Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (> 22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (< 8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the K-m was 39.9 mM, the V-max was 638 U mg(-1), and the K-cat/K-m was 1.79 x 104 M-1 s(-1), compared to a K-m of 76.0 mM, a V-max. of 423 U mg(-1), and a K-cat/K-m of 0.62 x 104 M-1 s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Site-directed mutagenesis and kinetic studies of the West Nile virus NS3 protease identify key enzyme-substrate interactions

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ae-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in VWNV NS3 protease at S1 (V115A/F, D129A/ E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe-K, Co-K and Zn-K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (alpha and P) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the beta-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron-zinc enzymes.

Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand

We examined the transmission efficiency of 2 strains of Wolbachia bacteria that cause cytoplasmic incompatibility in field populations of Aedes albopictus by polymerase chain reaction assay. We found mainland and island populations throughout Thailand to be superinfected with group A and B bacteria. Of 320 Wolbachia-positive adult mosquitoes, 97.5% were infected with both groups. Single infected individuals of each Wolbachia group were encountered in nearly equal numbers. We screened 550 offspring from 80 field-collected mothers and found the transmission efficiency of group A Wolbachia to be 96.7% and that of group B Wolbachia to be 99.6%. Mothers that did not transmit both Wolbachia infections to all of their offspring were significantly larger in size than those with perfect transmission fidelity. We discuss our findings in relation to the prospects of the use of Wolbachia as a gene-driving mechanism.

An introduction to efficiency and productivity analysis

The second edition of An Introduction to Efficiency and Productivity Analysis is designed to be a general introduction for those who wish to study efficiency and productivity analysis. The book provides an accessible, well-written introduction to the four principal methods involved: econometric estimation of average response models; index numbers, data envelopment analysis (DEA); and stochastic frontier analysis (SFA). For each method, a detailed introduction to the basic concepts is presented, numerical examples are provided, and some of the more important extensions to the basic methods are discussed. Of special interest is the systematic use of detailed empirical applications using real-world data throughout the book. In recent years, there have been a number of excellent advance-level books published on performance measurement. This book, however, is the first systematic survey of performance measurement with the express purpose of introducing the field to a wide audience of students, researchers, and practitioners. Indeed, the 2nd Edition maintains its uniqueness: (1) It is a well-written introduction to the field. (2) It outlines, discusses and compares the four principal methods for efficiency and productivity analysis in a well-motivated presentation. (3) It provides detailed advice on computer programs that can be used to implement these performance measurement methods. The book contains computer instructions and output listings for the SHAZAM, LIMDEP, TFPIP, DEAP and FRONTIER computer programs. More extensive listings of data and computer instruction files are available on the book's website: (www.uq.edu.au/economics/cepa/crob2005).

Eco-efficiency for the Dairy Processing Industry - Fact Sheet Sumary

This Toolkit was developed for the Australian dairy processing industry on behalf of Dairy Australia. At the conclusion of the project, industry participants gained exclusive access to a comprehensive Eco-Efficiency Manual, which outlined many of the opportunities available to the industry. Summary fact sheets were also prepared as publicly available resources and these are available for download below

Eco-efficiency for the Dairy Processing Industry

This manual has been developed to help the Australian dairy processing industry increase its competitiveness through increased awareness and uptake of eco-efficiency. The manual seeks to consolidate and build on existing knowledge, accumulated through projects and initiatives that the industry has previously undertaken to improve its use of raw materials and resources and reduce the generation of wastes. Where there is an existing comprehensive report or publication, the manual refers to this for further information. Eco-efficiency is about improving environmental performance to become more efficient and profitable. It is about producing more with less. It involves applying strategies that will not only ensure efficient use of resources and reduction in waste, but will also reduce costs. This chapter outlines the environmental challenges faced by Australian dairy processors. The manual explores opportunities for reducing environmental impacts in relation to water, energy, product yield, solid and liquid waste reduction and chemical use.

Catalytic activities and coking characteristics of oxides-supported Ni catalysts for CH4 reforming with carbon dioxide

Catalytic activities and deactivation characteristics of oxides-supported nickel catalysts for the reaction of methane reforming with carbon dioxide were investigated. The dynamic carbon deposition on various nickel catalysts was also studied by a thermogravimetric method. Among the catalysts prepared, Ni/La2O3, Ni/alpha-Al2O3, Ni/SiO2, and Ni/CeO2 showed very high CH4 and CO2 conversions and moderate deactivation whereas Ni/MgO and Ni/TiO2 had lower conversions when the Ni reduction was conducted at 500 degrees C. When Ni/MgO catalyst was reduced at 800 degrees C, it exhibited not only comparable conversions of CH4 and CO2 with other active catalysts but also much longer period of stability without deactivation. The amount of carbon deposited in Ni-based catalysts varied depending on the nature of support and followed the order of Ni/La2O3 > Ni/alpha-Al2O3 > Ni/SiO2 > Ni/MgO > Ni/CeO2 at 700 degrees C. The carbons formed on the catalyst surface showed different structural and chemical properties, and these in turn affected the catalytic activity of the catalysts.

Correlation between carbon isotope discrimination and transpiration efficiency in lines of the C-4 species Sorghum bicolor in the glasshouse and the field

Transpiration efficiency, W, the ratio of plant carbon produced to water transpired and carbon isotope discrimination of leaf dry matter, Delta(d)' were measured together on 30 lines of the C-4 species, Sorghum bicolor in the glasshouse and on eight lines grown in the field. In the glasshouse, the mean W observed was 4.9 mmol C mol(-1) H2O and the range was 0.8 mmol C mol(-1) H2O The mean Delta(d) was 3.0 parts per thousand and the observed range was 0.4 parts per thousand. In the field, the mean W was lower at 2.8 mmol C mol H2O and the mean Delta(d) was 4.6 parts per thousand. Significant positive correlations between W and Delta(d) were observed for plants grown in the glasshouse and in the field. The observed correlations were consistent with theory, opposite to those for C-4 species, and showed that variation in Delta(d) was an integrated measure of long-term variation in the ratio of intercellular to ambient CO2 partial pressure, p(i)/p(a). Detailed gas exchange measurements of carbon isotope discrimination during CO2 uptake, Delta(A) and p(i)/p(a) were made on leaves of eight S. bicolor lines. The observed relationship between Delta(A) and p(i)/p(a) was linear with a negative slope of 3.7 parts per thousand in Delta(A) for a unit change in p(i)/p(a). The slope of this linear relationship between Delta(A) and p(i)/p(a) in C-4 species is dependent on the leakiness of the CO2 concentrating mechanism of the C pathway, We estimated the leakiness (defined as the fraction of CO2 released in the bundle sheath by C-4 acid decarboxylations, which is lost by leakage) to be 0.2. We conclude that, although variation in Delta(d) observed in the 30 lines of S. bicolor is smaller than that commonly observed in C-4 species, it also reflects variation in transpiration efficiency, W. Among the eight lines examined in detail and in the environments used, there was considerable genotype x environment interaction.

Radiation use efficiency increases when the diffuse component of incident radiation is enhanced under shade

Theoretical analyses have shown the radiation use efficiency of maize, soybean, and peanut to increase with a decrease in the level of incident radiation and an increase in the proportion of diffuse radiation. This study compared the growth and radiation use efficiency of Panicum maximum cv. Petrie (green panic) and Bothriochloa insculpta cv. Bisset (creeping bluegrass) beneath shading treatments (birdguard and solarweave shadecloths) with that in full sunlight. A level of incident radiation reduced by 25% under birdguard shadecloth decreased final yield and final leaf area index, but increased canopy leaf nitrogen concentration and radiation use efficiency (19-14%) (compared with the full sun treatment). A similar level of reduced incident radiation under solarweave shadecloth (which provided an increased proportion of diffuse radiation), increased final yield and radiation use efficiency (46-50%). An understanding of the effects of composition of incident radiation on radiation use efficiency of tropical grasses enables more accurate estimation of potential pasture growth in shaded environments. It also has impact upon crop production in glasshouses and greenhouses.