of Lys-1249 remained 65.95% in substrate of CMC-Na 1.0% containing ethanol 6.0%。2, After cellulase purified and separated by native-PAGE, the polyacrylamide gel was put on CMC-Na agar plate to detect the hydrolysis transparent strip. It was proved to be a feasible method of identification celullase system components. Becaues this method had characteristics of direct (directly observed hydrolysis transparent strip) and sensitivity (0.1-0.2 μg/mL enzyme protein was able to be detected). By this method,3 enzyme components of strain Lys-1249 were identified as P1, P2, P3 respectively. Also each of them had only one subunit, of which the molecular weight of P1 was 65.1Ku, the molecular weight of P2 was 43.7Ku, the molecular weight of P3 was 48.0Ku by methids of SDS-PAGE.After digestion by trypsin, the the fragment of endo-cellulase components P1, P2, P3 of strain Lys-1249 were submited to Swissprot database and Uniprot protein database for identification and analysis. The results indicated that the P1, P2 of strain Lys-1249 matched well with Glucose-6-phosphate isomerase of Bacillus subtilis (strain.168) in significant similarity by scoring method. But no related protein matched well with P3 of strain Lys-1249。...