Crystallization and preliminary crystallographic analysis of LipC12, a true lipase isolated through a metagenomics approach.

LipC12, a true lipase from family I.1 of bacterial lipases which was previously isolated through a metagenomics approach, contains 293 amino acids. Among lipases of known three-dimensional structure, it has a sequence identity of 47% to the lipase from Pseudomonas aeruginosa PAO1. Recombinant N-terminally His(6)-tagged LipC12 protein was expressed in Escherichia coli, purified in a homogenous form and crystallized in several conditions, with the best crystals being obtained using 2.0 M sodium formate and 0.1 M bis-tris propane pH 7.0. X-ray diffraction data were collected to 2.70 Å resolution. The crystals belonged to the tetragonal space group P4(1)22, with unit-cell parameters a = b = 58.62, c = 192.60 Å.

Isolation of a novel lipase from a metagenomic library derived from mangrove sediment from the south Brazilian coast.

A novel gene coding for a LipA-like lipase with 283 amino acids and a molecular mass of 32 kDa was isolated and characterized from a metagenomic library prepared from mangrove sediment from the south Brazilian coast. LipA was 52% identical to a lipolytic enzyme from an uncultured bacterium and shared only low identities (< or =31%) with lipases/esterases from cultivable microorganisms. Phylogenetic analysis showed that LipA, together with an orthologous protein from an uncultured bacterium, forms a unique branch within family I of true lipases, thereby constituting a new lipase subfamily. Activity determination using crude extracts of Escherichia coli bearing the lipA gene revealed that this new enzyme has a preference for esters with short-chain fatty acids (C < or = 10) and has maximum activity against p-nitrophenyl-caprate (chain length C10, 0.87 U/mg protein). The optimum pH of LipA was 8.0, and the enzyme was active over a temperature range of 20 to 35 degrees C, with optimum activity against p-nitrophenyl-butyrate at 35 degrees C and pH 8.0.

A broad pH range and processive chitinase from a metagenome library.

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked ß(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

A broad pH range and processive chitinase from a metagenome library

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.