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Purification and Characterization of ?-1,3-Glucanase from the Antagonistic Fungus Trichoderma reesei SRI WAHYUNI BUDIARTI; SITI MUSLIMAH WIDYASTUTI; SEBASTIAN MARGINO
HAYATI Journal of Biosciences Vol. 16 No. 3 (2009): September 2009
Publisher : Bogor Agricultural University, Indonesia

Trichoderma enzymes that inhibit fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal root rot pathogen Ganoderma philippii. This experiment was aimed to purify and characterize the ?-1,3-glucanase of T. reesei. Extracellular ?-1,3-glucanase was produced by growing mycoparasite T. reesei isolate T13 in colloidal chitin and sucrose as carbon sources. The enzyme was then purified to its homogeneity by precipitation with ammonium sulfate, followed by gel filtration chromatography and chromatofocusing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 12% was used to confirm the purity of enzyme at each stage of preparation and to characterize purified protein. The results showed that T. reesei produced at least three extracellular ?-1,3-glucanases. Estimation of molecular weight based on SDS-PAGE 12% have three isoform of ?-1,3-glucanase were 90 kDa for ?-1,3-glucanase-I, 75 kDa for ?-1,3-glucanase-II, and 64 kDa for ?-1,3-glucanase-III. Their optimum pH and temperature were 5 and 50 oC, respectively. Key words: ?-1,3-glucanase, Trichoderma reesei, Ganoderma philippii

Superoxide Dismutase of Micrococcus sp. S2 and Its Involve in Paraquat Detoxification Sebastian Margino; Erni Martani; Medhina Magdalena
Indonesian Journal of Biotechnology Vol 12, No 1 (2007)
Publisher : Universitas Gadjah Mada

As an active ingredient of herbicide, paraquat will induce formation of superoxide radicals. The previousresearch succeeded in isolating paraquat degrading bacteria from peat soil, Micrococcus sp. S2, that tolerant to highconcentration of paraquat. An anti-oxidative enzyme, namely superoxide dismutase (SOD, EC.1.15.1.1), wasbelieved to be responsible for the paraquat tolerance. This research was conducted to study the characteristic of theSOD synthesize by Micrococcus sp. S2 and its ability on neutralize superoxide which arise from paraquat reoxidation.To observe the effect of paraquat on Micrococcus sp. S2, the bacteria was grown in 10% Luria Bertani brothmedium amended with several concentrations of paraquat, from 0 (control) up to 100 mg/ml. Within incubationtime of 72 hours, bacterial growth, activity of superoxide dismutase and paraquat residue were analyzed. Theisozymes of superoxide dismutase were distinguished using two kinds of specific inhibitor, namely HO and KCN. 2 2The results showed that paraquat significantly inhibit the growth of Micrococcus sp. S2. The higher paraquatcocentration in the medium caused the higher growth inhibition. However, the bacteria is still survive in the mediumcontaining toxic herbicide, and this ability was suggested related to superoxide dismutase activity in removing thesuperoxide radicals. Analysis using gel electrophoresis indicated that at least three types of SOD isozyme weresynthesized by Micrococcus sp. S2; they were Ferri-SOD (Fe-SOD), Mangani-SOD (Mn-SOD), and the last one wassuspected to be the Cupro Zinc-SOD (CuZn-SOD). The Mangani-SOD was suspected to play an important roles ondetoxifying superoxide which arise from paraquat oxidation.Keywords : Micrococcus sp.S2, paraquat, superoxide dismutase, isozymes

Production of Poly-α-hydroxybutyrate (PHB) from Sago Starch by The Native Isolate Bacillus megaterium PSA10 Nur Arfa Yanti; Langkah Sembiring; Sebastian Margino
Indonesian Journal of Biotechnology Vol 14, No 1 (2009)
Publisher : Universitas Gadjah Mada

A new bacterial strain that produces amylase and poly-a-hidroxybutyrate (PHB) using sago starch as carbon source was characterized and identified to be member of the Bacillus megaterium group based on phenotypic characteristics and 16S rDNA gene sequences. Amylase activity was determined spectrophotometrically on the basis of substrate concentration reduction. PHB production was quantified with UV spectrophotometer. The polymer produced by B. megaterium PSA10 was identified by Fourier Transform Infrared spectroscopy (FTIR). The result of the study showed that the amylase specific activity B. megaterium PSA10 was 593,61 DUN/mg protein and PHB production from sago starch was 52,28 % of cell dry weight (CDW). FTIR analysis of the polymer indicated that the strain B.megaterium PSA10 was a potent PHB producer.

Purification and Characterization of Streptomyces sp. IK Chitinase Sebastian Margino; Agustinus Joko Nugroho; Widya Asmara
Indonesian Journal of Biotechnology Vol 15, No 1 (2010)
Publisher : Universitas Gadjah Mada

Streptomyces sp. IK isolated from compost inoculants, could produce extra cellular chitinase in a medium containing 0.2% (w/v) colloidal chitin, fermented for 96 hours at 30oC. The enzyme was purified by a combination of ammonium sulphate precipitation and DEAE-Cellulose anion-exchange chromatography. On SDS-polyacrylamide gel electrophoresis analysis, the purified enzyme showed a mass of 71 kDa. Chitinase was optimally active at pH of 6.7 and at 37oC. Km value and Vmax of the protein for colloidal chitin were 2.92 mg/ml and 4.26 ìg/h, respectively.Key words : chitinase, Streptomyces, purification, characterization

Isolation and Purifi cation of Chitinase Bacillus sp. D2 Isolated from Potato Rhizosfer Sebastian Margino; Chatarina Behar; Widya Asmara
Indonesian Journal of Biotechnology Vol 17, No 1 (2012)
Publisher : Universitas Gadjah Mada

Potato Cyst Nematodes (Globodera rostochiensis) is one of the important potato’s pests and caused economic looses up to 70% in the several centrals of potato plantations in Indonesia. Potato Cyst Nematodes (PCN) shell component of egg shell containing chitin (inner layer) and vitelline/protein (outer layer), so the purpose of research was to fi nd out of chitin degrading bacteria for controlling of egg’s PCN by cutting of their life cycle. The results showed that Bacillus sp. D2 isolated from potato rhizosphere could produce extra cellular chitinase in the medium containing of 0.20% colloidal chitin and fermented for 72 hours. Result of chitinase purifi cation using ammonium sulphate precipitation and DEAE-Cellulose ion-exchange chromatography showed a specifi c activity 2691,052 U/mg and analyzing using SDS-PAGE 12.5% resulted in molecular weight 30 kDa. The apparent Km and Vmax of chitinase towards colloidal chitin were 2 mg/ml and 2.2 μg/h, respectively.

A Study on Production of Poly-β-Hydroxybutyrate Bioplastic from Sago Starch by Indigenous Amylolytic Bacteria Nur Arfa Yanti; Langkah Sembiring; Sebastian Margino; Nurhayani H. Muhiddin
Indonesian Journal of Biotechnology Vol 18, No 2 (2013)
Publisher : Universitas Gadjah Mada

Bacillus sp. PSA10 and Bacillus sp. PPK5 were two indigenous strain amylolytic bacteria from SoutheastSulawesi that have ability to produce bioplastic poly-β-hydroxybutyrate (PHB) from sago starch. The study wasattempted to determine the mechanism of PHB production by bacteria amylolytic was grown on sago starchcontainingmedia. Two amylolytic bacteria i.e. Bacillus sp. PSA10 and Bacillus sp. PPK5 was grown for 168 hin a mineral salts medium with sago starch as carbon source. Growth of amylolytic bacteria was monitoredby cell dry weight. Extraction of PHB was done by N-hexane acetone-diethyl ether method and PHB contentwas quantifi ed with UV spectrophotometer at 235 nm. Glucose level was determined by using kit of glucoseGOD 10” and was quantifi ed with spectrophotometer at 500 nm. Sago starch concentration was determinedby phenol method using specthrophotometer at 490 nm. The result of the study showed that Bacillus sp.PSA10 was produced PHB up to 66,81 % (g PHB/g cell dry weight) at 48 h and Bacillus sp. PPK5 up to 24,83% (g PHB/g cell dry weight) at 84 h. Bacillus sp. PSA10 has ability to converse sago starch to be PHB directlywithout glucose accumulation in the media, whereas Bacillus sp. PPK5 have to accumulate glucose as productof sago starch hydrolysis to produce of PHB. PHB synthesis by Bacillus sp. PHB production on sago starchof the Bacillus sp. PSA10 was found to be growth-associated whereas Bacillus sp. PPK5 was found to be nongrowth-associated. Therefore, two indigenous amylolytic bacteria were having of difference in biosynthesismechanism of PHB in sago starch medium and their characteristics of PHB synthesis should be consideredin developing cultivation methods for the effi cient production of PHB. Keywords : Production, PHB, Amylolytic bacteria, Sago starch.

Purification and Characterization of Protease From Bacillus sp. TBRSN- 1 Sebastian Margino; J. Jumi'ati; N. Ngadiman
Indonesian Journal of Biotechnology Vol 18, No 2 (2013)
Publisher : Universitas Gadjah Mada

Potato Cyst Nematode (PCN), Globodera rostochiensis, is one of the important potato’s pests and causedeconomic looses up to 70% in the several centrals of potato plantations in Indonesia. PCN’s shell componentof egg shell containing chitin (inner layer) and viteline/ protein (outer layer). The purpose of this researchwas to purify of protease Bacillus sp. TBRSN-1, isolate from tomato’s rhizosfer in Yogyakarta province. Thepurifi ed protease could be used for cutting the life cycle of PCN. Results showed that Bacillus sp. TBRSN-1could produce extracellular protease and purifi cation using DEAE-cellulose ion-exchange chromatographyand Sephacryl S-300 gel fi ltration chromatography resulted in specifi c activity 4.31 fold and 1.68% recovery.Analysing using SDS-PAGE 12.5% and molecular weight 48.1 kDa. Km and Vmax values of the protease forcasein substrate were 7.83 mg/ml and 4.03 μg/h, respectively. The optimum activity at the temperature30oC and pH 7.0. Keywords : protease, purifi cation, indigenous Bacillus sp. TBRSN-1

Poly-β-Hydroxybutyrate (PHB) Production By Amylolytic Micrococcus sp. PG1 Isolated From Soil Polluted Arrowroot Starch Waste Sebastian Margino; Erni Martani; Andriessa Prameswara
Indonesian Journal of Biotechnology Vol 19, No 1 (2014)
Publisher : Universitas Gadjah Mada

Poly-β-hydroxybutyrate (PHB) production from amylolytic Micrococcus sp. PG1. Poly-β-hydroxybutyrate(PHB) is an organic polymer, which synthesized by many bacteria and serves as internal energy. PHB ispotential as future bioplastic but its price is very expensive due to glucose usage in PHB industry. Thedevelopment of PHB production using starch as an alternative carbon source has been conducted to reducethe dependence of glucose in PHB production. In this study, amylolytic bacteria from arrowroot processingsite were screened quantitavely based on amylase specifi c activity and PHB producing ability. The result of thestudy showed that among of 24 amylolytic isolates, 12 isolates of them were able to accumulate PHB rangedfrom 0,68-11,65% (g PHB/g cdw). The highest PHB production from substrate arrowroot starch was PG1 andafter optimization resulted in increasing of PHB production up to 16,8% (g PHB/g cdw) 40 hours incubationtime. Based on morphological, biochemical and physiological characters, the PG1 isolate was identifi ed asMicrococcus sp. PG1. Result of the FTIR analysis of produced polymer by Micrococcus sp. PG1 was indicatedas poly-β- hydroxybutyrate (PHB)

Kondisi Optimum untuk Produksi Kitinase dari Streptomyces Rkt5 dan Karakterisasi pH dan Suhu Enzim Yurnaliza Yurnaliza; Sebastian Margino; Langkah Sembiring
Biota : Jurnal Ilmiah Ilmu-Ilmu Hayati Vol 13, No 3 (2008): October 2008
Publisher : Universitas Atma Jaya Yogyakarta

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24002/biota.v13i3.2571

Chitinase is chitin degrading enzyme which is produced by Streptomyces Rkt 5 is isolated microorganism from peanut rhizosfer. This enzyme and its microorganism can be used in many agricultural, medicine and industrial purposes. The aim of the research was to find out the optimum condition for production of chitinase and to characterize of pH and temperature to chitinase activity. Optimalizing production the research had 4 treatments. The optimum conditions were achieved at mineral liquid medium containing with chitin 0,2% (w/v) as inducer, 10% (v/v) inoculum, pH 7 and 48 hours incubation. The crude enzyme was partially purified by salting out with 70% ammonium sulfate resulted in 3.31 time more purity enzyme than the crude one. This enzyme had maximum activity at 50oC and pH 5.5.

Optimasi Produksi Poli-β-Hidroksibutirat (PHB) oleh Bacillus sp. PSA10 Nur Arfa Yanti; Sebastian Margino; Langkah Sembiring
Biota : Jurnal Ilmiah Ilmu-Ilmu Hayati Vol 15, No 3 (2010): October 2010
Publisher : Universitas Atma Jaya Yogyakarta

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24002/biota.v15i3.2587

A new strain characterized as Bacillus sp. PSA10 was found to produce poly-β-hydroxybutyrate (PHB) at concentration of 52.28% (g PHB/g dry cell weight) in shaken flask culture, using sago starch as a carbon source. This research is aimed to determine the optimum culture condition of PHB production Bacillus sp. PSA10 at laboratory scale. Optimization of PHB production was conducted in this research, in terms of inoculum concentration, concentration of the major components in minimal medium, environmental condition and incubation time. The result showed that optimum conditions for the production of PHB by Bacillus sp. PSA10 were achieved at minimal medium (Ramsay medium) with 5% (v/v) inoculum concentration, 2% (w/v) sago starch, 1.0 g/l (NH4)2SO4, 6.7 g/l Na2HPO4.7H2O, and 0 g/l KCl. The optimum environmental conditions were achieved with initial pH 7, temperature 37oC, agitation speed at 150 rotary per minute (rpm) and the best of incubation time was 48 hour. Under this optimum condition, the maximum PHB production by Bacillus sp. PSA10 increased from 52.28% to 71.35% (g PHB/g dry cell weight) at 48 hour cultivation. Therefore, Bacillus sp. PSA10 is potential to apply for PHB production from sago starch at industrial scale.

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