A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters

Construction and characterization metagenomic library

Fosmids to constructing metagenomics libraries

more than 40,000 fosmid clones = 1.6 Gb of DNA

Screening and identification of lipolytic clones

Ability of the clones growing on LB chloramphenicol plates.

New transformants characterized by hydrolysis halos (2 to 14 mm)

Incubate for 48 h at 37 °C

Five enzymes; Fos-est1, Fos-est2, Fos-est3, Fos-est4 and Fost-est5

Sub-cloning and sequence

Identify the genes that encodes the enzymes

Each fragment was cloned into pBluescript II SK(+)

the potencial genes were identified on ORF analysis and BlasP alignments

Phytogenetic relationships (lipolytic enzymes)

The enzymes can be grouped in 3 families

EST4 family V, a/n hydrolase fold
EST3 family VIII, b-lactamases
EST5 family VII carboxylesterase

Heterologous expression

Lipolytic genes amplified cloned into pET-28a(+) vector witj 6x

overexpressed in E. coli Top10F′/pLLP-OmpA with a C-terminal His6-tag

High expression levels of the enzymes

EST4 purified to electrophoretic homogeneity trough nickel affinity chromatography

Substrate specificity

catagorized as lipases that hydrolyze ester bonds or emulsified lipid substrate

EST4 tasted its ability to hydrolyze esters

Effect of temperature and pH/ metal ions and orgnic solvents

Ideal pH at 8, high activity at alkaline conditions

T range 20 to 60°C(increase activity at 45°C)

EST4 activity decrease with hydrophilic organic solvents

No stimulatin in activity , metal ions decreses the esterase activity

increase in esterase activity with 0.5% of Tween

Methods

Chemical and reagents

Phanta super-fidelity DNA polymerase for DNA amplification , CIP, restriction enzymes (NotI and Sau3AI)

Bacteria strains, plasmids and growth conditions

E.coli EPI1300-T1R and pCC1FOS vectors used to contruct the library

E. coli Top10Fʹ/pLLP-OmpA as recombinant protein

E. coli DH5α and the pBluescript II SK(+) for subcloning and sequencing

DNA extraction and purufucation from marine mud

DNA isolated using Mo Bio Power Soil DNA isolation kit

To remove co-extracted substances was purified with ethanol

Metagenomix library

constructed with CopyControl pCC1FOS Fosmid Library Production Kit

For lipolytuc activity screening, diluted, incubated on LB agar with Chl and emulsified tributryn at 37°C for 3 days

subcloning and sequence

Clones with clear halos selected as positive was sequenced

Cloning and expression of lipolytic

C.terminal His-tag in recombinant target protein . recombinant expression plasmids transformed in E.coli

Purification of EST4

Purufucate with buffer A, supernatant in NTA column , wash the column with imidazole. Fraction contain the target protein

Effects of detergents, metal ions and organic solvents

Concentration of 1 and 5mM

organic solvents investigated using method of Li el at

Synthesis of short-chain flavor esters

Trasesterificaation reaction was alcohol and vinyl acetate

Analized by gas chromatography

analytical methods

Usinf 6890 gas chromatograph

Sepatation was on a HP-5 capillary column