Ratio Of Crude Extract Research Articles

Three-phase partitioning and immobilization of Bacillus methylotrophicus Y37 cellulase on organo-bentonite and its kinetic and thermodynamic properties

AbstractIn this study, for the first time Bacillus methylotrophicus Y37 cellulase was purified and recovered in a single step by three-phase partitioning (TPP). The optimal purification parameters for TPP were 40% ammonium sulfate saturation (m/v) with a 1.0:1.0 (v/v) ratio of crude extract:t-butanol, which gave 5.8-fold purification with 155% recovery of cellulase. Non-covalent immobilization of the partitioned cellulase was performed using bentonite as a support material. The activity observed in the 20th experiment was 100%. The optimal pH values and temperatures determined for the free enzyme and the immobilized enzyme were 5.0 and 6.0 and 45°C and 50°C, respectively. The Arrhenius activation energy (Ea) of the immobilized enzyme was lower than that of the free enzyme, whereas the Michaelis–Menten constant (Km) and maximum velocity (Vm) of the immobilized enzyme increased. The turnover number (kcat) and the catalytic performance (kcat/Km) demonstrated the improved catalytic properties of the immobilized enzyme compared to the free enzyme. Immobilization of cellulase is thermodynamically preferred.

Three phase partitioning as a rapid and efficient method for purification of plant-esterase from wheat flour

Abstract Three-phase partitioning (TPP) was used to purify plant-esterase from wheat flour. Effect of various process parameters has been evaluated and plant-esterase was purified to 11.35-fold by optimized single step TPP system (50%, (w/v) (NH4)2SO4 saturation, 1:1 (v/v) ratio of crude extract: t-butanol at pH 4).The enzyme was found to be exclusively partitioned in the aqueous phase. Using TPP system, plant-esterase quickly purified to homogeneity with very high purity and activity. On the basis of single factor research, purification process was optimized by using response surface method, established a new type of high efficient purification plant-esterase method. To the best of our knowledge, this is the first report for purification and characterization of plant-esterase by using three phase partitioning (TPP). The results indicated that, TPP is a simple, quick, economical and very attractive process for purification of plant-esterase compared to conventional chromatographic protocols.

Antioxidant Activities ofCentella asiaticaExtract-loaded Bovine Serum Albumin Nanoparticles in Simulated Gastrointestinal System Study

Bioavailability of active compounds extracted from herbs is generally limited to be adsorbed or expressed to target organisms due to several physical and chemical factors. Nanoparticle encapsulation techniques was developed to carry bioactive macromolecules ofCentella asiatica(Buabok) in the form ofC. asiaticaExtract-loaded Bovine Serum Albumin Nanoparticles (CBNPs) to improve bioavailability. In this study, the antioxidant activities of CBNPs andC. asiaticacrude extract were evaluated by using DPPH radical scavenging assay in the simulated gastrointestinal system, including mastication, stomach, duodenum, and ileum conditions to provide the environment which similar toin vivosystem in terms of chemical and physical parameters. CBNPs were prepared by mixing ofC. asiaticacrude extract and BSA at different ratio of 1:2, 1:3, and 1:4. The results showed that the highest antioxidant activity of CBNP was observed when the ratio of crude extract and BSA at 1:2 was used at pH 2.0 or in the simulated stomach condition. The denaturation or unfolding of BSA in the simulated stomach occurred when pH was low could lead to the release of active compounds at certain area in gastrointestinal system.

One-step recovery of latex papain from Carica papaya using three phase partitioning and its use as milk-clotting and meat-tenderizing agent

Three Phase Partitioning (TPP) system as an elegant non-chromatographic and bulk separation method was successfully applied for the extraction and recovery of papain from the latex of Carica papaya. The optimized parameters of TPP allowed achieving a purification fold of 11.45 and activity recovery of 134% with 40% (NH4)2SO4, 1.0:0.75 ratio of crude extract: t-BuOH at pH and temperature of 6.0 and 25 °C, respectively. The recovered papain had a molecular weight of 23.2 kDa and revealed maximum activity at pH 6.0 and temperature of 50 °C. The maximum values of Km and Vmax parameters were 10.83 mg mL−1 and 33.33 U mL−1, respectively. The protease with 4 isoforms was stable at 40–80 °C and a pH range of 6.0–7.5 against numerous metal ions and none of them inactivated the recovered protease. Moreover, 10 mM Ca2+ improved 2-folds the activity and half-life of the protease at temperatures from 30 to 50 °C. The milk-clotting activity tests revealed high stability of latex papain at storage, namely at −20 °C compared to 4 °C and 25 °C for up than 5 weeks. As a meat tenderizing agent, it showed promising role under different treatments by improving the texture of tough meat. The findings indicated that one-step TPP system is a simple, quick, economical and very attractive process for fast recovery of latex papain compared to other proposed protocols.

Partial Purification and Biochemical Characterization of ?-Glucosidase from Corn by Three-Phase Partitioning

α-Glucosidase was first purified from corn by three-phase partitioning(TPP) and then characterized. Ammonium sulfate and t-butanol were used in order to obtain three phases. Effect of different process parameters such as; ammonium sulfate concentration, enzyme to t-butanol ratio and pH required for efficient purification of the corn α-glucosidase was studied to get highest purification fold and activity recovery. Optimum purification parameters of the TPP system were determined as 50% (w/v) ammonium sulfate saturation with 1.0:0.75 (v/v) ratio of crude extract: t-butanol at pH 4.5. Under optimized conditions α-glucosidase was purified with 3.8 purification fold and 60% activity recovery. The molecular weight of α-glucosidase was determined approximately as 29 kDa by using sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE). Characterization studies showed that, optimum pH and temperature of α-glucosidase were pH 4.5 and 55°C, respectively. The purified enzyme was found to be very stable at a temperature range of 25-60°C and a pH range of 3.5-5.5. Kinetic constants (KM and Vmax) were calculated from Lineweaver-Burk plot as 0.64 mM and 0.45 U, respectively. With 20 mM of CaCl2 concentration α-glucosidase activity was significantly increased to 130%. TPP is a useful strategy to concentrate and purify α-glucosidase for its applications.

Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3

The present study investigates the production and partial biochemical characterization of an extracellular thermostable xylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phase partitioning (TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in the presence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system using the optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purification fold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of 5.0–10. The optimum temperature for xylanase activity was 55 °C and the half-life time at this temperature was of 6 h. At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist to sodium dodecyl sulfate and β-mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. The recovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% was observed. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability was observed with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested as a potential candidate for application in some industrial processes. To the best of our knowledge, this is the first report of xylanase activity and recoverey using three phase partitioning from B. oceanisediminis.

Three phase partitioning, a scalable method for the purification and recovery of cucumisin, a milk-clotting enzyme, from the juice of Cucumis melo var. reticulatus

Cucumisin [EC 3.4.21.25] was first purified from Cucumis melo var. reticulatus juice by three-phase partitioning (TPP). Optimum purification parameters of the TPP system were determined as 60% ammonium sulfate saturation with 1.0:1.25 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 20°C, respectively. Cucumisin was purified with 4.61 purification fold and 156% activity recovery. The molecular weight of the recovered cucumisin was determined as 68.4kDa and its isoelectric point is 8.7. Optimum pH and temperature of cucumisin were pH 9.0 and 60–70°C, respectively. The protease was very stable at 20–70°C and a pH range of 2.0–12.0. Km and Vmax constants were 2.24±0.22mgmL−1 and 1048±25μ Mmin−1, respectively. The enzyme was stable against numerous metal ions and its activity was highly enhanced by Ca2+, Mg2+, and Mn+2. Cucumisin activity was 2.35-folds increased in the presence of 5mM of CaCl2. It was inactivated by Co2+, Cd2+, Zn2+ and Fe2+ and dramatically by PMSF. Cucumisin milk-clotting activity was highly stable when stored under freezing (−20°C) compared at 4°C and 25°C. Finally, TPP revealed to be a useful strategy to concentrate and purify cucumisin for its use as a milk-clotting enzyme for cheese-making.

Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes

The present work describes for the first time an elegant non-chromatographic method, the three phase partitioning for the purification and recovery of zingibain, a milk-clotting enzyme, from Zingiber officinale rhizomes. Factors affecting partitioning efficiency such as (NH4)2SO4 saturation, crude extract to t-butanol ratio and pH on zingibain partitioning were investigated. Optimal purification parameters were 50% (NH4)2SO4 saturation with 1.0:1.0 ratio of crude extract:t-butanol at pH 7.0, which gave 14.91 purification fold with 215% recovery of zingibain. The enzyme was found to be exclusively partitioned in the aqueous phase. The enzyme showed a prominent single band on SDS-PAGE. It is a monomeric protein of 33.8kDa and its isoelectric point is 4.38. The enzyme exhibited maximal proteolytic activity at a temperature of 60°C and pH 7.0. It was found to be stable at 40–65°C during 2h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca2+, K+ and Na+. It was completely inhibited by heavy metal ions such as Cu2+ and Hg2+ and partially by Cd+. Zingibain milk-clotting activity (MCA) was found to be highly stable when stored under freezing (−20°C) for 30 days compared at 4°C.

Three-phase partitioning as an efficient method for the purification and recovery of ficin from Mediterranean fig (Ficus carica L.) latex

Three phase partitioning or TPP consists of proteins’ precipitation due to simultaneous presence of ammonium sulfate and t-butanol. The technique has been successfully used for the partial purification of ficin (EC 3.4.22.3) from Ficus carica latex. In this study, ficin of Mediterranean F.carica was for the first time partially purified and recovered in a single step using TPP. Optimal purification parameters of the system were 40% ammonium sulfate saturation (w/v) with 1.0:0.75 (v/v) ratio of crude extract: t-butanol at pH 7.0, which gave 6.04 purification fold with 167% recovery of ficin. The enzyme was found to be exclusively partitioned in the interfacial precipitate phase. The Tricine SDS–polyacrylamide gel electrophoresis analysis of the enzyme showed comparatively purification and protein molecular weight of the enzyme was nearly found to be 23.4kDa. Characterization of enzyme showed that optimal pH of purified enzyme was 6.5 and optimal temperature was 60°C. The enzyme was found to be stable at 40–70°C for 1h. This study showed that TPP is a simple, economical and quick method for ficin recovering from F.carica latex trees and can be performed in a purification process to be used successfully in food industries.

Purification, recovery, and characterization of chick pea (Cicer arietinum) β-galactosidase in single step by three phase partitioning as a rapid and easy technique

In this study chick pea β-galactosidase was first time purified and recovered in single step by three phase partitioning (TPP). Optimal purification parameters for TPP were 60% ammonium sulfate saturation (w/v) with 1:0.5 (v/v) ratio of crude extract:t-butanol at pH 6.8, which gave 10.1 purification fold with 133% recovery of β-galactosidase. SDS–PAGE analysis showed that protein has two subunits with molecular masses of 48 and 38kDa, respectively. Characterization of enzyme showed that optimal pH of purified enzyme was 2.8 and optimal temperature was 50°C. Enzyme was further characterized by the Arrhenius activation energy and Michael–Menten kinetic constants. Activation energy (Ea) was calculated by using Arrhenius equation and determined to be 15.52kcalmol−1. Km value of purified enzyme was estimated for the o-nitrophenyl β-d galactopyranoside (ONPG) substrate as 1.09mM, while its maximum velocity, Vm was 0.90U/mL/min at 37°C. TPP improved substrate affinity of enzyme by the increased flexibility during the partitioning. TPP is simple, easy and economic technique for purification and recovery of β-galactosidase from chick pea, and has a big potential use for industrial applications.