Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.     

Purification of pectinases by three-phase partitioning

Three-phase partitioning was used to purify pectinases from Aspergillus niger and tomato by addition of tert-butanol in the presence of ammonium sulphate. The yields of 76% (Aspergillus niger) and 183% (tomato) and purifications of 10-fold (Aspergillus niger) and 9-fold (tomato) were obtained. The final purified enzyme enzyme from tomato showed a single band on SDS-PAGE with a molecular weight of 46 kDa.     

Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) for purification of xylanase

It is shown that eudragit S-100, a copolymer of methylacrylic acid and methylmethacrylate, undergoes three-phase partitioning. It was found that 95% eudragit S-100 could be recovered as the interfacial precipitate by using 30% (w/v) ammonium sulfate, 1:1 ratio of t-butanol to polymer solution at 40 degrees C. Three-phase partitioning of proteins uses simultaneous addition of ammonium sulfate and t-butanol to precipitate proteins in an interfacial layer separating the aqueous phase and organic solvent. Exploiting the affinity of xylanases towards eudragit S-100, it was possible to purify xylanase from Aspergillus niger; 60% recovery of activity with 95-fold purification could be obtained by this process. The purified enzyme showed A single band on SDS-PAGE. The technique shows promise to develop into a general method that could be termed "macroaffinity ligand-facilitated three-phase partitioning (MLFTPP).     

Purification of a novel lipoxygenase from eggplant (Solanum melongena) fruit chloroplasts

A novel membrane lipoxygenase (LOX; EC 1.13.11.12) from eggplant ( Solanum melongena L. cv. Belleza negra) fruit chloroplasts has been purified 20-fold to a specific activity of 207 enzymatic units per mg of protein with a yield of 72%. The purification was carried out by sonicating the chloroplastic membranes in the presence of Triton X-114 followed by phase partitioning and anion exchange chromatography. The purified membrane LOX preparation consisted of a single major band with an apparent molecular mass of 97 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results obtained using intact chloroplasts indicate that the enzyme is not localized in the stroma. When the enzyme reacts with linoleic acid, it produces a single peak, which comigrates with standard 9-hydroperoxy-octadecadienoic acid. A physiological role for this chloroplastic LOX is proposed.     

Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems

The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a PEG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold increases in the partitioning to the PEG rich top-phase were obtained, when compared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, whereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegrates, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitioning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic whole cell disintegrate system. The results presented should be of general interest also for the improvement of the partitioning properties of other industrially interesting proteins including bulk enzymes.     

Three-phase partitioning of α-galactosidase from fermented media of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and comparison with conventional purification techniques

Simple, attractive and versatile technique, three-phase partitioning (TPP) was used to purify α-galactosidase from fermented media of Aspergillus oryzae. The various conditions required for attaining efficient purification of the α-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 α-galactosidase produced by Aspergillus oryzae and overall the proposed TPP technique resulted in 70% reduction of purification cost compared to conventional chromatographic protocols.     

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.     

Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots

Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme, with the exception of the sulfhydryl inhibitor p-chloromercuriphenyl-sulfonate, which was a strong inhibitor of ferricyanide reducing activity.     

Effective extraction and purification of beta-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning

Effective extraction of protein from bulk medium is an important technique in bioresearch. In the present study, we describe an extracellular beta-xylosidase from the fermentation supernatant of Trichoderma koningii G-39 that was successfully extracted and purified simultaneously in a single step by using an aqueous two-phase partitioning method. This two-phase system was prepared by dissolving suitable amount of poly(ethylene glycol) (PEG) and sodium dihydrogenphosphate (NaH(2)PO(4)) in aqueous solution. beta-Xylosidase was recovered with high yield and high concentration in the bottom salt-rich phase when 25% (w/v) PEG 1500 and 20-25% (w/v) NaH(2)PO(4) were applied. Based on a 1-liter scale extraction, the purity of the enzyme was enhanced at least 33-fold. The total activity increased 422% in comparison with that in the untreated filtrate. The effectiveness and simplicity may make this technique potentially useful in various applications. The transxylosylation activity of the enzyme purified by this technique was also investigated.     

Purification and characterization of an invertase produced by Aspergillus ochraceus TS.

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.     

Purification of alkaline phosphatase from chicken intestine by three-phase partitioning and use of phenyl-Sepharose 6B in the batch mode

Alkaline phosphatase from chicken intestine was purified from the crude preparation employing three-phase partitioning and by the use of phenyl Sepharose-6B in the batch mode. TPP uses a combination of ammonium sulphate and t-butanol to precipitate proteins from crude aqueous extracts. The precipitated protein forms interface between lower aqueous phase and upper organic solvent phase. The fold purification of the finally purified enzyme was 80 and the activity recovery was 61%. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was found to be around 67 kDa.     

Purification and properties of lactate dehydrogenase from Nocardia asteroides.

The lactate dehydrogenase (LDH) from Nocardia asteroides was purified to homogeneity by ammonium sulphate precipitation, gel filtration on Sephadex G-150 and DEAE-Sepharose column chromatography. The purified enzyme showed a single band in native condition which indicated its homogeneity. SDS-PAGE of the purified enzyme showed the presence of three bands which correspond to molecular weights of 60, 66 and 74 kDa. The pH and temperature optima of the purified enzyme were 9.5 and 50 degrees C, respectively. The metal ions Mn++, Fe++, Co++, Mg++ and Ca++, increased the purified LDH activity. On the other hand, enzyme activity was completely inhibited by CuCl2. Potassium chloride, ammonium sulphate and sodium chloride did not alter the enzyme activity. The purified enzyme exhibited a Km value of 1.6 x 10(-5) M for pyruvate.     

Biosynthesis of bacterial glycogen: purification and properties of Salmonella typhimurium LT-2 adenosine diphosphate glucose pyrophosphorylase.

The adenosine diphosphate glucose pyrophosphorylase from a Salmonella typhimurium LT-2 mutant, JP102, derepressed in the glycogen biosynthetic enzymes was purified to homogeneity. The enzyme was found to be identical with the parent wild-type enzyme with respect to regulatory properties, immunological reactivity, and kinetic constants for the allosteric effectors and for the substrate, adenosine triphosphate. The JP102 enzyme was composed of four identical subunits, each with a molecular weight of about 48,000. This was supported by the findings that (i) gel electrophoresis under denaturing conditions showed only one component; (ii) digestion with carboxypeptidase B released stoichiometric amounts of arginine, and (iii) amino-terminal sequencing showed a single sequence for the first 27 residues. The properties of the purified S. typhimurium enzyme were compared with the properties of the previously purified Escherichia coli B enzyme.     

Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase

Cinnamic acid 4-hydroxylase (CA4H) was purified from microsomes of manganese-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. The three-step purification procedure involved solubilization and phase partitioning in Triton X-114, followed by chromatography on DEAE-Trisacryl and hydroxylapatite columns. Purification was monitored using carbon monoxide and type I substrate binding properties of the enzyme. The protein, purified to electrophoretic homogeneity, showed an Mr of about 57,000 on SDS-PAGE. Polyclonal antibodies raised against this protein selectively reacted with a 57-kDa polypeptide on Western blots of induced Jerusalem artichoke microsomes. The antibody selectively and strongly inhibited CA4H activity from several plant species.     

Purification and characterization of bile salt hydrolase from Clostridium perfringens

Bile salt hydrolase (cholylglycine hydrolase, EC 3.5.1.24) has been purified to homogeneity (792-fold) from Clostridium perfringens using high performance DEAE-chromatography. The purified enzyme showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a relative molecular weight ca. 56,000. The intact enzyme had a relative molecular weight (Mr) of ca. 250,000 as determined by nondenaturing PAGE. The NH2-terminal sequence of bile salt hydrolase was determined to be Met-(Ser/Cys)-Arg-Thr-Lys-Leu-Val-Ileu-Thr-Ileu-Gly-Ala-Ser. The purified enzyme was active towards both glycine and taurine conjugates of cholate. The apparent Km and Vmax of the enzyme for glycocholate was estimated to be 0.5 mM and 107 nmol/min.mg protein, respectively. The pH optimum was in the range of 5.8 to 6.4. The enzyme was inhibited 85%, 81%, and 83% by 2 mM iodoacetate, p-chloromercuribenzoate, and phenylmethanesulfonylfluoride, respectively. Rabbit polyclonal antibody was prepared and used to demonstrate a single form of the enzyme in crude cell extracts.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Site-directed mutagenesis of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans

Using oligonucleotide-directed mutagenesis of the gene encoding the small subunit (rbcS) from Anacystis nidulans mutant enzymes have been generated with either Trp-54 of the small subunit replaced by a Phe residue, or with Trp-57 replaced by a Phe residue, whereas both Trp-54 and Trp-57 have been replaced by Phe residues in a double mutant. Trp-54 and Trp-57 are conserved in all amino acid sequences or the small subunit (S) that are known at present. The wild-type and mutant forms of Rubisco have all been purified to homogeneity. The wild-type enzyme, purified from Escherichia coli is indistinguishable from enzyme similarly purified from A. nidulans in subunit composition, subunit molecular mass and kinetic parameters (Vmax CO2 = 2.9 U/mg, Km CO2 = 155 microM). The single Trp mutants are indistinguishable from the wild-type enzyme by criteria (a) and (b). However, whereas, Km CO2 is also unchanged, Vmax CO2 is 2.5-fold smaller than the value for the wild-type enzyme for both mutants, demonstrating for the first time that single amino acid replacements in the non-catalytic small subunit influence the catalytic rate of the enzyme. The specificity factor tau, which measures the partitioning of the active site between the carboxylase and oxygenase reactions, was found to be invariant. Since tau is not affected by these mutations we conclude that S is an activating not a regulating subunit.     

Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato

Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato (Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20 kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.     

A new type of endo-xyloglucan transferase devoted to xyloglucan hydrolysis in the cell wall of azuki bean epicotyls

A new type of xyloglucan-degrading enzyme was isolated from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls and its characteristics were determined. The enzyme was purified to apparent homogeneity by Concanavalin A (Con A)-Sepharose, cation exchange, and gel filtration columns from a cell wall protein fraction extracted with 1 M sodium chloride. The purified enzyme gave a single protein band of 33 kDa on SDS-PAGE. The enzyme specifically cleaved xyloglucans and showed maximum activity at pH 5.0 when assayed by the iodine-staining method. An increase in reducing power in xyloglucan solution was clearly detected after treatment with the purified enzyme. Xyloglucans with molecular masses of 500 and 25 kDa were gradually hydrolyzed to 5 kDa for 96 h without production of any oligo- or monosaccharide with the purified enzyme. The purified enzyme did not show an endo-type transglycosylation reaction, even in the presence of xyloglucan oligosaccharides. Partial amino acid sequences of the enzyme shared an identity with endo-xyloglucan transferase (EXGT) family, especially with xyloglucan endotransglycosylase (XET) from nasturtium. These results suggest that the enzyme is a new member of EXGT devoted solely to xyloglucan hydrolysis.     

An improved purification procedure of alkaline phosphatase from calf intestine by applying partition in aqueous two-phase systems and dye-ligand chromatography.

Aqueous two-phase partitioning has been elaborated in order to improve the purification of alkaline phosphatase from calf intestine in larger scale. The laborious precipitation and centrifugation steps for the removal of the enzyme from the cell debris and from the bulk protein were replaced by this technique yielding a high recovery (88%) and a significant lower time requirement. For the preparation of 100.000 units (46 mg) of a homogeneous enzyme 2.0 kg of a system containing 200 g PEG 4000 and only 10 g dextran M 70 is necessary. Affinity partitioning in aqueous two-phase systems was used to screen 41 dyes for selecting a suitable ligand for the dye-ligand chromatography of the enzyme. In the case of alkaline phosphatase the results obtained by affinity partitioning coincide with the experimental requirements for the affinity chromatography of the enzyme. Procion Navy HE-R (Blue 171) exhibits a high affinity, selectivity and binding capacity for the enzyme compared with other dyes investigated. The purification procedure provided the same degree in purity (2200 U/mg) and yield (59%) if mucosa or chyme was applied as starting material. In the range of practical use the purified enzyme contains no detectable activities of DNAses (endonucleases) and DNA-nicking activities. The contamination with phosphodiesterase I (EC. 3.1.4.1.) is less than 0.01%.