Search

This study was carried out to investigate the purification and biochemical characterization of a new extracellular alkaliphilic and thermostable pectate lyase (Pel-20) isolated from Actinomadura keratinilytica strain Cpt20. Pure protein was obtained after sequential chromatographies on a fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC) columns. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 34125.11-Da. The enzyme had an NH 2 -terminal sequence of GFATNQGGTTGGAGGTLS, thus, sharing high homology with actinomycetes pectate lyase family. The results showed that this enzyme was completely inhibited by EDTA, which supports its belonging to the pectate lyase superfamily. It showed optimum activity at pH 10.5 and 70 °C. The thermoactivity and thermostability of Pel-20 were enhanced in the presence of 1 mM Ca 2+ . Its half-life times at 70, 80, 90, and 100 °C were 18, 12, 7, and 2 h, respectively. Its kinetic parameters, K m and V max values were 0.45 mM and 21,700 U/mg, respectively. Low-esterified pectin was the optimum substrate for the Pel-20. However, higher-esterified pectin was also weakly cleaved. Overall, the alkaliphilicity and thermostability properties of Pel-20 make it a potential candidate for future application in industrial bioprocesses. [ABSTRACT FROM AUTHOR]

Abstract: A thermoalkaline protease with a molecular weight of 22kDa was purified from the Bacillus cereus SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed the K m, V max and k cat to be 1.09mgml−1, 0.909mgml−1 min−1 and 3.11s−1, respectively, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 45–55°C. It was fully stable at 0.0–2.0% and moderately stable at 2.5–10.0% (w/v) sodium chloride. Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitory with regard to enzyme activity, whereas cysteine, β-mercaptoethanol, calcium, magnesium, manganese and copper at concentration of 1.0mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100, Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and 1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stable and retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is predominantly a β-pleated structure and a detailed structural composition showed ∼50% β-sheets. The CD-based conformational evaluation of the protease after incubation with modulators, metal ions, detergents and at different pH values, revealed that the change in the β-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30min. [Copyright &y& Elsevier]

Abstract: Ureases are used in beverage, fertilizer and pharmaceutical industries. We report purification, characterization and a simple method for immobilization of urease form Momordica charantia seeds. The purification of the enzyme is a two steps process involving anion exchange chromatography using DEAE–Sepharose matrix followed by size exclusion chromatography. The fold purification of the enzyme was found to be 6.5. The estimated molecular mass and K m of the enzyme were 174.5kDa and 34±0.5mM, respectively. The purified urease was immobilized on chitosan beads activated with glutaraldehyde. The activation of chitosan beads and immobilization were confirmed by Fourier Transform Infrared (FTIR) spectrometry and Scanning Electron Microscope (SEM), respectively. Moreover, Energy Dispersive X-ray (EDX) analysis shows increased percentage of nitrogen which further confirms immobilization. The efficiency of immobilization was found to be approximately 97%. The immobilized enzyme retains 60% activity till fourteen successive batches of enzymatic reactions which makes it a highly stable product. Comprehensively, the enzyme shows desirable properties for various applications. [Copyright &y& Elsevier]

Abstract: Dityrosine (DY) can be used as a biomarker to detect oxidative protein damage and selective proteolysis. It is generally prepared by horseradish peroxidase (HRP)-catalyzed oxidation of l-tyrosine (Y) followed by multistep chromatographic separations. In this study, we present an alternative method for the preparation of DY by HRP-catalyzed synthesis of N,N′-diBoc-dityrosine (DBDY) from N-Boc-l-tyrosine (BY). The presence of the tert-butoxycarbonyl (Boc) group ensured that the fraction of further oxidized by-products (e.g., trimers and pulcherosine) was quite low. The yield of DBDY (37.5%) was comparable to that reported for DY (> 26%). DBDY could be purified by a simple one-step silica column chromatography procedure that resulted in a purity of 89.7%. DBDY is considered to be better than DY for subsequent chemical reactions (for binding to polymers, amino acids, drugs, antibodies, etc.) because such reactions can be selectively performed by using the carboxylic acid and amine groups in the following sequence: first, the carboxylic acid groups are used; next, the Boc groups are removed; and finally, the amino groups are used. To prepare DY, the Boc groups in DBDY were simply and completely removed by treatment with trifluoroacetic acid. [ABSTRACT FROM AUTHOR]

Abstract: Two cold-adapted lipases (Lipase-A and Lipase-B in the paper) of mesophilic Geotrichum sp. SYBC WU-3 were purified by using (NH4)2SO4 fractionation, chromatography separation on a DEAE-cellulose-32 column and a Sephadex G100 column. The molecular mass of Lipase-A and Lipase-B were determined to be approximately 41.1 and 35.8kDa, respectively by SDS-PAGE. The optimum temperature for the activity of Lipase-A was found to be 20°C, and that of Lipase-B was 15°C. Lipase-A and Lipase-B had good stability when temperature was below 40°C. Both the optimum pH for the activity of the lipases was 9.5. Lipase-A retained about 80% of its activity when pH was between 3 and 6 and Lipase-B maintained over 80% activity in the pH range of 3–8. The two lipases showed hydrolysis efficiency to various p-nitrophenyl esters, but they were more active with shorter p-nitrophenyl esters (C2 and C4). [Copyright &y& Elsevier]

Abstract: A novel isolation and purification method was developed for producing 1,3-propanediol with high purity and high yield from the mixture containing 1,3-propanediol, 1,2-propanediol, glycerol, and glucose. The developed method, which consisted of phase separation and chromatography, was found to be a simple and efficient procedure for the isolation and purification of 1,3-propanediol from the other components in the mixture. The use of a phase separation process allows rapid separation of 1,3-propanediol from interfering compounds and dramatically reduces solvent usage compared to alternative methods. The overall purity and yield of 1,3-propanediol in this study were 98% and 82% in the purification process, respectively. [Copyright &y& Elsevier]