PRODUCTION, PURIFICATION, CHARACTERIZATION AND APPLICATION OF ORGANIC SOLVENT TOLERANT LIPASE FROM TRICHODERMA SP. BW45 IN PALM OIL HYDROLYSIS
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Date
2010
Authors
AL-LIMOUN, MUHAMAD ODEH ATYEH
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Abstract
In order to establish a successful process of enzymatic hydrolysis of palm oil, for the
production of palmitic acid and oleic acid, the following objectives have been
designated: Screening for potential microorganisms producing a significant amount of
extracellular organic solvent tolerant lipase from indigenous soil samples,
optimization of the physicochemical parameters required to achieve maximum lipase
production in shake flask system, purification of the enzyme using aqueous two phase
system coupled with Sephadex G-75 gel filtration, characterization of the pure
enzyme properties and application of the crude lipase preparation in palm oil
hydrolysis for the production of palmitic and oleic acids.
Microorganisms producing lipase were isolated from indigenous soil samples on NA
and PDA plates supplemented with tributyrin. A total of 21 lipolytic colonies were
further cultivated in submerged fermentation media and their crude extracts were
screened for organic solvent tolerant lipase. Two isolates were selected, bacterial
isolate BW16 and fungal isolate BW45, based on their lipase stability in presence of
broad range of hydrophobic organic solvents. The bacterial isolate BW16 was
identified as Bacillus megaterium species and the fungal isolate BW45 was identified
as Trichoderma species. The comparative characterization of both enzyme properties
revealed that isolate Bacillus megaterium BW16 lipase as water miscible organic
solvent tolerant enzyme. Meanwhile, lipase enzyme produced by Trichoderma sp.
BW45 as water immiscible organic solvent tolerant enzyme. Maximum activity of
Bacillus megaterium lipase was obtained at 45°C and pH 9.0. Trichoderma sp. BW45
lipase enzyme exhibited broader range of optimum temperature for maximum
hydrolytic activity in the range of 30 to 45°C at pH 6.0. Both enzymes were revealed
a moderate thermostability. Trichoderma sp. BW45 lipase enzyme exhibited broader
range of pH stability ranging from pH 4.0 to pH 9.0 compared to Bacillus
megaterium BW16 lipase which exhibited pH stability in the range of 7.0 to 10.0. In
addition to the characteristics mentioned above for Trichoderma sp. BW45 and
Bacillus megaterium BWI6, high initial productivity of Trichoderma sp. BW45
lipase enzyme (138.8 U/mL) compared to Bacillus megaterium (7.8 U/mL), the high
preference for long carbon chain triglycerides and the stability in the hydrophobic
organic solvents made the suitable choice for palm oil hydrolysis is the lipase
produced by Trichoderma sp. BW45. The physical and chemical parameters effect on
lipase production by Trichoderma sp. BW45 in shake flask system using stepwise
optimization approach was investigated. Maximum lipase activity of 621.0 ± 1.3
U/ml in the culture filtrate was obtained after 60 hr cultivation when the fermentation
medium (pH 8.0) composed of 0.5% (w/v) starch, 1 % (w/v) yeast extract, 0.3% (w/v)
Tween 80 and 0.5% (w/v) NaCI was inoculated with 1.4 x 105 spore suspension and
incubated at 25°C and 150 rpm agitation speed.
Partially pure enzyme solution with 2.78 folds enhancement in lipase purity and total
recovery of 56.4% was obtained in the bottom salt-rich phase using A TPS composed
of 11 % (w/w) PEG 6000 and 8.6% (w/w) potassium phosphate buffer (pH 6.0).
Further purification of the enzyme to homogeneity level was achieved by filtration
chromatography using sephadex G-75. SDS-PAGE electrophoresis showed a single
band protein with a relative molecular mass of 61 KDa. The purified enzyme
exhibited a pJ value approximately equal to 6.2 using immobilized pH gradient strip
and the enzyme displayed random regio-specificity in triolein hydrolysis. The pure
enzyme maintained the original activity after exposure to cyclohexane, n-hexane, nheptane
and isooctane. Meanwhile, with water miscible organic solvents the enzyme
exhibited reduction in the activity. However, the pure enzyme showed maximum
activity in the temperature range of 30 to 40°C and pH 6.5 and the enzyme
maintained 100% of the original activity in pH range of 4.0 to 7.0 and up to 40°C. No
inhibitory effects on the enzyme activity were observed when various metals and 1.0
mM of 2-Mercaptoethanol, EDTA and SDS were added. On the other hand, addition
of different concentrations of Tween 20 and Tween 80 to the assay reaction mixture
showed inhibitory effects on the enzyme activity and this inhibition in the enzyme
activities was increased with an increase in the concentration. The lipase showed a
preference to C4 acyl group of the p-nitrophenyl monoesters. On the other hand, the
enzyme showed higher preference specificity towards the synthetic triglyceride
triolein compared to tributyrin.
Maximum hydrolysis degree of palm oil of 94.86% ±0.58 was achieved after 48 hr
hydrolysis when 1: 1 aqueous to non-aqueous phase was operated at 25 ± 1 °C and 600
rpm stirring speed. The optimum concentration of enzyme in the aqueous phase was
found to be 434 U crude enzyme per gram palm oil adjusted to pH 6.0 using
phosphate buffer and the optimum non-aqueous phase contained 50 g of palm oil
dissolved in isooctane in the ratio of 1 :0.5 (w/v). Nevertheless, after optimization of
the hydrolysis process, 233.34 and 195.02 mg of palmitic acid and oleic acid,
respectively were detected in each mL of the non-aqueous phase using Gas
Chromatography analysis. The reusability of lipase was tested and it was found that
nearly 93.7% palm oil hydrolysis was achieved after the first recycle
Description
Keywords
CHARACTERIZATION , ORGANIC SOLVENT