| Cellulose,the most abundant renewable resource found in agricultural waste,exhibits significant developmental prospects.Nevertheless,its intricate structure and resistance to degradation necessitate the development of environmentally friendly and safe cellulose degradation technologies to effectively harness its potential.However,commercial cellulase production is costly,and enzymes often suffer from inadequate thermal stability.Consequently,research in this field primarily focuses on exploring microorganisms capable of cellulose degradation.Silkworm excrement is the main excreta of silkworms after consuming mulberry leaves,rich in difficult to degrade cellulose,and is one of the key factors restricting the utilization efficiency of silkworm sand resources.This study isolated and screened cellulose degrading bacteria from fermented silkworm excrement,and conducted morphological and molecular biological identification.Select strains with high cellulase activity as the research target,and study their enzyme production process conditions and enzymatic properties.Transcriptome technology was used to screen the key gene of cellulose degradation of isolated strains,and to study the cloning and expression of this gene in E.coli.The main research results were as follows:1.Six bacterial strains capable of cellulose degradation were isolated and selected from fermented sandworm casting samples utilizing the dilution plate method.Strain DC-11,identified as Bacillus subtilis through morphological observations,physiological and biochemical tests,and phylogenetic analysis of the 16 Sr DNA sequence after CMC-Na cultivation,Congo red staining,and filter paper enzyme activity assay,exhibited efficient cellulose degradation capabilities.The optimal growth temperature for strain DC-11 was determined to be 37°C,with a p H of 5.0.Notably,the activities of filter paper cellulase(FPase),endoglucanase(CMCase),and exoglucanase(CXase)reached 15.40 U/m L,11.91U/m L,and 20.61 U/m L,respectively.Moreover,in the bioaugmentation experiment,the cellulose degradation rate of the experimental group(TG group)treated with strain DC-11 was significantly higher at 29.97% compared to the control group(CK group)at 7.02%.Strain DC-11,characterized by its acid and heat tolerance,along with its high cellulase activity,demonstrates the promising potential for bioaugmentation in cellulose degradation processes.2.RNA-seq technology was employed to conduct transcriptomic analysis of strain DC-11 cultivated with glucose and CMC-Na as the exclusive carbon sources.The findings unveiled a total of 942 genes exhibiting upregulation and 1996 genes exhibiting downregulation in the CMC-Na group in comparison to the glucose group.Alignment of these differentially expressed genes with the GO,KOG,and KEGG databases unveiled a noteworthy GO term,“GO:0030248(cellulose binding)”,which is closely associated with cellulose degradation.Additionally,an important KOG category G(Carbohydrate transport and metabolism)linked to cellulose degradation,along with two vital pathways involved in cellulose degradation,“ko00500(starch and sucrose metabolism)” and “ko02060Phosphotransferase system(PTS)”,were identified.Notably,cellulase genes associated with cellulose hydrolysis displayed significant downregulation within the “ko02060Phosphotransferase system(PTS)” pathway in the CMC-Na group.The findings were further validated through q RT-PCR experiments,confirming the pivotal role of these differentially expressed genes in the metabolic pathways identified through transcriptomic analysis.These results shed light on the potential significance of these genes in the degradation of cellulose substrates,such as CMC-Na,by Bacillus subtilis strain DC-11.3.By employing whole-genome sequencing and gene engineering methodologies,the cellulase gene yto P from strain DC-11 was effectively cloned and expressed.The recombinant strain demonstrated optimal CMCase production at a p H of 5.0 and a temperature of 55°C.Notably,the cellulase production capacity of the recombinant strain exceeded that of the control strain.Following protein purification,the enzyme activity of the recombinant protein reached 16.98 U/m L under the optimal conditions of p H 5.0 and 55°C,with a concentration of 100 μg/m L.The above research results provide theoretical reference for the efficient degradation of cellulose biomass and the efficient utilization of silkworm excrement and even agricultural straw resources,and have great application value. |
