Abstract:

Objective To screen out the microbial strains with strong cellulose degrading enzyme activity for efficient fermentation of silage. Method The cellulase-producing strains were screened by carboxymethyl cellulose sodium plate from existing strain banks and their cellulase activity was determined. Morphological observation, physiological and biochemical analysis and 16S rRNA were used to identify this strain. The growth curve, growth temperature, pH and salt tolerance of strain FJAT-25102 were measured to study its growth characteristics. In further, the whole genome data of this strain was analyzed by bioinformatics to explore the cellulase genes. Result According to the ratio D/d between transparent circle D and colony diameter d, the strain with the highest D/d ratio was FJAT-25102. Further determination of its enzyme activity showed that its cellulase activity was 221.81 U/mL at 48 h. It was found that this strain had the ability to produce protease and amylase. Strain FJAT-25102 was identified as Micrococcus luteus by morphological, physiological, biochemical and phylogenetic analysis. Strain FJAT-25102 was in the growth initiation stage after 2 h of culture, and the OD₆₀₀ value reached the maximum at 26 h of culture. It grew between 20-50℃, pH 5-11 and 0%-5% NaCl concentration, and had certain high temperature resistance and saline-alkaline resistance. The genome of strain FJAT-25102 consisted of a ring chromosome with a size of about 2 570 649 bp and a GC content of 72.98%. The genome contained 2 378 predicted protein-coding sequences. All possible genes involved in cellulose degradation in strain FJAT-25102 included possible alpha-glucosidase GH13, endoglucanase GH74, and acetylxylanesterase CE1, CE7, and CE3. Among them, GH13 and GH74 regulated enzymes in the glycoside hydrolase family of FJAT-25102 may play an important role in the process of cellulose degradation. The contents of crude fiber, acid detergent fiber and neutral detergent fiber in the silage treated by strain FJAT-25102 significantly reduced. Conclusion The selected Micrococcus luteus FJAT-250102 may provide microbial strain resources for silage fermentation. The determination of its whole genome and investigation of cellulase genes may provide the basis for the subsequent research on the cellulase production mechanism of this strain.

Key words: cellulase, Micrococcus luteus, growth characteristics, genome, cellulose degradation genes