Using Soil Metagenome To Obtain Novel Lipolytic Enzyme

Lipolytic enzymes include lipases (EC 3.1.1.3), which hydrolyze long chain acylglycerols (≥C10) and esterases (EC 3.1.1.1), which hydrolyze short chain acylglycerol (≤C10).Lipolytic enzymes are important biocatalysts for various industrial applications because of their ability to substrate specificity and stereo-selective organic reactions. Recently, an increasing number of drugs and organic materials have been produced through enzyme-catalyzed reactions. The classical approach to finding useful lipolytic enzymes and other is pure culture-based, cumbersome screening of a wide variety of microorganisms for the desired lipolytic activity. However, it has been estimated that up to 99% of the bacteria present in environment cannot be cultured in laboratory by traditional culture techniques. This vast diversity of"yet to be cultured"bacteria represents a large gene pool for biotechnological exploitation. To access the genome resource of uncultured microorganisms, there have been developed several cultural-independent method. A significant uncultural approach is to screen directly novel biocatalysts from a"metagenome library"constructed by isolation of genomic DNA from whole microbes directly from soils and cloning them into various bacterial vectors. Using metagenomic libraries have successfully cloned genes from soil communities that code for lipolytic enzymes, proteases, oxidoreductases, amylases, antibiotics, antibiotic resistance enzymes.In this study, the metagenomic DNA were isolated indirectly from loam topsoil and further purification of the DNA was performed through direct extraction from the agarose gels. The yield of purified DNA was approximately 0.7μg/g loam topsoil. A metagenomic library from 66 g loam soil was constructed, which contain 8000 clones with an average DNA insert size of 3 kb. The metagenomic library represented about 2.4 Mb of soil DNA. Two unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. The nucleotide sequence of the positive clone was determined using primer walking method.Bioinformatic analysis indicated that each clone contained one lipolytic activity ORF,designated as e-1 and e-2, respectively. Esterase gene e-1 consisted of 834 bp encoding a polypeptide of 277 amino acids with a molecular mass of 29998.4 Da. The deduced amino acid sequence showed similarities with the carboxylesterase of alpha proteobacterium BAL199, which feature identities of 48%, indicating that they belong toα/βhydrolase 3 super family and prolyl oligopeptidase family. Residues essential for esterase activity, such as pentapeptide (GXSXG) and catalytic triad sequences, were uncovered. While the protein was overproduced mainly as inclusion body. E-1S had a temperature optimum at 50°C and a pH optimum at pH 9.0. The best substrate for the enzyme among the p-nitrophenyl esters (C2 to C16) examined was p-nitrophenyl butyrate (C4), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.Putative esterase e-2 consisted of 693 bp encoding a polypeptide of 230 amino acids with a molecular mass of 22789.5 Da. The deduced amino acid sequence showed similarities with the lipolytic enzyme of Ralstonia pickettii 12D, which feature identities of 42%, indicating that they belong to GDSL hydrolase family. N-terminal of E-2 containing the conserved sequence motifs of GDSL esterases/lipases, such as GDSL (residues 49-53)and the oxyanion hole. We also had constructed recombinant expressing plasmid to overproduce the E-2 proteins. However, E-2 has no lipolytic activity.