Production and Characterisation of Cell-Bound Lipases Secreted by a Newly Isolated Strain of Geotrichum Candidum
Indigenous lipolytic microorganisms were successfully isolated from soil samples collected from an oil palm plantation and were identified up to the generic level. Over seven hundred microbial colonies were screened and fifteen were found to be positive for the hydrolysis of triolein. Of these, t...
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| Format: | Thesis |
| Language: | English English |
| Published: |
1990
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| Online Access: | http://psasir.upm.edu.my/id/eprint/8347/1/FSMB_1990_5_A.pdf http://psasir.upm.edu.my/id/eprint/8347/ |
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| Summary: | Indigenous lipolytic microorganisms were successfully isolated from soil samples
collected from an oil palm plantation and were identified up to the generic level. Over
seven hundred microbial colonies were screened and fifteen were found to be positive for
the hydrolysis of triolein. Of these, three were yeast species, another three were strains
of Geotrichum candidum and the rest were bacteria. Studies on the lipolysis of various
natural oils on solid media and in liquid media by the yeasts and the G. candidum strains
showed that the latter were the most potent lipolytic organisms. One of the yeasts was
found to be weakly lipolytic. These organisms shared two common features: they were
not able to hydrolyse tributyrin and they hydrolysed palm kernel olein, which is a lauric
acid oil, poorly. Results obtained indicated that these organisms probably elaborated
extracellular lipases that possessed some degree of fatty acid specifiCity. The cultural conditions for the maximal hydrolysis of palm olein and for the
production of extracellular lipases, both soluble and cell-bound, in submerged culture
were determined for one of the G. candidum strains. The optimal pH for lipolysis and
maximal production of lipases occurred at pH 7.0 - 7.2. It was discovered that the soluble
lipase of this organism was produced constitutively. The cell-bound lipase, however, was
found to be an inducible enzyme where production took place only when an oil was added
to the culture medium. The type of oil used did not affect production significantly but the
presence of sugars and glycerol decreased lipase productivity markedly. High levels of
glycerol suppressed growth of the organism.
The cell-bound lipase was characterised and was shown to be most active at 43°C
and preferred p-nitrophenylcaprylate as the substrate. The kinetics of the hydrolysis of
various fatty acid esters of p-nitrophenolwere studied and the Km and Vmax values are
presented. When the enzyme was stored at 4°C, a second temperature optimum
developed at 30˚C. Continued storage resulted in an increase in the activity at 30˚C with
a concomittant decrease in activity at 43˚C. After 6 days, the temperature optimum at
430C was completely lost The shift in temperature optimum from 43˚C to 30˚C could be
quickly achieved by heating the ceU-bound lipase at 4O"C for 2 h.
The extraction of the cell-bound lipase of G. candidum was simply and easily
achieved by shaking induced cells in a buffer solution. Complete extraction could be
accomplished in 4-5 h and the total enzyme activity recovered was 4.6-fold greater than
what was initially measured and found to be bound to the ceUs. Magnesium ions
when added to the extraction buffer caused a delay in the release of the enzyme from the cells. The most efficient pH for extraction was pH 8.4. The extracted lipase was most
active at pH 7.8. This enzyme had two temperature optima : 33°C and 4O˚C. The
temperature optimum at 33°C was observed only upon storage of the enzyme extract at
4°C. When in the soluble form, the cell-bound lipase preferred p-nitrophenylpalmitate
as the substrate, instead of p-nitrophenylcaprylate. The Km and Vmax values of the
enzyme for this ester was 6.7 mm and 6.3 x 10₃ umol/min, respectively. The rate of
hydrolysis of olive oil exceeded the rate of hydrolysis of tributyrin by 4 times. The profiles
of the hydrolysis of a number of fatty acid esters of p-nitrophenol, olive oil and tributyrin
of the extracted lipase and those of several commercial lipases were obtained and
compared.
Purification of both the extracted lipase and soluble lipase was performed and the
results obtained are presented. Gel filtration of cell-bound extract and soluble lipase
extract on Sephadex G-150 revealed that the G. candidum produced at least two cellbound
lipase and two soluble lipase isozymes. The molecular weights of the bound lipases
were estimated to be 75,000 and 58,000-61,000, respectively. |
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