Hydrogen oxidation by membranes from autotrophically grown Alcaligenes eutrophus H16: role of the cyanide-resistant pathway in energy transduction

Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, aK _m for acetate of 160 mM, and ak _cat of 16,800 min^-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.Abbreviations Acetyl-P Acetyl-phosphate - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

Trehalose–Water–Salt Interactions Related to the Stability of β-Galactosidase in Supercooled Media

The conservation of desirable properties in foods and ingredients is often based on the maintenance of the amorphous metastable properties of the systems. Enzymes may be stabilized by drying in saccharide matrices, but a second excipient is generally required to improve sugar protective effects. The effect of electrolytes on the thermophysical properties of sugar systems is of special interest because of their major influence on water structure and their possible interactions with biomolecules. Salts affect the kinetics of very important changes in sugar systems such as crystallization. The purpose of the present work was to analyze fungal β-galactosidase stability in supercooled systems of trehalose-containing electrolytes (water soluble acetates, citrates, and chlorides of magnesium and potassium). The degree of sugar crystallization was also related to enzyme stability. Potassium citrate and acetate improved enzyme stability during freeze-drying and thermal treatment of samples at water activity (a _w) of 0.22. However, trehalose crystallization inhibition at a _w = 0.43 (which was about 50–60%, related to the system without salt) impaired enzyme protection. Certain salts may act retarding sugar crystallization, but in the presence of salts, trehalose crystallization is even more critical because the enzyme is confined in a highly salt-concentrated region. Thus, crystallization inhibition by sugar–salt combinations should be carefully conducted. Santagapita, Research Fellow, CONICET, Argentina. Buera, Member of CONICET, Argentina. An erratum to this article can be found at     

Assignment of the histidine 12-threonine 45 hydrogen-bonded proton in the NMR spectrum of ribonuclease A in H2O.

Described herein are proton nmr experiments on chemically modified derivatives of ribonuclease A designed to elucidate the origin of an exchangeable resonance, assigned previously to a histidine ring N proton that titrates between 11 to 13 ppm with a pK_a of 6.1 in H₂O solution. Histidines 48 and 105, which are distant from the active site, are eliminated as candidates for this resonance from inhibitor binding studies on the enzyme in acetate–water solutions. This exchangeable resonance titrates with modified pK_a's and constant area over the above pH range in His-119-N₁-carboxymethylated-RNase A and des-(121–124)-RNase A, thus eliminating the imidazole N₃ proton in the His 119-Asp 121 hydrogen bond. In His-12-N₁-carboxymethylated-RNase A, this resonance is also observable, but broadens on raising the pH above 7 and at elevated temperatures above neutrality. It exhibits a pH-independent chemical shift characteristic of the protonated state of histidine. On the basis of these findings, this exchangeable resonance, designated a, is assigned to the imidazole N₁ proton of His 12, which is hydrogen-bonded to the carbonyl oxygen of Thr 45 in the crystal.     

Two enzymatic reaction pathways in the formation of pyropheophorbide a

The demethoxycarbonyl reaction of pheophorbide a in plants and algae was investigated. Two types of enzyme that catalyze alternative reactions in the formation of pyropheophorbide a were found. One enzyme, designated `pheophorbidase (Phedase)', was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus). This enzyme catalyzes the conversion of pheophorbide a to a precursor of pyropheophorbide a, C-13<sup>2</sup>-carboxylpyropheophorbide a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield pyropheophorbide a. The activity of Phedase was inhibited by the reaction product, methanol. The other enzyme, termed `pheophorbide demethoxycarbonylase (PDC)', was highly purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. This enzyme had produced no intermediate as shown in the Phedase reaction, indicating that it converts pheophorbide a directly into pyropheophorbide a, probably by nucleophilic reaction. Phedase and PDC consisted of both senescence-induced and constitutive enzymes. The molecular weight of both Phedases was 113 000 and of senescence-induced PDC was 170 000. The K _m values against pheophorbide a for both Phedases were 14–15 μM and 283 μM for senescence-induced PDC. The activity of both Phedases was inhibited by the reaction product, methanol, whereas methanol had no specific effect on senescence-induced PDC. Phenylmethylsulfonic fluoride and N-ethylmaleimide inhibited the senescence-induced Phedase and PDC, respectively. Among the 23 species from 15 different families tested, Phedase activity was found in 10 species from three families. PDC activity was not detected in plants lacking Phedase activity, except for Chlamydomonas. Based on these findings, a likely conclusion is that at least two alternative pathways that are catalyzed by two different enzymes, Phedase and PDC, exist for the formation of pyropheophorbide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relation between electrophoretic esterase patterns and organophosphate resistance in Musca domestica

An Italian OP-resistant strain C turned out to be heterogeneous for a gene on the 5th chromosome causing a difference in the mobility of an esterase in electrophoresis. Individuals containing only band 1 or 2 are homozygous for either allele, individuals containing esterase 1 and 2 are heterozygous. Two substrains were derived, E₁ and E₂, homozygous for the allele for 1 and 2 respectively. These strains were found to be remarkably different in OP-resistance. E₁ is approximately equally resistant as strain C, and contains the breakdown enzyme degrading paraoxon and diazoxon. E₂ has a lower resistance and does not contain the breakdown enzyme. The presence of the breakdown enzyme and the 1 esterase are controlled by the same gene allele, but they are certainly not identical. For instance the 1 band is quite stable, and is in solution in normal homogenates, whereas the breakdown enzyme is very labile and particulate. Therefore one allele, a _C1, controls both band 1 and the breakdown enzyme, the other, a _C2, seems to control only band 2. A similar situation was found in a susceptible strain, bwb; ocra, which had previously been shown to be heterogeneous for the ali-esterase a. Two alleles of the a gene are present here: the a ⁺allele controls the presence of band 1 and ali-esterase a; the a ₂allele seems to control band 2 only. The electrophoretic speed of the breakdown enzyme was found to be approximately equal to that of esterase 2.

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Zusammenfassung Ein italienischer OP-resistenter Musca domestica-Stamm erwies sich als heterogen für ein Gen im 5. Chromosom, das einen Unterschied in der Beweglichkeit einer Esterase bei der Elektrophorese bedingt. Individuen, die nur die Bande 1 oder 2 enthalten, sind homozygot für jeweils das eine der beiden Allele. Individuen, welche die Esterase 1 und 2 enthalten, sind heterozygot. Es wurden zwei Unterstämme E₁ und E₂ abgezweigt, die jeweils für das Allel 1 und 2 homozygot sind. Diese Stämme erwiesen sich als bemerkenswert verschieden hinsichtlich der OP-Resistenz. E₁ ist annähernd so resistent wie Stamm C und enthält das Abbau-Enzym, welches Paraoxon und Diazoxon zerstört. E₂ hat eine geringere Resistenz und enthält kein Abbau-Enzym. Die Gegenwart des Abbau-Enzyms und der 1-Esterase werden von dem gleichen Genallel kontrolliert, sind aber sicher nicht identisch. Z.B. ist Bande 1 ganz stabil und in normalen Homogenisaten in Lösung, während das Abbau-Enzym sehr labil und ungelöst ist. Deshalb kontrolliert das eine Allel, a _C1, sowohl Bande 1 und das Abbau-Enzym, das andere, a _C2, anscheinend nur Bande 2. Eine ähnliche Situation wurde in dem anfälligen Stamm, bwb; ocra, gefunden, der sich bereits früher als heterozygot für die Ali-Esterase a erwiesen hatte. Dabei sind 2 Allele des a-Gens vorhanden: das a ⁺-Allel kontrolliet die Anwesenheit von Bande 1 und Ali-Esterase a; das a ₂-Allel scheint nur Bande 2 zu kontrollieren. Die Elektrophorese-Geschwindigkeit des Abbau-Enzyms erwies sich als annähernd so groß wie die der Esterase 2.

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When the first author died in October 1964, he left the material for this publication. It is a tribute to his accuracy that it was possible for the last author to prepare this paper from his notes.     

DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

Changes in protein secretion of Aspergillus niger caused by the reduction of the water activity by potassium chloride

The effect of the rapid reduction of the water activity (a_w) on the extracellular protein and amylolytic activity of Aspergillus niger was studied. An a_w value gradient from 0.90 to 0.99 in KCl solutions was applied for the mycelium treatment. It was found that the a_w reduction considerably influenced the protein secretion. This phenomenon was dependent on the age of the treated mycelium and the range of the a_w gradient. The highest protein and enzyme secretion yields were obtained at a_w = 0.98 using a 72-h old mycelium. In comparison with the non-treated mycelium, the increase in the secretion amounted to about 60% for the amylolytic activity and 37% for the soluble protein, respectively. It was shown that the mycelium incubated in KCl solutions of an a_w value from 0.90 to 0.99 had the ability for regeneration in fresh CZAPEK-DOX medium. The effect of the osmotic shock on the protein secretion was limited only for the treated cell population and declined in the mycelium which was regenerated after the transfer into the culture medium.     

Purification and Characterization of an Esterase Involved in Poly(vinyl alcohol) Degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45°C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K _m and V _max of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 μM, and 6.52 and 12.6 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

Influence of cytochrome composition on the energy conservation of acinetobacter calcoaceticus 69-V

Acinetobacter calcoaceticus 69-V contained cytochromes b, o, d and traces of cytochrome a₁ after growth on ethanol. Cytochromes o, d, and a₁ are known to be potentially capable of functioning as terminal oxidases. After growth on acetate only smaller amounts of cytochromes b and o were detectable. Cytochrome spectra of acetate plus glucose-grown cells resembled more those of ethanolgrown cells. Cytochrome spectra of acetate plus ethanol-grown cells were more similar to those of acetate-grown cells but already showed small amounts of cytochrome d. Ethanol-grown cells were energized only by the oxidation of glucose. But the velocity of ATP synthesis was so low that the P/O quotient amounted to practically zero. The stoichiometries of ATP synthesis of the otherwise-grown cells were generally higher for the oxidation of glucose than for the oxidations of succinate and D-lactate. Similar high P/O quotients were measured for the oxidations of glucose and ethanol in acetate plus ethanol-grown cells. No significant energization could be observed during the oxidations of gluconate and acetate. The findings indicate that electrons delivered by the PQQ-dependent glucose as well as by NAD⁺- dependent ethanol oxidations have probably a common place of entry into the respiratory chain. Glucose and NADH appeared to be energetically equivalent although at least two energetically different pathways might be involved in substrate oxidation.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.     

A density functional study towards substituent effects on anion sensing with urea receptors

Effects of substituents on anion binding in different urea based receptors have been examined using density functional (B3LYP/6-311+G**) level of theory. The complexes formed by a variety of substituted urea with a halide anion (fluoride) and an oxy-anion (acetate) have been calculated. The stronger complexes were predicted for receptors with fluoride ion than that of acetate ion, however, in water the preference was found to be reversed. The pK _a calculations showed the preferred sites of deprotonation for positional isomers, while interacting with anions. The position of the substituent in the receptor, however, could change the preferred sites of deprotonation compared to the site predicted with pK _a values.      

Chemical Modification Causes Similar Change in Dependence on Water Activity of Chymotrypsin Hydration and Catalysis in Hexane

Amino groups in alpha-chymotrypsin were reacted with pyromellitic anhydride, introducing 17 to 32 additional carboxyl groups. This modification causes a major change in the water adsorption isotherm of the lyophilized protein powder. Little water is bound by the modified enzyme at water activity (a_w) below 0.35, but it shows increased water binding at a_w over 0.5. This correlates with a similar change in the a_w dependence of the catalytic activity of the enzyme powder suspended in hexane, with a much steeper increase in activity of the modified chymotrypsin.     

Cold-adapted Features of Arginine Kinase from the Deep-sea Clam <Emphasis Type="Italic">Calyptogena kaikoi</Emphasis>

The heterodont clam Calyptogena kaikoi, which inhabits depths exceeding 3,500 m where low ambient temperatures prevail, has an unusual two-domain arginine kinase (AK) with molecular mass of 80 kDa, twice that of typical AKs. The purpose of this work is to investigate the nature of the adaptations of this AK for functioning at low temperatures. Recombinant C. kaikoi AK constructs were expressed, and their two-substrate kinetic constants (k _cat, K _a, and K _ia) were determined at 10°C and 25°C, respectively. When measured at 25°C, the K _ia values were tenfold larger than those for corresponding K _a values, while at 10°C, the K _ia values decreased remarkably, but the K _a values were almost unchanged. The Calyptogena two-domain enzyme has threefold higher catalytic efficiency, calculated by k _cat/(K _a^ARG·K _ia^ATP), at 10°C, than that at 25°C, reflecting adaptation for function at reduced ambient temperatures. The activation energy (E _a) and thermodynamic parameters were determined for Calyptogena two-domain enzyme and compared with those of two-domain enzymes from mesophilic Corbicula and Anthopleura. The value for E _a of Calyptogena enzyme were about half of those for mesophilic enzymes, and a larger decrease in entropy was observed in Calyptogena AK reaction. Although large decrease in entropy increases the ΔG ^o‡ value and consequently lowers the k _cat value, this is compensated with its lower E _a value thereby minimizing the reduction in its k _cat value. These thermodynamic properties, together with the kinetic ones, are also present in the separated domain 2 of the Calyptogena two-domain enzyme.     

Two additives to improve stability of immobilized lipase

Abstract

The presence of two different additives during non-covalent immobilization of lipase was studied. Lipase was immobilized via hydrophobic interactions on an amorphous silica with large pore size bearing octyl groups on the surface. Polyethyleneglycol (PEG) with different molecular weights (MW: 1500, 3000 and 10,000) were added to the suspension during enzyme immobilization, in an enzyme to PEG molar ratio of 1:10, and also 1:20 in the case of PEG1500. The activity after 15 d increased from 10% (absence of PEG) to values close to 40% in samples with PEG except the catalyst immobilized in the presence of 1:10 PEG1500, which kept fully active after 15 d incubation in toluene at 70?°C. The presence of water during storage of immobilized enzymes leads to significant activity loss. Saturated solutions of salts controlling the water activity of the systems were used to reduce in a controlled fashion the moisture of the systems: CaCl₂ (a_w=?0.037), MgCl₂ (a_w=?0.328), Mg(NO₃)₂ (a_w=?0.529), Na₂PO₄.12H₂O (a_w=?0.74) and KCl (a_w=?0.84). The immobilized lipase was suspended in saturated solutions of these salts, and then filtered and incubated in desiccators in the presence of the corresponding saturated salt solutions. Catalysts suspended and incubated in KCl or only suspended in phosphate kept some 20% activity after 33 d incubation whereas the maximal stability was achieved when the catalyst was suspended in phosphate and kept in a desiccator without salt solution. This catalyst kept around 50% activity after 33 d incubation. An inversely proportional relationship can be established between the stability achieved by the enzyme and the water content of the system.     

Intracytoplasmic membrane production inEscherichia coli O111a1: Nutritional parameters

Escherichia coli O111a₁ ceased growth prematurely and accumulated intracytoplasmic membrane at 42°C in an amino acids-mineral salts medium. The amount of membrane formed appeared to be proportional to the concentration of amino acids in the medium—the greater the concentration of amino acids in the medium, the greater the membrane production.E. coli O111a₁, did not grow at 42°C in glucose-, glycerol- or acetate-mineral salts medium, but mesosome-like structures were produced in glucose-grown cells and some intracytoplasmic membrane in cells grown on glycerol and acetate. Supplementation of the glucose medium with pantothenate and/or thiamine permitted normal growth. The vitamins did not restore growth of the mutant in glycerol or acetate, but intracytoplasmic membrane production was increased, especially in glycerol. Amino acids plus glucose supported normal growth with no membrane production. Glycerol and acetate had no effect on the growth in the amino acids medium, but stimulated the accumulation of membrane.     

Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

Summary An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm² in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, β-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlied-atmosphere chamber which was gassed with 95% O₂ and 5% CO₂. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30°C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, aflatoxin B₁, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.     

Twin-core packed-bed reactors for organic-phase enzymatic esterification with water activity control

A method for the removal of water and the control of water activity, a _w, during enzymatic esterification is the use of salt hydrate pairs. When this technique is used on a laboratory scale, the recovery and reuse of the salt are not critical. Potential problems, such as the reactivity of some salts, can also be overcome simply by substituting another salt. However, if this technique is to be used on a larger scale, economic constraints would require salt recovery and restric the range of salts that could be used. In this article a twin-core packed-bed reactor — used for the esterification of an equimolar mixture of decanoic acid and dodecanol catalysed by lipase from Candida rugosa — which facilitates salt recovery and permits a _w control without direct contact between immobilized enzyme and salt, has been described. a _w control was maintained by using suitable salt hydrate mixtures in the inner core of the reactor. The substrate mixture was esterified by pumping it through the outer core of the reactor, which contained enzyme immobilized on a macroporous polypropylene support. Complete conversion, albeit at different rates, was obtained with a _w buffering at 0.48 and 0.8 by using hydrates of Na₄P₂O₇ and Na₂HPO₄.