PENGHASILAN DAN PENGGUNAAN ENZIM PENGURAI LIGNIN OLEH PHANEROCHAETE CHRYSOSPORIUM ME 446 SURDS
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Date
1997-04
Authors
IBRAHIM, DARAH
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Abstract
The optimization of some governing parameters in the enzyme production
revealed that an increment of 50% was obtained using the medium
composition containing 0.1 % (w/v) carbon source; 2.4 mM . ammonium
dihydrogen phosphate and 0.4 mM veratryl alcohol. The cultivation was
performed at 35 - 37°C, with an agitation speed of 150 rpm and inoculum size
of 6 x 106 spores/ml. The production of lignin degrading enzymes by P.
chrysosporium was found to be significant in the presence of complex
polysaccharides such as barly straws and rice husks. The maximum lignin
peroxidase activity after four days of cultivation was 82.0 mU/ml, while the
activity of manganese.peroxidase was 58.0 - 63.0 mU/ml after seven days.
The higher production of lignin peroxidase and manganese peroxidase by free
cells of P. chrysosporium of about 520.0 mU/ml and 350.0 mU/ml, respectively,
was obtained when cultivated in a tubular air-lift bioreactor, when compared to
a looped air-lift bioreactor or a stirred tank bioreactor. The optimised
production parameters used in the bioreactor were aeration rate of 1.0 wm,
1.0% (w/v) of glucose and the inoculum size of 8 x 106 spores/ml. The
enzymes production was further improved by entrapping the P. chrysosporium
cells in nylon sponge using the same medium composition. A maximum
production of 950.0 mU/ml of lignin peroxidase and 620.0 mU/ml of
manganese peroxidase was obtained.
Solid state fennentation system (SSF) using rice husks as substrate was
performed. Using the optimised fermentation system of 200g rice husks as
substrate, water content of 0.4 ml/g substrate, 0.1 % (w/w) ammonium
dihydrogen phosphate, 0.5% (w/w) yeast extract, 0.068 gIl veratryl alcohol,
i'noculum size of 1.0 x 107 spores/ml and inCUbation at 30°C, a maximum
production of about 165.0 U/g lignin peroxidase and 110.0 U/g manganese
peroxidase were achieved after 10 days cultivation.
The lignin peroxidase was purified by means of ammonium sulphate
precipitation and an anion exchange chromatography, using a prepacked
Mono Q column. The resolution from Mono Q column resulted in six different
peaks with the total recovery of 51 %, of which 29% was peak 5 (P5)· Ps was
purified about 23 folds with the specific activity of about 11.7 U/mg protein.Purity
was perfonned using the polyacrylamide gel electrophoresis technique.
The molecular weight of Ps was estimated to be 42,000 Dalton by SOS-PAGE
and the isoelectric point was at pH 4.2. The manganese peroxidase was
purfied by means of ammonium sulphate precipitation and gel filtration
chromatography of Sephadex G-100 and agarose. The enzyme recovery of
40.9% was obtained, with about 16 folds with the specific activity of about 8.0
U/mg protein. The enzyme showed a broad band on SoS-PAGE which was
considered partially purified.
The decolourization of methylene blue, polyvinylamine sulfonate anthrapiridone
(Poly R-478) and a waste water from a local textile industry revealed that the
decolourization was related to the lignin peroxidase and manganese
peroxidase formation in the growth medium. However, only lignin peroxidase
was found responsible for the decolourization process. The decolourization
rate and chemical oxygen demand (COD) reduction of the local waste waters
occured during the secondary phase of the fungal growth and related to lignin
peroxidase formation.
The biodegradation of rice husks and oil palm trunk chips by P. chrysosporium
and its ligninolytic enzymes were investigated. P. chrysosporium produced
lignin peroxidase, manganese peroxidase, cellulase, xilanase and ~-
glucosidase in the growth medium, with rice husks of oil palm trunk chips. as
substrates in a SSF system. The ligninolytic degradation on rice husks
enabled silica extraction be performed at lower temperature of 323°C.
Similarly, the extraction of reducing sugar from oil palm trunk chips was highly
improved by sequential treatment of acid, lignin peroxidase and cellulase
hydrolysis. Under these optimum conditions, a maximum production of 78.6
g/g of reducing sugar was obtained.
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Keywords
ENZIM PENGURAI , PHANEROCHAETE CHRYSOSPORIUM