Methyl hexanoate

Authentic standards of hexanal, 2-hexenal, nonanal, benzaldehyde, methyl acetate, methyl hexanoate, ethyl hexanoate, ethyl octanoate, 2-heptanone, butyric acid, hexanoic acid, and n-alkane standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Authentic standards of octanal-d16, ethyl butanoate-d3 and n-hexyl-d13 alcohol, used as internal standards, were purchased from C/D/N Isotope (Pointe-Claire, Quebec, Canada). Nanopure water used in the study was obtained from a water purification system (Milli-Q Direct 8, Merck Millipore, Billerica, MA, USA).

Cell culture media were purchased from Becton-Dickinson (Franklin Lakes, NJ, USA); chemical reagents such as methanol, acetic acid and potassium phosphate from Fisher Scientific (Waltham, MA, USA); N-acyl-l-homoserine lactones (except N-(3-oxohexanoyl)-l-homoserine lactone, which was from Santa Cruz Biotechnology, Dallas, TX, USA), l-homoserine lactone hydrochloride (HSL), penicillin V, phenoxyacetic acid (POA), methyl phenoxyacetate (MPOA), methyl hexanoate, methyl decanoate, p-dimethylaminobenzaldehyde (PDAB), o-phthalaldehyde (OPA), kanamycin, and DMSO from Sigma-Aldrich (Burlington, MA, USA); methyl octanoate and methyl dodecanoate from Fluka (Burlington, MA, USA); methyl butyrate from Acros Organics (Waltham, MA, USA); methyl butyrylacetate from Alfa Aesar (Tewksbury, MA, USA); and methyl 3-oxodecanoate and methyl 3-oxododecanoate from Toronto Research Chemicals (Toronto, ON, Canada). 6-aminopenicillanic acid (6-APA) and natural aliphatic penicillins (penicillin K, penicillin F and penicillin dihydro-F) were provided by Antibióticos S.A. (León, Spain). All reagents used showed a purity level of at least ≥95%. Oligonucleotides were synthesized by Sigma-Aldrich (Burlington, MA, USA).

All triglyceride substrates were purchased from Sigma-Aldrich Canada (Oakville, ON) using the highest purity available. Analytical standards for methyl acetate, isoamyl acetate, methyl butyrate, methyl hexanoate, methyl octanoate, ethyl butyrate, propyl butyrate, butyl butyrate and amyl butyrate were also purchased from Sigma-Aldrich. Analytical grade methyl nonanoate was purchased from Chromatographic Specialties (Brockville, ON). All other chemicals were obtained commercially and were of analytical grade.
The strain Escherichia coli DH11S was employed for all subcloning procedures. The strain E. coli BL21 (DE3) was employed for the heterologous expression of the lipase gene. Expression vector pET16b (EMD Millipore) was used for the production of the N-terminal histidine-tagged recombinant protein. Plasmid pIAFD95A was used as template for the amplification of the lipIAF5-2 gene (Genbank EU660533) [22] (link).

Absolute ethanol (>99.5%) and sodium chloride (99.5%) were purchased from Scharlab (Barcelona, Spain). Ultrapure water (18 MΩ cm⁻¹) was obtained from a Milli-Q system (Millipore, Milford, MA, USA). The volatile standards, with purity above 99%, were supplied by Chemservice (West Chester, PA, USA) and Aldrich (Gillingham, UK): methyl butanoate, ethyl butanoate, methyl hexanoate, ethyl hexanoate, hexyl hexanoate, cis-3-hexenyl acetate, trans-2-hexenyl acetate, hexanal, trans-2-hexen-1-al, benzaldehyde, 1-hexanol, trans-2-hexen-1-ol, benzyl alcohol, linalool, geraniol, cis-nerolidol, nerol, mesifurane, furaneol, 2-methylbutanoic acid, 3-methylbutanoic acid, hexanoic acid, γ-nonalactone, and Δ-decalactone. 2-octanol (internal standard, IS) was obtained from Fluka (Madrid, Spain). Individual stock solutions at 1000 mg L⁻¹ for each compound and IS were prepared in ethanol and stored at −20 °C.

Pure reference standards of methyl-2-methylbutanoate (98.0%), 2-hexanol (97.0%), 3-methylbutanoic acid (97.5%), methyl butyl acetate (98.0%), methyl-2-methylpentanoate (99.5%), gamma-butyrolactone (98.0%), dimethyl malonate (97.0%), ethyl-2,3-dimethylbutanoate (99.5%), methyl-2-methyl acetoacetate (99.5%), methyl-2-hydroxy-2-methylbutanoate (98.0%), methyl hexanoate (99.5%), methyl-3-(methylthiol)-propanoate (99.5%), hexanoic acid (97.0%), trans-β-ocimene (98.0%), methyl-2-methylhexanoate (99.5%), ethyl hexanoate (98.0%), δ-lactone (98.0%), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (99.5%), methyl-3-hydroxyhexanoate (99.5%), 2,5-dimethyl-4-methoxy-3(2H)-furanone (98.0%), methyl octanoate (99.5%), octanoic acid (97.0%), y-octalactone (98.5%), δ-octalactone (98.0%), copaene (97.0%), methyl decanoate (99.5%), 2-methyl-4-vinyl phenol (99.5%), decanoic acid (97.0%), y-farnesene (98.0%), germacrene (98.0%), globulol (98.0%), spathulenol (98.0 5), (Z)-7-tetradecenal (97.0%), and octadecanal (99.5%) were purchased from Aldrich, Steinheim, Germany. Gamma-lactone (98.0%) and methyl dodecane (99.5%) were obtained from Parchem, New Rochelle, NY and Achemica Corp. Aigle, Switzerland, respectively. The n-alkane standard (C₇–C₃₀) was obtained from Sigma-Aldrich Chemicals Co. (St. Louis, MO). Other chemicals were of analytical grade.