title: Isolation of an organic solvent-tolerant lipolytic enzyme from uncultivated microorganism.
abstract: Although the use of lipases as biocatalysts has frequently been proposed, it is yet scarcely being implemented in industrial processes. This is mainly due to the difficulties associated with the discovery and engineering of new enzymes and the lack of versatile screening methods. In this study, we screened the available strategy from a metagenomic pool for the organic solvent-tolerant lipase with enhanced performance for industrial processes. A novel lipase was identified through functional screening from a metagenomic library of activated sludge in an Escherichia coli system. The gene encoding the lipase from the metagenomic pool, metalip1, was sequenced and cloned by PCR. Metalip1 encoding a polypeptide of 316 amino acids had typical residues essential for lipase such as pentapeptide (GXSXGG) and catalytic triad sequences (Ser160, Asp260, and His291). The deduced amino acid sequence of metalip1 showed high similarity to a putative lipase from Pseudomonas sp. CL-61 (80 %, ABC25547). Metalip1 was expressed in E. coli BL21 (DE3) with a his-tag and purified using a Ni-NTA chelating column and characterized. This enzyme showed high expression level and solubility in the heterologous E. coli host. This enzyme was active over broad organic solvents. Among organic solvents examined, dimethyl formamide was the best organic solvent for metalip1. We showed that function-based strategy is an effective method for fishing out an organic solvent-tolerant lipase from the metagenomic library. Also, it revealed high catalytic turnover rates, which make them a very interesting candidate for industrial application.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany, chroh@kaeri.re.kr.
authors.forename: Changhyun
authors.initials: C
authors.lastname: Roh
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
mesh_terms: D000595:Amino Acid Sequence; D064349:Genome, Microbial / Q000235:genetics*; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008066:Lipolysis; D056186:Metagenomics; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D009930:Organic Chemicals; D011994:Recombinant Proteins; D012997:Solvents; D008049:Lipase