Expression, purification and characterization of recombinant phosphomannomutase and GDP-alpha-D-mannose pyrophosphorylase from Salmonella enterica, group B, for the synthesis of GDP-alpha-D-mannose from D-mannose.

The genes rfbK and rfbM from the rfb cluster (O-antigen biosynthesis) of Salmonella enterica, group B, encoding for the enzymes phosphomannomutase (EC 5.4.2.8) and GDP-alpha-D-mannose pyrophosphorylase (EC 2.7.7.13) were overexpressed in E.coli BL21 (DE3) with specific activities of 0.1 U/mg and 0.3-0.6 U/mg, respectively. Both enzymes were partially purified to give specific activities of 0.26 U/mg and 2.75 U/mg, respectively. Kinetic characterization of the homodimeric (108 kDa) GDP-alpha-D-mannose pyrophosphorylase revealed a K(m) for GTP and mannose-1-P of 0.2 mM and 0.01 mM with substrate surplus inhibition constants (Kis) of 10.9 mM and 0.7 mM, respectively. The product GDP-alpha-D-mannose gave a competitive inhibition with respect to GTP (Ki 14.7 microM) and an uncompetitive inhibition with respect to mannose-1-P (Ki 115 microM). Both recombinant enzymes were used for repetitive batch synthesis of GDP-alpha-D-mannose staring from D-mannose and GTP. In three subsequent batches 581 mg (960 mumol) GDP-alpha-D-mannose was synthesized with 80% average yield. The overall yield after product isolation was 22.9% (329 mumol, 199 mg).

A continuous microtiter plate assay for screening nucleotide sugar-synthesizing nucleotidyltransferases.

A continuous microtiter plate nucleotidyltransferase substrate screening assay (NUSSA) is described which allows the identification of nucleotide sugar-synthesizing enzyme activities. The assay is accomplished by the determination of the common product of these enzymes PPi with a PPi-dependent phosphofructokinase. A subsequent enzyme reaction cascade leads to the production of 2 mol NAD per mol PPi. PPi-dependent phosphofructokinase was purified from potato with respect to contaminating enzyme activities which would disturb NUSSA performance. NUSSA allows the quick, simultaneous, and comprehensive check of different sugar 1-phosphate and nucleoside triphosphate substrates using purified pyrophosphorylases or crude extracts of plants, microorganisms, and mammalian tissues. Moreover, NUSSA will assist to evaluate these enzymes for the synthesis of important nucleotide sugars which serve as substrates of glycosyltransferases in carbohydrate syntheses.

Predicting lifetimes of materials and material structures.

The mechanical strength of brittle materials under stress is of prime importance in applications where allowable design stress, lifetime, and reliability are critical issues. A proper analysis should enable an engineer to select an allowable design stress that will permit a brittle component to function for the expected lifetime with an acceptable low probability of failure. It is the purpose of this paper to provide the background for assessing the mechanical reliability of brittle materials under tensile strength.

Critique of test methods for lifetime predictions.

Failure predictions for ceramics depend on the experimental parameters that measure the strength distribution (m and sigma 0) and the time-dependency of strength (n). These parameters can be determined by measuring strength as a function of stressing rate in a test environment that simulates the service environment. To minimize the uncertainty in these experimental parameters, at least 30 samples per stressing rate should be tested over a stressing rate range of at least 3 orders of magnitude. The uncertainty in these experimental parameters can be taken into account in design calculations by the use of appropriate safety factors. Thus, through well designed experiments coupled with a reliability analysis, rational design decisions can be made that ensure the successful use of ceramics in demanding structural applications.

Use of fracture mechanics theory in lifetime predictions for alumina and bioglass-coated alumina.

The fatigue behavior of alumina and bioglass-coated alumina was determined in air and biological test environments by the dynamic fatigue test technique in which strength is measured as a function of stressing rate. The good correlation found between the test data and fracture mechanics theory indicates that fatigue failure is controlled by the slow crack growth of preexisting flaws and that fracture mechanics theory can be used in making failure predictions for alumina and bioglass-coated alumina in biological environments. Thus, it is believed that lifetime predictions can be made for ceramic implants on the basis of short-term test data utilizing fracture mechanics principles.