Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1

Pollution of terrestrial surfaces and aquatic systems by hexavalent chromium, Cr(VI), is a worldwide public health problem. A chromium resistant bacterial isolate identified as Exiguobacterium sp. GS1 by 16S rRNA gene sequencing displayed high rate of removal of Cr(VI) from water. Exiguobacterium sp. GS1 is 99% identical to Exiguobacterium acetylicum. The isolate significantly removed Cr(VI) at both high and low concentrations (1–200 μg mL⁻¹) within 12 h. The Michaelis–Menten K _m and V _max for Cr(VI) bioremoval were calculated to be 141.92 μg mL⁻¹ and 13.22 μg mL⁻¹ h⁻¹, respectively. Growth of Exiguobacterium sp. GS1 was indifferent at 1–75 μg mL⁻¹ Cr(VI) in 12 h. At initial concentration of 8,000 μg L⁻¹, Exiguobacterium sp. GS1 displayed rapid bioremoval of Cr(VI) with over 50% bioremoval in 3 h and 91% bioremoval in 8 h. Kinetic analysis of Cr(VI) bioremoval rate revealed zero-order in 8 h. Exiguobacterium sp. GS1 grew and significantly reduced Cr(VI) in cultures containing 1–9% salt indicating high salt tolerance. Similarly the isolate substantially reduced Cr(VI) over a wide range of temperature (18–45  °C) and initial pH (6.0–9.0). The T _opt and initial pH_opt were 35–40  °C and 7–8, respectively. Exiguobacterium sp. GS1 displayed a great potential for bioremediation of Cr(VI) in diverse complex environments.     

Site-directed mutagenesis of cysteine to threonine in Proteus vulgaris urease active site increases enzyme activity and stability

Cysteine-319 belongs to the flexible flap at the active site of Proteus vulgaris urease. Replacing this cysteine by threonine resulted in a 20-fold increase of specific activity. Temperature stability increased, susceptibility to inhibition by dipyridyl disulfide decreased, and pH optimum shifted from 8 to 6.9. K _m (35 to 12 mM) and V_max (47.4 to 1.8 mol min^–1) were substancially altered. Both variants of the enzyme were irreversibly inhibited by phenylmethanesulfonyl fluoride.     

Kinetics of sulfate uptake by freshwater and marine species ofDesulfovibrio

Apparent half-saturation constants (K _m) and maximum uptake rates (V _max) for sulfate were determined in four species ofDesulfovibrio of freshwater and marine origin using a³⁵S-sulfate tracer technique. The lowerstK _m (5 M) was found in the freshwater speciesDesulfovibrio vulgaris (Marburg) and the highestK _m (77 M) in the marine speciesDesulfovibrio salexigens. Maximum specific rates of sulfate uptake (i.e.,V _max) were proportional to the growth rates observed in batch cultures. The halophilicDesulfovibrio salexigens did not change itsK _m andV _max between 1 and 6,000 M SO ₄ ^2- , and apparently did not induce a low-affinity uptake system at high sulfate concentrations. The low half-saturation constants measured for sulfate uptake explain why high rates of bacterial sulfate reduction occur in surface sediments of freshwater lakes, and why sulfate reduction can be a quantitatively important process in anaerobic carbon mineralization in low-sulfate environments. The results shows that extremely low sulfate concentrations must occur before sulfate reduction is completely outcompeted by methanogenesis.Abbreviations MPB methane producing bacteria - SRB sulfate reducing bacteria     

Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions

The basis for the outcome of competition between sulfidogens and methanogens for H₂ was examined by comparing the kinetic parameters of representatives of each group separately and in co-culture. Michaelis-Menten parameters (V _max and K _m) for four methanogens and five sulfate-reducing bacteria were determined from H₂-depletion data. Further, Monod growth parameters (_max, K _s, Y _H2) for Desulfovibrio sp. G11 and Methanospirillum hungatei JF-1 were similarly estimated. H₂ K _m values for the methanogenic bacteria ranged from 2.5 M (Methanospirillum PM1) to 13 M for Methanosarcina barkeri MS; Methanospirillum hungatei JF-1 and Methanobacterium PM2 had intermediate H₂ K _m estimates of 5 M. Average H₂ K _m estimates for the five sulfidogens was 1.2 M. No consistent difference among the V _max estimates for the above sulfidogens (mean=100 nmol H₂ min^-1 mg^-1 protein) and methanogens (mean=110 nmol H₂ min^-1 mg^-1 protein) was found. A two-term Michaelis-Menten equation accurately predicted the apparent H₂ K _m values and the fate of H₂ by resting co-cultures of sulfate-reducers and methanogens. Half-saturation coefficients (K _s) for H₂-limited growth of Desulfovibrio sp. G11 (2–4 M) and Methanospirillum JF-1 (6–7 M) were comparable to H₂ K _m estimates obtained for these organisms. Maximum specific growth rates for Desulfovibrio sp. G11 (0.05 h^-1) were similar to those of Methanospirillum JF-1 (0.05–0.06 h^-1); whereas G11 had an average yield coefficient 4 x that of JF-1. Calculated _max and V _max/K _m values for the methanogens and sulfidogens studied predict that the latter bacterial group will process more H₂ whether these organisms are in a growing or resting state, when the H₂ concentration is in the first-order region.     

Uptake of iron byGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from⁵⁹FeCl₃ showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentK _m = 3 m,V _max = 0.054 nmol · mg^–1 · min^–1). The maximal rate of Fe²⁺ uptake was higher than Fe³⁺ (V _max = 0.25 nmol · mg^–1 · min^–1) but theK _m was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (K _m = 2 M,V _max = 0.22 nmol · mg^–1 · min^–1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either⁵⁷FeCl₃ or⁵⁷FeCl₂ accumulated unknown ferric and ferrous binding metabolites.     

β-Linked disaccharides stimulate,but do not act as primer for,β(1–3)glucan synthase activity ofNeurospora crassa

-Linked disaccharides (laminaribiose and cellobiose) stimulated(1–3)glucan synthase activity ofNeurospora crassa by reducing the K_{m app} for the substrate while not changing the V_max. Laminaribiose and cellobiose werelinear activators with a K_{a app} of 0.32 mM and K_{a app} of 1.7 mM, respectively. Laminaribiose was not found to be incorporated into product, i.e., did not act as a primer covalently bound to product.     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp.

Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K₂CrO₄ ml^–1 on nutrient agar, 25mgml^–1 in nutrient broth, and up to 10mgml^–1 in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing. Uptake of chromate was greater in living cells than in heat-killed on dried cells. CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000gml^–1 after 24h while CrT-13 reduced 41%, 14% and 9%. Other heavy metals at low concentrations did not affect these reductions. At 150 and 300gml^–1 in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13.Revisions requested 17 May 2004; Revisions received 2 July 2004     

Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd⁰), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd⁰, immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd⁰ and `chemical Pd⁰' were packed into continuous-flow reactors, and challenged with a solution containing 100 m Cr(VI) (pH 7) at a flow rate of 2.4 ml h^–1. Agar-immobilized chemical Pd⁰ columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd⁰ maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.     

The influence of salinity on the kinetics of NH inf4 sup+ uptake in Spartina alterniflora

Summary The effects of short- and long-term exposure to a range in concentration of sea salts on the kinetics of NH _inf4 ^sup+ uptake by Spartina alterniflora were examined in a laboratory culture experiment. Long-term exposure to increasing salinity up to 50 g/L resulted in a progressive increase in the apparent K_m but did not significantly affect V_max (mean V_max=4.23±1.97 mole·g^–1·h^–1). The apparent K_m increased in a nonlinear fashion from a mean of 2.66±1.10 mole/L at a salinity of 5 g/L to a mean of 17.56±4.10 mole/L at a salinity of 50 g/L. These results suggest that the long-term effect of exposure to total salt concentrations within the range 5–50 g/L was a competitive inhibition of NH _inf4 ^sup+ uptake in S. alterniflora. No significant NH _inf4 ^sup+ uptake was observed in S. alterniflora exposed to 65 g/L sea salts. Short-term exposure to rapid changes in salinity significantly affected both V_max and K_m. Reduction of solution salinity from 35 to 5 g/L did not change V_max but reduced K_m by 71%. However, exposing plants grown at 5 g/L salinity to 35 resulted in an decrease in V_max of approximately 50%. Exposure of plants grown at 35 g/L to a total sea salt concentration of 50 g/L for 48h completely inhibited uptake of NH _inf4 ^sup+ . For both experiments, increasing salinity led to an increase in the apparent K_m similar to that found in response to long-term exposure. Our data are consistent with a conceptual model of changes in the productivity of S. alterniflora in the salt marsh as a function of environmental modification of NH _inf4 ^sup+ uptake kinetics.     

Chromate tolerance in strains of Rhodosporidium toruloides modulated by thiosulfate and sulfur amino acids

Cr(VI) tolerance was studied in four strains of Rhodosporidium toruloides and compared with that of a fifth strain, DBVPG 6662, isolated from metallurgical wastes and known to be Cr(VI) resistant. Tolerance was studied in relation to different species of sulfur (sulfates, thiosulfates, methionine, cysteine) at different concentrations. Djenkolic acid, a poor source of sulfur and an activator of sulfate transport, was also considered. In synthetic medium all strains except the Cr(VI)-resistant one started to be inhibited by 10 g ml^– (0.2 mm) Cr(VI) as K₂Cr₂O₇. DBVPG 6662 was inhibited by 100 g ml^– (2.0 mm) Cr(VI). In Yeast Nitrogen Base without amino acids (minimal medium), supplemented with varying concentrations of chromate, all Cr(VI)-sensitive strains accumulated concentrations of total chromium (from 0.8 to 1.0 g mg^– cell dry wt) after 18 h of incubation at 28 °C. In minimal medium supplemented with 10 g ml^– Cr(VI), the addition of sulfate did not significantly improve the yeast growth. Cysteine at m levels increased tolerance up to 10 g ml^–, whereas methionine only reduced the Cr(VI) toxicity in the strain DBVPG 6739. Additions of djenkolic acid resulted in increased Cr(VI) sensitivity in all strains. The best inorganic sulfur species for conferring high tolerance was thiosulfate at concentrations up to 1 mm. In all cases increased Cr(VI) tolerance was due to a significantly reduced uptake in the oxyanion by the cells and not to the chemical reduction of Cr(VI) to Cr(III) by sulfur compounds.     

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (P_i). The first is synthesized on germination in medium containing 50 M P_i and has a K_m of approx. 30 M (V_max=7–9 nmol P_i/h·10⁶ cells). The second is synthesized under conditions of P_i-deprivation and has a higher affinity for P_i (K_m=1–2 M), but a lower V_max (0.5–2 nmol P_i/h·10⁶ cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of P_i in the medium. The K_m of the lower affinity system is 3 mM (V_max=20 nmol phosphite/h·10⁶ cells) and that of the higher affinity system is 0.6 mM (V_max=12 nmol/h·10⁶ cells). P_i and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit P_i transport, only M concentrations of P_i are required to inhibit phosphite transport. A third system of uptake for P_i also exists, since when phosphate-deprived cells are presented with mM concentrations of P_i, they transport the anion at a very high rate (around 100 nmol/h·10⁶ cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.     

Quantitative determination of calcium-activated myosin adenosine triphosphatase activity in rat skeletal muscle fibres

Summary A quantitative histochemical technique was developed for determining the kinetics of the calcium-activated myosin ATPase (Ca²⁺-myosin ATPase) reaction in rat skeletal muscle fibres. Using this technique, the maximum velocity (V_max) and the apparent Michaelis-Menten rate constant for ATP (K_app) of the Ca²⁺-myosin ATPase reaction were measured in type-identified fibres of the rat medial gastrocnemius (MG) muscle. The V_max and the K_app of the Ca²⁺-myosin ATPase reaction were lowest in type I fibres and highest (i.e., approx. two times greater) in type IIb fibres. The K_app in type IIa fibres was similar to that in type I. However, the V_max was 1.5 times greater in type IIa fibres, compared to type I fibres. Evidence is presented to suggest that the type IIb fibre population in the MG does not represent a single myosin isozyme. In addition, the broad range of V_max and K_app values indicates that there is marked heterogeneity in the myosin heavy chain and myosin light chain composition of myosin isozymes among individual fibres.     

Evaluation of chromium(VI) removal behaviour by two isolates of Synechocystis sp. in terms of exopolysaccharide (EPS) production and monomer composition

Chromium(VI) removal and its association with exopolysaccharide (EPS) production in cyanobacteria were investigated. Synechocystis sp. BASO670 produced higher EPS (548 mg L⁻¹) than Synechocystis sp. BASO672 (356 mg L⁻¹). While the EC₅₀ of the Cr(VI) for Synechocystis sp. BASO670 and Synechocystis sp. BASO672 were determined as 11.5 mg L⁻¹, and 2.0 mg L⁻¹, respectively, there was no relation between Cr(VI) removal and EPS production. Synechocystis sp. BASO672, which has higher EPS value, removed (33%) more Cr(VI) than Synechocystis sp. BASO670. Monomer compositions of EPS of each of the isolates were determined differently. Synechocystis sp. BASO672 which removed higher Cr(VI), had higher values of uronic acid and glucuronic acid (192 μg/mg and 89%, respectively). Our results showed that EPS might play a role in Cr(VI) tolerance. Monomer composition, especially uronic acid and glucuronic acid content of EPS may have enhanced Cr(VI) removal.     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg²⁺ and in the presence of Mg²⁺ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl^->HCO ₃ ^- >Br^->malate>acetate>SO ₄ ^2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH ₄ ⁺ Na⁺>K⁺). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na⁺ and K⁺. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in V_max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO₃ but other inhibitors tested (azide, oligomycin, orthovanadate, K₃[Cr(oxalate)₆] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATP_F free ATP - ATP_T total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - K_m apparent Michaelis constant - MgATP complex of Mg²⁺ and ATP - Mg _F ²⁺ free Mg²⁺ - Mg _T ² total Mg²⁺ - MES 2-(N-Morpholino)ethanesulphonic acid - Na₂EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - P_i inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - V_max maximum velocity     

Chromium uptake,retention and reduction in photosynthetic <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Photosynthetic Euglena gracilis grown with different K₂CrO₄ concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl₃. Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K _m of 1.1 mM, V _m of 16 nmol (h × 10⁷ cells)⁻¹, and K _i sulfate of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K _m = 0.83 mM, V _m = 15.9 nmol (h × 10⁷cells)⁻¹ and K _i chromate = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 × 10⁷ cells at 1 mM K₂CrO₄) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 × 10⁷ cells at 1 mM CrCl₃). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

Biochemical and catalytic properties of endo-1,4-β-xylanases from Thermomyces lanuginosus (wild and mutant strains)

Endoxylanases from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 (cellulase free wild and mutant strains), were purified to homogeneity by anion-exchange and molecular-sieve chromatographic methods. The purified enzymes were monomers with molecular masses of 22 kDa (wild type) and 24 kDa (mutant), estimated by SDS-PAGE and gel filtration. As glycoproteins, the purified enzymes had 0.74% (wild type) and 11.8% (mutant) carbohydrate contents, and pI values of 5.8 and 6, respectively. The optimal pH and temperature values of wild type xylanase were determined to be pH 7 and 60 °C, whereas pH 6.7 and 70 °C, were optimal for the purified mutant enzyme (K _m and V _max values of 3.7 mg ml^–1 and 670 mol min^–1 xylose compared to the kinetic values of the purified wild type xylanase –5.1 mg ml^–1 and 385 mol min^–1 xylose). Inhibition studies suggested the possible involvement of histidine, tryptophan residues and carboxylic groups in the binding or catalysis.