Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase.

The beta-glucosidase from Aspergillus niger (CMI CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. (1)H-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by 2-deoxy-2-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K(I) = 35.4 mM, through the trapping of a covalent glycosyl enzyme intermediate. The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW. Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 438-442).

Synthesis of isopropyl-1-thio-beta-D-glucopyranoside (IPTGlc), an inducer of Aspergillus niger B1 beta-glucosidase production.

Production of beta-glucosidase in Aspergillus niger B1 is subjected to catabolic repression by glucose. Aspergillus niger B1 grown on bran as a carbon source secreted beta-glucosidase. The maximum level of the enzyme was reached after 7 d of fermentation. Addition of 1% glucose to the medium suppressed beta-glucosidase production to undetectable levels. In this study, the organic synthesis of a potential inducer of beta-glucosidase production by A. niger B1's reported. Isopropyl-1-thio-beta-D-glucopyranoside (IPTGlc) was synthesized using a two-step organic synthesis protocol. The H-NMR data agreed with those reported previously for the galactoside analog. When IPTGlc was added 24 h after inoculation at a final concentration of 0.4 mM, similar levels of beta-glucosidase were reached 3 to 4 d earlier as compared to fermentation without IPTGlc induction. In practice, this may translate to a more efficient method of producing beta-glucosidase from this fungus.

Photoacoustic and fluorescence measurements of the chilling response and their relationship to carbon dioxide uptake in tomato plants.

The response of tomato plants to various chilling treatments was studied using two approaches for the measurement of photosynthetic activity. One involved the use of a portable fluorometer for the measurement of in-vivo chlorophyll fluorescence, while the other employed a newly introduced photoacoustic system which allowed changes in oxygen evolution to be followed in a leaf disc. A strong correlation was found between results obtained by each system and those obtained by a conventional open gas-exchange system for the determination of CO2 uptake. Both systems of measurements could readily distinguish between the effects of chilling in the dark (at 3° C for 18 h) and chilling at high photon flux density (2000 µmol m(-2) s(-1) for 5h at 5° C). Chilling in the dark had practically no effect on the quantum yield of oxygen evolution, chlorophyll fluorescence or CO2 uptake, while chilling at excessively high photon flux density resulted in a sharp reduction (50-70%) in the quantum yields obtained. The results support the view that photosystem II cannot be the primary site of damage by chilling in the dark, although it is significantly affected by chilling at high light intensity.

Effect of carbon dioxide on photorespiration.

The isotopic CO(2) technique for measuring photorespiration was shown to be a valid technique for measuring the unidirectional inward and outward fluxes of CO(2) from a sunflower (Helianthus annuus L.) leaf in the light. The rate of photorespiration was decreased little as the CO(2) concentration was increased from 20 to 1,150 microliters per liter. This finding contradicts the widely held assumption that photorespiration is suppressed at high CO(2) concentrations. Some discussion regarding this apparent conflict is presented.

Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

Response of carbon dioxide fixation to water stress: parallel measurements on isolated chloroplasts and intact spinach leaves.

Application of water stress to isolated spinach (Spinacia oleracea) chloroplasts by redutcion of the osmotic potentials of CO(2) fixation media below -6 to -8 bars resulted in decreased rates of fixation regardless of solute composition. A decrease in CO(2) fixation rate of isolated chloroplasts was also found when leaves were dehydrated in air prior to chloroplast isolation. An inverse response of CO(2) fixation to osmotic potential of the fixation medium was found with chloroplasts isolated from dehydrated leaves-namely, fixation rate was inhibited at -8 bars, compared with -16 or -24 bars.Low leaf water potentials were found to inhibit CO(2) fixation of intact leaf discs to almost the same degree as they did CO(2) fixation by chloroplasts isolated from those leaves. CO(2) fixation by intact leaves was decreased by 50 and 80% when water potentials were reduced from -7.1 to -9.6 and from -7.1 to -17.6 bars, respectively. Transpiration was decreased by only 40 and 60%, under the same conditions. However, correction for the increase in leaf temperature indicated transpiration decreases of 57 and 80%, similar to the relative decreases in CO(2) fixation.Despite the 4-fold increase in leaf resistance to CO(2) diffusion in the gas phase when the water potential of leaves was reduced from -6.5 to -14.0 bars, an additional increase of about 50% in mesophyll resistance was obtained. CO(2) concentration at compensation also increased when leaf water potential was reduced.

Carbon dioxide compensation points of leaves and stems and their relation to net photosynthesis.

The interactions between CO(2) and H(2)O vapor exchange of the leaf and respirant organs like stems were studied in tobacco plants. The results were analyzed according to a suggested model. Good agreement between open and closed system measurements supported the validity of the model.The measured over-all resistance to CO(2) of a leaf and a stem enclosed in a measuring cuvette was the same as the measured resistance of the leaf when measured alone provided the resistance of the stem to CO(2) is relatively high. The combined CO(2) compensation concentration of a leaf and stem having high resistance to CO(2) was higher than the CO(2) compensation point of the leaf alone, by the magnitude of rate of CO(2) evolution from the stem multiplied by the overall resistance of the leaf.CO(2) evolution into CO(2)-free air was found to be higher in light than in dark in leaves, while the reverse was true for stems. It was concluded that normally the CO(2) compensation point of a leaf is unaffected by stomata and boundary layer resistance while the combined CO(2) compensation point of a leaf and a stem differs in its nature since it represents a steady state of photosynthesis in which stem contribution, I(a), is equal to net photosynthesis, I(s). Interpretation of the experimental data shows tht respiration in the light is unaffected by external CO(2) concentration (at the range of 0-300 mul liter) and by intensity of photosynthesis.

Decrease in net photosynthesis caused by respiration.

CO(2) exchange between air and leaf is conceived as a current along a resistor with a respiratory current, unaffected by CO(2) concentration, entering the resistor. The conclusion follows that a plant placed in an atmosphere free of CO(2) will increase the CO(2) concentration to the compensation concentration along a curve determined by the resistor and the volume of the atmosphere. This was verified. Also a photosynthesis rate calculated from the parameters of the observed curve agreed with an independent observation of photosynthesis in CO(2)-free air. The decrease in net photosynthesis caused by respiration is, according to the model, the CO(2) compensation concentration divided by the concentration in the atmosphere.