PENGHASILAN, PENULENAN DAN PENCIRIAN ENZIM DEPOLIMERASE POLIHIDROKSIALKANOAT BERANTAISEDERHANA DARIPADA PSEUDOMONAS sp.

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Date
2005-07
Authors
WAN CHIK, MOHD HAYAZI
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Twenty six bacterial isolates with the ability to degrade medium chain length polyhydroxyalkanoates (MCL PHA), were obtained from several areas in Pulau Pinang, Malaysia. In this study, three methods have been carried out to isolate local MCL PHA depolymerase producer, namely the screening method using isolation medium containing MCL PHA as the sole carbon source, the degradation index method and the enzyme assay method. Among these isolates, NT-Dp B which showed the best ability to produce MCL PHA was selected for further investigation. Identification test showed that the bacterium was Pseudomonas sp. The bacterium was unable to degrade short chain length polyhydroxyalkanoates (SCL PHA) such as poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), [P(3HB-co-3HV)]. Further studies on the bacteria ability to produce PHA showed that the bacteria was capable of accumulating MCL PHA from carbon sources such as glucose, 4-hydroxybutyric acid, succinate and oleic acid. Pseudomonas sp. was able to produce extracellular depolymerase capable of degrading MCL PHA. After optimizing the media composition and culture condition using shake flask, the enzyme activity obtained was 0.749 mg/mUminutes, which was 20 fold higher compared to before the optimization process. The medium composition for optimized production of the depolymerase enzyme consisted of 2.5 giL MCL PHA, 6.4 XXIV giL urea, 4.0 giL KH2P04, 2,5 giL K2HP04, 1.0 giL MgS04.1H20, 3% (v/v) ferric ammonium citrate and calcium chloride solution. Whereas, the physical parameters for optimized depolymerase production were culture temperature at 30°C, pH medium at 6.8, inoculums size of 0.030 giL and agitation at 200 rpm. The purification process was successfully carried out and separated the enzymes from other kind of proteins. The purified depolymerase enzyme had molecular weight of 22.4 kDa and isoelectric point was at pH 8.8. The enzyme showed optimum activity at 40°C and was stable from 30°C to 35°C. The optimum pH was 8.5, and the enzyme was stable at pH 9.5 in glycineNaOH buffer. The enzyme Km was 0.74 mg/ml whereas the Vmaxs was 0.27 mg/mL/minutes.
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PENGHASILAN, PENULENAN DAN PENCIRIAN ENZIM DEPOLIMERASE POLIHIDROKSIALKANOAT BERANTAISEDERHANA
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