Freeze-thaw system for thermostable β-Galactosidase isolation from Gedong Songo Geobacillus sp. isolate

The effective isolation of intracellular enzymes from thermophilic bacteria is challenging because of their sturdy membrane. On the other hand, the low-cost and nontoxic method is essential for industrial food enzymes. The freeze-thaw cycles using acetone-dry ice as a frozen system was studied for efficient isolation of thermostable b-galactosidase from Geobacillus sp. dYTae-14. This enzyme has been known for application in the dairy industry to reduce the lactose content. In this study, the freeze-thaw method was performed with cycle variations 3, 5, and 7 cycles. Acetone-dry ice (-78°C) is used as a frozen system and boiling water for thawing. The b-galactosidase activity was assayed using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate and protein content determined with the Lowry method. The results show that the most effective freeze-thaw is five cycles. The enzyme’s highest specific activity is 3610.13 units/mg proteins at 40-60 % ammonium sulfate saturation, with a purity value of 2.52.

Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.

Partial Purification and Characterization of Polyphenol Oxidase from the Wild Edible Mushroom Lepiota Procera Using Three-Phase Partitioning

Polyphenol oxidase (PPO) from the wild edible mushroom Lepiota procera is partially purified and biochemically characterized using three-phase partitioning (TPP), which is an easily applied and effective method. This method includes ammonium sulfate saturation at different concentrations, t-butanol addition (1:1; 1:5), and adjustment of pH. Optimum purification parameters with 20% ammonium sulfate saturation and 1:1 t-butanol conditions led to the highest activity at bottom phase with 8.4 fold purification. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the molecular weight of the purified enzyme to be 35 kDa. The partially purified PPO presented a high level of activity with L-DOPA (Michaelis-Menten constant, Km – 0.12 mM), followed by caffeic acid (0.27 mM) and 4-methylcatechol (0.46 mM) and it is classified as a catecholase type of PPO. The enzyme had peak activity at a temperature of 40 °C and a pH value of 7.0. These results demonstrate a new enzyme source and easy purification method, useful for various industrial applications.

Conjugation of Peroxidase from Brassica oleracea gongylodes for Use as a Label- Prospect of a Novel Enzyme Tag for Immunoassay Systems

Peroxidase tagged proteins are being used successfully as immune-histological probes for the demonstration of tissue antigens, and in enzyme amplified immunoassay systems for the quantitative determination of soluble and insoluble antigens. The glycoprotein nature of peroxidases can be exploited for conjugation to proteins of interest. Peroxidase extracted from the bulbs of Brassica oleracea gongylodes was salted out at 40-80% ammonium sulfate saturation and activated by treatment with 1-Fluoro-2,4-dinitro benzene (FDNB) and periodate. Treatment with 0.08% FDNB and 12.5mM periodate was optimized for activation of the enzyme. The treated enzyme was found to conjugate successfully to immunoglobulin fractions harvested from egg yolk (IgY), human plasma and goat serum. Enzyme conjugated to IgY fraction showed improvement in its pH stability and temperature stability. The affinity of the enzyme for its substrate phenol did not alter to a significant extent upon activation and conjugation. The conjugates exhibited high affinity towards phenol, bromocresol purple and bromothymol blue in comparison to HRP conjugates prepared using the same protocol. Int. J. Appl. Sci. Biotechnol. Vol 5(1): 59-65

Identification and Purification of Cholera Like Toxin from Environmental Isolate of Vibrio cholerae

The presence and prevalence of V. cholerae were investigated in forty five water samples collected from different locations of Tiger River/ Baghdad city. Twenty one isolates were isolated by adopting a simple isolation techniques. The final identification revealed that only three isolates were confirmed as V. cholerae. They were named 1J, 1R and Dial 131 which are all serogrouped as non-O1. Toxin Coregulated Pili (TCP) and heat labile enterotoxin (LT) were determined in only the environmental isolate 1J while non of the isolates produced heat stabile toxin (ST). The purification scheme was improved, few steps were adopted to include back extraction of ammonium sulfate, saturation between 80-20%, desalting through Sephadex G25, and gel filtration using Sephadex G100 which highly increase the specific activity.

Effect of alloxan diabetes on a Ca2+-activated proteinase in rat skeletal muscle

The effect of making rats diabetic by alloxan injection on activity of the muscle Ca2+-activated proteinase (CAF) was investigated. Groups of four to seven control or alloxan-injected rats were killed 10 min (0 day) and 10, 17, and 24 days after a second alloxan injection. The second alloxan injection was given 3 days after the first. CAF activity was assayed in fractions precipitated between 0 and 45% ammonium sulfate saturation (P0-45 crude CAF fractions) that had been prepared so as to remove the protein inhibitor of CAF. Gel permeation, followed by DEAE-cellulose chromatography of pooled P0-45 crude CAF fractions from each time-treatment group, demonstrated that the assays used in this study were specific for CAF activity. Muscle CAF activity was up to 50% higher in alloxan-injected rats than in control rats, regardless of whether activity was expressed per gram sarcoplasmic protein, per gram contractile protein, or per gram skeletal muscle fresh weight. Alloxan injection diminished rate of muscle mass accumulation but did not change the proportion of sarcoplasmic or contractile protein in skeletal muscle. Hence, alloxan injection decreased the rate of contractile protein deposition. The elevation of muscle CAF activity in alloxan-injected rats is consistent with the proposed role of CAF in initiating metabolic turnover of myofibrillar proteins but does not prove this role nor exclude participation of other proteinases.

Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits

Partially purified thrombopoiesis-stimulating activity was prepared from the plasma of thrombocytopenic rabbits using ammonium sulfate precipitation and DEAE cellulose, Sephadex, and carboxymethyl cellulose chromatography. The protein fraction precipitated by an ammonium sulfate saturation of 60%-80%, previously shown to contain thrombopoiesis-stimulating activity, was used as starting material. Column chromatography was carried out at room temperature at pH 5.6. Under these conditions, thrombopoiesis-stimulating activity (thrombopoietin) was retained by DEAE cellulose (0/03 M citrate- phosphate buffer) and carboxymethyl cellulose (0/003 M citrate- phosphate buffer), and eluted with 0.4 M NaCl. Thrombopoietin was retarded by Sephadex G-100; the ratio of the elution volume to the void volume was 1.32:1. Immunoelectrophoretic analysis of partially purified thrombopoietin indicated that following removal of most of the albumin by DEAE chromatography, only proteins with the mobilities of beta- globulins and albumin and traces of other anodally migrating proteins were detectable in the fractions that contained thrombopoiesis- stimulating activity. Thrombopoietin was not dialyzable and was stable from at least pH 5.6 to 7.5. It was approximately 1000-fold purified following sequential chromatography with DEAE and carboxymethyl cellulose. Although the three fractions described reproducibly stimulated thrombopoiesis, as measured by increased levels of selenomethionine-75Se (75SeM) in the circulating platelets, platelet counts did not increase.

Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits

Partially purified thrombopoiesis-stimulating activity was prepared from the plasma of thrombocytopenic rabbits using ammonium sulfate precipitation and DEAE cellulose, Sephadex, and carboxymethyl cellulose chromatography. The protein fraction precipitated by an ammonium sulfate saturation of 60%-80%, previously shown to contain thrombopoiesis-stimulating activity, was used as starting material. Column chromatography was carried out at room temperature at pH 5.6. Under these conditions, thrombopoiesis-stimulating activity (thrombopoietin) was retained by DEAE cellulose (0/03 M citrate- phosphate buffer) and carboxymethyl cellulose (0/003 M citrate- phosphate buffer), and eluted with 0.4 M NaCl. Thrombopoietin was retarded by Sephadex G-100; the ratio of the elution volume to the void volume was 1.32:1. Immunoelectrophoretic analysis of partially purified thrombopoietin indicated that following removal of most of the albumin by DEAE chromatography, only proteins with the mobilities of beta- globulins and albumin and traces of other anodally migrating proteins were detectable in the fractions that contained thrombopoiesis- stimulating activity. Thrombopoietin was not dialyzable and was stable from at least pH 5.6 to 7.5. It was approximately 1000-fold purified following sequential chromatography with DEAE and carboxymethyl cellulose. Although the three fractions described reproducibly stimulated thrombopoiesis, as measured by increased levels of selenomethionine-75Se (75SeM) in the circulating platelets, platelet counts did not increase.