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