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" Production, purification, characterization of selected microbial lipases and their application for interesterification of butter fat "
F. S. K. Pabai
S. Kermasha
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Latin Dissertation
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English
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| Record Number
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1113324
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TLpq304398464
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| Main Entry
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F. S. K. Pabai
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S. Kermasha
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| Title & Author
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Production, purification, characterization of selected microbial lipases and their application for interesterification of butter fat\ F. S. K. PabaiS. Kermasha
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| College
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McGill University (Canada)
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| Date
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1997
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1997
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| Degree
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Ph.D.
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| Page No
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179
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| Abstract
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The screening, biomass production of lipase-producing microorganisms from several sources, as well as the purification, characterization and utilization of the enzymatic extracts for the interesterification of butter fat were investigated. Pseudomonas fragi CRDA 323 and Aspergillus niger CBS 131.52 were considered to be good lipase producers, whereas, those from Pseudomonas putida ATCC 795 and Rhizopus oryzae ATCC 34612 as weak ones; all four microorganisms produced maximal amount of extracellular lipases by batch fermentation after three-four days of incubation in a continuously stirred tank reactor. The lipases were partially purified by ammonium sulfate precipitation and characterized with respect to pH, kinetic parameters and molecular size. The lipases from P. fragi and P. putida were optimal at pH 8.5 and 8.0, respectively, whereas those from A. niger and R. oryzae were optimal at pH 7.5. The A. niger lipase had the lowest V{max} value usd\rm(0.51 \times 10\sp3\ U\ min\sp{-1});usd R. oryzae the highest usd\rm (1.86 \times 10\sp3\ U\ min\sp{-1}).usd The K{m} values for P. fragi, P. putida, A. niger and R. oryzae lipases were 0.70, 1.18, 0.97 and 0.98 mg ml respectively. Interesterification of butter fat by the partially purified enzymatic extracts in a microemulsion free co-surfactant system containing sorbitol monostearate and polyoxyethylene sorbitan monostearate in the ratio 48:52 (V/V) decreased the water activity as well as the hydrolytic activity. The P. fragi lipase had the highest interesterification yield value (43%) and the R. oryzae lipase the lowest (4%). In addition, P. fragi lipase exhibited the highest decrease (18%) in long-chain hypercholesterolemic fatty acids (C12:0, C14:0 and C16:0) at the sn-2-position; the P. putida lipase demonstrated the least favorable changes in specificity at the same position. Continuous cultivation technique was developed to investigate the screening for lipase-producing microorganisms from four commercial starters suitable for the degradation of domestic wastes; using this technique, three strains of lipase-producing bacteria were isolated within a relatively short period (12 days). Among the three strains isolated, that from P. fluorescens BW96CC1 was considered to be a potentially good candidate for further studies. Hence, a biomass of this newly isolated lipase extract (FI) from P. fluorescens BW96CC1 was partially purified by ammonium sulfate precipitation (FII) and successive ion-exchange (FIIIa) and size-exclusion (FIVa) chromatographies. The purified enzymatic fractions were characterized with respect to effects of pH on lipase activity and stability, optimum temperature and stability upon storage, kinetic parameters, electrophoresis, effects of metal ions and chemical reagents and substrate specificity. The results showed that the purified lipase fraction (FIVa) has a preferential specificity for hydrolysing triacylglycerols having long-chain saturated fatty acids (C12:0 to C18:0) as well as the unsaturated fatty acid (C18:1). The interesterification of butter fat by the purified fraction (FIVa) exhibited an important increase (31%) in the proportions of hypercholesterolemic fatty acyl moieties (C12:0, C14:0 and C18:0) at the sn-2 position, with a concomitant decrease of 4 and 19% in these same fatty acids at the sn-1 and sn-3 positions, respectively; furthermore, the results showed that this fraction favorably redistributed hypocholesterolemic fatty acids (C18:0 and C18:1) among the sn-1 and sn-3 positions of butter fat triacylglycerols.
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Aspergillus niger
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Biological sciences
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Butter fat
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Interesterification
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Lipases
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Pseudomonas fragi
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