The Research On Detection Of Genes Encoding Lignin-Degrading Enzymes By DNA Sensor And Composting Community Diversity

Composting technology is applied to disposal of the municipal solid waste increasingly. The metabolic process during microbial degradation of lignin is completed together by a series of enzymes in the compost system, in which manganese peroxidase and cellobiose dehydrogenase play the key role. It is better to know the synergy between the two enzymes by using DNA biosensor. This study was to prepare DNA probes immobilized biosensor for detecting manganese peroxidase and cellobiose dehydrogenase encoding genes from Phanerochaete chrysosporium. The feasibility for simultaneous detection of two genes was discussed and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis was used to study the composting microbial community diversity.First, the detection of manganese peroxidase encoding genes from Phanerochaete chrysosporium was studied. Mercapto-modified probe self-assembled on the gold electrode surface and the process of immobilization and hybridization of ssDNA were characterized by impedance spectroscopy and cyclic voltammetry. The linear relationship of electrochemical signals and concentration of target DNA was obtained by chronoamperometry and the range of detection, specificity, various factors were also studied. The amperometric current linearly related to the common logarithm of the target nucleic acid concentration in the range from 0.001 to 400 nM and the detection limit of biosensor was 0.001 nM.Next, mercapto-modified two lignin-degrading enzymes—manganese peroxidase(MnP) and cellobiose dehydrogenase(CDH) oligonucleotide probes self-assembled on the surface of the gold electrode. The sandwich hybridization between target sequences and complementary sequences displayed high sensitivity and good molecular recognition. Horseradish peroxidase-streptavidin (HRP-SA) conjugate and laccase-streptavidin (LAC-SA) conjugate were applied for enzyme-amplifeid amperometric measurement. The two target genes were simultaneously quantified in the presence of corresponding substrate. The amperometric current responses to HRP and LAC catalyzed reactions were linearly related to the common logarithm of two target nucleic acids concentrations in the range from 0.01 to 40 nM and 0.1 to 40 nM. The correlation coefficient of MnP and CDH was 0.9884 and 0.9881 and the detection limits were 0.01 nM to 0.05nM, respectively. Each of the concentration was done three times, and the average values of the relative standard deviations (RSD) were 4.30% and 3.52%, respectively. The DNA biosensor exhibited good selectivity, sensitivity, precision and stability.Finally, composting microbial community diversity and succession were analysed by PCR-DGGE. Physicochemical parameters such as pH, temperature, organics and C/N ratio were measured, which indicated a typical composting. It showed that DGGE bands in different periods of composting appeared diversities. Temperature appeared to play a key role in determining the microbial composition present at respective stages of composting.