Uncultured Microbial Cellulase Gene Cloning, Identification And Expression

The study on uncultured microorganism is an important research field in microbiology. And it is the only method to clone the genes from the uncultured microorganisms by constructing metagenomic DNA library with the DNA directly extracted from the environment.But the methods for extracting and purifying DNA from soil are still imperfect. Although there are several reports on the construction of metagenomic DNA library with the DNA extracted from soil, it is hard to find suitble methods to extract and purify DNA from compost. There is no report on the construction of metagenomic DNA library with the DNA extracted from compost owning to the lack of methods of purifying DNA from compost.This report offers a two step purification method for successfully purifying the DNA from soil and compost. The method is composed of the first step of polyvinylpolypyrrolidone+Sephadex G200 chromatography and the second step of electroelution. Purified DNA by this method can be used further manipulation such as restriction endonucleases digestion, PCR, ligation and transformation.A soil metagenomic DNA Cosmid library (TD1) containing 50,000 clones was constructed. The total length of insert DNA in the TD1 library is about 1.84×109bp. And another compost metagenomic DNA Cosmid library (DU1) containing 100,000 clones was constructed. The total length of insert DNA in the DU1 library is about 3.4×109bp.Four positive clones were isolated from DU1 library expressing CMCase activity. Two clones were chosen for further study. The CMCase genes (umcel9A and nmcel9E) in the two clones were sequenced with the strategy of primer walking. GenBank database searching shows that the product of umcel9A gene has highest identity (54%) with the endo-l,4-beta-D-glucanase of Xanthomonas campestris pv. campestris and the product of umcel9B gene has highest identity (48%) with hypothetical protein of Microbulbifer degradans 2-40. Domain analysis with SMART tools showed that Umcel9A and Umcel9Bhave a glycosyl hydrolase family 9 catalytic domain.The coding regions of umcel9A and umcel9B were amplified by PCR and cloned into expression vector pQE30 and pQE32, respectively. Most of the expressed product of wncel9A gene formed inclusion bodies while the expressed products of umcel9B gene were soluble. After purify the recombinant Umcel9A and Umcel9B, their enzyme properties were characterized. The optimum temperature of umce19A is 40℃ and its optimum pH value is 6.6. The Km value of Umcel9A to CMC is 1850mmol/l. The optimum temperature of umce19B is 45℃ and its optimum pH value is 7.0. The Km value of Umcel9B to CMC is 555mmol/l.