Introduction

Current industrial practices have led to an enormous generation of various crude fatty materials as waste that are difficult to treat and valorise (Fickers et al., 2005). Among these, the important ones are lipid waste from slaughterhouses. Slaughterhouse lipid waste, tallow, is a fat-rich material and has been used as raw material for the production of low value products like soap and detergents. Recovery of high value - added products from the above solid wastes has been largely a neglected field due to the survival of microorganisms in such highly hydrophobic substrates as a result of which the bio - transformations into value added products becomes difficult. It has been reported that fermentation of lipids under acidic conditions favour the surface attachment of the micro organisms onto substrate. Hence, there has been a constant search for the screening of extremophiles for lipid hydrolysis with the emphasis to produce high value added products such as highly active and stable lipases. In the present study, the lipolytic strain, Pseudomonas gessardii with high yield coefficient and high lipase yield was isolated from the tallow acclimatized soil. There are no reports available in literature on the utilization of slaughterhouse lipid waste for the production of lipase. Thus, the present study was focused on the production of lipase from Pseudomonas gessardii using slaughterhouse lipid waste as a substrate.

Substrate

Slaughterhouse waste, namely goat tallow, was used as a substrate in the present study. It is a whitish solid substance rich in fat with a mild odour which was obtained from a slaughterhouse in Chennai.

Isolation and identification of P. gessardii

Lipase producing microbial cultures was isolated from the tallow acclimatized soil. 10 g of tallow was buried in black soil for a period of 3 weeks. During this period, the soil microbes utilize the tallow as a nutrient (carbon) source. The decomposed tallow along with contaminated soil were dispersed in nutrient broth (NB) of composition peptone (5.0 g/l); yeast extract (1.5 g/l); beef extract (1.5 g/l); NaCl (0.5 g/l) and was incubated for 48 h at 37^oC. The cultured broth was plated on tributyrin agar and incubated at 37^oC for 24 h. The colonies showing clear zones were picked out from the plate and inoculated into the basal medium containing the goat tallow for the maximum yield of lipase. Among the fifteen isolated strains, one strain exhibited a better lipolytic activity than the others and thus it was selected for the production of lipase in our study. The strain was identified by 16S ribosomal DNA (16S rDNA) sequencing and phylogenetical analysis. Based on nucleotides homology (16S rDNA sequencing) and phylogenetical analysis, the organism was identified as Pseudomonas gessardii. The 1466 bp sequence was submitted to GenBank (NCBI) and the accession number “FJ943496” obtained. The BLAST algorithm was used to search for homologous sequences in GenBank.

Production, purification and characterization of acidic lipase from goat tallow using P. gessardii

P. gessardii was grown in the basal medium containing KH₂PO₄ (1.0 g/L); NH₄(SO₄)₂ (0.5 g/L); CaCl₂ (1.0 g/L); 0.31 % (w/v) goat tallow and 1% (v/v) gum arabic at pH 3.5. The maximum lipase production (52 U/ml) was found at the beginning of the stationary phase in the presence of goat tallow as a substrate at pH 3.5 and temperature 37^oC. The glycerol and fatty acid formation during the hydrolysis of goat tallow is presented in Fig. 1.

Fig. 1. Effect of time on the formation of glycerol and fatty acids from the goat tallow

This shows the maximum concentration of 830 mg/L and 7152 mg/L of glycerol and fatty acid respectively, which was produced from the 12444 mg/L of lipid substrate. Such high concentration of fatty acids might be formed due to the affinity of lipase towards the polar group of lipid substrate, goat tallow, because lipase attaches onto glyceride bond leading to the formation of 3 fatty acid molecules for every one molecule of glycerol. This higher yield of free fatty acid is possible only with high polar lipases which has high selectivity in solvating the glycerol moiety. The lipase was characterised by high polar/apolar amino acid ratio of 10.02. This is the highest ratio compared to other lipases in the literature. The maximum lipase production was observed at a pH of 3.5, thus the lipase production may be regarded as extremely acidic lipase. The purified P. gessardii acidic lipase was able to tolerate a broad range of pH from 1.0 to 6.0 with maximum lipase activity at pH 3.5. The enzyme remained stable with in the pH range of 1.0 to 5.5 (Fig. 2).

Fig. 2. Relative lipase activity and stability of purified acidic lipase at different pH

On the other hand many Pseudomonas sp. reported in the literature were known for alkaline lipases, which were stable in the pH range of 8.5 to 10 (Lin et al., 1996 ; Ogino et al., 2000; Rahman et al., 2005). There is no report on the production of lipase from bacterial strain, P. gessardii using goat tallow and other lipid substrates. The acidic lipases (less than pH 4.0) have wide potential for application in medicinal field as a substitute for pancreatic lipases in enzyme therapy (Sani, 2006).

The crude acidic lipase was purified and the specific activity of the purified acidic lipase was 1473 U/mg protein. The molecular weight of purified acidic lipase was 94 kDa corresponding to a well defined single band which is the characteristic feature of monomeric enzyme (Fig. 3). The purified acidic lipase from fungal source (Aspergillus niger) was reported to have lower molecular weight of 32.2 kDa (Mhetras et al., 2009). The high molecular weight acidic lipase produced by P. gessardii is due to the fact that the substrate goat tallow used in the present study was of high molecular weight consisting of 18 oleic acid molecules arranged in a chain and it is embedded in palmitic acid residue. It is known that the essential characteristic of enzymes to cleave or solvate the high molecular weight substrates must possess more number of active sites i.e., with large number of peptide linkages. Thus, enzymes with larger number of peptide bonds are characterised by high molecular weight. This is reflected by high protein (116 mg) and total amino acid content (102 mg) of P. gessardii compared to other lipases. 

Fig. 3. SDS-PAGE of purified acidic lipase from P. gessardii.
Lane 1: Molecular mass standards (14.3 kDa to 94.7 kDa).
Lane 2: Lipase (10 µg), purified by ammonium sulfate precipitation in combination with ion
exchange chromatography and gel filtration chromatography.

Conclusion

The extremely acidic lipase producing strain, Pseudomonas gessardii was isolated from tallow acclimatized black soil and identified by 16S rDNA sequencing. The presented lipase is a novel, extremely acid tolerant, biocatalyst from P. gessardii using goat tallow as a substrate, with wide industrial applications potential. Slaughterhouse waste, goat tallow, can be considered as a potential lipid substrate to produce extremely acidic lipase with high lipase activity. The maximum lipase production was observed at a pH of 3.5, for 48 h with the temperature of 37^oC. The purified lipase was highly stable under extremely acidic conditions (pH 1.0 to 5.5). The acidic lipase was characterised by high polar/apolar amino acid ratio than the other Pseudomonas lipases reported in the literature. The purified lipase from P. gessardii has a high molecular weight enzyme. The acidic lipase can be exploited in the hydrolysis of lipid wastes from slaughterhouses, households and oleochemical industries.

References:

Fickers, P., Benetti, P. H., Wache, Y., Marty, A., Mauersberger, S., Smit, M. S. and Nicaud, J. M. (2005) Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Research. 5, 527 - 543.

Lin, S. F., Chiou, C. M., Yeh, C. M. and Tsai, Y. C. (1996) Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111. Appl. Environ. Microbiol. 62, 1093 - 1095.

Mhetras, N. C., Bastawde, K. B. and Gokhale, D. V. (2009) Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207. Biores. Technol. 100, 1486 - 1490.

Ogino, H., Nakagawa, S., Shinya, K., Muto, T., Fujimura, N., Yasudo, M. and Ishikawa, H. (2000) Purification and characterization of organic solvent tolerant lipase from organic solvent tolerant Pseudomonas aeruginosa LST-03. J. Biosci. Bioeng. 89, 451 - 457.

Rahman, R. N. Z. R. A., Baharum, S. N., Basri, M. and Salleh, A. B. (2005) High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5. Analytical Biochemistry. 341, 267 - 274.

Sani, D. G. (2006) Step up: Bacterial lipase to substitute pancreatic lipase for enzyme therapy. Available from: http://microbepundit.blogspot.com/2006/10/step-up-bacterial-lipase-to- substitute.html. 

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