Three-phase Partitioning Technique Research Articles

Enhancing the efficiency and functionality of xylanase from Bacillus sp. RTS11: Optimization, purification, characterization, and prospects in kraft pulp bleaching.

Bacillus sp. RTS11, a xylanolytic strain, was isolated from the Algerian desert rocks. Genetic analysis revealed a remarkable 98.69% similarity to Bacillus pumilus. We harnessed optimization techniques, including Plackett-Burman screening and Box-Behnken optimization design, to amplify xylanase production and activity. The outcome of these efforts was an optimized medium that yielded an impressive xylanase production titer of 448.89 U, a threefold increase compared to the non-optimized medium (146 U). The Purification of xylanase was achieved through the three-phase partitioning technique, employing t-butanol and various chromatographic methods. Notably, anion exchange chromatography led to isolating a highly pure enzyme with a molecular weight of 60 kDa. The xylanase exhibited its peak activity at a temperature of 60°C and a pH of 9.0. When applied to pulp pretreatment, 20 U/g of xylanase demonstrated a substantial increase in the release of phenolic and chromophore compounds while reducing sugar content in the pulp. Furthermore, this versatile xylanase shows its ability to efficiently hydrolyze a variety of agro-industrial residues, including wheat bran, corn and grape waste, wheat straw, and sugarcane bagasse. These findings underscore the significant potential of this xylanase enzyme in biobleaching processes and the utilization of agro-industrial waste, opening up exciting avenues for sustainable and environmentally friendly industrial applications.

Insolubilization of Tramates versicolor laccase as cross-linked enzyme aggregates for the remediation of trace organic contaminants from municipal wastewater

The novelty of this study deals with the biocatalytic treatment of trace organic contaminants (TrOCs) from municipal wastewater by insolubilized laccase. Laccase from Trametes versicolor was aggregated by three-phase partitioning technique followed by cross-linking with glutaraldehyde to produce insolubilized laccase as cross-linked enzyme aggregates (CLEAs). The optimal conditions for CLEAs preparation include ammonium sulphate concentration of 83% (w/v), crude to t-butanol ratio of 1.00: 1.05 (v/v), pH 5.3, and glutaraldehyde concentration of 20 mM obtained via statistical design. The efficiency of insolubilization of the CLEAs laccase based on the kcat/km ratio was approximately 4.8-fold greater than that of free laccase. The developed CLEAs showed greater resistance to product inhibition mediated by ABTS than the free enzyme and exhibited excellent catalytic activity even after the tenth successive cycle. Further, free laccase and the synthesized CLEAs laccase were utilized to treat five analgesics, two NSAIDS, three antibiotics, two antilipemics, and three pesticides in the municipal wastewater. Under the batch process with operating conditions of pH 7.0 and 20 °C, 1000 U/L of CLEAs, laccase removed 11 TrOCs in the range of about 20–99%. However, the inactivated CLEAs only adsorbed 2–25% of TrOCs. It was observed that acetaminophen, mefenamic acid, trimethoprim, and metolachlor depicted almost complete removal with CLEAs laccase. The performance of CLEAs laccase in a perfusion basket reactor was tested for the removal of TrOCs from municipal wastewater.

Valorization of waste Syzygium cumini seed kernels by three-phase partitioning extraction and evaluation of in vitro antioxidant and hypoglycemic potential

Diabetes is one of the leading causes of morbidity and mortality worldwide. Replacement of the chemically synthesized medicines with phytomedicines is of great importance. Syzygium cumini fruit has around 30% hard seed kernel part that is considered solid waste and can be a good source of bioactive phenolic compounds. Thus, this work was performed to valorize Syzygium cumini kernels powder (SCKP) using a three-phase partitioning technique (TPP) and evaluate in vitro antioxidant and hypoglycemic activities. The extraction yield of catechin, gallic acid, TFC, and TPC obtained through TPP was 0.52, 33.24 mg/g, 7.78 mg CE/g, and 82.66 mg GAE/g, respectively, in 45 min, 40% ammonium sulfate, 1:1 (V/V) extraction slurry to t-butanol, 30 ± 2 °C temperature, pH 4, 1:20 (g/mL) SCKP to water and SCKP size 106 µm. IC50 values 12.15, 9.33, and 7.55 in µg/mL for TPP extract were obtained from DPPH, α-amylase, and α-glucosidase in vitro assays, respectively. TPP for extraction of bioactive compounds was found superior over traditional extraction techniques (devoid of t-butanol and ammonium sulfate). The extract obtained shows enormous antioxidant and hypoglycemic potential for use in nutraceutical and pharmaceutical industries. Highlights First report on three-phase partitioning (TPP) extraction of catechin and gallic acid from SCKP. Effective extraction of total phenolic and flavonoid compounds. Proved the enormous antioxidant and hypoglycemic potential of SCKP’s TPP extract. TPP extraction, the selective, efficient, and economically viable option.

Encapsulation and characterization of cellulase purified with three-phase partitioning technique

Purification of Trichoderma reesei cellulase by three-phase partitioning (TPP) technique and encapsulation of the enzyme in calcium alginate gel were studied. These two basic experiments were optimized with all variables and finally optimum conditions for both purification and encapsulation studies were determined. Ideal TPP condition for the purification of T. reesei cellulase was determined as; 50% (w/v) ammonium sulphate saturation, 1:1 (v/v) enzyme: t-butanol ratio, pH 4.5 and 0.1 mg of the protein. By using this condition, the cellulase enzyme was successfully purified with 113% activity yield and 1.42 purification fold. As a result of the encapsulation studies, optimum immobilization condition for cellulase was determined as 0.5 mg enzyme (protein amount) and 2% (w/v) alginate, 0.5% (w/v) chitosan in 0.15 M CaCl2, 3 h for immobilization, 2.0% (v/v) glutaraldehyde and 2 h for cross-linking. The enzyme was immobilized with 82% activity yield by using this conditions. The cellulase, which was concentrated in the middle phase of TPP, could also immobilized with this condition. The optimal temperature of the free and immobilized cellulase were determined as 65 °C and the optimum pHs were found as pH 4.0 and pH 5.0, respectively. The immobilized cellulase was also used three times with high activity yields. The biochemical characterization and stability studies confirm that this immobilized enzyme show promise for industry that could be safely used especially both in the feed and food industry and food processing.

Extraction of peroxidase from bitter gourd (Momordica charantia ) by three phase partitioning with dimethyl carbonate (DMC) as organic phase

Abstract Three phase partitioning (TPP) is most renowned technique used for extraction and purification of natural products. In previous studies of TPP, t -butanol is mainly used as an organic phase. This is the first report that explores ability of dimethyl carbonate (DMC) in the field of TPP as an alternate solvent for t -butanol. In the present study TPP process with t -butanol and DMC as organic phase along with different salts was applied to waste bitter gourd powder to obtained peroxidase enzyme. DMC was found to be compatible with most of salts such as ammonium sulphate and sodium citrate and explored as more efficient solvent than t -butanol. This TPP system provides 4.84 fold purity of peroxidase enzyme at optimum source concentration of 0.15 g/mL, with a system comprising DMC as organic phase, sodium citrate (20%) as salt, agitation speed 120 rpm, pH 7, temperature 30 °C and extraction time of 3 h. Present study has aimed for extraction and separation of peroxidase from bitter gourd waste with TPP technique and ensures the scope of carbonated solvents in extraction and purification of proteins.

Extraction of acetyl 11-keto-β-boswellic acid (AKBA) from Boswellia serrata plant oleo gum resin using novel three phase partitioning (TPP) technique

The oleo gum resin of Boswellia serrata belonging to family Burseraceae, is Ayurvedic medicine and has been used for the treatment of inflammatory diseases and arthritic diseases in many countries. Acetyl 11-keto-β-boswellic acid (AKBA) is a potent anti-inflammatory agent obtained from oleo gum resin of Boswellic serrata. Our aim is to extract maximum amount of AKBA from Boswellic serrata by using Three Phase Partitioning (TPP) technique. In the TPP system, the effect of different process parameters such as ammonium sulfate concentration, extraction time, slurry to t-butanol ratio, solute to t-butanol ratio, pH and temperature, was studied in order to enhance the extraction of AKBA. Under the optimized conditions of ammonium sulfate concentration 0–50% (w/v), extraction time 180min, slurry to t-butanol ratio 1:1 (v/v), solute to solvent ratio 1:25 (w/v), pH 6 and temperature 50°C, the maximum yield of AKBA was found to be (80±0.33mg/g). TPP technique gave higher extraction yield of AKBA compared to conventional Soxhlet extraction (18±0.37mg/g), batch extraction (20±0.45mg/g), ultrasound assisted extraction (UAE) (24.23±0.28mg/g). Thus, TPP can be recommended as an attractive and simple technique for the extraction of AKBA from Boswellic serrata.

A novel extraction of trichosanthin from Trichosanthes kirilowii roots using three-phase partitioning and its in vitro anticancer activity

Context: Three-phase partitioning (TPP), a unique technique which has been explored for protein separation, was used for extraction of trichosanthin (TCS).Objective: TPP was used to optimize the TCS extraction and to determine its anticancer activity.Materials and methods: The process consists of the simultaneous addition of t-butanol and ammonium sulfate to the aqueous slurry of Trichosanthes kirilowii Maxim (Cucurbitaceae) root powder. The extraction of TCS was optimized with respect to the concentration of ammonium sulfate loading, the ratio of t-butanol to slurry, extraction time and pH. The anticancer activity was performed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay in vitro.Results: The extraction time with this technique is lower in comparison to conventional solvent extraction. The optimized protocol resulted in maximum recovery of 98.68% (w/w) protein within 1 h. The in vitro cytotoxic activity of the TCS was evaluated against HepG2 and WRL 68 cancer cell line and results showed that TCS possesses quite highly significant anticancer activity having IC50 values of 10.38 and 15.45 μmol/l, respectively, comparable to standard drugs.Conclusion: This framework is utilized as a basis for optimization for protein separation using TPP technique which is economical and eco-friendly.

Preparation and characterization of porous cross linked laccase aggregates for the decolorization of triphenyl methane and reactive dyes

The production of porous cross-linked enzyme aggregates (p-CLEAs) is a simple and effective methodology for laccase immobilization. A three-phase partitioning technique was applied to co-precipitate laccase and starch, followed by cross-linking with glutaraldehyde and removal of starch by α-amylase to create pores in the CLEAs. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. The half-life of free laccase at 55°C was calculated to be 1.3h, while p-CLEAs did not lose any activity even after 14h. p-CLEAs also exhibited improved storage stability, catalytic efficiency and could be recycled 15 times with 60% loss of activity. The catalysts decolorized triphenylmethane and reactive dyes by 60–70% at initial dye concentrations of 2 and 0.5gL−1, respectively, without any mediators. These results suggest the potential of CLEA technology in waste-water treatment.

Purification of α-galactosidase from pepino ( Solanum muricatum) by three-phase partitioning

Three-phase partitioning (TPP) is a bioseparation technique used for the extraction, concentration and purification of biomolecules. The technique consists of simultaneous addition of a salt (generally ammonium sulfate) to the crude extract followed by the addition of an organic solvent (generally t-butanol). In the present study, α-galactosidase was purified from pepino ( Solanum muricatum) fruit by TPP. The influence of various process parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) on α-galactosidase partitioning is investigated to get highest purity fold and yield. The results showed that, 50% (w/v) ammonium sulfate saturation with 1:1.5 crude extract to t-butanol ratio at pH 5.25 gave 6.2-fold purification with 127% activity recovery of α-galactosidase. Characterization and determination of biochemical properties of the partitioned α-galactosidase were also aimed. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed considerable purification and the molecular weight of the enzyme was nearly found to be as 38 kDa. Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and stability were studied. The optimum temperature and pH of the partitioned α-galactosidase were found to be 50 °C and pH 5.5, respectively. The enzyme was very stable at the temperature ranged from 37 to 45 °C and also in a pH range of 4.5–7.0. The kinetic constants; K m and V max were determined to be 0.37 mM and 0.46 U, respectively. The results indicated that, TPP technique is very attractive process for purification of pepino α-galactosidase and the characteristic properties of the enzyme partitioned by TPP make the enzyme good candidates for its several industrial applications.

Corynebacterium Pseudotuberculosis Secreted Antigen-Induced Specific Gamma-Interferon Production by Peripheral Blood Leukocytes: Potential Diagnostic Marker for Caseous Lymphadenitis in Sheep and Goats

In the current study, the applicability of the quantification of gamma interferon (IFN-γ) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis-secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-γ enzyme-linked immunosorbent assay was used to quantify IFN-γ production. Goats and sheep with CLA had higher IFN-γ levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-γ levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-γ is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.

Three-phase partitioning of hydrolyzed Levan

During the fructose and polymerization to synthesize levan, smaller fructooligosaccharide (FOS) molecules are produced. FOS can also be obtained by levan hydrolysis. Three-phase partitioning (TPP) is a separation technique that has been used for polysaccharide precipitation and gathers t-butanol and ammonium sulphate to exclude the polymer from the aqueous solution. In this work TPP was tested to separate levan and FOS from aqueous solution. The FOS used was obtained from Zymomonas mobilis levan acid hydrolysis and fractionation with ethanol. The yield of low TPP fractions was higher than those obtained from the native levan. The F-90 exhibited a higher yield than other fractions. However, when applying the TPP technique to lightest fraction not precipitated by ethanol at 90% (F>90), the intermediate phase was not possible to be visualize. These results have potential application because they show that by using the levan TPP separation it is possible to separate low-molecular weight sugar.

Three-phase partitioning of α-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques

Simple, attractive and versatile technique, three-phase partitioning (TPP) was used to purify alpha-galactosidase from fermented media of Aspergillus oryzae. The various conditions required for attaining efficient purification of the alpha-galactosidase fractions were optimized. The addition of n-butanol, t-butanol, and isopropanol in the presence of ammonium sulfate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The single step of three-phase partitioning, by saturating final concentration of ammonium sulfate (60%) with 1:1 t-butanol, gave activity recovery of 92% with 12-fold purification at second phase of TPP. The final purified enzyme after TPP showed considerable purification on SDS-PAGE with a molecular weight of 64 kDa. The enzyme after TPP showed improved activity in organic solvents. Results are compared with conventional established processes for the purification of alpha-galactosidase produced by Aspergillus oryzae and overall the proposed TPP technique resulted in 70% reduction of purification cost compared to conventional chromatographic protocols.

Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted–secreted proteins of Corynebacterium pseudotuberculosis

The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary ( n) or tertiary ( t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.

A Method for Large Scale Purification of Turnip Peroxidase and Its Characterization

Purification of peroxidase has been carried out since 1960 from different sources and with different methods. Ion exchange, affinity, hydrophobic, and metal affinity chromatography are known, to our knowledge. The present method, developed in this study, is three-phase partitioning, a novel technique to separate protein directly from a large volume of crude suspension. It has been observed that interfacing phase with a metal makes this technique highly selective. Turnip peroxidase purified with this method has 512 units/mg with 20.3% recovery. The natural proteins containing histidine or cystine are often purified by immobilized metal affinity chromatography. The purification of turnip peroxidase with the three-phase partitioning technique is based on immobilized metal affinity chromatography and is used for large-scale purification. The present method, described here, would prove its value in purifying an industrially important enzyme on a large scale from a crude suspension. The enzyme purified with this technique showed two bands on SDS- PAGE, which showed a molecular weight of approx. 39KD. Enzyme showed maximum purification with Cu++ metal and had a maximum activity at pH 6.0. The enzyme has an affinity towards hydrogen peroxide as its substrate in the presence of orthodianisidine as a chromogenic substrate. Enzyme activity was enhanced with calcium and magnesium, whereas sodium, potassium, and manganese inhibit the enzyme activity.