Isolation Of Cellulase Genes From Metagenomes By A Novel PCR Method

With the rapid development of metagenomic technology, It provides a convenient way for mining enzymes and active prducts from the uncultured microorganisms. And a large number of enzymes can be used in industrial such as lipase, protease, cellulase. There is urgent need for enzymes which have diversity enzymatic properties in industrial applications. It encourage people to develop new enzyme molecules to meet the needs of practical application, but also to promote the development of screening methods which effective and efficient to mining new enzyme molecules. Therefore, this paper attempts to build efficient metagenomic enzyme PCR screening method and application to screening cellulase from metagenomes.In this study, we constructed a soil metagenomic library (WK-s016) using silt soil and a new cellulase gene (GHF9s016) was obtain form this fosmid library using function-based screening method. A new method, termed metagenomic gene specific multi-primer PCR (MGSM-PCR), is built that uses multiple gene specific primers derived from an isolated cellulase gene (GHF9s016) from the constructed metagenomic library (WK-s016) rather than degenerate primers designed based on a known enzyme family. The utility of MGSM-PCR was shown by applying it to search for homologues of the glycoside hydrolase family9cellulase in metagenomic DNA. A total of127homologous genes were amplified with combinatorial multi-primer reactions from34soil DNA samples. Multiple alignments revealed extensive sequence diversity among these captured sequences with sequence identity varying from26to99.7%. These results indicated that significantly diverse homologous genes were indeed readily accessible when using multiple metagenomic gene specific primers. This method will provide a potential new enzyme sequence screening methods.Meanwhile, The sequence-based screening method were used to parallel screening the Glycosyl Hydrolase Family9E sub-family cellulase gene. Two pairs of primers were designed by Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) online primer design software acoording to the E sub-family of E1and E2cellulase amino acid sequence in the database. The degenerate PCR were carried out to amplification of metagenomic DNA and successfully obtain49E sub-family new cellulases family gene fragments:29El cellulases,20 E2cellulases. It proved that there are a lot of diversity cellulase genes in the soil metagenomic DNA.