scholarly journals New Lipase for Biodiesel Production: Partial Purification and Characterization of LipSB 25-4

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Aysel Ugur ◽  
Nurdan Sarac ◽  
Rukiye Boran ◽  
Berk Ayaz ◽  
Ozgur Ceylan ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Nitrogen Sources ◽  
Biodiesel Production ◽  
Partial Purification ◽  
Period Effect ◽  
Carbon And Nitrogen ◽  
Ammonium Sulphate Precipitation ◽  
Thermophilic Conditions

The lipolytic activities of 300 Streptomyces isolates were determined in Tributyrin and Rhodamine-B Agar. Lipase activities were also measured with p-nitrophenyl palmitate (p-NPP) as a substrate. The strain of Streptomyces bambergiensis OC 25-4 used in this study was selected among 300 strains of Streptomyces from MUCC as the best lipase producer. The incubation conditions were optimized and the inoculum amount, incubation period, effect of carbon and nitrogen sources, and rates of MgSO4 and CaCO3 were investigated. LipSB 25-4 (the lipase produced by S. bambergiensis OC 25-4 strain) was partially purified with ammonium sulphate precipitation, dialysis, and gel filtration chromatography 2.73-fold and with 92.12 U/mg specific activity. The optimal pH and temperature for LipSB 25-4 were determined as 8.0 and 50°C, respectively. The lipase has high stability in all pH and temperature values used in this study. While LipSB 25-4 was slightly activated in the presence of β-mercaptoethanol, it was slightly reduced by PMSF. The enzyme conserved approximately 75% of its activity at the end of 60 h, in the presence of methanol and ethanol. Since LipSB 25-4 displays high activity in the thermophilic conditions and stability in the presence of organic solvents, this lipase can catalyse the biodiesel production from olive oil by the transesterification reactions.

PURIFICATION AND CHARACTERIZATION OFESTERASE ENZYME ISOLATED FROM CONTAMINATED SOIL SAMPLE

2019 ◽  
pp. 1-3
Author(s):  
S. Chithra ◽  
K. Silambarasi ◽  
I. Yasmin
Keyword(s):  
Soil Sample ◽  
Ammonium Sulphate ◽  
Contaminated Soil ◽  
Specific Activity ◽  
Nitrogen Sources ◽  
Partial Purification ◽  
Carbon And Nitrogen ◽  
Crude Enzyme Preparation ◽  
Ammonium Sulphate Precipitation ◽  
Highly Active

Isolation and partial purification of esterase from contaminated soil sample was the main aim of this study. The production medium for organism was optimized by using different pH,Temperature,Carbon and Nitrogen sources.The esterase enzyme was highly active and stable from pH 5.0 to 9.0 with an optimum at pH 9.Its optimum temperature was 35°C. The best carbon and nitrogen sources were mannitol and yeast extract. Esterase was partially purified by ammonium sulphate precipitation,dialysis.The specific activity of partially purified esterase obtained from ammonium sulphate fractionation is found to be 8.6485U/mg and the fractionation is 5.4 fold purified from the crude enzyme preparation yielding 17.5U/mg from the crude protein. This result showed that Bacillus subtilis under study is a good producer of extra cellular esterase,which can be beneficial for industries

Purification and Partial Characterization of Cellulase Produced by Aspergillus niger Cultured on Vitellaria paradoxa shells

2017 ◽  
Vol 6 (2) ◽  
Author(s):  
Abdulhakeem Olarewaju Sulyman ◽  
Yusuf A Iyanda ◽  
Afolabi Olaniyi Opasola ◽  
OtunOla Adedayo ◽  
Raliat Abimbola Aladodo
Keyword(s):  
Aspergillus Niger ◽  
Specific Activity ◽  
Gel Filtration ◽  
Inoculum Size ◽  
Partial Characterization ◽  
Effect Of Temperature ◽  
Vitellaria Paradoxa ◽  
Endoglucanase Activity ◽  
Ammonium Sulphate Precipitation

This research investigated the purification and partial characterization of cellulase produced by Aspergillus niger cultured on Vitellaria paradoxa shells. Cellulase (endoglucanase) from A. niger was produced under optimum fermentation conditions at 35 °C, pH 4.7, V. paradoxa, 4 g/L, inoculum size of 10 mm and the fermentation media incubated for 120 hours. The crude endoglucanase obtained were partially purified by subjecting to ammonium sulphate precipitation, dialysis and gel filtration chromatography for further purification. The effect of temperature and pH on the activity of purified endoglucanase was determined. Cellulase was purified to 734.33 folds by Sephadex G-100 column chromatography with a specific activity and yield of 4.406 U/mg and 63.03% respectively. Fractions 4 and 7 contained the highest endoglucanase activity out of 18 fractions collected and the two fractions were pooled for further analysis. The activity of purified endoglucanase was optimum at a temperature of 40 °C and pH 5. Therefore, the purified endoglucanase produced may be explored in detergent industry.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

2017 ◽  
Vol 37 (1) ◽  
pp. 31
Author(s):  
Fitria Fitria ◽  
Nanik Rahmani ◽  
Sri Pujiyanto ◽  
Budi Raharjo ◽  
Yopi Yopi
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Centrifugal Filter ◽  
Sds Page ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial  fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to  conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the  xylooligosaccharide types from xylan hydrolysis by this enzyme.  Based on this research, the optimum time for enzyme production  occurred at 96 hours with the enzyme activity of 6.275 U/mL and  enzyme specific activity of 5.093 U/mg. The specific activities were  obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0  U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of  xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram  showed that the molecular weight of xylanase protein were about  25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan  (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian  ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji  hidrolisis untuk mengetahui jenis xilooligosakarida yang  dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96  dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil  pemekatan dengan amicon® ultra-15 centrifugal filter devices,  kromatografi filtrasi gel dan kromatografi penukar anion  mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas  tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang  dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

scholarly journals Kinetic studies of partially purified lipase from marine actinomycete streptomyces acrimycini ngp 1

2020 ◽  
Vol 7 (1) ◽  
pp. 63-68
Author(s):  
Vishnupriya B ◽  
Anbarasi G
Keyword(s):  
Specific Activity ◽  
Coastal Region ◽  
Kinetic Studies ◽  
Biodiesel Production ◽  
Partial Purification ◽  
Native Page ◽  
Ammonium Sulphate Precipitation ◽  
South Indian ◽  
Percent Recovery ◽  
Marine Actinomycete

This study was focused on partial purification and characterization of lipase from Streptomyces acrimyciniNGP 1, isolated from marine sediment of south Indian coastal region. In purification steps, 4.53 fold purification was achieved after 85% ammonium sulphate precipitation with 0.97 percent recovery. In further purification steps, 1.33 fold purification was achieved by Sephadex G-100 chromatography with 1.61 percent of recovery. The specific activity of purified enzyme was 1525 U/mg. Zymogram of crude enzyme on native-PAGE presented bands with lipase activity of molecular weight and Isoelectric point were 50 kDa and 7.4 respectively. These features render this lipase of interest as a biocatalyst for applications such as biodiesel production and detergent formulations.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

scholarly journals Extraction, Characterization and Partial Purification of L-Asparaginase from the Leaves of Arachis Hypogaea L.

2019 ◽  
Vol 16 (3) ◽  
pp. 681-691
Author(s):  
Niranjana. J Niranjana ◽  
Kandasamy Arun Gandhi ◽  
D. Sunmathi ◽  
P. Nanthavanan
Keyword(s):  
Metal Ions ◽  
Arachis Hypogaea ◽  
Specific Activity ◽  
Lymphoblastic Leukemia ◽  
Gel Filtration ◽  
Size Exclusion ◽  
Partial Purification ◽  
Crude Enzyme Extract ◽  
Ammonium Sulphate Precipitation ◽  
Arachis Hypogaea L

L-asparaginase has been a promising therapeutic agent in the treatment of acute lymphoblastic leukemia and lymphoma. In recent times, due to the side effects of commercially available bacterial L-asparaginase and its unavoidable importance, plants are being explored as the source of L-asparaginase. The enzyme L-asparaginase was partially purified from Arachis hypogaea L. The crude enzyme extract was subjected to different purification steps including ammonium sulphate precipitation, dialysis followed by separation on Sephadex G-100 gel filtration (size exclusion chromatography) to obtain partially pure form of L - asparaginase. The enzyme was partially purified to 118 folds and contained specific activity of 4686.86 U/mg with 9.85% yield. SDS-PAGE electrophoresis of the partially purified enzyme revealed that it was a single protein with molecular weight of 70 kDa. The study on physiochemical properties showed that L - asparaginase from Arachis hypogaea L. was potassium-dependent in nature, where its optimum pH of enzyme activity was found to be 8.0 and temperature as 40°/50°C with reaction time of 15 - 20 minutes. Also it was observed that the L-asparaginase activity increased with the presence of metal ions such as Na+, Mg++, making it an enzyme dependent on metal ions for its reaction. In addition to this, it was revealed that the enzyme was partially inhibited in presence of certain chelators. The specificity of L-asparaginase obtained from Arachis hypogaea L. with lack of urease activity and minimal glutaminase activity along with less cytotoxicity on human blood indicated it as an efficient chemotherapeutic agent that could be investigated further in future studies.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
Author(s):  
I Ishii-Karakasa ◽  
H Iwase ◽  
K Hotta ◽  
Y Tanaka ◽  
S Omura
Keyword(s):  
Enzyme Activity ◽  
Culture Medium ◽  
Gel Filtration ◽  
Partial Purification ◽  
Gastric Mucus ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Optimum Ph ◽  
New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

2012 ◽  
Vol 10 (1) ◽  
Author(s):  
Ismat Bibi ◽  
Haq Nawaz Bhatti
Keyword(s):  
Lignin Peroxidase ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Gel Filtration ◽  
Veratryl Alcohol ◽  
Exchange Chromatography ◽  
Different Temperatures ◽  
Scale Experiment ◽  
Menten Kinetic

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

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