Large-scale purification of halophilic enzymes by salting-out mediated chromatography

Leicht, Wolfgang; Pundak, Shlomo

doi:10.1016/0003-2697(81)90472-3

Cited by 25 publications

(13 citation statements)

References 15 publications

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“…Initial attempts to purify GCR from extracts of H. halobium by techniques that have proven useful in the purification of other halobacterial enzymes were unsuccessful. Ion-exchange chromatography on DEAE-cellulose and hydrophobic chromatography on several alkyl-agarose resins (n-butyl-, n-octyl-and n-dodecylagarose) in buffer containing (NH4)2SO4 (14,18) and hydroxylapatite chromatography in buffer containing NaCl (24) each gave less than fourfold purification (data not shown). Also, several affinity resins were tested for their ability to bind GCR in buffers containing molar levels of either NaCl or (NH4)2SO4.…”

Section: Resultsmentioning

confidence: 98%

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The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

Sundquist

Fahey

1988

J Bacteriol

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An NADPH-specific disulfide reductase that is active with bis-'y-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-y-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2" resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2O resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native Disulfide-specific reductases play a central role in the maintenance of aerobic cell cytoplasms in a chemically reduced state. Glutathione reductase (EC 1.6.4.2) is an NADPH-dependent disulfide reductase that is present in aerobic eubacteria and eucaryotic cells and maintains the peptide glutathione (y-Glu-Cys-Gly) predominantly in its reduced or thiol form (13, 39). Reduced glutathione is found at millimolar levels in cells and is a coenzyme for several enzymes that reduce intracellular disulfides and detoxify potent oxidants, such as peroxides (8,15,21). These processes yield the disulfide form of glutathione, which is cycled back to the thiol by glutathione reductase.Glutathione may not be the only thiol antioxidant utilized in aerobic cells; several aerobic bacteria that lack glutathione have been shown to produce other low-molecularweight thiols and disulfide reductase activities (23,32,35). One such group of bacteria are the halophilic archaebacteria, which have been shown to lack reduced glutathione but contain millimolar levels of -y-glutamylcysteine (,y-Glu-Cys) (23). This finding suggested that the halobacteria may employ a redox metabolism similar to that established for glutathione but based on -y-Glu-Cys. Central to this metabolism would be a disulfide reductase that is active with -y-Glu-Cys disulfide (bis-y-glutamylcystine), and the detection of an NADPH-dependent reductase activity in crude extracts of Halobacterium halobium indicated that this enzyme was present (23). In this paper we report the complete purification of an NADPH-specific bis-y-glutamylcystine reductase (GCR) from extracts of H. halobium. The characteristics of GCR and the possible functions of -y-Glu-Cys in the halobacteria are also discussed.Purification of most halobacterial enzymes requires the use of high-ionic-strength buffers (,u > 2 M) to retain enzyme activity (17). Chromatographic methods have been devel-* Corresponding author.oped that function well in buffers containing molar levels of NaCl or (NH4) 2SO4 (14,18,22,24), and a small number of halobacterial enzymes have been purified to homogeneity. Our limited success in purifying the GCR from H. halobium by established methods led us to examine additional approaches to purification in high-salt buffers. Immobilizedmetal-ion affinity chromatography (also known as metal chelate affinity chromatography) (26, 27), a technique used...

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Section: Resultsmentioning

confidence: 98%

“…oped that function well in buffers containing molar levels of NaCl or (NH4) 2SO4 (14,18,22,24), and a small number of halobacterial enzymes have been purified to homogeneity. Our limited success in purifying the GCR from H. halobium by established methods led us to examine additional approaches to purification in high-salt buffers.…”

mentioning

confidence: 99%

The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

Sundquist

Fahey

1988

J Bacteriol

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“…In this work we will call these bacteria H. marismortui, although this has not yet been definitively established. Further details on the characterization and growth of the bacteria and largescale enzyme purification have been given elsewhere [32,33].…”

Section: Taxonomy Of the Halobacterium From The Dead Seamentioning

confidence: 99%

“…In parallel procedures about 200 mg of homogeneous glutamate dehydrogenase (h) could be obtained [32,33].…”

Section: Growth Of Halobacteria and Large-scale Purification O J Malamentioning

confidence: 99%

Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 1. Conformation and interaction with Water and Salt between 5 M and 1 M NaCl Concentration

Pundak

Eisenberg

1981

Eur J Biochem

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Large-scale growth of extreme halophilic bacteria from the Dead Sea and purification of malate dehydrogenase (and other proteins) in quantities of hundreds of milligrams makes possible a detailed study of the adaptation to high salt. Halophilic malate dehydrogenase is stable at 20 "C in NaCl solutions between 2.5 -5 M. Below 2.5 M NaCl time-dependent inactivation, paralleled by structural changes, sets in. Within the time scale of the sedimentation, diffusion and circular dichroism experiments discussed here, it was possible to analyze data corresponding to the active halophilic malate dehydrogenase between 1 M and 5 M NaCl. The striking observation was that rather minor conformational changes were observed over the whole range, yet the special properties of the halophilic enzyme seem to be related to its capacity of associating with unusually large amounts of water and of salts, quite distinct from non-halophilic counterparts. These special properties seem to be related to the intact structure of the protein. Some parallel properties of halophilic glutamate dehydrogenase are also discussed.Extreme halophilic bacteria grow best in 20 -30 % NaCl 11 -31. These obligate halophiles are red-pigmented organisms which occur naturally inwaters of high salinity such as theDead Sea, for instance. The extreme halophilic bacteria adapt themIselves to life at high salinity by having internal concentrations (of salt that equal or exceed even the high concentrations in which they grow. They lyse at low salt concentrations. For recent reviews of the unusual properties of the halobacteria, some of which bear closer resemblance to eukaryotes rather than to prokaryotes, we refer to Kushner [4] and to Bailey ,md Morton [5]. Striking compositional properties, which have also been extensively studied in our laboratory are that halophilic proteins possess a molar excess of 16-18 % negatively charged amino acids [6 -81, whereas in non-halophilic proteins the excess is only 7-9%. The content of hydrophobic amino acids is significantly low, yet the content of borderline hydrophobic amino acids (e.g. alanine and threonine) is higher than in non-halophilic counterparts.Halophilic proteins carry out identical functions to their nonhalophilic counterparts, yet in a medium in which one parameter (salt concentration) differs greatly. The halophilic character is an intrinsic property of the proteins and does not depend on additional factors [7,9,10]. The proteins are stabilized by multimolar salt concentrations and the stabilization effect of different salts follows the Hofmeister lyotropic series. At high salt concentrations (3-5 M NaCl or KC1) enzymatic activity of halophilic proteins is observed whereas non-halophilic enzymes tend to be inactivated and to precipitate [11,12]. At low salt concentrations (< 2 M NaCI) halophilic enzymes denature. Denaturation involves unfolding, dissociation and inactivation, processes which may

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“…Salting-out chromatography on Sepharose CL-4B has been used by several investigators for fractionating proteins from halophiles (17,21,23). The basis of the adsorption of proteins onto Sepharose is not fully known, although it has been attributed to hydrophobic interactions (21) and surface effects (31).…”

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confidence: 99%

Superoxide dismutase from the extremely halophilic archaebacterium Halobacterium cutirubrum

May¹,

Dennis²

1987

J Bacteriol

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Halobacterium cutirubrum, a member of the archaebacteria, contains one superoxide dismutase (EC 1.15.1.1). This enzyme functions in the high-ionic-strength intracellular environment and protects the organism against the toxic effects of the superoxide anion. The enzyme has been purified to about 90% homogeneity by a four-step procedure which never removes it from conditions of high ionic strength. The subunits of the purified enzyme have a molecular weight of 25,000 and are possibly in tetrameric association. Halobacteria are aerobic, mesophilic heterotrophs which inhabit brines containing, ideally, 4 to 5 M NaCl (8, 16). As members of the archaebacteria, they are only distantly related to eubacteria and eucaryotes (33). The halobacteria are phylogenetically related to the methanogenic archaebacteria and are believed to have evolved from anaerobic, nonhalophilic ancestors (26, 33). Two major steps were required for this evolution. One was the adaptation to high salt concentrations; this was achieved by raising the intracellular salt concentration to near saturation and altering enzyme structures to function in this environment (3,14,15). The second was the adaptation to oxygen and aerobic respiration, which necessitated the development of mechanisms to protect against toxic oxygen radicals such as the superoxide anion.One key enzyme involved in this protection is superoxide dismutase (SOD) (9,20). It catalyzes the dismutation of superoxide to peroxide and molecular oxygen (6). On the basis of metal content, three types of SOD have been described: copper-zinc SOD in eucaryotic cytoplasm, manganese SOD in eubacteria and mitochondria, and iron SOD in eubacteria (29). To date, SOD has been isolated from two species of archaebacteria, Methanobacterium bryantii (11), an anaerobe, and Thermoplasma acidophilum (27) Cell culture. H. cutirubrum was grown to mid-log phase (A460 = 1.0) in rich medium as described previously (12).SOD purification. Hydroxylapatite chromatography and gel filtration were performed at 20°C. All other steps were performed at 4°C, and all buffers contained 1 mM 2-mercaptoethanol. Cells from 4 liters of culture were pelleted at 4,000 x g for 20 min and then suspended in 40 ml of 3.0 M ammonium sulfate-50 mM sodium phosphate (pH 7.0). The cells were lysed by five 2-min sonications (0.15 relative output in a Fisher model 150 dismembrator). The lysate was centrifuged at 44,000 x g for 12 h. The supernatant was dialyzed against lysis buffer for 4 h and recentrifuged at 114,000 x g for 2 h. The supernatant was adjusted to 2.5 M ammonium sulfate and adsorbed onto a column (60 by 2.6 cm) of Sepharose CL-4B which had been equilibrated with 2.25 M ammonium sulfate-50 mM sodium phosphate (pH 7.0). The column was washed with 700 ml of buffer, and the SOD activity was then eluted with a 1.3-liter decreasing linear ammonium sulfate gradient (2.25 to 1.40 M in phosphate buffer; Fig. 1). Fractions containing SOD activity were pooled and applied to a column (35 by 2.6 cm) of DEAESepharose CL-6B which had been equi...

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Large-scale purification of halophilic enzymes by salting-out mediated chromatography

Cited by 25 publications

References 15 publications

The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 1. Conformation and interaction with Water and Salt between 5 M and 1 M NaCl Concentration

Superoxide dismutase from the extremely halophilic archaebacterium Halobacterium cutirubrum

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