Screening The Food Industry Genes From Metagenomelibrary And Cloning And Expression The Dehalogenase

Metagenomics is a new field of research in which genetic material of genome D NA or cDNA were recovered from environmental samples. Compared with the traditi onal researches,metagenomics can reveal more details on diverse genes of microscopi c life, which is very helpful in food industry or pharmaceutical researches.The microorganisms survived in an alkaline environment for a long time evolved a unique mechanism, there may have basophilic enzyme gene in microorganism, study on the diversity of the alkalophilic microorganism and the functional gene screening will enrich our knowledge about the metabolism under extreme environments and explore enzymes with industrial potential.Haloalkane dehalogenases (HLDs) are key enzymes for degrading halogenated al iphatic pollutants. It can degrade chlorides including dichlorophenoxyacetic acid, tric hlorophenol, hexachloro-cyclohexane soprocide and mustard gas. So it was wildy use d in organo-chlorine pesticide degradation and chemical weapon elimination. In additi on, pesticide residues in crops, vegetables, tea and Chinese herbology could be checke d out by HLDs. Three are many disadvantages in HLD catalytic activity, stability and productive rate. Thus it is necessary to find out new haloalkane dehalogenases. The m ain results in this paper were shown as follows:1.Total soil DNA was extracted from soils in Ruanjiang, Hunan Province.16S rRNA gene was sequenced for microbial biodiversity research.there are four kinds of bactorias including Proteobacteria, Firmicutes,Bacteroidetes and Euryarchaeota. The similarity among BacBl,Shewanella putrefaciens and Shewanella hafniensis was 98%.One of the major bacterias, Methanosarcina mazei,was Anaerobic methanogenic archaea.lt can take H2/CO2,acetate, methyl alcohol, methylamine and trimethylamine as carbon sources to produce methane.This bactoria was scarely found in the environment of pH 9.0. Whether it can produce methane need a further test.2.Rich microbes were found in the samples and there were significant differences amo ng different sample pools.Metagenomic plasmid libraries were constructed firstly. An d then 36 amylase (4 of them were high expressed),9 lipases(2 lipases were high expr essed),4 protease(1 protease was expressed) and 4 cellulose (1 cellulose was high exp ressed) were coloned.3.Five HLDs genes were coloned from Huangia manganoxydans 8047 which obtained in Deep-sea Hydrothermal Vents and were high expressed in E.coli. it was easily purified and showed a stable property at high temperature.Enzymatic analyzation of DhmB showed that the most prompt reaction temperature was 65℃ and chlorides including dichlorophenoxyacetic acid, trichlorophenol, hexachloro-cyclohexane soprocide and mustard gas could be degraded.The similarity among DhmB and all the Dehalogenation enzyme genes was 63%(Aurantimonas manganoxydans SI85-9A1),which showed DhmB was a new gene.Huangia manganoxydans8047 can live at low temperatues, however, the dehalogenase can act at high temperature.