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Karossi, A.T.
Research Center for Chemistry - LIPI
Computer Science & IT

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PURIFICATION AND CHARACTERIZATION OF WHITE RADISH (RAPHANUS SATIVUS L.VAR LONG WHITE) PEROXIDASE FROM CELL CULTURE EXTRACT Pudjiraharti, -; Karossi, A.T.
Teknologi Indonesia Vol 32, No 2 (2009)
Publisher : LIPI Press

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/jti.v32i2.8

Abstract

Horseradish peroxidase (HRP) has been widely used as a component of clinical diagnostic reagent for Enzyme Linked I mmunosorbent Assay (ELISA) technique. White radish as one of Brassicaceae family members was found to have high peroxidase activity. In this study, white radish (Raphanus sativus L. var Long White) was used for the production of peroxidase by cell suspension culture technique. Isolation of the enzyme was conducted by ammonium sulfate precipitation followed by purification using DEAE-Cellulose column chromatography eluted with 0.01 M phosphate buffer, pH 7.5 and 0-0.5 M NaCl gradient. A major peak of protein having the highest enzyme activity and purity 25 folds compared to the crude was resulted. SDS-Polyacrilamide gel electrophoresis showed one main band with molecular weight of 47.000 Da. This white radish peroxidase enzyme is very efficient by demonstrating maximum activity at temperature 55oC and pH 7.5 as well as a Km 76.6 ?g/mL and V max 275 ?g/mL/minute toward hydrogen peroxide substrate and pyrogallol hydrogen donor.
HORSERADISH PEROXIDASE (HRP) ACTIVITY OF ARMORACHIA LAPATHIFOLIA SUBCULTURED CALLUS IN SOLID AND LIQUID MEDIA Budiwati, Thelma A.; Pudjiraharti, S.; Karossi, A.T.
Teknologi Indonesia Vol 32, No 1 (2009)
Publisher : LIPI Press

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/jti.v32i1.51

Abstract

Peroxidase is an enzyme which catalyzes the oxidation of organic compound done by peroxide. The peroxidase enzyme can be produced by plants or microorganisms. One of the peroxidase enzymes is Horseradish Peroxidase (HRP). This commercial, HRP is produced through the extraction of Horseradish root (Armorachia lapathifolia) besides it can also be produced through tissue culture. The callus induction was carried out in Linsmaier-Skoog media containing naftalen acetic acid (NAA) and benzyl amino purin (BAP) as the growth hormone. Incubation was conducted at room temperature (2428oC) without lighting anymore for three weeks, At the age of three weeks some of the calli were subcultured in solid media, and the rests were subcultured in liquid media containing growth hormone NAA and kinetin. Incubation was in a shaking incubator at 120 rpm, without light, at the room temperature for 40 days. The sampling was conducted every 10 days. The results of the research showed that the 8th (SC8) and the 10th (SC10) subcultures produced the highest extracellular and intracellular enzyme activity at the age of 20 and 30 days. The extracellular enzyme activity was 38.06 U/ml with specific activity 22.36 U/mg protein and the total activity of 79.92 U. While the intracellular enzyme activity was 253,82 U/g callus, with specific activity of 39.05 U/mg protein and total activity of 649.77 U.
SAGO STARCH AS FERMENTATION MEDIUM FOR PRODUCTION OF AND SUBSTRATE FOR CARBOHYDRATE ENZYMES Karossi, A.T.
Teknologi Indonesia Vol 32, No 2 (2009)
Publisher : LIPI Press

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (16.042 KB) | DOI: 10.14203/jti.v32i2.7

Abstract

It is well known that sago starch extracted from sago palm (Metroxylon sp.) is abundantly available especially in eastern part of Indonesia. On the other hand, amylases and related enzymes are recognized as the most important industrial enzymes, specifically in the field of food industries. Therefore, fermentation experiments were carried out to utilize sago starch as medium component to produce extracellular alpha amylase and glucoamylase as well as glucose isomerase, which is the later being an intracellular enzyme. Aspergillus oryzae was selected fermenting microorganism to produce the extracellular alpha amylase and intracellular glucose isomerase, while Rhizopus oryzae was for producing extracellular glucoamylase. The investigation included optimum fermentation process condition for enzymes production, both in Ehrlenmeyer shaken flasks and stirred tank fermentors, then was followed by isolation with ammonium sulfate fractionation, concentration employing polysulfon membrane, purification with chromatography technique, and characterization of the enzymes. In fact, sago starch could function as a medium component in fermentation producing amylases as well as a substrate for the enzymes. The biomass of the Aspergillus contained glucose isomerase activity. Experiments with fermentor indicated that alpha amylase was best produced at agitation rate of 500 rpm, while glucoamylase was at 350 rpm, both at 1.5 vvm aeration. The SDS-PAGE of the purified enzymes found that the alpha amylase and glucoamylase had molecular weights of 53 and 36 kDalton respectively, while the glucose isomerase indicated to have two subunits with molecular weight each of 55 and 60 kDalton. As a substrate for alpha amylase and glucoamylase, sago starch exhibited higher affinity compared with arenga, corn, and cassava starches although it was still inferior to soluble starch.
Co-Authors Budiwati, Thelma A. Pudjiraharti, - Pudjiraharti, S.