Structure-activity relationships for xyloglucan oligosaccharides with antiauxin activity

This work was designed to investigate the structural features required for a branched xyloglucan nonasaccharide (XG9; composition: glucose₄xylose₃galactose₁fucose₁) to exhibit anti-auxin activity in the pea (Pisum sativum L.) stem segment straight-growth bioassay. Oligosaccharides were prepared by cellulase-catalyzed hydrolysis of Rosa xyloglucan, and tested for auxin antagonism. The quantitatively major hepta-, octa-, and decasaccharides (XG7, XG8, and XG10) showed no antiauxin activity at the concentrations tested and did not interfere with the antiauxin effect of 10⁻⁹ molar XG9 when coincubated at equimolar concentrations. The results indicate that the XG9-recognition system in pea stem segments is highly discriminating. A terminal α-l-fucose residue is essential for the antiauxin activity of XG9 and a neighboring terminal β-d-galactose residue can abolish the activity; possible reasons for the effect of the galactose residue are discussed. A sample of XG9 extensively purified by gel-permeation chromatography followed by paper chromatography in two solvent systems still exhibited antiauxin activity with a concentration optimum around 10⁻⁹ molar. This diminishes the likelihood that the antiauxin activity reported for previous nonsaccharide preparations was due to a compound other than XG9.     

Further studies of the ability of xyloglucan oligosaccharides to inhibit auxin-stimulated growth

The structural features required for xyloglucan oligosaccharides to inhibit 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments have been investigated. A nonasaccharide (XG9) containing one fucosyl-galactosyl side chain and an undecasaccharide (XG11) containing two fucosyl-galactosyl side chains were purified from endo-β-1,4-glucanase-treated xyloglucan, which had been isolated from soluble extracellular polysaccharides of suspension-cultured sycamore (Acerpseudoplatanus) cells and tested in the pea stem bioassay. A novel octasaccharide (XG8′) was prepared by treatment of XG9 with a xyloglucan oligosaccharide-specific α-xylosidase from pea seedlings. XG8′ was characterized and tested for its ability to inhibit auxin-induced growth. All three oligosaccharides, at a concentration of 0.1 microgram per milliliter, inhibited 2,4-dichlorophenoxyacetic acid-stimulated growth of pea stem segments. XG11 inhibited the growth to a greater extent than did XG9. Chemically synthesized nona- and pentasaccharides (XG9, XG5) inhibited 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stems to the same extent as the same oligosaccharides isolated from xyloglucan. A chemically synthesized structurally related heptasaccharide that lacked a fucosyl-galactosyl side chain did not, unlike the identical heptasaccharide isolated from xyloglucan, significantly inhibit 2,4-dichlorophenoxyacetic acid-stimulated growth.     

Abscission: role of cellulase

Cellulase (β-1,4-glucan-glucanohydrolase EC 3.2.1.4) activity increased during abscission and was localized in the cell separation layer of Phaseolus vulgaris L. cv. Red Kidney (bean), Gossypium hirsutum L. cv. Acala 4-42 (Cotton) and Coleus blumei Benth. Princeton strain (Coleus) abscission zone explants. Cellulase activity was optimum at pH 7, was reduced by one-half after heating to 55° for 10 min, and was associated with the soluble components of the cell. Explants treated with aging retardants (indoleacetic acid, ⁶N-benzyladenine, and coumarin), CO₂, actinomycin D or cycloheximide had less cellulase activity than untreated controls. Ethylene increased cellulase activity of aged explants after a 3-hr lag period but had no effect on cellulase activity of freshly excised explants. It was concluded that 1 of the roles of ethylene in abscission is to regulate the production of cellulase which in turn is required for cell separation.     

Inhibition of auxin-stimulated growth of pea stem segments by a specific nonasaccharide of xyloglucan

Hemicellulose extracted from cell walls of suspension-cultured rose (Rosa Paul's Scarlet) cells was digested with cellulase from Trichoderma viride. The quantitatively major oligosaccharide products, a nonasaccharide and a heptasaccharide derived from xyloglucan, were purified by gel permeation chromatography. The nonasaccharide was found to inhibit the 2,4-dichlorophenoxy-acetic-acid-induced elongation of etiolated pea (Pisum sativum) stem segments. This confirms an earlier report (York et al., 1984, Plant Physiol. 75, 295–297). The inhibition of elongation by the nonasaccharide showed a maximum at around 10^-9M with higher and lower concentrations being less effective. The heptasaccharide did not significantly inhibit elongation at 10^-7–10^-10M and also did not affect the inhibition caused by the nonasaccharide when co-incubated with the latter.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - XG xyloglucan - XG7 xyloglucan heptasaccharide (Glc₄·Xyl₃) - XG9 xyloglucan nonasaccharide (Glc₄·Xyl₃·Gal·Fuc)     

Multiphasic Kinetics of Transformation of 1,2,4-Trichlorobenzene at Nano- and Micromolar Concentrations by Burkholderia sp. Strain PS14

The transformation of 1,2,4-trichlorobenzene (1,2,4-TCB) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with Burkholderia sp. strain PS14. 1,2,4-TCB was metabolized from nano- and micromolar concentrations to below its detection limit of 0.5 nM. At low initial 1,2,4-TCB concentrations, a first-order relationship between specific transformation rate and substrate concentration was observed with a specific affinity (a⁰_A) of 0.32 liter · mg (dry weight)⁻¹ · h⁻¹ followed by a second one at higher concentrations with an a^o_A of 0.77 liter · mg (dry weight)⁻¹ · h⁻¹. This transition from the first-order kinetics at low initial 1,2,4-TCB concentrations to the second first-order kinetics at higher 1,2,4-TCB concentrations was shifted towards higher initial 1,2,4-TCB concentrations with increasing cell mass. At high initial concentrations of 1,2,4-TCB, a maximal transformation rate of approximately 37 nmol · min⁻¹ · mg (dry weight)⁻¹ was measured, irrespective of the cell concentration.     

Temperature-dependent Response to Indoleacetic Acid Is Altered by NH(4) in Cultured Cotton Ovules

Cut carnations (Dianthus caryophyllus L. cv. `Improved White Sim') were exposed to ultra high purity ¹⁴C₂H₄ (20 μl/1) during flower opening and senescence to study its incorporation and metabolism. During treatment precautions were taken to exclude inhibitory volatiles from rubber serum stoppers which were identified as CS₂ and COS. As with the pea seedling (Nature 1975, 255:144-147), cut carnations incorporated ¹⁴C₂H₄ into ethanol-soluble tissue metabolites and oxidized the hormone to ¹⁴CO₂. Oxidation increased from 0.5 to 3 dpm · mg dry wt⁻¹·6 hr⁻¹ during the period of flower opening and early petal wilt. As severe petal wilt set in, and the ovary increased in size and dry weight, oxidation increased to a peak of nearly 29 dpm · mg dry wt⁻¹·6 hr⁻¹. Concomitant with this peak was a similar rise in the rate of ¹⁴C₂H₄ incorporation into the petals, peduncle, bracts, and sepals. Much higher rates of incorporation were found for the reproductive and receptacle tissues. Incorporation into these tissues steadily increased during flower opening reaching a peak of over 160 dpm · mg dry wt⁻¹ · 6 hr⁻¹ just before full bloom. This peak preceded a peak of endogenous ethylene production while the ¹⁴C₂H₄ oxidation peak followed it.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

In-vivo formation of xyloglucan nonasaccharide: A possible biologically active cell-wall fragment

The in-vivo formation of a specific nonasaccharide of xyloglucan was investigated. This nonasaccharide has been reported to have biological activity, inhibiting auxin-induced growth in pea stem segments. Cell-suspension cultures of spinach were grown in the presence of [³H]arabinose and [³H]fucose, and the culture-filtrates were examined for oligosaccharides by gelpermeation chromatography and by paper chromatography. Sixteen [³H]pentose-containing oligosaccharides were found, including twelve that contained the sequence [³H]xylosyl-(16)-glucose, which is diagnostic of xyloglucan. In addition, [³H]fucose-containing oligosaccharides of at least three sizes were found. Radiochemical evidence is presented that one of these oligosaccharides was labelled with both [³H]fucose and with [³H]pentose, and was identical with the major xyloglucan-derived nonasaccharide associated with anti-auxin activity. It was largely present in the form of acylated (possibly acetylated) derivatives. It accumulated extracellularly to a steady-state concentration of about 4.3·10^-7M. This is the first report of the production of a biologically-active oligosaccharide by living plant cells.Abbreviations BAB butanone/acetic acid/H₃BO₃-saturated water (9:1:1) - BAW butan-1-ol/acetic acid/water (12:3:5) - BPW butan-1-ol/pyridine/water/(4:3:4) - DP degree of polymerisation - FAW ethyl acetate/acetic acid/water (10:5:6) - EPW ethyl acetate/pyridine/water (8:2:1) - k _av elution volume relative to Blue Dextran (k _av.=0.0) and glucose (k _av.=1.0) - XG7 XG9 minus the fucose and galactose residues - XG9 the particular xyloglucan nonasaccharide illustrated in Fig. 1 - W water-saturated phenol     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

Warm growth temperatures decrease soybean cholinephosphotransferase activity

The activity of cytidine 5′-diphosphate (CDP) choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) in developing soybean (Glycine max L. var Williams 82) seeds was 3 to 5 times higher in cotyledons grown at 20°C than in those grown at 35°C. Some characteristics of the enzyme from cotyledons cultured at 20 and 35°C were compared. In preparations from both growth temperatures, the enzyme showed a pH optimum of 7, K_m of 7.0 micromolar for CDP-choline, and an optimum assay temperature of 45°C. Both enzyme preparations were stimulated by increasing concentrations of Mg²⁺ or Mn²⁺, up to 10 millimolar and 50 micromolar, respectively, though Mn²⁺ produced lower activities than Mg²⁺. Enzymes from both 20 and 35°C show the same specificity for exogenous diacylglycerol. No metabolic effectors were detected by addition of heat treated extracts to the assay mixture. The above findings suggest that the higher enzyme activity at 20°C can be attributed to a higher level of the enzyme rather than to the involvement of isozymes or metabolic effectors. Enzyme activity decreased rapidly during culture at 35°C, indicating a rapid turnover of the enzyme. The level of temperature modulation was found to be a function of seed developmental stage.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Studies on the congenitally goitrous sheep. The iodinated compounds of serum, and circulating thyroid-stimulating hormone

1. A group of normal and congenitally goitrous Merino sheep were investigated to identify the metabolic defect present in the abnormal animals. 2. Protein-bound iodine concentrations of serum from goitrous animals (average 5·7μg./100ml.) were higher than normal (average 4·2μg./100ml.; P 0·001), but the hormonal iodine measured as butanol-extractable ¹³¹I was low in the serum of goitrous (average 40·3% of protein-bound ¹³¹I) compared with that of normal (84·2%; P 0·02) sheep. The non-hormonal iodine of the serum of goitrous sheep appeared to include iodotyrosines and iodinated protein. 3. Starch-gel-electrophoretic separations of sera from normal and goitrous sheep after ¹³¹I injection (100–500μc) showed no qualitative differences in the radioactivity of protein components. No significant differences in thyroxine-binding in vitro by serum proteins of normal and goitrous sheep were observed. 4. The clearance rates of ¹³¹I-labelled iodotyrosines (t_½ 1·2–2·9hr.) and iodothyronines (t_½ 33·5–47·4hr.) were similar in normal and goitrous sheep. 5. The concentration of circulating thyroid-stimulating hormone was significantly higher (P<0·01 in three sheep, P<0·05 in one sheep) in goitrous sheep. 6. The congenital goitre appears to be due to compensatory hypertrophy of the gland resulting from an inability to synthesize an adequate supply of thyroid hormone.     

Erythrocruorin of Marphysa sanguinea. Isolation of some physical, physicochemical and other properties

1. The polychaete worm Marphysa sanguinea has a circulating erythrocruorin of mol.wt. about 2·4×10⁶ (S⁰_20,w 58·2s, D_20,w 2·06×10⁻⁷ cm.²/sec). This is the predominant form existing at pH 6–8 and (non-protein) I 0·10–0·21, and also at approx. pH 6·7 and I 0·15–3·00. 2. The pigment contains 2·24% of protohaem. 3. The 58s protein has an electrophoretic mobility of 8·08×10⁻⁵ cm.²/v/sec. at pH 8·12, I 0·21 and 0°. The isoelectric point of suspended particles is 4·63 at I 0·16 and 21·5°. 4. At very low ionic strength and pH 6·7 (unbuffered) the 58s pigment associates reversibly to 97s and 150s forms, which are probably dimer and tetramer species. 5. At pH 10·0 and I 0·025, it dissociates irreversibly to give a small amount of 2–4s non-haem-containing protein and much 9s haem-enriched protein. These and the 58s pigment may correspond to structures found in Levin's (1963) electron-microscope studies of other erythrocruorins. 6. Absorption spectra of the 58s oxygenated erythrocruorin and the deoxygenated and carbon monoxide derivatives have been obtained.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70°C. Under optimized reaction conditions, the kinetic properties (V_max, 0.4 μmol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of C. thermocellum cellulose-solubilizing activity were comparable to those reported for T. reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 μM). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag²⁺ and Hg²⁺ effectively inhibited C. thermocellum but not T. reesei cellulase at <20 μM, whereas Ca²⁺, Mg²⁺, and Mn²⁺ inhibited T. reesei but not C. thermocellum cellulase at >10 mM. Both enzymes were inhibited by Cu²⁺ (>20 mM), Zn²⁺ (>1.0 mM), and ethylene glycol-bis(β-aminoethyl ether)- N,N-tetraacetic acid (>10 mM). T. reesei but not C. thermocellum cellulose-solubilizing activity was 20% inhibited by glucose (73 mM) and cellobiose (29 mM). Both cellulases preferentially cleaved the internal glycosidic bonds of cellooligosaccharides. The overall rates of cellooligosaccharide degradation were higher for T. reesei than for C. thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotriose were equivalent.     

Xyloglucan mobilisation and purification of a (XLLG/XLXG) specific β-galactosidase from cotyledons of Copaifera langsdorffii

The storage xyloglucan of germinating seeds of Copaifera langsdorffii is degraded by the action of β-galactosidase, endo-β-glucanase, α-xylosidase and β-glucosidase, producing free galactose, glucose and xylose. One of the β-galactosidases from cotyledons of germinating seeds of C. langsdorffii was purified by ion exchange and gel chromatography (Biogel P-60), leading to a single polypeptide (molecular mass 40 kDa). The enzyme has optimum activity at pH 3.2 (stable from pH 2.3 to 6.0) and is active on p-NP-β-gal (K_m 3.5 mM) and lactose but not on o-NP-β-gal or p-NP-β-gal. Small amounts of galactose were released from xyloglucan of seeds of C. langsdorffii, Tamarindus indica and less from Hymenaea courbaril. No galactose was released after incubation with β-1,4-linked galactan from Lupinus angustifolius cotyledons. Much higher activity was observed on oligosaccharides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichoderma viride cellulase. The purified β-galactosidase attacked XLLG and XLXG specifically, producing a mixture of XXXG and XXLG (unsubstituted glucose is assigned G; glucose branched with xylose is assigned X and if galactose is branching xylose, the trisaccharide is assigned L). Considering the recent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents β-glucosidase from acting on GLXG or GLLG but not on GXLG or GXXG, the β-galactosidase isolated in this work seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance (L) for further hydrolysis of the oligosaccharides and therefore essential for completion of xyloglucan mobilisation.     

Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers

The wide variety of bacteria in the environment permits screening for more efficient cellulases to help overcome current challenges in biofuel production. This study focuses on the isolation of efficient cellulase producing bacteria found in organic fertilizers and paper mill sludges which can be considered for use in large scale biorefining. Pure isolate cultures were screened for cellulase activity. Six isolates: S1, S2, S3, S4, E2, and E4, produced halos greater in diameter than the positive control (Cellulomonas xylanilytica), suggesting high cellulase activities. A portion of the 16S rDNA genes of cellulase positive isolates were amplified and sequenced, then BLASTed to determine likely genera. Phylogenetic analysis revealed genera belonging to two major Phyla of Gram positive bacteria: Firmicutes and Actinobacteria. All isolates were tested for the visible degradation of filter paper; only isolates E2 and E4 (Paenibacillus species) were observed to completely break down filter paper within 72 and 96 h incubation, respectively, under limited oxygen condition. Thus E2 and E4 were selected for the FP assay for quantification of total cellulase activities. It was shown that 1% (w/v) CMC could induce total cellulase activities of 1652.2±61.5 and 1456.5±30.7 μM of glucose equivalents for E2 and E4, respectively. CMC could induce cellulase activities 8 and 5.6X greater than FP, therefore CMC represented a good inducing substrate for cellulase production. The genus Paenibacillus are known to contain some excellent cellulase producing strains, E2 and E4 displayed superior cellulase activities and represent excellent candidates for further cellulase analysis and characterization.     

Purification and Characterization of a Secreted Laccase of Gaeumannomyces graminis var. tritici

We purified a secreted fungal laccase from filtrates of Gaeumannomyces graminis var. tritici cultures induced with copper and xylidine. The active protein had an apparent molecular mass of 190 kDa and yielded subunits with molecular masses of 60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2,6-dimethoxyphenol (K_m = 2.6 × 10⁻⁵ ± 7 × 10⁻⁶ M), catechol (K_m = 2.5 × 10⁻⁴ ± 1 × 10⁻⁵ M), pyrogallol (K_m = 3.1 × 10⁻⁴ ± 4 × 10⁻⁵ M), and guaiacol (K_m = 5.1 × 10⁻⁴ ± 2 × 10⁻⁵ M). In addition, the laccase catalyzed the polymerization of 1,8-dihydroxynaphthalene, a natural fungal melanin precursor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These findings indicate that this laccase may be involved in melanin polymerization in this phytopathogen’s hyphae and/or in lignin depolymerization in its infected plant host.