Screening Of Cellulose Degrading Bacteria And Studies On Cellulase Characteristics

Cellulose is the most abundant organic material with most extensive source in nature,and is the most important renewable resource on the Earth.In Heilongjiang Province,plant cellulose is resourceful,but most is burned and abandoned,causing serious environmental pollution.In order to degrade and transform the cellulose to small moleculars or other nutrient mateirals,the key problem is to screen and separate the preponderant strains.This study includes screening of strains,optimizing fermentation conditions,researching on cellulase characteristics and genome.The main contents and results are as follows:1.Screening of cellulose degrading bacteriaThe cellulase-producing bacterium named DL was isolated from the feces of giant pandas,used the methods of sodium carboxymethylcellulose?CMC-Na?as unique carbon source and iodine staining,disintegration of filter paper and measurement of cellulase activity.Based on the morphological observation,physiological and biochemical characteristics and the result of sequences alignment of 16S r DNA,strain DL was identified as Paenibacillus cookii LZ033.The strain of Paenibacillus cookii LZ033 was found to be a spore-forming,gram-positive aerobic bacterium.The mice were fed with Paenibacillus cookii LZ033,once poured into rat's stomach with paste of bacterial liquid 0.2mg/10g,concentration was 3×10⁸CFU/mL,3×10⁷CFU/m L,3×10⁶CFU/mL,and set the positive control group.There was no intoxication occurred in mice after 14 days,let alone death.Liver,spleen and thymus no abnormal changes.2.Opimization of fermentation conditionsAfter optimizing Paenibacillus cookii LZ033 by submerged fermentation,we get the best condition of medium:glucose 0.99%,peptone 0.97%,MgSO₄ 0.076%,KH₂PO₄ 0.31%.Under the optimum condition,the activity of cellulose was up to 92.2U/mL.After optimizing Paenibacillus cookii LZ033 by submerged fermentation,we get the best condition of culture:medium initial pH 6.6,culture temperature 35?,rotation speed 121r/min,broth content 120 mL/250 mL flask.Under the optimum condition,the activity of cellulose was up to 97.8U/mL.3.Research on cellulase characteristicsThe high stability of cellulase was found at temperature 60?⁷0?.The high stability of cellulase was found at pH3⁴.6.The optimal reaction temperature of cellulase was 70?.The optimal reaction pH of cellulase was 3.Fe²⁺,Mn²⁺can promote the activity of cellulase at the concentration of 1mmol/L while Mg²⁺,Zn²⁺,Co²⁺,Cu²⁺can inhibit its activity.Fe²⁺,Mn²⁺were helpful to improve the cellulase-producing capacity of the strain.3.Genome analysisUsed the Illumina/Miseq sequencing platform,the basic genomic information was analyzed.The results showed that Paenibacillus cookii LZ033 had genome size of 3978050bp and the GC content was 46.46%.Based on the genomic data,4049 coding sequences were predicted by Gene Mark and annotated by BLAST sequence similarity searches against the COG,GO and KEGG databases,4049 genes from Paenibacillus cookii LZ033,and the average length of 878bp accounting for 88.7%of the length of the genome.10 Cellulase genes were annotated in Paenibacillus cookii LZ033 by comparing withe the database of COG,GO and KEGG.Including the encoding genes of?-glucosidase and endoglucanase.Paenibacillus cookii LZ033 has an intact glycolysis/gluconeogenesis pathway,citrate cycle?TCA cycle?,pentose phosphate pathway and starch and sucrose metabolism.The complete primary metabolism pathway can provide the needs of physiological activities.And multi-function mechanism studies of Paenibacillus cookii LZ033 also need a long accumulation process,and some new characterics will be verified in the future.