Organic solvent resistant microorganism which secretes lipolytic enzyme

MARTINEZ ALEJANDRA; TORRES SEBASTIÁN; DEL CASTILLO LOURDES; CASTRO GUILLERMO R.

Orlando, Florida, USA

Congreso; American Society for Microbiology (ASM) – 101st General Meeting; 2001

American Society for Microbiology (ASM)

Lipases are hydrolases acting on water poor soluble substrates such as triglycerides and esters. Non-aqueous enzymology has become an area of interest to chemical and pharmaceutical industries. Recent reports have shown that enzymes produced by organic solvent resistant microorganisms were stable in presence of organic solvents.  The aim of this work was to isolate thermoresistant and organic solvent resistant microorganisms that produce lipolytic enzyme. Heat shocked soil samples (80ºC, 10 min) were cultured in LB medium supplemented with NH4NO3 and tributyrin at  55ºC. Screening for microorganims lipolytic activity was performed on tributyrin agar. Organic solvent resistant bacteria were selected in liquid medium with isoamyl alcohol  (from 0.1 to 1%) at 55ºC. Enzyme activity was assayed using p-nitrophenyl derivatives. Effects of organic solvents on hydrolytic activity were carried out using six hydrophylic solvents. Strain characterization was made using biochemical and molecular approaches (API system and PCR-based techniques: 16S amplification, sequence analysis, ITS-PCR and t-RNA-PCR). Considering the highest lipolytic activity and organic solvent resistance (it was able to grow in presence of 0,6 % of isoamyl alcohol) strain S-86 was selected. From morphological, biochemical and molecular characteristics, strain S-86 was identified as Bacillus licheniformis. Optimal growth temperature was 50ºC (m = 3,12 h-1 in medium without solvent; m = 1,55 h-1 in medium with 0.4 % of isoamyl alcohol). Enzyme production was maximal at the stationary phase in both media (with and without isoamyl alcohol), and it was the highest in  the medium with 0.4% organic solvent at 50ºC (5.37 U/mg). Relative enzyme activities at 50% solvent composition were in glycerol (81%), ethanol (54%), isopropanol (50%), 1,3-propanediol (38%), acetone (22%) and propanol (4%). Optimal temperature for enzyme activity was between 60 to 70ºC.  Because of its organic solvent resistant and high activity, the enzyme from Bacillus licheniformis S-86 can be used as biocatalytic in non-aqueous media.