scholarly journals PEMURNIAN ENZIM SEFALOSPORIN-C ASILASE DAN OPTIMASI PROSES KROMATOGRAFI PENUKAR ION

2018 ◽  
Vol 5 (2) ◽  
pp. 119
Author(s):  
Uli Julia Nasution ◽  
Silvia Melinda Wijaya ◽  
Ahmad Wibisana ◽  
Anna Safarrida ◽  
Indra Rachmawati ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Ammonium Sulphate ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Cephalosporin C ◽  
Α Subunit ◽  
Ammonium Sulphate Precipitation ◽  
Cephalosporin C Acylase ◽  
Sds Page

Purification of Cephalosporin-C Acylase and Its Optimization of Ion-Exchange ChromatographyABSTRACTCephalosporin-C acylase (CCA) has an important role in the one-step conversion of cephalosporin-C into 7-ACA. Purification process aims to increase specific activity of CCA enzyme. Purification began with cell lysis, ammonium sulphate precipitation, dialysis, ion exchange chromatography (IEC) and size exclusion chromatography. IEC optimization of elution step was also done to compare gradient and isocratic elusion. Purification was capable to increase the enzyme purity upto 33.66 fold, with specific activity of 3.00 U/mg and the yield reached 41.41%. Optimization of elusion during IEC showed that isocratic protein elusion was more efficient (taking shorter time, 3 column volume (CV) only) than that of gradient batch (up to 9 CV). SDS-PAGE analysis demonstrated that the recombinant CCA enzyme existed in two types, active enzyme containing α-subunit (25 kDa) and β-subunit (58 kDa), and inactive enzyme (83 kDa) as precursor. Furthermore, 30% ammonium sulphate saturated precipitation was able to precipitate this inactive CCA.Keywords: 7-ACA,CCA, cephalosporin C, protein purification, specific activity ABSTRAKSefalosporin-C asilase (CCA) merupakan enzim yang berperan penting dalam konversi satu tahap sefalosporin-C menjadi 7-ACA. Proses purifikasi merupakan salah satu cara untuk meningkatkan aktivitas spesifik enzim CCA. Proses purifikasi dimulai dari memecah sel, diikuti dengan tahap presipitasi menggunakan amonium sulfat, dialisis, kromatografi penukar ion (IEC) dan kromatografi eksklusi. Optimasi proses IEC pada tahap elusi juga dilakukan untuk membandingkan elusi enzim CCA secara gradien dan isokratik. Proses purifikasi pada penelitian ini mampu meningkatkan kemurnian enzim hingga 33,66 kali, dengan aktivitas spesifik sebesar 3,00 U/mg dan perolehan enzim sebesar 41,41%. Hasil optimasi IEC pada proses elusi secara isokratik lebih efisien dari segi waktu (hanya membutuhkan 3 kolom volume (CV) dibandingkan dengan secara gradien (sampai 9 CV). Hasil SDS-PAGE menunjukkan bahwa CCA rekombinan merupakan enzim dengan 2 macam bentuk yaitu enzim aktif, yang terdiri dari subunit α (25 kDa) dan β (58 kDa), dan enzim tidak aktif berupa prekursor (83 kDa). Proses presipitasi menggunakan amonium sulfat 30% tersaturasi dapat mengendapkan prekursor CCA.Kata Kunci: 7-ACA, aktivitas spesifik, CCA, purifikasi protein, sefalosporin C

Purification of a Fibrinolytic Enzyme from Bacillus Sp. Isolated from Budu

2012 ◽  
Vol 59 (1) ◽  
Author(s):  
Nurulhanis Ahmad Sanusi ◽  
Haryati Jamaluddin
Keyword(s):  
Ammonium Sulphate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Culture Broth ◽  
Fibrinolytic Enzyme ◽  
Total Protein Content ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page

Bacillus sp. strain B1 producing wild type fibrinolytic enzyme was isolated from Budu. The fibrinolytic enzyme was collected from the supernatant of Bacillus sp. strain B1 culture broth and purified to electrophoretic homogeneity through a combination of various purification schemes, which include ammonium sulphate precipitation, followed by anion exchange chromatography using DEAE–Sepharose Fast Flow and gel filtration chromatography on Sephadex G–75 column. During ammonium sulphate precipitation screening, it was observed that the crude enzyme from Bacillus sp. strain B1 precipitated at 40% and 50% of ammonium sulphate saturation respectively. The fibrinolytic enzyme was purified 58.5–fold with a final yield of 0.51%. The specific activity was determined to be 1.17 Units/mg using plasmin as standard and the final total protein content was 8.58 mg/ml. After the successive purification steps, the estimated molecular mass of fibrinolytic enzymes from strain B1 was estimated via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE analysis showed a single band at 45 kDa corresponding to the purified fraction with fibrinolytic activity.

scholarly journals PURIFICATION OF β-GLUCOSIDASE PRODUCED FROM Trichoderma viride USING COW DUNG AS CARBON SOURCE

2020 ◽  
Vol 4 (2) ◽  
Author(s):  
S. T. Tyohemba ◽  
S. Aliyu ◽  
N. N. Ndukwe ◽  
G. G. Memi ◽  
U. O. Edem
Keyword(s):  
Specific Activity ◽  
Trichoderma Viride ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Broad Peak ◽  
Cow Dung ◽  
Elution Profile ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Peak Fraction

β-glucosidases have characteristics of biotechnological interest and have thus become important industrial enzymes.In this study, β-glucosidase produced by Trichoderma viride from cow dung was subjected to a three step purification process involving ammonium sulphate precipitation, gel filtration by Sephadex G-100 and ion exchange chromatography by DEAE-Sephadex A-25. The elution profile on Sephadex G-100 resulted in a single broad peak (fractions 9-21) which had a yield of 3.7% and a purification fold of 4.29 with a specific activity of 25.70 µmol/min/mg proteins while the elution profile on DEAE-Sephadex A-25 resulted in a single broad peak (fraction 8-14) which had a yield of 2.76% and a purification fold of 22.14 with a specific activity of 132.41µmol/min/mg of protein. The purified enzyme was obtained as a single band and had a molecular mass of 51.8 kDa on SDS-PAGE. This results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

scholarly journals Purification and Characterization of Cellulase from Termite Ametermes eveuncifer (Silverstri) Soldiers

2016 ◽  
Vol 9 (1) ◽  
pp. 1 ◽  
Author(s):  
B. S. Fagbohunka ◽  
R. E. Okonji ◽  
Ayinla Zainab Adenike
Keyword(s):  
Ion Exchange ◽  
Specific Activity ◽  
Ion Exchange Chromatography ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Percentage Yield ◽  
Termite Soldiers

Cellulase enzyme was purified and characterized from termite soldiers (Ametermes eveuncifer) using 70% ammonium sulphate precipitation, ion exchange chromatography and affinity chromatography. The enzyme isolated had a specific activity of 5.04 U/mg with a percentage yield of 11.7%. The enzyme showed maximum activity at 500C and pH 8. The enzyme was not inhibited by Ba2+ at a concentration of 1mM and Pb2+ at 10 mM concentration but was inhibited by other metal ions at 1 mM and 10 mM concentrations of their salts (NaCl, KCl, MnCl2, and NiCl2),

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

2021 ◽  
Vol 9 (2) ◽  
pp. 24-30
Keyword(s):  
Metal Ions ◽  
Sugarcane Bagasse ◽  
Specific Activity ◽  
Gel Filtration ◽  
Value Added ◽  
Cost Effective ◽  
Fermentation Medium ◽  
Exchange Chromatography ◽  
Sds Page ◽  
The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

scholarly journals Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A

1993 ◽  
Vol 295 (3) ◽  
pp. 813-819 ◽  
Author(s):  
A Varro ◽  
G J Dockray
Keyword(s):  
Ion Exchange ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Specific Activity ◽  
Brefeldin A ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Gastrin Cells ◽  
Tryptic Fragment ◽  
C Terminus

The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation.

scholarly journals Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

PURIFICATION AND CHARACTERISATION OF FIBRINOLYTIC ENZYMES FROM ENDOPHYTIC FUNGI AND LIGNOSUS RHINOCERUS

2016 ◽  
Vol 78 (6-5) ◽  
Author(s):  
Zainon Mohd Noor ◽  
Mohd Sidek Ahmad ◽  
Zaidah Zainal Ariffin
Keyword(s):  
Ion Exchange ◽  
Endophytic Fungi ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fibrinolytic Enzymes ◽  
Molecular Weights ◽  
Ammonium Sulphate Precipitation ◽  
Fusarium Sp ◽  
Different Sources

Three enzymes FH3, S13 and LR1 from three different sources showed fibrinolytic activities. Two were from endophytic fungal cultures and one from the sclerotium of Lignosus rhinocerus mushroom (LR1). FH3, S13 cultures and LR1, the crude extract of the sclerotium were concentrated and purified by ammonium sulphate precipitation, ion-exchange chromatography and gel-filtration. The molecular weights of the FH3, S13 and LR1 purified enzymes were estimated to be approximately 34kDa, 34kDa and 10kDa, respectively. Maximum fibrinolytic activities were observed for FH3 at pH 7 and 30°C, S13 at pH 8 and 40°C and LR1 at pH 6 and 40°C.  In our earlier paper we identified FH3 as Fusarium sp. and S13 as Penicilium citrinum. 

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