Screening And Analysis Of Halogenase And Esterase In Metagenomic Library

Soil was such a diverse ecosytem,that millions of microbes were contained in just one gram of soil.Microbe was one of the main sources of bioactive natural products,for example antibiotics,anticancer drugs and biological enzymes.However,only<1%of microbes could be cultured under standard laboratory condition.Metagenomic method was to regard the whole soil genome as a unit rather than culture soil microorganisms first,and the whole genome was soil metagenome.Metagenomic technology could transfer the gene of uncultured microbal into cultured microbal,then research on gene function and natural products expression could be carried on.As a result,new genes and natural products would be found.Through direct lysis,the environmental total DNA of soil samples(sited in Yunnan)was obtained.The humus was removed by centrifugation and electrophoresis.The purified eDNA need dialysis and concentration before being used to construct metagenomic library.Finally,YNF3(10,000,000 clones)and YNF8(100,000 clones)metagenomic cosmid libraries were constructed,with an average insert size of 37.3 kb and 38.2 kb,respectively.The halogen substituents could deeply affect the biological activity of many natural products.The halogenated process of natural products was usually catalyzed by halogenase.Through phylogenetic analysis of halogenase related gene,the possibility of producing compounds could be inferred.Through sequence-based screening of clones containing halogenase gene,we could gain the chance to get access to the halogenated natural products.Sixteen halogenated clones were obtained through sequence based screening of YNF3 and YNF8 library.Phylogenetic analysis showed that the sixteen clones had the potential to produce halogenated compound.Esterase was belonged to hydrolase and decomposed water soluble short chain fatty acid esters(<10 carbon atoms).Esterase showed many applications in different aspects,such as fat modification to improve the taste and nutritional value in food industry,remove the asphalt of pulp,as additive in detergents,production of biodiesel.Through functional screening,a new esterase EstGXl was obtained from YNF8 library,and biochemical characteristics of EstGXl were studied.The preference substrate was C4,while no hydrolysis of C14 was detected.It suggested that EstGXl was a carboxylesterase.EstGX1 was an alkaline eaterase with an optimum pH 9.0.The optimum temperature of EstGX1 was 46.4?.EstGXl still hold more than 20%of relative activity at 4?.After incubation at 50? for 50 min,EstGXl still hold more than 60%of relative activity.EstGX1 was tolerant to several surfactants(such as Tween-20,Triton X-100)and organic solvents(such as ethanol,isopropanol,DMSO).The Km value of EstGXl was 45.12 ?M.The specific activity of EstGX1 was 57.23 U.mg-1 and Vmax was 8.07 ?mol.min-1.EstGX1 was tolerant to several surfactants which indicated that it could be a potential candidate in washing industry.