Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase

Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure-hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27 [EC] , 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions.     

Accumulation of nitrite and sulfite in yeast cells and synergistic depletion of the intracellular ATP content

Summary When nitrite or sulfite are applied to yeast cells below pH 5.0, an enormous intracellular accumulation occurs. It is assumed that nitrite and sulfite penetrate the cell membrane in their undissociated forms as nitrous acid (pK = 3.3) or sulfurous acid (pK =1.8), respectively. Due to the neutral intracellular pH they are trapped inside the cell in their anionic forms, which are impermeable to the cell membrane. It has previously been shown that sulfite causes a rapid depletion of the ATP content of yeast cells [Schimz, K. L. and Holzer, H. (1979) resp. Hinze et al. as above]. Similarly, millimolar concentrations of nitrite decrease the ATP level to less than 10% of the initial value. Nitrite and sulfite in combination deplete the ATP content of yeast cells much stronger than expected for the sum of the separate effects of these compounds (synergistic effect).

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Akkumulation von Nitrit und Sulfit in Hefezellen und synergistischer Abfall des intrazellulären ATP-Gehalts

Zusammenfassung Wenn Hefezellen mit Nitrit oder Sulft bei pH-Werten unter 5,0 inkubiert werden, beobachtet man eine starke intracelluläre Akkumulation von Nitrit, bzw. Sulfit. Es ist anzunehmen, daß Nitrit und Sulfit in ihrer undissoziierten Form als salpetrige Säure (pK=3,3) bzw. schweflige Säure (pK=1,8) penetrieren und dann in den Zellen durch Neutralisation zu den anionischen Formen, die die Zellmembran nicht mehr permeieren können, abgefangen werden. Ähnlich dem früher beschriebenen raschen Abfall des ATP-Gehaltes nach Zusatz von Sulfit in Hefe [Schimz, KL und Holzer H (1979) Arch Microbiol 121:225–229] und in Bakterien [Hinze H, Maier K, Holzer H (1981) Z Lebensm Unters Forsch 172:389-392] verursacht auch Nitrit einen raschen Abfall des ATP-Gehaltes in Hefe auf weniger als 10 % des Anfangswertes. Werden Nitrit und Sulfit in Kombination verabreicht, so beobachtet man einen wesentlich stärkeren Abfall des ATP-Gehaltes als er aus der Summe der Einzeleffekte von Nitrit, bzw. Sulfit zu erwarten wäre (Synergistischer Effekt).

     

Control of yeast neutral trehalase by distinct polyphosphates and ribonucleic acid

Summary The activity of yeast trehalase when assayed at pH 7 in a crude extract was found to increase 2- to 3-fold upon incubation with 0.1 % (v/v) polyethyleneimine or other polycations such as polylysine (0.075-mMol) and calf thymus histories (0.08 mMol). Incubation with 3 mM-Mn²⁺ and 5 mM-Ca²⁺ also led to 3- and 1.6-fold increases in trehalase activity, respectively. The activities of 11 other enzymes assayed in the crude yeast extract did not increase after addition of polyethylene imine. At concentrations of polyethylenemine that maximally stimulated trehalase activity, 97% of the total RNA present in the crude extract, 40% of total protein, and 60% of the polyphosphate (assayed as inorganic phosphate liberated during 7 min incubation at 95 °C and pH O) were found to be precipitated. A similar finding was made with trehalase-stimulating concentrations of Mn²⁺. Activation of trehalase by polyethylene imine rendered this enzyme susceptible to inhibition by a preparation of total yeast RNA, inorganic polyphosphates, and related polyanions. We present further evidence that the removal of a distinct RNA and/or polyphosphate is the basic principle of polyetyleneimine-induced activation of trehalase.A more pronounced stimulation of trehalase activity (4-fold) could be obtained by enzymatic phosphorylation with ATP in the presence of cyclic AMP and Mg²⁺ as described by van Solingen and van der Plaat (1975) [9]. Thus, trehalase 2-fold activated by polyethylene imine was further activated by another 2-fold increase by enzymatic phosphorylation. Conversely, no additional stimulation by polyethylene imine was obtained for the maximally active, phosphorylated enzyme. We conclude that phosphorylation-mediated activation of trehalase is a two-step event, one being the removal of an inhibitor, which can be achieved by polyethylene imine or related cations independent on phosphorylation. The most likely candidate for the inhibitor appears to be a distinct RNA.

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Kontrolle der neutralen Hefen-Trehalase durch bestimmte Polyphosphate und RNA

Zusammenfassung Die in einem rohen Extrakt aus Hefe bei pH 7 gemessene Aktivität von Trehalase nimmt bei der Inkubation mit 0,1% Polyethylenimin oder mit anderen Polykationen, wie Polylysin (0,075 mmol) oder Histon aus Kalbsthymus (0,08 mmol) auf das zwei- bis dreifache zu. Auch die Inkubation mit 3 mmol Mn²⁺ oder 5 mmol Ca²⁺ führt zu einer 3-, bzw. 1,6fachen Zunahme der Trehalase-Aktivität. Die Aktivität von 11 anderen Enzymen, die im rohen Extrakt aus Hefe bestimmt wurden, nimmt nach Zusatz von Polyethylenimin nicht zu. Bei der Inkubation mit Polyethylenimin, das eine maximale Stimulierung der Trehalase-Aktivität bewirkt, werden 97% der gesamten Ribonucleinsäure, 40% des gesamten Proteins und 60% des Polyphosphats (bestimmt als anorganisches Phosphat, das in 7 min bei 95 °C und pH O freigesetzt wird) präcipitiert. Eine gleichartige Präcipitation wurde bei der Inkubation mit Trehalase-stimulierenden Konzentrationen von Mn²⁺ beobachtet. Mit Polyethylenimin aktivierte Trehalase wird durch Ribonucleinsäure aus Hefe, anorganisches Polyphosphat und verwandte Polyanionen gehemmt. Vermutlich führt die Entfernung einer bestimmten Ribonucleinsäure-Fraktion und/oder von Polyphosphat zu der Polyethylenimin-induzierten Aktivierung der Trehalase.Wie von van Solingen und van der Plaat 1975 beschrieben [9], wird eine 4fache Stimulierung von Trehalase durch enzymatische Phosphorylierung mit ATP in der Gegenwart von cyclischem AMP und Mg²⁺ erreicht. Das so durch Phosphorylierung maximal aktivierte Enzym kann durch Zusatz von Polyethylenimin nicht weiter aktiviert werden. Das mit Polyethylenimin 2fach aktivierte Enzym kann jedoch noch einmal 2fach weiter aktiviert werden durch enzymatische Phosphorylierung. Aus diesen Beobachtungen wird geschlossen, daß die durch enzymatische Phosphorylierung bewirkte Aktivierung der Trehalase ein zweistufiger Vorgang ist: Zuerst findet die Entfernung eines Inhibitors statt (dies kann auch mit Polyethylenimin oder mit verwandten Kationen unabhängig von der enzymatischen Phosphorylierung erreicht werden), hierauf folgt weitere Aktivierung durch Konformationsänderung des Enzyms. Wahrscheinlich handelt es sich bei dem durch Phosphorylierung bzw. Behandlung mit Polyethylenimin abgetrennten Inhibitor um eine gewisse Ribonucleinsäure-Fraktion.

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Abbreviation PEI polyethylene imine The followingenzymes are mentioned in the text: Alcohol dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (1.1.1.12), Malate dehydrogenase (1.1.1.37), Glucose-6-phosphate dehydrogenase (1.1.1.49), Glutamate dehydrogenase (NAD) (1.4.1.2), Glutamate dehydrogenase (NADP) (1.4.1.4), Aspartate -ketoglutarate aminotransferase (2.6.1.1), 6-Phosphofructokinase (2.7.1.11), Neutral trehalase (3.2.1.28), Pyruvate decarboxylase (4.1.1.1), Phosphoenolpyruvate carboxykinase (4.1.1.49).This paper is dedicated to Prof. Dr. Karl Decker on the occasion of his 60th birthday     

Crystallization of Fe-Si-B metallic glasses studied by X-ray synchrotron radiation

We have studied the crystallization kinetics of Fe_90-x Si _x B₁₀ amorphous alloys withx ranging from 7 to 21, by synchrotron X-ray radiation. Using energy- dispersive X-ray diffraction, the kinetics of the different crystalline phases evolving during isothermal annealing were followed. These crystalline phases were identified as precipitation of-Fe(Si) and/or Fe₃Si in the amorphous matrix. At a later time or at a higher temperature, Fe₂B starts to crystallize (x < 21 ). Only at low iron concentration (x = 21) was the second phase different, namely Fe₅SiB₂ The hypo- and hyper-eutectic Fe-Si-B glasses were found to crystallize differently. The crystallization processes are discussed in some detail.     

Mechanical aspects of the rare-earth effect

In order to gain a better understanding of the reactive-element effect (REE), the improvement of the oxidation behavior of chromia- or alumina-scale-forming alloys by the addition of small amounts of elements with higher affinity to oxygen than the scale-forming element, it is necessary to clearly distinguish between isothermal oxidation and the behavior of the metal/oxide composite system during cooling. An approach is presented based on fracture-mechanical considerations which correlates critical differential strain between scale and substrate, fracture toughness of the metal/scale interface, scale thickness, defect size and interfacial amplitude. This approach allows a quantitative assessment of the REE for scale adhesion, and although the necessary experimental data are yet lacking, it describes the reported REE in a qualitatively correct manner.     

Combined hydration of tricalcium silicate and β-dicalcium silicate

The mutual interaction of tricalcium silicate (C₃S) and β-dicalcium silicate (β-C₂S) in their combined hydration was studied. The rate of β-C₂S hydration was accelerated significantly in the presence of C₃S. The rate of C₃S hydration was retarded, but only in the presence of large amounts of β-C₂S. The stoichiometric composition and the pore structure of the hydrates formed was altered only unsignificantly when both compound hydrated simultaneously.     

Analytical studies on the temperature distribution in spiral plate heat exchangers: Straightforward design formulae for efficiency and mean temperature difference

The temperature distribution in spiral plate heat exchangers has been calculated numerically to obtain the efficiency and the logarithmic mean temperature difference (LMTD) correction factors F as a function of the number of transfer units N, the number of turns n, and the heat capacity rate ratio C. It has been found that the LMTD correction factors, when plotted against the number of transfer units per turn N/n, fall approximately on a single curve. That curve for balanced countercurrent operation (C = −1) can be very closely represented by the simple formula F = (n/N)tanh(N/n). From that simple analytic representation of our numerical results it was concluded that a simpler physical model might exist to represent the overall behaviour of a spiral plate heat exchanger equally well. In fact, a countercurrent cascade of n cocurrent heat exchangers does result exactly in the above-mentioned formula for the LMTD correction factor. From that model the F-factors for other heat capacity rate ratios C (−1 < C 0) can also be calculated and they are in sufficient agreement with the numerical results.     

The action of various lime soap dispersants

The course of the lime soap-dispersing action of various surfactants has been examined by use of a titrimetric method. Two types of dispersants can be distinguished. With the addition of a surfactant of the first type, the concentration of the dispersed fatty acid increases gradually. If a surfactant of the second type is used, a definite increase of the fatty acid concentration first occurs after the addition of considerable amounts of the dispersant. Ethylene oxide adducts and sodium triethylene glycol monolauryl ether sulfate behave like dispersants of the first type whereas sodium dodecylbenzenesulphonate and sodium lauryl sulfate belong to those of the second type. The dispersing action of both types of surfactants is discussed.     

An Asymptotic Preserving Method for Linear Systems of Balance Laws Based on Galerkin’s Method

We apply the concept of asymptotic preserving schemes (SIAM J Sci Comput 21:441–454, 1999) to the linearized \(p\) -system and discretize the resulting elliptic equation using standard continuous Finite Elements instead of Finite Differences. The fully discrete method is analyzed with respect to consistency, and we compare it numerically with more traditional methods such as Implicit Euler’s method.