Anak Agung Istri Ratnadewi, Anak Agung Istri
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Jember. Center for Development of Advanced Sciences and Technology, Universitas Jember

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EXPLORATION OF BROTH CHICKEN GUT AS GROWTH MEDIA OF Escherichia coli BL21 pET-Endo FOR ENDO-Î’-1,4-D-XYLANASE PRODUCTION Ratnadewi, Anak Agung Istri; Alidion, Moch. Yoris; Santoso, Agung Budi; Oktavianawatia, Ika
ALCHEMY Jurnal Penelitian Kimia Vol 13, No 2 (2017): Alchemy Jurnal Penelitian Kimia
Publisher : UNIVERSITAS SEBELAS MARET (UNS)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.20961/alchemy.13.2.4356.191-204

Abstract

Endo-β-1,4-D-xylanase is a hydrolytic enzyme that breakdown the 1.4 chain of xylan polysaccharide. We have succes to transform the plasmid pET-Endo gene encoding endo-1,4-β-D-xylanase from Bacillus sp. originally from termites abdominal to E. coli BL21. The clone was ready for large scale of enzyme production. To reduce production cost, we look for subtitute media for the expensive Luria Berthani broth. Chicken guts broth is good alternative while rich of protein and very cheap. The content of N dissolved chicken guts broth reaches 87 % of LB broth. Growth of E. Coli BL21 in Chicken guts broth and LB broth (as control) was observed by Optical Density (OD) using spectrofotometer. Concentration of glucose added in broth and incubation temperature was varied. The result showed that optimal growth was in addition of 1.5 % glucose and incubated at  37 oC for 16 h. This optimal condition was used to grow E. coli BL21 pET-Endo for xylanase production. Enzyme purification was done by Ni-NTA affinity chromatography. Highest protein yield was 0.076 mg/mL obtained in 100 mM imidazole elucidation. The activity and specific activity of xylanase were estimated as 0.042 U/mL and 0.556 U/µg, respectively. The purification factor was 3.16 time and the molecular weight of enzyme was ± 30, 000 Dalton
Optimization pH of Enzymatic Hydrolysis of Endo-1,4-β-Xylanase for Xylooligosaccharides Production Ratnadewi, Anak Agung Istri; Kurniawan, Andika Ade; Handayani, Wuryanti
UNEJ e-Proceeding Indonesian Protein Society (IPS), International Seminar and Workshop 2014
Publisher : UNEJ e-Proceeding

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Abstract

Xylooligosaccharides (XOS) with polymerisation degree between 2 till 10 monomer  have prebiotic effect for better digestion system. In this research, production of XOS was performed by enzymatic hydrolysis of xylan with endo-1,4-β-xylanases enzyme. Endo 1,4-β-xylanases enzyme was aqcuired from Bacillus sp. isolated from termite’s abdominal.Meanwhile oat xylan was used as substrate. Optimal condition of enzymatic hydrolysis was evaluated at pH: 4, 5, 6, and 7 with incubation time from 5 to 20 h.pH 5 was optimum pH to produce XOS from 0.8 % xylan oat at 40 oC.The hydrolysis product purified and analyzed by thin layer chromatographyyielding spot of xylobiose, xylotriose, xilotetraose and xilopentaose. Further analysis by HPLC indicated dominant xilopentaosa X5 (3522 ppm) among  the other XOS   X2 (14 ppm), X3 (43 ppm) and X4 (15 ppm). Keywords: Xylooligosaccharides, endo-1,4-β- endoxylanase
Transformation of Plasmid pET Endo-1,4-β-xilanase from E. coli TOP10 to E. coli BL21 Santoso, Agung Budi; Kurniawati, Eka Yuni; Ratnadewi, Anak Agung Istri
UNEJ e-Proceeding Indonesian Protein Society (IPS), International Seminar and Workshop 2014
Publisher : UNEJ e-Proceeding

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Abstract

In this research we successfully transmited Plasmid pET- Endo from E.coli TOP10 to E.coli BL21. Plasmid pET Endo is recombinant plasmid base on pET-30a(+)  inserted with gene of endo-1,4-β-xilanase isolated from bacillus subtilis sp which is originally living in termite abdomen. First step is isolation of plasmid pET-Endo from E.coli TOP10 with alkaline lyses. Denaturation and renaturation of DNA occurred then separated with centrifugation. Plasmid pET-Endo is smaller than Chromosome DNA. Agarose gel electrophoresis confirmed that Plasmid pET-Endo has isolated. Electrogram of pET-Endo show the band in 6022 bp compare to empty plasmid pET-30a(+) 5422 bp. E.Coli BL21 got pretreatment with  CaCl2 solution to make cell competent for transformation. Isolated pET-Endo inserted to E.coli BL21 with heat shock method. Resistance test with antibiotic has done to know the result of transformation. E.coli BL21 contained plasmid pET-Endo will survive in kanamycin agar media. Colony of E.coli BL21 then cultured in liquid media and examinee it’s growth phase. IPTG as gene inducer given after 2,5 hour of inoculation. Crude enzyme tested for xilanase activity and well proved. Isolation of plasmid pET-Endo then running in Agarose gel electrophoresis also confirm good result of transformation.
OPTIMASI KONSENTRASI SUBSTRAT XILAN AMPAS TAHU TERHADAP ENDO-Î’-1,4-D-XYLANASE UNTUK MEMPRODUKSI XILOOLIGOSAKARIDA Ratnadewi, Anak Agung Istri; Handayani, Wuryanti; Avida, Siti Nur
Jurnal Kimia Riset Vol 1, No 2 (2016): Desember
Publisher : Universitas Airlangga

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (496.779 KB) | DOI: 10.20473/jkr.v1i2.3084

Abstract

AbstrakAmpas tahu merupakan limbah samping dari proses pengolahan tahu dan susu kedelai. Ampas tahu berpotensi sebagai sumber xilan. Xilan digunakan sebagai substrat endo-β-1,4-D-xilanase untuk menghasilkan xilooligosakarida. Penelitian ini digunakan xilan ampas yang telah dihilangkan lemak dan protein tanpa penghilangan lignin (X1nD). Xilan ampas tahu tanpa penghilangan lemak dan protein tetapi dilakukan penghilangan lignin (X2D). Enzim yang digunakan adalah endo-β-1,4-D-xilanase dari isolat Bacillus sp. asal abdomen rayap. Optimasi variasi konsentrasi substrat bertujuan untuk mengetahui konsentrasi optimum dalam menghasilkan xilooligosakarida. Produk hidrolisis yang dihasilkan dianalisis menggunakan metode Miller untuk mengetahui total gula pereduksi. Produk hidrolisis konsentrasi optimum dianalisis menggunakan Kromatografi Lapis Tipis (KLT) untuk mengetahui komponen penyusun xilooligosakarida. Substrat X1nD dan X2D optimum pada konsentrasi 6% dan 5% dengan total gula pereduksi sebesar 0,196 mg/ml dan 0,211 mg/ml. Komponen penyusun xilooligosakarida ampas tahu berupa xilotriosa (X3), xilotetraosa (X4), dan xilopentaosa (X5).Kata Kunci: Ampas tahu, endo-β-1,4-D-xilanase, xilan, xilooligosakarida. AbstractOkara is a waste byproduct of the processing of tofu and soy milk. Okara potential as a source of xylan. Xylan is used as the substrate endo-1,4-β-D-xylanase to produce xyloologosaccharide. This study used okara xylan had eliminated fat and protein without removal of lignin (X1nD). Okara xylan out without the removal of fat and protein but do removal of lignin (X2D). The enzyme used is endo-1,4-β-D-xylanase of isolates of Bacillus sp. From abdominal termites. Optimization of substrate concentration variation aims to determine the optimum concentration in generating xyloologosaccharide. Hydrolysis products were analyzed using Miller method to determine total reducing sugars. The optimum concentration of hydrolysis products were analyzed using Thin Layer Chromatography (TLC) to determine the components of xyloologosaccharide. X1nD and X2D optimum substrate at a concentration of 6% and 5% to the total reducing sugars of 0.196 mg/ml and 0.211 mg/ml. Xyloologosaccharide of okara components of the pulp out the form xylotriose (X3), xylotetraose (X4), and xylopentaose (X5).Keywords: Okara, endo-1,4-β-D-xylanase, xylan, xyloologosaccharide
The Analysis of Pb and Cu levels in Canned fish and Sauces for the Storage Time Erfiandika, Hefinda; Asnawati, Asnawati; Ratnadewi, Anak Agung Istri
Jurnal ILMU DASAR Vol 15 No 2 (2014)
Publisher : Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Jember

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (361.887 KB) | DOI: 10.19184/jid.v15i2.835

Abstract

Lead (Pb) is pollutant that found in canned foods which derived from the soldering between the can and the lead. Copper (Cu) is one of material which one tin packaging can be oxidized and dissolved in acidic foods. Pb and Cu are not dangerous at lowest but it can cause botulism in gross. The storage time can affect the solubility of the metals. The purpose of the research is to know levels metal Pb and Cu in fishes and sauces canned and compared with limit BPOM. The limit of BPOM in canned fish for metal Pb is 0,3 ppm and for metal Cu is 5 ppm. The steps of the method are optimization the method of destruction and the measurement using Atomic Absorption Spectrofometry (SSA). The result shows that the storage time  give effect to the greater of metal Pb and Cu in fish and sauce.The content of metal Pb in all sample exceeded the limit of BPOM. The content of metal Cu in sample A does not exceed in fish and sauce. The first month of sample B does not exceed, but the sixth month up to the twenty fourth month exceed the limit of BPOM in fish and sauce. The precision in all the measurement have on average  <2 %, it  shows that all the measurements are good repetition. Keywords: Lead , Copper, Atomic Absorption  Spectrofometry, fish, Sauce 
Optimization pH of Enzymatic Hydrolysis of Endo-1,4-β-Xylanase for Xylooligosaccharides Production Ratnadewi, Anak Agung Istri; Kurniawan, Andika Ade; Handayani, Wuryanti
UNEJ e-Proceeding Indonesian Protein Society (IPS), International Seminar and Workshop 2014
Publisher : UPT Penerbitan Universitas Jember

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

Xylooligosaccharides (XOS) with polymerisation degree between 2 till 10 monomer  have prebiotic effect for better digestion system. In this research, production of XOS was performed by enzymatic hydrolysis of xylan with endo-1,4-β-xylanases enzyme. Endo 1,4-β-xylanases enzyme was aqcuired from Bacillus sp. isolated from termite’s abdominal.Meanwhile oat xylan was used as substrate. Optimal condition of enzymatic hydrolysis was evaluated at pH: 4, 5, 6, and 7 with incubation time from 5 to 20 h.pH 5 was optimum pH to produce XOS from 0.8 % xylan oat at 40 oC.The hydrolysis product purified and analyzed by thin layer chromatographyyielding spot of xylobiose, xylotriose, xilotetraose and xilopentaose. Further analysis by HPLC indicated dominant xilopentaosa X5 (3522 ppm) among  the other XOS   X2 (14 ppm), X3 (43 ppm) and X4 (15 ppm). Keywords: Xylooligosaccharides, endo-1,4-β- endoxylanase
Transformation of Plasmid pET Endo-1,4-β-xilanase from E. coli TOP10 to E. coli BL21 Santoso, Agung Budi; Kurniawati, Eka Yuni; Ratnadewi, Anak Agung Istri
UNEJ e-Proceeding Indonesian Protein Society (IPS), International Seminar and Workshop 2014
Publisher : UPT Penerbitan Universitas Jember

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

In this research we successfully transmited Plasmid pET- Endo from E.coli TOP10 to E.coli BL21. Plasmid pET Endo is recombinant plasmid base on pET-30a(+)  inserted with gene of endo-1,4-β-xilanase isolated from bacillus subtilis sp which is originally living in termite abdomen. First step is isolation of plasmid pET-Endo from E.coli TOP10 with alkaline lyses. Denaturation and renaturation of DNA occurred then separated with centrifugation. Plasmid pET-Endo is smaller than Chromosome DNA. Agarose gel electrophoresis confirmed that Plasmid pET-Endo has isolated. Electrogram of pET-Endo show the band in 6022 bp compare to empty plasmid pET-30a(+) 5422 bp. E.Coli BL21 got pretreatment with  CaCl2 solution to make cell competent for transformation. Isolated pET-Endo inserted to E.coli BL21 with heat shock method. Resistance test with antibiotic has done to know the result of transformation. E.coli BL21 contained plasmid pET-Endo will survive in kanamycin agar media. Colony of E.coli BL21 then cultured in liquid media and examinee it’s growth phase. IPTG as gene inducer given after 2,5 hour of inoculation. Crude enzyme tested for xilanase activity and well proved. Isolation of plasmid pET-Endo then running in Agarose gel electrophoresis also confirm good result of transformation.
Purification of Xylooligosaccharide From Casavva Pulp by Ultrafiltration Method Ratnadewi, Anak Agung Istri; Fauziyah, Kamelia Rizqi; Indarti, Dwi
BERKALA SAINSTEK Vol 9 No 2 (2021)
Publisher : Universitas Jember

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.19184/bst.v9i2.23388

Abstract

Xylan is the main component of hemicellulose. Xylan can be extracted from agricultural waste, such as cassava pulp. Xylan is used as an endo-β-1,4-D-xylanase substrate to produce impure xylooligosaccharides (XOS)This study aims to purify XOS from cassava pulp using the ultrafiltration method. The components of XOS obtained from the enzymatic hydrolysis were analyzed using thin layer chromatography (TLC) and densitometry methods. In addition, the XOS was purified by the ultrafiltration method using a cellulose membrane with a Molecular Weight Cut Off (MWCO) of 12 kDa. The permeate obtained from the purification results was also analyzed using TLC and densitometry. The results of this study indicated that the components in XOS cassava pulp before and after purification by the TLC method were X5 and X6, while the XOS components before and after purification by the densitometric method were X3, X4 and X5.