Interaction of loratadine with serum albumins studied by fluorescence quenching method

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)] = 10:1. In the linear range (0–50 μmol L^− 1) quenching constants were calculated using Stern–Volmer equation. Temperature in the range 298 K–310 K had a significant effect (p < 0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.     

Human growth hormone-specific aptamer identification using improved oligonucleotide ligand evolution method

LETEG is a method developed and used for the separation and purification of proteins employing a single-step ligand (aptamers) evolution in which aptamers are eluted with an increasing temperature gradient. Using recombinant human growth hormone (rhGH) as the test purification target, and after avoiding cross reactions of aptamers with Bacillus subtilis extracellular proteins by negative SELEX, the effects of time and pH on aptamer binding to rhGH were investigated. The highest binding efficiency of aptamers on rhGH-immobilized microparticles was obtained at pH 7.0. The aptamers that interacted with rhGH were eluted by a multi-stage step-up temperature gradient in ΔT = 10 °C increments within the range T = 55–95 °C; and the strongest affinity binding was disrupted at T = 85 °C where C_Apt = 0.16 μM was eluted. The equilibrium binding data obtained was described by a Langmuir-type isotherm; where the affinity constant was K_D = 218 nM rhGH. RhGH was separated from the fermentation broth with 99.8% purity, indicating that the method developed is properly applicable even for an anionic protein.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).     

Rapid cloning,expression and purification of a novel high-activity alkaline phosphatase with detoxification of lipopolysaccharide

Lipopolysaccharide (LPS) is a bacterial endotoxin leading to endotoxemia. Its virulence factor ‘diphosphoryl lipid A’ can be abolished by alkaline phosphatase (AP). A novel AP gene (without introns) was cloned from Saccharomyces boulardii ATCC MYA-796 with a GenBank accession number KF471017, and the recombinant AP (rAP) was expressed as a soluble protein in Pichia pastoris X-33 with a yield of 43.66 mg/l at the end of 120 h of induction in a shaker flask. After purification by affinity-column chromatography, the purity of rAP was over 90%. The optimal reaction conditions of rAP were pH 9.6, temperature at 60 °C and 2 mM Mg²⁺ in diethanolamine buffer, and EDTA was a potent inhibitor of rAP activity. The specific activity of rAP was 9912.01 U/mg under the optimal conditions. Furthermore, rAP showed a broad dephosphorylation activity to LPS over a broad pH range (pH 2–10) in vitro and peaked at pH 4 in Tris–HCl buffer. After LPS dephosphorylated by rAP was injected intraperitoneally into mice, the serum level of tumor necrosis factor (TNF)-α was significantly reduced compared to that of the LPS group (p < 0.01). These findings suggest that rAP has great potential to cure diseases caused by LPS.     

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

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.     

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9 ± 2.6 mg/g beads. Adsorbed trypsin was eluted by using (0.1 M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.     

A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats

Marine Chlorella ellipsoidea protein was hydrolyzed using Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin and papain. Alcalase-proteolytic hydrolysate exhibited the highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight (below 5 kDa, 5–10 kDa and above 10 kDa). The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from marine C. ellipsoidea, which was composed of 4 amino acids, Val–Glu–Gly–Tyr (MW: 467.2 Da, IC₅₀ value: 128.4 μM), was isolated. Lineweaver–Burk plots suggest that the peptide purified acts as a competitive inhibitor against ACE and stable against gastrointestinal enzymes of pepsin, trypsin and α-chymotrypsin. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHRs) also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results suggest that marine C. ellipsoidea would be an attractive raw material for the manufacture of antihypertensive nutraceutical ingredients.     

Modelling and simulation of continuous L (+) lactic acid production from sugarcane juice in membrane integrated hybrid-reactor system

Modelling and simulation was done for a two-stage membrane-integrated hybrid reactor system for continuous production of L (+) lactic acid under non-neutralizing conditions. The model captures microbial conversion of sugar cane juice to lactic acid under substrate–product inhibitions with downstream purification by nanofiltration. All the major phenomena and the governing parameters like fluid flow, feed dilution, substrate–product inhibitions, Donnan and steric effects during micro and nanofiltration for cell recycle, product separation and purification have been reflected in the modelling. The model describes a green, integrated continuous process of direct lactic acid production starting with a cheap, renewable carbon source. The highest lactic acid concentration achieved after the final stage of nanofiltration was 66.97 g/L at 13 kg/cm² operating pressure when the overall productivity reached 12.40 g/(L h). The developed model could successfully predict production, purification and transport of lactic acid through two stage membrane modules. Performance of the model was very good as indicated in the high overall correlation coefficient (R² > 0.980) and the low relative error (RE < 0.1).     

Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction

This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose? solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20 mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06 mg IgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1 ± 0.25 mg Fab/ml moist resin displaying an affinity constant K_d = (2.6 ± 0.3) × 10^?6 M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH₁-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Combinational biosynthesis of dual-functional streptavidin-phycobiliproteins for high-throughput-compatible immunoassay

The development of fluorescent tools with desired fluorescence and efficient targeting is of great importance in the high-throughput immunoassay. Here we report combinational biosynthesis of new dual-functional streptavidin-phycobiliproteins (SA-PBPs) in Escherichia coli by fusing streptavidin with phycobiliproteins. These recombinant proteins can achieve a purity over 95% after one-step purification, and their maximum absorption and fluorescence emission wavelength are at 556 nm and 568 nm for SA-PCA-PEB (streptavidin-phycocyanin α subunit-phycoerythrin), and 624 nm and 646 nm for SA-PCA-PCB (streptavidin-phycocyanin α subunit-phycocyanobilin), respectively. The potential application of these dual-functional SA-PBPs in immunoassay was evaluated using “sandwich” ELISA method for detection of two biomarkers of liver cancers, i.e. α-fetoprotein (AFP) and carcinoembryonic antigen (CEA). As a result, both SA-PCA-PCB and SA-PCA-PEB showed a good linear function with AFP & CEA within 0–50 ng/ml concentrations. Their limits of detection (LODs) for AFP and CEA were 0.25 ng/mL and 0.28 ng/ml using SA-PCA-PEB, and 1.01 ng/ml and 1.12 ng/ml using SA-PCA-PCB respectively. These results indicate that these novel dual-functional SA-PBPs are useful tools for a wide variety of immunoassay, and may have the advantages in their higher expandability and compatibility with existing and future immunoassay technologies.     

Expanded bed adsorption of an alkaline lipase from Pseudomona cepacia

An extracellular lipase was isolated from Pseudomona cepacia by expanded bed adsorption on an Amberlite 410 ion-exchange resin. Enzyme characterization and hydrodynamic study of a chromatography column were done. Enzyme purification was done at three condition of expanded bed height (H): at one and half (6 cm), at two (8 cm) and at three (12 cm) times the fixed bed height (H₀ = 4 cm). The results showed that the experimental data was fitted to the Richardson and Zaki equation, and the comparison between the experimental and calculated terminal velocities showed low relative error. In enzyme purification for better condition, a purification factor of about 80 times was found at 6 cm of expanded bed height, or 1.5 times of expansion degree. Purified lipase had an optimal pH and a temperature of 8 and 37 °C, respectively.     

Schoolhouse wastewater purification in a LWA-filled hybrid constructed wetland in Estonia

This paper analyses the purification efficiency and mass removal of organic material, suspended solids, nitrogen and phosphorus in a hybrid constructed wetland (CW) system treating wastewater from a basic school in Paistu, Estonia. The CW consists of two subsurface flow filter beds using lightweight aggregates (LWA): a two-chamber vertical subsurface flow (VSSF) filter bed followed by a horizontal subsurface flow (HSSF) filter bed, with a total area of 432 m². This CW was constructed in summer 2002 by the Centre for Ecological Engineering in Tartu (CEET). Eighteen series of water samples (from 30.10.2003 to 15.10.2005) were undertaken. The analyses show the outstanding purification effect of the system: for BOD₇ the average purification efficiency is 91%; for total suspended solids (TSS)—78%, for total P—89%, for total N—63%, and for NH₄N—77%. The average outlet values for the above-listed parameters were 5.5, 7.0, 0.4, 19.2 and 9.1 mg L⁻¹, respectively. According to our results, the purification parameters meet the standards set by the Water Act of Estonia for wastewater treatment plants of 2000–9999 PE: 15, 25, and 1.5 mg L⁻¹ for BOD₇, TSS and total P, respectively. The results show that hybrid CW systems consisting of subsurface flow filter beds can work efficiently in conditions of changing hydraulic loading and relatively cold climate. We did not find significant differences between the removal efficiency, mass removal, and values of the first-order rate-constant k for most water quality indicators during the warm (May–October) and cold (November–April) periods. Locally produced LWA as a filter material in CWs has shown good hydraulic conductivity and phosphorus sorption capacity (k = 17.1 ± 12.4 m yr⁻¹). The Paistu CW, with its proper design and outstanding purification results, can be considered one of the best systems in Estonia.     

Synthesis and characterization of 123I-CMICE-013: A potential SPECT myocardial perfusion imaging agent

Coronary artery disease (CAD) is a major cause of death in Canada and the United States. Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is a useful diagnostic test in the management of patients with CAD. The widely used SPECT MPI agents, ^99mTc sestamibi and ^99mTc tetrofosmin, exhibit less than ideal pharmacokinetic properties with decreasing uptake with higher flows. ¹²³I has a similar energy as ^99mTc, an ideal half life, and is readily available from cyclotrons. The objective of this study was to develop an ¹²³I labeled MPI agent based on rotenone, a mitochondrial complex I inhibitor, as an alternative to currently available SPECT MPI agents. Methods: ¹²³I-CMICE-013 was synthesized by radiolabeling rotenone with ¹²³I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, followed by RP-HPLC purification. The product was formulated in 5% EtOH in 10 mM NaOAc pH 6.5. The inactive analog ¹²⁷I-CMICE-013 was isolated and characterized by NMR and mass spectrometry, and the structure determined. Micro-SPECT imaging studies were carried out in normal and infarcted rats. Biodistribution studies were performed in normal rats at 2 h (n = 6) and 24 h (n = 8) post injection (p.i.). Results: ¹²³I-CMICE-013 was isolated with >95% radiochemical purity and high specific activity (14.8–111 GBq/μmol; 400–3000 mCi/μmol). Structural analysis showed that rotenone was iodinated at 7′-position, with removal of the 6′,7′-double bond, and addition of a hydroxy group at 6′-position. MicroSPECT images in normal rats demonstrated homogeneous and sustained myocardial uptake with minimal interference from lung and liver. Absent myocardial perfusion was clearly identified in rats with permanent left coronary artery ligation and ischemia-reperfusion injury. In vivo biodistribution studies in normal rats at 2 h p.i. showed significant myocardial uptake (2.01 ± 0.48%ID/g) and high heart to liver (2.98 ± 0.93), heart to lung (4.11 ± 1.04) and heart to blood (8.37 ± 3.97) ratios. At 24 h p.i., the majority of ¹²³I-CMICE-013 was cleared from tissues, and a significant amount of tracer was found in the thyroid, indicating in vivo deiodination of the tracer. Conclusion: ¹²³I-CMICE-013 is a promising new radiotracer for SPECT MPI with high myocardial uptake, very good target to background ratios and favorable biodistribution characteristics.     

Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged d-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 μmol Cu²⁺/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of d,l-p-hydroxyphenylhydantoin (d,l-HPH) to N-carbamoyl-d-hydroxyphenylglycine, an intermediate for the production of d-hydroxylphenylglycine. Under optimal conditions, 60 °C and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation.     

Concurrent purification and antioxidant activity of phycobiliproteins from Lyngbya sp. A09DM: An antioxidant and anti-aging potential of phycoerythrin in Caenorhabditis elegans

The present study probes into the purification of phycobiliproteins, and characterization of their in vitro anti-oxidant activity. Moreover, the study also demonstrates the use of antioxidant virtue of phycoerythrin in moderating the phenomenon of aging in Caenorhabditis elegans. Phycoerythrin, phycocyanin and allophycocyanin were purified successfully from Lyngbya sp. A09DM by ammonium sulfate fractionation appended with Triton X-100 intercession. The success of protocol was examined by a series of biochemical characterization like SDS-PAGE, native-PAGE, UV–visible spectroscopy and fluorescence spectroscopy ensuring purity, integrity and functionality of purified phycoerythrin, phycocyanin and allophycocyanin. Purified phycobiliproteins were evaluated for antioxidant and metal ion chelating activity by various in vitro antioxidant assay systems. Results showed significant and dose-dependent antioxidant as well as metal chelating potential of all phycobiliproteins in decreasing order of phycoerythrin > phycocyanin > allophycocyanin. Expansion in lifespan and improvement in pharyngeal pumping of C. elegans were noticed upon pre-treatment with phycoerythrin (100 μg ml⁻¹). Moreover, phycoerythrin mediated increase in worm survival under oxidative stress revealed that the life expansion effect of phycoerythrin on nematode is in part by an action of its antioxidant virtue. These results collectively added up evidence in favor of the ‘free-radical theory of aging’. The present report, for the first time, describes antioxidant potential of phycoerythrin and its use in extending life-span of C. elegans.     

Purification,characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis

Beef tallow, a slaughter house waste was used as a substrate for lipase production, employing Pseudomonas gessardii. The strain, P. gessardii was isolated from the beef tallow acclimatized soil. The crude lipase activity at 139 U/ml by volume was obtained at optimized conditions of pH 5.0 and temperature of 37 °C. After purification, a 7.59-fold purity of lipase with specific activity of 1120 U/mg protein and molecular mass of 92 kDa was obtained. The purified lipase showed maximum activity and stability at pH 5.0 and 30 °C. Ca²⁺ had a stimulatory effect on the lipase activity compared to the other metal ions studied. The relative activity was enhanced with the addition of Triton X-100 with lower hydrophilic–lipophilic balance (HLB) value as 13.0 and DMSO with the lowest partition coefficient (log P) value, as 1.378. The amino acid composition and the functional groups of lipase were confirmed by HPLC and FT-IR spectroscopy. The purified lipase had the highest hydrolytic activity towards slaughterhouse wastes and vegetable oils. This work provides a potential biocatalyst for the wide applications in oleochemical and biotechnological industries.     

Separation and purification of laccases from two different fungi using aqueous two-phase extraction

Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)–phosphate aqueous two-phase system at 25 °C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and 0.013 g g⁻¹ of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.