• Food Science and Preservation
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• v.7 no.2
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• pp.201-206
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• 2000

${\beta}$-Galactosidase was extracted and purified from strawberry. The purified ${\beta}$-Galactosidase from strawberry was investigated their physicochemical characteristics. ${\beta}$-Galactosidase was purified 25.74 fold from strawberry. The purification procedure include ammonium sulfate fraction, acetone powder treatment and gel and ion exchange chromatography. Yield of the enzyme purification was 18.11%. The purified enzyme has native molecular weight of 116,000 dalton. Vmax value and Km value of ${\beta}$-Galactosidase were 0.077 mM ONPG/ml/15mim and 1.75x10-2mM, respectively. The optimum temperature and pH of ${\beta}$-Galactosidase were 43$^{\circ}$C and pH 4.0, respectively. The ${\beta}$-Galactosidase activity was stable below 50$^{\circ}$C and at pH 4.0 to pH 6.0. Among the metal ions Ca and Mg were did not affect, whereas K, Cu and Zn show a little effect on the enzyme activity. The ${\beta}$-Galactosidase activities were inhibited by treatment with EDTA and SDS.

• Journal of Microbiology
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• v.35 no.2
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• pp.134-140
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• 1997

Botrytis cinerea T91-1 has been shown to produce at least four different polygalacturonases into a liquid medium containing citrus pectin, a carbon sousrce. One of the enzymes, which had an apparent molecular weight of 66 kDa estimated by denatured polyacrylamide gel electrophoresis, was purified to electrophoretic homogeneity by a series of procedures including a cetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. The molecular weight of native enzyme was determined to be 64 kDa by gel permeation chromatography indicating the enzyme to be a single polypeptide chain. By viscometric analysis, the enzyme was revealed as exo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as $Ca^{2+}$, $Mg^{2+}$, and Cu$^{2+}$. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was 5$0^{\circ}C$. And the enzyme showed optimal pH values between 4.0 and 5.0. The enzyme was stable upto 12 hours in the range of pH 3 to 8 and at temperature below 3$0^{\circ}C$.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.82-86
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• 1998

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins in E. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated buy centrifugation, resulting in highly purified proteins in the supernatant.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.593-598
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• 2005

A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.11
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• pp.1559-1563
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• 2002

The ovarian follicular fluid was found to contain steroidogenesis stimulatory protein similar to albumin from human and buffalo. Therefore, the albumins from various species, commercial and purified, were studied for their steroidogenic effect on progesterone secretion by granulosa cells from buffalo ovaries, during culture. A dose of $20{\mu}g$ of bovine serum albumin was optimum to exhibit maximum progesterone secretion on day 6 of culture, in medium ($350{\mu}l$) containing $10^5$ cells. Among commercial albumins, chicken albumin showed highest effect on progesterone secretion, which was followed by albumins from goat, bovine, human, sheep and rat, respectively at day 6 of culture. The albumins were also purified from blood serum of buffalo, goat and rat using salt fractionation, ion-exchange chromatography, gel filtration and SDS-PAGE. The highest stimulatory effect on progesterone secretion was shown by albumin purified from buffalo blood serum and lowest by that from rat blood. Comparatively the buffalo and goat albumins were more biologically active than commercial albumins. The presence of some active molecules conjugated with freshly purified albumins may be responsible for better stimulatory effect.

• Bulletin of the Korean Chemical Society
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• v.32 no.12
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• pp.4253-4257
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• 2011

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (CIEF) were employed to characterize and compare ricin E purified from the small grain seeds of Ricinus communis with ricin D isoform. During the purification of ricin E using ion-exchange and size-exclusion chromatography, SDS-CGE was found to be useful for monitoring the efficiencies of purification steps. SDS-CGE showed that the molecular size of ricin E was not significantly different from that of ricin D, which was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CIEF was useful for discriminating ricin E from ricin D based on their isoelectric points (pI). The pI values of ricin E and D were 8.6-8.8 and 7.0-7.4, respectively. This study demonstrates the usefulness of SDS-CGE and CIEF for the characterization of ricin toxins.

• KSBB Journal
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• v.14 no.3
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• pp.279-283
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• 1999

We have compared the breakthrough behavior of lysozyme contained in fresh han egg white on various cation exchagers, and the adsorbent, known by the trade name Cellufine C-200 (Amicon), has shown the best performance. The effects of ion strength, pH, and linear flow rate on the breakthrough behavior were examined using the Cellufine C-200 adsorbent. The optimal conductivity, pH and linear flow rate were determined from the breakthrough behavior and found to be 2.75 mS/cm, 7.0, and 0.635 cm/min, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1369-1379
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• 2010

This study investigated purification and milk-clotting activity of the enzymes produced by Bacillus subtilis var, natto and Rhizopus oligosporus compared with that of commercial rennet. The clotting time, viscosity, tension and microstructure of the curd and electrophoretic patterns of milk proteins were determined. The milk-clotting activity/proteolytic activity ratios (MCA/PA ratio) of B. subtilis, R. oligosporus and commercial rennet were also compared. The results revealed that the curd formed by the commercial rennet had the highest viscosity and curd tension and the shortest clotting time among the three enzymes. However, curd produced by Rhizopus enzymes was ranked as second. From the MCA/PA ratio and electrophoretogram analyses it could be concluded that the enzyme produced by B. subtilis had the highest proteolytic activity, while the commercial rennet had the highest milk-clotting activity. Observations of microstructures of SEM showed that the three-dimensional network for curd formed by commercial rennet was denser, firmer and more smooth. The milk-clotting activity, specific activity, purification ratio and recovery of the purified enzymes produced by both the tested organisms were also determined with ion exchange chromatography and gel filtration.

• BMB Reports
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• v.38 no.4
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• pp.407-413
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• 2005

Calcium and zinc binding protein, calprotectin is a multifunctional protein with broad spectrum antimicrobial and antitumoural activity. It was purified from human neutrophil, using a two-step ion exchange chromatography. Since surface hydrophobicity of calprotectin may be important in membrane anchoring, membrane penetration, subunits oligomerization and some biological roles of protein, in this study attempted to explore the effect of calcium in physiological range on the calprotectin lipophilicity. Incubation of human calprotectin ($50\;{\mu}g/ml$) with different calcium concentrations showed that 1-anilino-8-naphthalene sulfonic acid (ANS) fluorescence intensity of the protein significantly elevates with calcium in a dose dependent manner, suggesting an increase in calprotectin surface hydrophobicity upon calcium binding. Our study also indicates that calcium at higher concentrations (6, 8 and 10 mM) induces aggregation of human calprotectin. Our finding demonstrates that the starting time and the rate constant of calprotectin aggregation depend on the calcium concentration.