Pseudomonas induces salinity tolerance in cotton (Gossypium hirsutum) and resistance to Fusarium root rot through the modulation of indole-3-acetic acid

Abiotic stresses cause changes in the balance of phytohormones in plants and result in inhibited root growth and an increase in the susceptibility of plants to root rot disease. The aim of this work was to ascertain whether microbial indole-3-acetic acid (IAA) plays a role in the regulation of root growth and microbially mediated control of root rot of cotton caused by Fusarium solani. Seed germination and seedling growth were improved by both NaCl and Mg₂SO₄ (100 mM) solutions when treated with root-associated bacterial strains Pseudomonas putida R4 and Pseudomonas chlororaphis R5, which are able to produce IAA. These bacterial strains were also able to reduce the infection rate of cotton root rot (from 70 to 39%) caused by F. solani under gnotobiotic conditions. The application of a low concentration of IAA (0.01 and 0.001 μg/ml) stimulated plant growth and reduced disease incidence caused by F. solani (from 70 to 41–56%, respectively). Shoot and root growth and dry matter increased significantly and disease incidence was reduced by bacterial inoculants in natural saline soil. These results suggest that bacterial IAA plays a major role in salt stress tolerance and may be involved in induced resistance against root rot disease of cotton.     

Salt tolerant Methylobacterium mesophilicum showed viable colonization abilities in the plant rhizosphere

The source of infection has always been considered as an important factor in epidemiology and mostly linked to environmental source such as surface water, soil, plants and also animals. The activity of the opportunistic pathogens associated with plant root, their adaptation and survival under hostile environmental condition is poorly understood. In this study the salt tolerance ability of Methylobacterium mesophilicum and its colonization in the root and shoot of plants under severe drought and salt stress conditions were investigated. The colonization of plant by M. mesophilicum was investigated in a gnotobiotic sand system, and their survival in pots with saline soil. Bacterial strain was found to colonize rhizosphere of cucumber, tomato and paprika grown under normal and salt stress condition and reached up to 6.4 × 10⁴ and 2.6 × 10⁴ CFU/g root. The strain was resistant to Gentamicin, Ampicillin, Amoxicillin plus Clavulanic acid, Cefotaxime, neomycin, penicillin and was also tolerant to salinity stress (up to 6% NaCl). These abilities play important roles in enabling persistent colonization of the plant surface by M. mesophilicum strains. In conclusion, this study provides background information on the behaviour of opportunistic pathogen M. mesophilicum on plants and their survival in harsh environmental conditions.     

Avocado roots treated with salicylic acid produce phenol-2,4-bis (1,1-dimethylethyl), a compound with antifungal activity

We demonstrated the ability of salicylic acid (SA) to induce a compound in avocado roots that strengthens their defense against Phytophthora cinnamomi. The SA content of avocado roots, before and after the application of exogenous SA, was determined by High-Performance Liquid Chromatography (HPLC). After 4 h of SA feeding, the endogenous level in the roots increased to 223 μg g⁻¹ FW, which was 15 times the amount found in control roots. The methanolic extract obtained from SA-treated avocado roots inhibited the radial growth of P. cinnamomi. A thin layer chromatographic bioassay with the methanolic extract and spores of Aspergillus showed a distinct inhibition zone. The compound responsible for the inhibition was identified as phenol-2,4-bis (1,1-dimethylethyl) by gas chromatography and mass spectrometry. At a concentration of 100 μg/mL, the substance reduced germinative tube length in Aspergillus and radial growth of P. cinnamomi. A commercial preparation of phenol-2,4-bis (1,1-dimethylethyl) caused the same effects on mycelium morphology and radial growth as our isolate, confirming the presence of this compound in the root extracts. This is the first report of the induction of this compound in plants by SA, and the results suggest that it plays an important role in the defense response of avocado.     

Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots

Saline environments of terrestrial halophytes are often prone to waterlogging, yet the effects on halophytes of combined salinity and waterlogging have rarely been studied. Either salinity or hypoxia (low O₂) alone can interfere with K⁺ homeostasis, therefore the combination of salinity or hypoxia is expected to impact significantly on K⁺ retention in roots. We studied mechanisms of tolerance to the interaction of salinity with hypoxia in Puccinellia ciliata and Thinopyrum ponticum, halophytic grasses that differ in waterlogging tolerance. Plants were exposed to aerated and stagnant saline (250 mM NaCl) treatments with low (0.25 mM) and high (4 mM) K⁺ levels; growth, net ion fluxes and tissue ion concentrations were determined. P. ciliata was more tolerant than T. ponticum to stagnant-saline treatment, producing twice the biomass of adventitious roots, which accumulated high levels of Na⁺, and had lower shoot Na⁺. After 24 h of saline hypoxic treatment, MIFE measurements revealed a net uptake of K⁺ (∼40 nmol m⁻² s⁻¹) for P. ciliata, but a net loss of K⁺ (∼20 nmol m⁻² s⁻¹) for the more waterlogging sensitive T. ponticum. NaCl alone induced K⁺ efflux from roots of both species, with channel blocker tests implicating GORK-like channels. P. ciliata had constitutively a more negative root cell membrane potential than T. ponticum (−150 versus −115 mV). Tolerance to salinity and hypoxia in P. ciliata is related to increased production of adventitious roots, regulation of shoot K⁺/Na⁺, and a superior ability to maintain negative membrane potential in root cells, resulting in greater retention of K⁺.     

Thiol-peptide level and proteomic changes in response to cadmium toxicity in Oryza sativa L. roots

In the present study, rice seedlings were exposed to a range of Cd concentrations (0.1 μM, 1 μM, 10 μM, 100 μM and 1 mM) for 15 days and a combination of different molecular approaches were used to evidence Cd effects and to assess the plants’ ability to counteract metal toxicity. At a macroscopical level, only the highest Cd concentration (1 mM) caused a complete plant growth inhibition, whereas the lowest concentrations seemed to stimulate growth. At genome level, the amplified fragment length polymorphism (AFLP) technique was applied to detect DNA sequence changes in root cells, showing that all the Cd concentrations induced significant DNA polymorphisms in a dose-dependent manner. Data also evidenced the absence of preferential mutation sites.Plant responses were analysed by measuring the levels of gluthatione (GSH) and phytochelatins (PCs), the thiol-peptides involved in heavy metal tolerance mechanisms. Results showed a progressive increase of GSH up to 10 μM of Cd treatment, whereas a significant induction only of PC₃ was detected in roots of plants exposed to 100 μM of Cd. As suggested by the proteome analysis of root tissues, this last concentration strongly induced the expression of regulatory proteins and some metabolic enzymes. Furthermore, the treatment with 10 μM of Cd induced changes in metabolic enzymes, but it mainly activated defence mechanisms by the induction of transporters and proteins involved in the degradation of oxidatively modified proteins.     

Interaction between Meloidogyne incognita and Rhizoctonia solani on green beans

The interaction between Meloidogyne incognita (race 2) and Rhizoctonia solani (AG 4) in a root rot disease complex of green beans (Phaseolus vulgaris) was examined in a greenhouse pot experiment. Three week-old seedlings (cv. Contender) were inoculated with the nematode and/or the fungus in different combinations and sequences. Two months after last nematode inoculation, the test was terminated and data were recorded. The synchronized inoculation by both pathogens (N + F) increased the index of Rhizoctonia root rot and the number of root galls; and suppressed plant growth, compared to controls. However, the severity of root rot and suppression of plant growth were greater and more evident when inoculation by the nematode preceded the fungus (N → F) by two weeks. Nematode reproduction (eggs/g root) was adversely affected by the presence of the fungus except by the synchronized inoculation. When inoculation by nematode preceded the fungus, plant growth was severely suppressed and roots were highly damaged and rotted leading to a decrease of root galls and eggs.     

The impact of heavy metals on the activity of some enzymes along the barley root

Barley seedlings 48 h after the onset of germination on filter paper treated for 24 h by 1 mM cadmium (Cd), 3 mM nickel (Ni) or 0.5 mM mercury (Hg) showed similar approximately 45% root growth inhibition. Although root growth inhibition was similar, loss of cell viability evaluated, as Evans blue uptake was distinct among Cd, Ni and Hg treated roots. While Cd and Hg caused cell death along the whole barley root (0–8 mm), Ni induced significant loss of cell viability only in root cells 6–8 mm distance from the root tip. Our results suggest that different metabolic processes are activated in different parts of barley root in relation to distance from the root tip during heavy metal (HM) treatment. Some of them are characteristic for several HMs such as inhibition of ascorbic acid oxidase or glutathione-S-transferase stimulation, while others are specific for individual HMs, e.g. activation of acid phosphatase and lipoxygenase by Cd and Hg, or inhibition of ascorbate peroxidase by Ni and Hg treatment.     

Diversity and heavy metal tolerance of endophytic fungi from six dominant plant species in a Pb–Zn mine wasteland in China

The diversity and metal tolerance of endophytic fungi from six dominant plant species in a Pb–Zn mine wasteland in Yunnan, China were investigated. Four hundred and ninety-five endophytic fungi were isolated from 690 tissue segments. The endophytic fungal colonization extent and isolation extent ranged from 59 % to 75 %, and 0.42–0.93, respectively, and a positive correlation was detected between them. Stems harboured more endophytic fungi than leaves in each plant species, and the average colonization extent of stems was 82 %, being significantly higher than that of leaves (47 %) (P ≤ 0.001, chi-square test). The fungi were identified to 20 taxa in which Phoma, Alternaria and Peyronellaea were the dominant genera and the relative frequencies of them were 39.6 %, 19.0 % and 20.4 %, respectively. Metal tolerance test showed that 3.6 mM Pb²⁺ or 11.5 mM Zn²⁺ exhibited the greatest toxicity to some isolates and they did not grow on the metal-amended media. In contrast, some isolates were growth stimulated in the presence of tested metals. The isolates of Phoma were more sensitive to Zn²⁺ than the isolates of Alternaria and Peyronellaea. However, the sensitivity of isolates to Pb²⁺ was not significantly different among Phoma, Alternaria, Peyronellaea and other taxa (P > 0.05, chi-square test). Our results suggested that fungal endophyte colonization in Pb–Zn polluted plants is moderately abundant and some isolates have a marked adaptation to Pb²⁺ and Zn²⁺ metals, which has a potential application in phytoremediation in this area.     

Effect of natural maize phytochemicals on Aspergillus section Flavi sclerotia characteristics under different conditions of growth media and water potential

The effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) at concentrations of 1–20 mM (CA) and 1–25 mM (FA) on sclerotial production by Aspergillus flavus and Aspergillus parasiticus were evaluated. Studies on sclerotium number and size were carried out in different growth media and water potentials (MPa). High concentrations of CA (20 mM, ?0.75 MPa; 10 mM, ?3.5 MPa) and FA (10, 20, 25 mM, ?0.75 and ?3.5 MPa) significantly reduced sclerotial production of Aspergillus strains. Overall, CA at concentrations of 10 and 20 mM on Czapek Dox medium (CD), maize meal extract agar (MMEA) and maize meal extract agar with sucrose and NaNO₃ (MMEA S/N) inhibited sclerotium most in the four species assayed. The data show that the sclerotia characteristics of A. flavus and A. parasiticus were influenced by natural phytochemicals and modifications of growth media and water potential. CA and FA could be used at high concentrations to prevent the survival of Aspergillus species in grain.     

Purification of a laccase from fungus combs in the nest of Odontotermes formosanus

A new enzyme was isolated from the fungus combs in the nest of Odontotermes formosanus and identified as a laccase. The single laccase was purified with a purification factor of 16.83 by ammonium sulphate precipitation and anion exchange chromatography, to a specific activity of 211.11 U mg⁻¹. Its molecular mass was 65 kDa. The optimum pH value and temperature were 4.0 °C and 10 °C with ABTS as the substrate, respectively. The enzyme activity stabilized at temperatures between 10 °C and 30 °C and decreased rapidly when the temperature was above 30 °C. The V_max and K_m values were 3.62 μmol min⁻¹ mg⁻¹ and 119.52 μM, respectively. Ethanol concentration affected laccase activity, inhibiting 60% of enzyme activity at a concentration of 70%. Metal ions of Mg²⁺, Ba²⁺ and Fe²⁺ showed inhibition on enzyme activity of 17.2%, 5.3% and 9.4%, respectively, with the increase of metal ions concentration from 1 mM to 5 mM. Especially Fe²⁺ strongly inhibited enzyme activity up to 89% inhibition at a concentration of 1 mM.     

Integrating insecticides and Trichogramma ostriniae to control European corn borer in sweet corn: Economic analysis

We compared the economics of controlling European corn borer (Ostrinia nubilalis) in sweet corn by using the egg parasitoid, Trichogramma ostriniae, alone and integrated with insecticidal sprays. An initial experiment in 2003 compared T. ostriniae alone against insecticide alone and a second set of experiments conducted over 3 years (2006–2008) compared (1) insecticide alone [Insecticide]; (2) no insecticide, no T. ostriniae [Untreated Check]; (3) T. ostriniae alone [T. ostriniae 1X]; and (4) T. ostriniae + insecticide [Integrated]. In 2007 and 2008, a fifth treatment was added consisting of three approximately weekly releases of T. ostriniae [T. ostriniae 3X]. Parasitism of O. nubilalis eggs was higher in plots receiving T. ostriniae; O. nubilalis eclosion was lower with T. ostriniae; there was no interaction of T. ostriniae and insecticide on parasitism, O. nubilalis eclosion, or total O. nubilalis larvae at harvest time. Partial crop budgets were conducted for each treatment. In three of the 4 years, Untreated Checks had the highest sweet corn ear damage. Ear damage after a single release of T. ostriniae was statistically no different than using insecticides. In two of the three years, the Integrated treatment (T. ostriniae 1X + insecticide) generated the largest increase in profitability. The insecticide only treatment generated the second best increase in profitability. When comparing a single release of T. ostriniae to the insecticide only, the latter provided a better combination of efficacy and profitability. The breakeven costs of the T. ostriniae justified their use relative to the Untreated Check treatment, but not when compared to the Insecticide treatment. The breakeven costs for T. ostriniae in the Integrated treatment exceeded the actual cost in two out of three years, suggesting again that conventional growers could benefit from integrating T. ostriniae with insecticidal treatments. Projected profitability based on ear packout obtained by combining data among years suggests that in general, for low prices and yields, the increase in profit is quite modest. In the lowest price-yield combination, the change in profit is T. ostriniae 3X > Integrated > Insecticide > T. ostriniae 1X ? Untreated Check. At high prices and high yields, the differences between the management options when compared to Untreated Check are considerable. In the highest price-yield combination, the change in profit is T. ostriniae 3X > Integrated ? Insecticide > T. ostriniae 1X ? Untreated Check.     

Short and long-term uptake of Hg in white lupin plants:Kinetics and stress indicators

The intrinsic characteristics of white lupin regarding biomass production and tolerance to abiotic stresses could make it a good candidate to be used in degraded mine soils containing mercury (Hg), but white lupin behaviour in response to Hg has to be previously evaluated. With this aim, kinetic parameters of Hg uptake in short and long-term experiments, and Hg resistance of white lupin plants using several stress indicators were studied. The plants were grown with increasing Hg doses in nutrient solutions (0, 5 and 10 μM). Hg uptake showed an active component in Hg influx, suggesting the existence of a low affinity root transporter that can be used for Hg uptake into white lupin root cells. K_m and V_max values obtained for the saturable component were 217.7 ± 27.6 μM and 3.78 ± 0.18 μmol Hg g FW^?1 h^?1. Hg accumulation was concentration–time-dependent, showing a saturable behaviour for the lower doses but a linear behaviour for the highest ones. A high ability of Hg absorption by white lupin was observed both in short and long-term uptake experiments. The highest Hg dose reduced biomass production especially in the shoots. Moreover, increases in chlorophylls, malondialdehyde, total thiols and phenols were observed in Hg-stressed plants. The enhancement of total thiols and phenols levels in roots reduced oxidative stress for the 5 μM dose, but not for higher Hg levels. The deleterious effect of Hg was less marked in root tissues, in spite of their accumulation of very high Hg amounts (99%) because of, at least in part, a combined increase in total thiols and phenolics able to minimize oxidative stress. Our results suggested that phenolic content in roots could be used as a new and easy-to-use indicator of Hg presence. On the whole, white lupin showed a certain ability to survive in Hg-contaminated media and it would be possible to include it in combined decontamination strategies.     

MTs in Palaemonetes argentinus as potential biomarkers of zinc contamination in freshwaters

Aquatic invertebrates take up and accumulate essential and non-essential trace metals even when both are likely to be poisonous. In order to study the potential of the metallothioneins (MTs) as biomarkers of metal contamination in native shrimp Palaemonetes argentinus, organisms have been exposed at 0, 5, 50 and 500 μg L⁻¹ of zinc for 96 h. Moreover, accumulation and subcellular distribution of this essential metal were evaluated. A significant Zn accumulation was observed in different body sections. Higher Zn levels occurred in cephalothorax compared to abdomen, especially at the highest exposure concentration (500 μg Zn L⁻¹). A clear differential subcellular metal distribution between cephalothorax and abdomen was also observed. In cephalothorax Zn was similarly distributed between the soluble and insoluble fractions; while in abdomen, when total Zn increased, insoluble metal augmented more markedly than the soluble one. Cytosolic Zn levels increased more in cephalothorax than in abdomen of shrimps exposed to 500 μg Zn L⁻¹ when compared to control. Finally, a significant induction of MTs was observed in cephalothorax at 500 μg Zn L⁻¹. A potential role for MTs as biomarkers in P. argentinus should be further studied to enhance the sensitivity of the response, although it is likely that MTs play a key role in metal detoxification since the increase of these proteins is linked to metal challenge.     

Salt tolerance in the halophyte Salicornia dolichostachya Moss: Growth,morphology and physiology

Salinization of agricultural land is an increasing problem. Because of their high tolerance to salinity, Salicornia spp. could become models to study salt tolerance; they also represent promising saline crops. The salinity-growth response curve for Salicornia dolichostachya Moss was evaluated at 12 salt concentrations in a hydroponic study in a greenhouse and at 5 different seawater dilutions in an outside setting. Salt concentrations ranged between 0 mM and 500 mM NaCl (≈seawater salinity). Plants were grown for six weeks and morphological and physiological adaptations in different tissues were evaluated.S. dolichostachya had its growth optimum at 300 mM NaCl in the root medium, independent of the basis on which growth was expressed. The relative growth rate (RGR) in the greenhouse experiment was comparable with RGR-values in the outdoor growth experiment. Leaf succulence and stem diameter had the highest values at the growth optimum (300 mM NaCl). Carbon isotope discrimination (δ¹³C) decreased upon salinity. S. dolichostachya maintained a lower leaf sap osmotic potential relative to the external solution over the entire salinity range, this was mainly accomplished by accumulation of Na⁺ and Cl⁻. Glycine betaine concentrations did not significantly differ between the treatments. Na⁺:K⁺-ratio and K⁺-selectivity in the shoots increased with increasing salinity, both showed variation between expanding and expanded shoot tissue. We conclude that S. dolichostachya was highly salt tolerant and showed salt requirement for optimal growth. Future growth experiments should be done under standardized conditions and more work at the tissue and cellular level needs to be done to identify the underlying mechanisms of salt tolerance.     

Expression,refolding and purification of a human interleukin-17A variant

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T₇ promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris–HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2–8 °C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.     

Silicon supply in soilless cultivations of zucchini alleviates stress induced by salinity and powdery mildew infections

In the present study, the hypothesis was tested as to whether silicon supplied via the nutrient solution is capable of enhancing the tolerance of hydroponically grown zucchini squash (Cucurbita pepo L. cv. ‘Rival’) to salinity and powdery mildew infections. Two experiments were conducted involving a low (2.2 dS m^?1, 0.8 mM NaCl) and a high salinity level (6.2 dS m^?1, 35 mM NaCl) in combination with a low (0.1 mM) and a high (1.0 mM) Si level in the nutrient solution supplied to the crop. The exposure of the plants to high external salinity restricted significantly the vegetative growth as well as the fruit yield of zucchini due to a reduction of both the number of fruits per plant and the mean fruit weight. However, the inclusion of 1 mM of Si in the salinized nutrient solution mitigated the salinity-associated suppression of both growth and yield. Part of the growth and fruit yield suppression at high salinity was due to restriction of net photosynthesis. The stomatal conductance was also restricted by salinity, whereas the substomatal CO₂ concentration was not affected by the NaCl or Si treatments. The supply of 1 mM of Si via the nutrient solution mitigated the inhibitory effect of salinity on net photosynthesis and this effect was associated with lower Na and Cl translocation to the epigeous plant tissues. Furthermore, the supply of Si via the nutrient solution suppressed appreciably the expansion of a powdery mildew (Podosphaera xanthii) infection in the leaves at both salinity levels. These results indicate that the supply of at least 1 mM of Si via the nutrient solution is capable of enhancing both tolerance to salinity and resistance to powdery mildew in soilless cultivations of zucchini squash.     

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO₂ in air for 40–44 h. No significant difference (P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased (P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced (P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser (P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced (P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.     

Deletion and site-directed mutagenesis of laccase from Shigella dysenteriae results in enhanced enzymatic activity and thermostability

A putative laccase gene was cloned from Shigella dysenteriae W202 and expressed in Escherichia coli as a soluble fusion protein with high yield. The purified product (Wlac) was characterized as the CueO-like laccase from E. coli, a monomer of molecular mass 55 kDa, with a maximum activity of 24.4 U/mg (K_m = 0.086) and a pH optimum of 2.5, in a standard assay using ABTS (2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonate) as the substrate. Activity was stable at 0–25 °C but inhibited above 40 °C. Purified Wlac was completely inhibited by 200 mM EDTA and partially by 32 mM SDS, 50 mM NaN₃ and 60 mM thioglycolic acid. Activity was stimulated by Cu²⁺; other metal ions had only slight or negative effects. Two mutated variants, WlacS and WlacD, were obtained by substituting Glu 106 with Phe 106, and adding a deletion of an α-helix domain (from Leu 351 to Gly 378). WlacS had a 2.2-fold (52.9 U/mg) and WlacD a 3.5-fold (85.1 U/mg) higher enzyme activity than the wild-type laccase and WlacD showed greater thermostability at higher temperatures. Sce VMA intein-associated fusion proteins maintained ～80% of total enzyme activity. Thus, deletion and site-directed mutagenesis of laccases are capable of promoting both enzymatic activity and thermostability.     

A highly selective ginsenoside Rb1-hydrolyzing β-d-glucosidase from Cladosporium fulvum

G-I, a highly selective β-glucosidase, was purified from phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva). G-I was a monomer with native molecular weight of 85 kDa and pI value of 4.2. The maximal activity to p-nitrophenyl-β-d-glucopyranoside (pNPG) occurred at pH 6.0 and 45 °C at which the K_m against pNPG was 0.18 mM and V_max was 46.7 μmol nitrophenol/min/mg. G-I was highly stable within pH 4.0–11.0 and below 40 °C. It was inhibited by Co²⁺, Cu²⁺ and Zn²⁺ (50 mM), but showed resistance to sodium dodecyl sulfonate (SDS, 250 mM). G-I was highly active against β-linked disaccharide cellobiose, gentiobiose and sophorose, but exhibited very low activities against other aryl-glycosides, methyl-α-glycosides and disaccharides trehalose and sucrose. Moreover, G-I specifically hydrolyzed β-(1 → 6)-glucosidic linkage at the C-20 site of ginsenoside Rb₁ to produce ginsenoside Rd, without attack on other β-d-glucosidic linkages. The oligopeptide fragments of G-I were sequenced by nanoESI-MS/MS and showed similarity to the sequences from the glycoside hydrolase family 3. G-I is different to G-II (a glycosidase previously purified from the same fungus) in composition and molecular weight. It shows more stable and higher selectivity than G-II.