Phylogenetic Distribution And Cloning Expression Of Chitinase Genes In Activated Sludge

Chitin,poly(?-(1?4)-N,acetyl-D-glucosamine),is important natural polymer that is second only to cellulose in nature.As its degradation product,due to its small molecular weight and enhanced solubility,chitin oligosaccharide is not only more feature-rich,but also easier to utilize.Biodegradation of chitin is a way to easily control the product and mildly obtain chitin oligosaccharides,and chitinase is the key enzyme in this process.Many microorganisms have been reported to produce chitinases,which are secreted by both bacteria and fungi.The traditional method for obtaining a novel chitinase is to screen microorganisms using a conditioned medium,and then a pure culture is used for enzyme production.Since current culture methods are unable to effectively culture most of the microorganisms in the natural environment,conventional methods severely limit the utilization of chitinase resources in microorganisms.In order to eliminate the restriction of the culture method on the development of chitinase,this study used sequence-based metagenomics combined with heterologous protein expression to study the chitinase gene in activated sludge.The results are as follows:1)The phylogenetic tree constructed by genomic ribosomal protein genes shows that 29 out of 74 bacteria phyla have chitinase genes predicted,and the number of microorganisms with chitin-degrading function exceeds expectations.2)According to the results of the original activated sludge metagenomic analysis,tere are a large number of potential chitinase genes in the activated sludge,and the transcriptome results indicate that most of them are expressed.3)This study selected genes for heterologous protein expression based on abundance changes,transcription levels,classificatlion of source bacterna,and phylogenetic relationships.Nine of the ten successfully expressed recombinant proteins were able to degrade chitin,indicating that the method for functional gene screening used in this study is effective and useful.5)Microorganisms containing potential chitinase genes in Nitrosomonadales are concentrated in the genus Nitrosomonas.The growth of Nitrosomonas europaea Winogradsky 1892 in different media showed that the bacteria could not grow with chitin as the sole nitrogen source,and the function of chitinase for the bacteria was not energy intake.In conclusion,this study clarified the distribution of chitinase genes in activated sludge,obtained a variety of new chitinases,and verified the feasibility of sequence-based metagenomics combined with heterologous expression.Therefore,our study provides a methodological approach for exploring other functional genes.