Laser-Based Measurement of Refractive Index Changes: Kinetics of 2,3-Epoxy-1-propanol Hydrolysis.

ERIC Educational Resources Information Center

Spencer, Bert; Zare, Richard N.

1988-01-01

Describes an experiment in which a simple laser-based apparatus is used for measuring the change in refractive index during the acid-catalyzed hydrolysis of glycidol into glycerine. Gives a schematic of the experimental setup and discusses the kinetic analysis. (MVL)

[3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

NASA Technical Reports Server (NTRS)

Hall, P. J.; Bandurski, R. S.

1986-01-01

[3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

DFT STUDY OF THE HYDROLYSIS OF SOME S-TRIAZINES

EPA Science Inventory

The acid-catalyzed hydrolysis of atrazine and related 2-chloro-s-triazines to the corresponding 2-hydroxy-s-triazines was investigated using the B3LYP hybrid density functional theory method. Gas-phase calculations were performed at the B3LYP/6-311++G(d,p)//B3LYP/6-31G* level of ...

Acid-catalyzed autohydrolysis of wheat straw to improve sugar recovery.

PubMed

Ertas, Murat; Han, Qiang; Jameel, Hasan

2014-10-01

A comparison study of autohydrolysis and acid-catalyzed autohydrolysis of wheat straw was performed to understand the impact of acid addition on overall sugar recovery. Autohydrolysis combined with refining is capable of achieving sugar recoveries in the mid 70s. If the addition of a small amount of acid is capable of increasing the sugar recovery even higher it may be economically attractive. Acetic, sulfuric, hydrochloric and sulfurous acids were selected for acid-catalyzed autohydrolysis pretreatments. Autohydrolysis with no acid at 190 °C showed the highest total sugar in the prehydrolyzate. Enzymatic hydrolysis was performed for all the post-treated solids with and without refining at enzyme loadings of 4 and 10 FPU/g for 96 h. Acid-catalyzed autohydrolysis at 190 °C with sulfurous acid showed the highest total sugar recovery of 81.2% at 4 FPU/g enzyme charge compared with 64.3% at 190 °C autohydrolysis without acid. Copyright © 2014 Elsevier Ltd. All rights reserved.

The computational analysis and modelling of substitution effects on hydrolysis of formanilides in acidic aqueous solutions

NASA Astrophysics Data System (ADS)

Lukeš, Vladimír; Škorňa, Peter; Michalík, Martin; Klein, Erik

2017-11-01

Various para, meta and ortho substituted formanilides have been theoretically studied. For trans and cis-isomers of non-substituted formanilide, the calculated B3LYP vibration normal modes were analyzed. Substituent effect on the selected normal modes was described and the comparison with the available experimental data is presented. The calculated B3LYP proton affinities were correlated with Hammett constants, Fujita-Nishioka equation and the rate constants of the hydrolysis in 1 M HCl. Found linear dependences allow predictions of dissociation constants (pKBH+) and hydrolysis rate constants. Obtained results indicate that protonation of amide group may represent the rate determining step of acid catalyzed hydrolysis.

Phospholipids and products of their hydrolysis as dietary preventive factors for civilization diseases.

PubMed

Parchem, Karol; Bartoszek, Agnieszka

2016-12-31

The results of numerous epidemiological studies indicate that phospholipids play an important role in the prevention of chronic diseases faced by contemporary society. Firstly, these compounds are responsible for the proper functioning of cell membranes, by ensuring liquidity and permeability, which is pivotal for normal activity of membrane proteins, including receptors. These mechanisms are at the core of prevention of cancer, autoimmune or neurological disorders. Secondly, structure and properties of phospholipids cause that they are highly available source of biologically active fatty acids. Thirdly, also products of endogenous hydrolysis of phospholipids exhibit biological activity. These include lysophospholipids formed as a result of disconnecting free fatty acid from glycerophospholipids in the reaction catalyzed by phospholipase A, phosphatidic acid and hydrophilic subunits released by the activity of phospholipase D. The bioactive products of hydrolysis also include ceramides liberated from phosphosphingolipids after removal of a hydrophilic unit catalyzed by sphingomyelinase. Phospholipids are supplied to the human body with food. A high content of phospholipids is characteristic for egg yolk, liver, pork and poultry, as well as some soy products. Particularly beneficial are phospholipids derived from seafood because they are a rich source of essential fatty acids of the n-3 family.

Switching catalysis from hydrolysis to perhydrolysis in P. fluorescens esterase

PubMed Central

Yin, De Lu (Tyler); Bernhardt, Peter; Morley, Krista L.; Jiang, Yun; Cheeseman, Jeremy D.; Purpero, Vincent; Schrag, Joseph D.; Kazlauskas, Romas J.

2010-01-01

Many serine hydrolases catalyze perhydrolysis – the reversible formation of per-acids from carboxylic acids and hydrogen peroxide. Recently we showed that a single amino acid substitution in the alcohol binding pocket - L29P - in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. Angew. Chem. Intl. Ed. 2005, 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two x-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active-site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of ε-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction – hydrolysis of peracetic acid to acetic acid and hydrogen peroxide – occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed two fold higher kcat, but Km also increased so the specificity constant, kcat/Km, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate), but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of ε-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties, but binds ε-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones. PMID:20112920

Synthesis of the Fatty Esters of Solketal and Glycerol-Formal: Biobased Specialty Chemicals.

PubMed

Perosa, Alvise; Moraschini, Andrea; Selva, Maurizio; Noè, Marco

2016-01-30

The caprylic, lauric, palmitic and stearic esters of solketal and glycerol formal were synthesized with high selectivity and in good yields by a solvent-free acid catalyzed procedure. No acetal hydrolysis was observed, notwithstanding the acidic reaction conditions.

The Tautomeric Half-reaction of BphD, a C-C Bond Hydrolase Kinetic and Structural Evidence Supporting a Key Role for Histidine 265 of the Catalytic triad

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horsman, Geoff P.; Bhowmik, Shiva; Seah, Stephen Y.K.

2010-01-07

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA ({lambda}{sub max} is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum ({lambda}{sub max} is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T.,more » and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate ({lambda}{sub max} is 506 nm) with a similar rate, 1/{tau} {approx} 500 s{sup -1}. The crystal structure of the S112A:HOPDA complex at 1.8-{angstrom} resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7{angstrom} away.« less

Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features.

PubMed

Liljeblad, Arto; Kallio, Pauli; Vainio, Marita; Niemi, Jarmo; Kanerva, Liisa T

2010-02-21

Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper.

Nerve Agent Sensing Biopolymer Wipe

DTIC Science & Technology

2003-04-01

3. Urease and BChE (at two concentrations) activity as function of pH. ..... 10 Figure 4. Reaction scheme Agentase nerve agent sensor...11 Figure 5. Signal development in Agentase’s Traffic Light Sensor Construct.......... 11 Figure 6. Effect of BChE/ urease ...between two competing enzyme reactions. BChE catalyzed butyrylcholine hydrolysis results in the production of acid (decreasing pH) while urease - catalyzed

Mechanism of 1,4,5,8-naphthalene tetracarboxylic acid dianhydride hydrolysis and formation in aqueous solution.

PubMed

Barros, T C; Cuccovia, I M; Farah, J P S; Masini, J C; Chaimovich, H; Politi, M J

2006-01-07

The study of highly conjugated, carbonyl-containing molecules such as 1,4,5,8-naphthalene tetracarboxylic dianhydride, III, is of interest since reactivity differences and transmission of electronic effects through the conjugated framework can be evidenced. The kinetics of hydrolysis of III in aqueous solution were determined from 5 M acid to pH 10. In basic solution hydrolysis of III yields, sequentially, 1,4,5,8-naphthalene diacid monoanhydride, II, and 1,4,5,8-naphthalene tetracarboxylic acid, I. The second order rate constant for alkaline hydrolysis is 200 fold higher for the first ring opening. The water-catalyzed hydrolysis of III yields a pH-dependent mixture of ionic forms of I and II. The rate constant for water-catalyzed hydrolysis of III is 25 fold higher than that for II. In concentrated acid the rates for reaching equilibrium (I, II and III) increase and III is the major product. The pK(a)s of I (3.24, 5.13 and 6.25) and II (3.05, 5.90) were determined by potentiometric, fluorescence and UV spectroscopy titrations and by quantitative fit of the kinetic and equilibrium data. The apparent, pH-dependent, equilibrium constants, K(EqII), for anhydride formation between I and II were obtained from the UV spectra. The quantitative fit of kinetic and equilibrium data are consistent with the assumption that anhydride formation only proceeds with the fully protonated species for both I and II and permitted the estimation of the equilibrium constants for anhydride formation, K(EqII). The value of K(EqII) (I <==> II) between pH 1 and 6 was ca. 5. Geometry optimization calculations in the gas phase of the reactions of III in alkaline, neutral and acid conditions, at the DFT level of theory, gave electronic distributions that were qualitatively consistent with the experimental results.

Enzymatic saccharification and lactic acid production from banana pseudo-stem through optimized pretreatment at lowest catalyst concentration

PubMed Central

Idrees, Muhammad; Adnan, Ahmad; Malik, Farnaz; Qureshi, Fahim Ashraf

2013-01-01

This work estimates the potential of banana pseudo-stem with high cellulosic content 42.2-63 %, for the production of fermentable sugars for lactic acid production through statistically optimized pretreatment method. To evaluate the catalyzed pretreatment efficiency of banana pseudo stem based on the enzymatic digestibility, Response Surface Methodology (RSM) was employed for the optimization of pretreatment temperature and time using lowest concentrations of H2SO4, NaOH, NaOH catalyzed Na2S and Na2SO3 that seemed to be significant variables with P<0.05. High F and R2 values and low p-value for hydrolysis yield indicated the model predictability. The optimized condition for NaOH was determined to be conc. 1 %, temperature 130 oC for 2.6 hr; Na2S; conc. 1 %, temperature 130 oC for 2.29 hr; Na2SO3; conc. 1 %, temperature 130 oC for 2.41 hr and H2SO4; conc. 1 %, temperature 129.45 oC for 2.18 hr, produced 84.91 %, 85.23 %, 81.2 % and 76.02 % hydrolysis yield, respectively. Sulphuric acid provided 33+1 gL-1 reducing sugars in pretreatment step along with 38+0.5 gL-1 during enzymatic hydrolysis. Separate hydrolysis and fermentation of resulting sugars showed that the conversion of glucans into lactic acid reached 92 % of the theoretical yield of glucose. PMID:26966423

Synthesis, properties, and application in peptide chemistry of a magnetically separable and reusable biocatalyst

NASA Astrophysics Data System (ADS)

Liria, Cleber W.; Ungaro, Vitor A.; Fernandes, Raphaella M.; Costa, Natália J. S.; Marana, Sandro R.; Rossi, Liane M.; Machini, M. Teresa

2014-11-01

Enzyme-catalyzed chemical processes are selective, very productive, and generate little waste. Nevertheless, they may be optimized using enzymes bound to solid supports, which are particularly important for protease-mediated reactions since proteases undergo fast autolysis in solution. Magnetic nanoparticles are suitable supports for this purpose owing to their high specific surface area and to be easily separated from reaction media. Here we describe the immobilization of bovine α-chymotrypsin (αCT) on silica-coated superparamagnetic nanoparticles (Fe3O4@silica) and the characterization of the enzyme-nanoparticle hybrid (Fe3O4@silica-αCT) in terms of protein content, properties, recovery from reaction media, application, and reuse in enzyme-catalyzed peptide synthesis. The results revealed that (i) full acid hydrolysis of the immobilized protease followed by amino acid analysis of the hydrolyzate is a reliable method to determine immobilization yield; (ii) despite showing lower amidase activity and a lower K cat/ K m value for a specific substrate than free αCT, the immobilized enzyme is chemically and thermally more stable, magnetically recoverable from reaction media, and can be consecutively reused for ten cycles to catalyze the amide bond hydrolysis and ester hydrolysis of the protected dipeptide Z-Ala-Phe-OMe. Altogether, these properties indicate the potential of Fe3O4@silica-αCT to act as an efficient, suitably stable, and reusable catalyst in amino acid, peptide, and protein chemistry as well as in proteomic studies.

Regulatory link between steryl ester formation and hydrolysis in the yeast Saccharomyces cerevisiae.

PubMed

Ploier, Birgit; Korber, Martina; Schmidt, Claudia; Koch, Barbara; Leitner, Erich; Daum, Günther

2015-07-01

Steryl esters and triacylglycerols are the major storage lipids of the yeast Saccharomyces cerevisiae. Steryl esters are formed in the endoplasmic reticulum by the two acyl-CoA:sterol acyltransferases Are1p and Are2p, whereas steryl ester hydrolysis is catalyzed by the three steryl ester hydrolases Yeh1p, Yeh2p and Tgl1p. To shed light on the regulatory link between steryl ester formation and hydrolysis in the maintenance of cellular sterol and free fatty acid levels we employed yeast mutants which lacked the enzymes catalyzing the degradation of steryl esters. These studies revealed feedback regulation of steryl ester formation by steryl ester hydrolysis although in a Δtgl1Δyeh1Δyeh2 triple mutant the gene expression levels of ARE1 and ARE2 as well as protein levels and stability of Are1p and Are2p were not altered. Nevertheless, the capacity of the triple mutant to synthesize steryl esters was significantly reduced as shown by in vitro and in vivo labeling of lipids with [(14)C]oleic acid and [(14)C]acetate. Enzymatic analysis revealed that inhibition of steryl ester formation occurred at the enzyme level. As the amounts and the formation of sterols and fatty acids were also decreased in the triple mutant we concluded that defects in steryl ester hydrolysis also caused feedback inhibition on the formation of sterols and fatty acids which serve as precursors for steryl ester formation. In summary, this study demonstrates a regulatory link within the steryl ester metabolic network which contributes to non-polar lipid homeostasis in yeast cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Ruthenium hydroxide supported on magnetic nanoparticles: a benign aqueous protocol for hydration of nitriles

EPA Science Inventory

Amides are an important class of compounds in the chemical and pharmaceutical industry1,2. Conventionally, amides have been synthesized by the hydration of nitriles, catalyzed by strong acids3 and bases4. Many by-products such as carboxylic acids are produced due to hydrolysis of...

A: The Progression of a Catalytic Immune Response. B: Molecular Recognition of Anions by Silica Bound Sapphyrin

DTIC Science & Technology

1994-08-01

Diels - Alder reactions (58-60), Claisen rearrangements (43-45), olefin isomerization (73), a O-elimination (74), an asymmetric ketone reduction (54...phosphorothioate hapten3 ........ 19 Figure 5. Carboxylic acid hydrolysis .................... 21 Figure 6. Reaction coordinates for antibody catalyzed ...and catalyze the reaction. Thus, it is important to design transition analogs that closely mimic the transition state in every possible chemical

VvMJE1 of the grapevine (Vitis vinifera) VvMES methylesterase family encodes for methyl jasmonate esterase and has a role in stress response

USDA-ARS?s Scientific Manuscript database

The known members of the plant methyl esterase (MES) family catalyze hydrolysis of a C-O ester linkage of methyl esters of several phytohormones including indole-3-acetic acid, salicylic acid, and jasmonic acid. The genome of grapevine (Vitis vinifera) was found to contain 15 MES genes, designated V...

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations.

PubMed

Patelski, J; Pioruńska-Stolzmann, M

1985-01-01

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.

Efficient production of fermentable sugars from oil palm empty fruit bunch by combined use of acid and whole cell culture-catalyzed hydrolyses.

PubMed

Li, Qingxin; Ng, Wei Ting; Puah, Sze Min; Bhaskar, Ravindran Vijay; Soh, Loon Siong; MacBeath, Calum; Parakattil, Pius; Green, Phil; Wu, Jin Chuan

2014-01-01

Empty fruit bunch (EFB) of oil palm trees was converted to fermentable sugars by the combined use of dilute acids and whole fungal cell culture-catalyzed hydrolyses. EFB (5%, w/v) was hydrolyzed in the presence of 0.5% H2 SO4 and 0.2% H3 PO4 at 160 °C for 10 Min. The solid fraction was separated from the acid hydrolysate by filtration and subjected to enzymatic hydrolysis at 50 °C using the whole cell culture of Trichoderma reesei RUT-C30 (2%, w/v), which was prepared by cultivation at 30 °C for 7 days to reach its maximal cellulase activity. The combined hydrolyses of EFB gave a total sugar yield of 82.0%. When used as carbon sources for cultivating Escherichia coli in M9 medium at 37 °C, the combined EFB hydrolysates were shown to be more favorable or at least as good as pure glucose for cell growth in terms of the higher (1.1 times) optical density of E. coli cells. The by-products generated during the acid-catalyzed hydrolysis did not seem to obviously affect cell growth. The combined use of acid and whole cell culture hydrolyses might be a commercially promising method for pretreatment of lignocellulose to get fermentable sugars. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Interchromosomal Associations that Alter Nf1 Gene Expression can Modify Clinical Manifestations of Neurofibromatosis 1

DTIC Science & Technology

2008-09-01

intracellular portion of the EGFR and stimulates PLD2 activity. PLD2 catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and...ARF4 can bind with EGFR and activate PLD2. The phosphatidic acid (PA) produced by PLD2 can recruit Sos, which can then colocalize and activate

Comparing the performance of Miscanthus x giganteus and wheat straw biomass in sulfuric acid based pretreatment.

PubMed

Kärcher, M A; Iqbal, Y; Lewandowski, I; Senn, T

2015-03-01

The objective of this study was to assess and compare the suitability of Miscanthus x giganteus and wheat straw biomass in dilute acid catalyzed pretreatment. Miscanthus and wheat straw were treated in a dilute sulfuric acid/steam explosion pretreatment. As a result of combining dilute sulfuric acid- and steam explosion pretreatment the hemicellulose hydrolysis yields (96% in wheat straw and 90% in miscanthus) in both substrates were higher than reported in literature. The combined severity factor (=CSF) for optimal hemicellulose hydrolysis was 1.9 and 1.5 in for miscanthus and wheat straw respectively. Because of the higher CSF value more furfural, furfuryl alcohol, 5-hydroxymethylfurfural and acetic acid was formed in miscanthus than in wheat straw pretreatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis.

PubMed

Song, Xiangfei; Zhang, Shujun; Wang, Yefei; Li, Jingwen; He, Chunyan; Yao, Lishan

2016-06-01

One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15-35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme's capability in hydrolyzing the soluble substrate. Copyright © 2016 Elsevier Inc. All rights reserved.

Pilot-scale study on the acid-catalyzed steam explosion of rice straw using a continuous pretreatment system.

PubMed

Chen, Wen-Hua; Tsai, Chia-Chin; Lin, Chih-Feng; Tsai, Pei-Yuan; Hwang, Wen-Song

2013-01-01

A continuous acid-catalyzed steam explosion pretreatment process and system to produce cellulosic ethanol was developed at the pilot-scale. The effects of the following parameters on the pretreatment efficiency of rice straw feedstocks were investigated: the acid concentration, the reaction temperature, the residence time, the feedstock size, the explosion pressure and the screw speed. The optimal presteaming horizontal reactor conditions for the pretreatment process are as follows: 1.7 rpm and 100-110 °C with an acid concentration of 1.3% (w/w). An acid-catalyzed steam explosion is then performed in the vertical reactor at 185 °C for 2 min. Approximately 73% of the total saccharification yield was obtained after the rice straw was pretreated under optimal conditions and subsequent enzymatic hydrolysis at a combined severity factor of 0.4-0.7. Moreover, good long-term stability and durability of the pretreatment system under continuous operation was observed. Copyright © 2012 Elsevier Ltd. All rights reserved.

DNA-Catalyzed Amide Hydrolysis.

PubMed

Zhou, Cong; Avins, Joshua L; Klauser, Paul C; Brandsen, Benjamin M; Lee, Yujeong; Silverman, Scott K

2016-02-24

DNA catalysts (deoxyribozymes) for a variety of reactions have been identified by in vitro selection. However, for certain reactions this identification has not been achieved. One important example is DNA-catalyzed amide hydrolysis, for which a previous selection experiment instead led to DNA-catalyzed DNA phosphodiester hydrolysis. Subsequent efforts in which the selection strategy deliberately avoided phosphodiester hydrolysis led to DNA-catalyzed ester and aromatic amide hydrolysis, but aliphatic amide hydrolysis has been elusive. In the present study, we show that including modified nucleotides that bear protein-like functional groups (any one of primary amino, carboxyl, or primary hydroxyl) enables identification of amide-hydrolyzing deoxyribozymes. In one case, the same deoxyribozyme sequence without the modifications still retains substantial catalytic activity. Overall, these findings establish the utility of introducing protein-like functional groups into deoxyribozymes for identifying new catalytic function. The results also suggest the longer-term feasibility of deoxyribozymes as artificial proteases.

Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.

PubMed

Park, Kyung-Eui; Kim, Jun-Dal; Nagashima, Yusuke; Kako, Koichiro; Daitoku, Hiroaki; Matsui, Motoki; Park, Gwi Gun; Fukamizu, Akiyoshi

2014-01-01

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.

Pyrimidine Nucleosides with a Reactive (β-Chlorovinyl)sulfone or (β-Keto)sulfone Group at the C5 Position, Their Reactions with Nucleophiles and Electrophiles, and Their Polymerase-Catalyzed Incorporation into DNA

PubMed Central

2018-01-01

Transition-metal-catalyzed chlorosulfonylation of 5-ethynylpyrimidine nucleosides provided (E)-5-(β-chlorovinyl)sulfones A, which undergo nucleophilic substitution with amines or thiols affording B. The treatment of vinyl sulfones A with ammonia followed by acid-catalyzed hydrolysis of the intermediary β-sulfonylvinylamines gave 5-(β-keto)sulfones C. The latter reacts with electrophiles, yielding α-carbon-alkylated or -sulfanylated analogues D. The 5′-triphosphates of A and C were incorporated into double-stranded DNA, using open and one-nucleotide gap substrates, by human or Escherichia coli DNA-polymerase-catalyzed reactions. PMID:29732453

A Catalytic Mechanism for Cysteine N-Terminal Nucleophile Hydrolases, as Revealed by Free Energy Simulations

PubMed Central

Lodola, Alessio; Branduardi, Davide; De Vivo, Marco; Capoferri, Luigi; Mor, Marco; Piomelli, Daniele; Cavalli, Andrea

2012-01-01

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors. PMID:22389698

Synthesis of 5,5-Diphenyl-4-penten-2-One: A Variation on a Classic Organic Synthesis Laboratory

ERIC Educational Resources Information Center

Alber, Joshua P.; DeGrand, Michael J.; Cermak, Diana M.

2011-01-01

The Grignard reaction and the addition of protecting groups are standard reactions in an organic chemistry course. Organic students learn about the "quench" step of the Grignard reaction using acid and water and the acid-catalyzed hydrolysis to remove the protecting group, yet in the lecture students find these two reactions to be confusing in…

Hydrolase BioH knockout in E. coli enables efficient fatty acid methyl ester bioprocessing.

PubMed

Kadisch, Marvin; Schmid, Andreas; Bühler, Bruno

2017-03-01

Fatty acid methyl esters (FAMEs) originating from plant oils are most interesting renewable feedstocks for biofuels and bio-based materials. FAMEs can also be produced and/or functionalized by engineered microbes to give access to, e.g., polymer building blocks. Yet, they are often subject to hydrolysis yielding free fatty acids, which typically are degraded by microbes. We identified BioH as the key enzyme responsible for the hydrolysis of medium-chain length FAME derivatives in different E. coli K-12 strains. E. coli ΔbioH strains showed up to 22-fold reduced FAME hydrolysis rates in comparison with respective wild-type strains. Knockout strains showed, beside the expected biotin auxotrophy, unchanged growth behavior and biocatalytic activity. Thus, high specific rates (~80 U g CDW -1 ) for terminal FAME oxyfunctionalization catalyzed by a recombinant alkane monooxygenase could be combined with reduced hydrolysis. Biotransformations in process-relevant two-liquid phase systems profited from reduced fatty acid accumulation and/or reduced substrate loss via free fatty acid metabolization. The BioH knockout strategy was beneficial in all tested strains, although its effect was found to differ according to specific strain properties, such as FAME hydrolysis and FFA degradation activities. BioH or functional analogs can be found in virtually all microorganisms, making bioH deletion a broadly applicable strategy for efficient microbial bioprocessing involving FAMEs.

Formic acid catalyzed hydrolysis of SO3 in the gas phase: a barrierless mechanism for sulfuric acid production of potential atmospheric importance.

PubMed

Hazra, Montu K; Sinha, Amitabha

2011-11-02

Computational studies at the B3LYP/6-311++G(3df,3pd) and MP2/6-311++G(3df,3pd) levels are performed to explore the changes in reaction barrier height for the gas phase hydrolysis of SO(3) to form H(2)SO(4) in the presence of a single formic acid (FA) molecule. For comparison, we have also performed calculations for the reference reaction involving water assisted hydrolysis of SO(3) at the same level. Our results show that the FA assisted hydrolysis of SO(3) to form H(2)SO(4) is effectively a barrierless process. The barrier heights for the isomerization of the SO(3)···H(2)O···FA prereactive collision complex, which is the rate limiting step in the FA assisted hydrolysis, are found to be respectively 0.59 and 0.08 kcal/mol at the B3LYP/6-311++G(3df,3pd) and MP2/6-311++G(3df,3pd) levels. This is substantially lower than the ~7 kcal/mol barrier for the corresponding step in the hydrolysis of SO(3) by two water molecules--which is currently the accepted mechanism for atmospheric sulfuric acid production. Simple kinetic analysis of the relative rates suggests that the reduction in barrier height facilitated by FA, combined with the greater stability of the prereactive SO(3)···H(2)O···FA collision complex compared to SO(3)···H(2)O···H(2)O and the rather plentiful atmospheric abundance of FA, makes the formic acid mediated hydrolysis reaction a potentially important pathway for atmospheric sulfuric acid production.

Triazine-Substituted and Acyl Hydrazones: Experiment and Computation Reveal a Stability Inversion at Low pH.

PubMed

Ji, Kun; Lee, Changsuk; Janesko, Benjamin G; Simanek, Eric E

2015-08-03

Condensation of a hydrazine-substituted s-triazine with an aldehyde or ketone yields an equivalent to the widely used, acid-labile acyl hydrazone. Hydrolysis of these hydrazones using a formaldehyde trap as monitored using HPLC reveals that triazine-substituted hydrazones are more labile than acetyl hydrazones at pH>5. The reactivity trends mirror that of the corresponding acetyl hydrazones, with hydrolysis rates increasing along the series (aromatic aldehyde

Modeling and optimization of phospholipase A₁-catalyzed hydrolysis of phosphatidylcholine using response surface methodology for lysophosphatidylcholine production.

PubMed

Lim, Chang Wan; Kim, Byung Hee; Kim, In-Hwan; Lee, Moon-Won

2015-01-01

Modeling the phospholipase A1 (PLA1 )-catalyzed partial hydrolysis of soy phosphatidylcholine (PC) in hexane for the production of lysophosphatidylcholine (LPC) and optimizing the reaction conditions using response surface methodology were described. The reaction was performed with 4 g of PC in a stirred batch reactor using a commercial PLA1 (Lecitase Ultra) as the biocatalyst. The effects of temperature, reaction time, water content, and enzyme loading on LPC and glycerylphosphorylcholine (GPC) content in the reaction products were elucidated using the models established. Optimal reaction conditions for maximizing the LPC content while suppressing acyl migration, which causes GPC formation, were as follows: temperature, 60°C; reaction time, 3 h; water content, 10% of PC; and enzyme loading, 1% of PC. When the reaction was conducted with 40 g of PC under these conditions, the reaction products contained 83.7 mol % LPC and were free of GPC. LPC had a higher total unsaturated fatty acid content than original PC had and was mainly composed of linoleic acid (78.0 mol % of the total fatty acids). © 2014 American Institute of Chemical Engineers.

Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling*

PubMed Central

Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

2015-01-01

The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the “lipolysome.” Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

Kinetics of Maleic Acid and Aluminum Chloride Catalyzed Dehydration and Degradation of Glucose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Ximing; Hewetson, Barron B.; Mosier, Nathan S.

We report the positive effect of maleic acid, a dicarboxylic acid, on the selectivity of hexose dehydration to 5-hydroxymethyfurfural (HMF) and subsequent hydrolysis to levulinic and formic acids. We also describe the kinetic analysis of a Lewis acid (AlCl3) alone and in combination with HCl or maleic acid to catalyze the isomerization of glucose to fructose, dehydration of fructose to HMF, hydration of HMF to levulinic and formic acids, and degradation of these compounds to humins. The results show that AlCl3 significantly enhances the rate of glucose conversion to HMF and levulinic acid in the presence of both maleic acidmore » and HCl. In addition, the degradation of HMF to humins, rather than levulinic and formic acids, is reduced by 50% in the presence of maleic acid and AlCl3 compared to HCl combined with AlCl3. The results suggest different reaction mechanisms for the dehydration of glucose and rehydration of HMF between maleic acid and HCl.« less

Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

NASA Technical Reports Server (NTRS)

Mullins, D. W., Jr.; Lacey, J. C., Jr.

1983-01-01

The kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine, from N-acetylglycyl adenylate anhydride and glycine in aqueous solution at 25 C, and at various PH's are reported. The reaction is of interest in that over a physiologically relevant pH range (6-8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrlysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

Reactivity of Dimeric Tetrazirconium(IV) Wells-Dawson Polyoxometalate toward Dipeptide Hydrolysis Studied by a Combined Experimental and Density Functional Theory Approach.

PubMed

Ly, Hong Giang T; Mihaylov, Tzvetan; Absillis, Gregory; Pierloot, Kristine; Parac-Vogt, Tatjana N

2015-12-07

Detailed kinetic studies on the hydrolysis of glycylglycine (Gly-Gly) in the presence of the dimeric tetrazirconium(IV)-substituted Wells-Dawson-type polyoxometalate Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4] · 57H2O (1) were performed by a combination of (1)H, (13)C, and (31)P NMR spectroscopies. The catalyst was shown to be stable under a broad range of reaction conditions. The effect of pD on the hydrolysis of Gly-Gly showed a bell-shaped profile with the fastest hydrolysis observed at pD 7.4. The observed rate constant for the hydrolysis of Gly-Gly at pD 7.4 and 60 °C was 4.67 × 10(-7) s(-1), representing a significant acceleration as compared to the uncatalyzed reaction. (13)C NMR data were indicative for coordination of Gly-Gly to 1 via its amide oxygen and amine nitrogen atoms, resulting in a hydrolytically active complex. Importantly, the effective hydrolysis of a series of Gly-X dipeptides with different X side chain amino acids in the presence of 1 was achieved, and the observed rate constant was shown to be dependent on the volume, chemical nature, and charge of the X amino acid side chain. To give a mechanistic explanation of the observed catalytic hydrolysis of Gly-Gly, a detailed quantum-chemical study was performed. The theoretical results confirmed the nature of the experimentally suggested binding mode in the hydrolytically active complex formed between Gly-Gly and 1. To elucidate the role of 1 in the hydrolytic process, both the uncatalyzed and the polyoxometalate-catalyzed reactions were examined. In the rate-determining step of the uncatalyzed Gly-Gly hydrolysis, a carboxylic oxygen atom abstracts a proton from a solvent water molecule and the nascent OH nucleophile attacks the peptide carbon atom. Analogous general-base activity of the free carboxylic group was found to take place also in the case of polyoxometalate-catalyzed hydrolysis as the main catalytic effect originates from the -C═O···Zr(IV) binding.

Hydrolysis optimization and characterization study of preparing fatty acids from Jatropha curcas seed oil.

PubMed

Salimon, Jumat; Abdullah, Bashar Mudhaffar; Salih, Nadia

2011-11-01

Fatty acids (FAs) are important as raw materials for the biotechnology industry. Existing methods of FAs production are based on chemical methods. In this study potassium hydroxide (KOH)-catalyzed reactions were utilized to hydrolysis Jatropha curcas seed oil. The parameters effect of ethanolic KOH concentration, reaction temperature, and reaction time to free fatty acid (FFA%) were investigated using D-Optimal Design. Characterization of the product has been studied using Fourier transforms infrared spectroscopy (FTIR), gas chromatography (GC) and high performance liquid chromatography (HPLC). The optimum conditions for maximum FFA% were achieved at 1.75M of ethanolic KOH concentration, 65°C of reaction temperature and 2.0 h of reaction time. This study showed that ethanolic KOH concentration was significant variable for J. curcas seed oil hydrolysis. In a 18-point experimental design, FFA% of hydrolyzed J. curcas seed oil can be raised from 1.89% to 102.2%, which proved by FTIR and HPLC.

Hydrolysis of substance P in the presence of the osteosarcoma cell line SaOS-2: release of free amino acids.

PubMed

Cavazza, Antonella; Marini, Mario; Roda, L Giorgio; Tarantino, Umberto; Valenti, Angela

2011-12-01

The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

Site- and species-specific hydrolysis rates of heroin.

PubMed

Szöcs, Levente; Orgován, Gábor; Tóth, Gergő; Kraszni, Márta; Gergó, Lajos; Hosztafi, Sándor; Noszál, Béla

2016-06-30

The hydroxide-catalyzed non-enzymatic, simultaneous and consecutive hydrolyses of diacetylmorphine (DAM, heroin) are quantified in terms of 10 site- and species-specific rate constants in connection with also 10 site- and species-specific acid-base equilibrium constants, comprising all the 12 coexisting species in solution. This characterization involves the major and minor decomposition pathways via 6-acetylmorphine and 3-acetylmorphine, respectively, and morphine, the final product. Hydrolysis has been found to be 18-120 times faster at site 3 than at site 6, depending on the status of the amino group and the rest of the molecule. Nitrogen protonation accelerates the hydrolysis 5-6 times at site 3 and slightly less at site 6. Hydrolysis rate constants are interpreted in terms of intramolecular inductive effects and the concomitant local electron densities. Hydrolysis fraction, a new physico-chemical parameter is introduced and determined to quantify the contribution of the individual microspecies to the overall hydrolysis. Hydrolysis fractions are depicted as a function of pH. Copyright © 2016 Elsevier B.V. All rights reserved.

The Chemistry of Paper Preservation: Part 5. Permanent Paper

ERIC Educational Resources Information Center

Carter, Henry A.

2007-01-01

The acid-catalyzed hydrolysis of cellulose accounts for the deterioration of paper in library books and other written records. To combat this threat to our written heritage, new permanent papers have been developed that are relatively chemically stable and undergo a very slow rate of deterioration. Many of these new papers are manufactured from…

A COMPUTATIONAL CHEMISTRY STUDY OF THE ENVIRONMENTALLY IMPORTANT ACID-CATALYZED HYDROLYSIS OF ATRAZINE AND RELATED 2-CHLORO-S-TRIAZINES

EPA Science Inventory

Many chlorine-containing pesticides, for example 2-chloro-s-triazines, are of great concern both environmentally and toxicologically. As a result, ascertaining or predicting the fate and transport of these compounds in soils and water is of current interest. Transformation pathwa...

Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of “hunger hormone” ghrelin

NASA Astrophysics Data System (ADS)

Yao, Jianzhuang; Yuan, Yaxia; Zheng, Fang; Zhan, Chang-Guo

2016-02-01

Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway.

Hydrolysis of VX and related compounds by organophosphorus hydrolase. Final report, Februray-December 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kolakowski, J.E.; DeFrank, J.J.; Lai, K.

1995-11-01

Organophosphorus Hydrolase (OPH) is a fully characterized and cloned enzyme, derived from Pseudomonas diminuta, consisting of 365 amino acids with a total molecular weight of 38,0(X). The enzyme has a leader sequence of 29 amino acids which has been removed in the construction used in this study. OPH was evaluated for its effectiveness in catalyzing the S-(2-diisopwpylaminoethyl) methylphosphonothioate (VX) and its analogs.

Modeling evolution of hydrogen bonding and stabilization of transition states in the process of cocaine hydrolysis catalyzed by human butyrylcholinesterase.

PubMed

Gao, Daquan; Zhan, Chang-Guo

2006-01-01

Molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the prereactive enzyme-substrate complex, transition states, intermediates, and product involved in the process of human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)-cocaine. The computational results consistently reveal a unique role of the oxyanion hole (consisting of G116, G117, and A199) in BChE-catalyzed hydrolysis of cocaine, compared to acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine. During BChE-catalyzed hydrolysis of cocaine, only G117 has a hydrogen bond with the carbonyl oxygen (O31) of the cocaine benzoyl ester in the prereactive BChE-cocaine complex, and the NH groups of G117 and A199 are hydrogen-bonded with O31 of cocaine in all of the transition states and intermediates. Surprisingly, the NH hydrogen of G116 forms an unexpected hydrogen bond with the carboxyl group of E197 side chain and, therefore, is not available to form a hydrogen bond with O31 of cocaine in the acylation. The NH hydrogen of G116 is only partially available to form a weak hydrogen bond with O31 of cocaine in some structures involved in the deacylation. The change of the estimated hydrogen-bonding energy between the oxyanion hole and O31 of cocaine during the reaction process demonstrates how the protein environment can affect the energy barrier for each step of the BChE-catalyzed hydrolysis of cocaine. These insights concerning the effects of the oxyanion hole on the energy barriers provide valuable clues on how to rationally design BChE mutants with a higher catalytic activity for the hydrolysis of (-)-cocaine. 2005 Wiley-Liss, Inc.

Modeling Evolution of Hydrogen Bonding and Stabilization of Transition States in the Process of Cocaine Hydrolysis Catalyzed by Human Butyrylcholinesterase

PubMed Central

Gao, Daquan; Zhan, Chang-Guo

2010-01-01

Molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the prereactive enzyme-substrate complex, transition states, intermediates, and product involved in the process of human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (−)-cocaine. The computational results consistently reveal a unique role of the oxyanion hole (consisting of G116, G117, and A199) in BChE-catalyzed hydrolysis of cocaine, as compared to acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine. During BChE-catalyzed hydrolysis of cocaine, only G117 has a hydrogen bond with the carbonyl oxygen (O31) of the cocaine benzoyl ester in the prereactive BChE-cocaine complex, and the NH groups of G117 and A199 are hydrogen-bonded with O31 of cocaine in all of the transition states and intermediates. Surprisingly, the NH hydrogen of G116 forms an unexpected hydrogen bond with the carboxyl group of E197 side chain and, therefore, is not available to form a hydrogen bond with O31 of cocaine in the acylation. The NH hydrogen of G116 is only partially available to form a weak hydrogen bond with O31 of cocaine in some structures involved in the deacylation. The change of the estimated hydrogen bonding energy between the oxyanion hole and O31 of cocaine during the reaction process demonstrates how the protein environment can affect the energy barrier for each step of the BChE-catalyzed hydrolysis of cocaine. These insights concerning the effects of the oxyanion hole on the energy barriers provide valuable clues on how to rationally design BChE mutants with a higher catalytic activity for the hydrolysis of (−)-cocaine. PMID:16288482

Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication

PubMed Central

Lapenta, Fabio; Montón Silva, Alejandro; Brandimarti, Renato; Lanzi, Massimiliano; Gratani, Fabio Lino; Vellosillo Gonzalez, Perceval; Perticarari, Sofia; Hochkoeppler, Alejandro

2016-01-01

DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics. PMID:27050298

The effect of high temperature sol-gel polymerization parameters on the microstructure and properties of hydrophobic phenol-formaldehyde/silica hybrid aerogels.

PubMed

Seraji, Mohamad Mehdi; Sameri, Ghasem; Davarpanah, Jamal; Bahramian, Ahmad Reza

2017-05-01

Phenol-formaldehyde/silica hybrid aerogels with different degree of hydrophobicity were successfully synthesized via high temperature sol-gel polymerization. Tetraethoxysilane (TEOS) and methyltriethoxysilane (MTES) were used as precursor and co-precursor of the hydrophobic silica-based phase, respectively. The hydrolysis step of silica based sols were conducted by acid catalyzed reactions and HCl was used as hydrolysis catalyst. The chemical structure of prepared hybrid aerogels was characterized by Fourier Transform Infrared spectroscopy (FT-IR). The effect of MTES/TEOS proportion and catalyst content on the morphology and microstructure of samples were investigated by FE-SEM and C, Si mapping analysis. The acid catalyzed hydrolysis of TEOS and MTES sols leads to formation of a sol with primarily silica particles in the organic-inorganic hybrid sol and varying colloid growth mechanisms were occurred with change in MTES and HCl molar ratio. With the increasing of MTES content, the microstructure of samples changed from uniform colloidal network, core-shell structure to polymeric structure with a huge phase separation. The increasing of HCl mole fraction leads to smaller particle size. Moreover, the shrinkage of samples was decreased and water contact angles of the resulted aerogels were increased from 40 to 156.8° with the increases of MTES content. Copyright © 2017 Elsevier Inc. All rights reserved.

Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides

PubMed Central

2015-01-01

Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R2CO, four bonds away from the site of hydrolysis. Electron-rich acyl R2 groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent’s Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis. PMID:24617596

Polymethylsilsesquioxanes through base-catalyzed redistribution of oligomethylhydridosiloxanes

DOE Office of Scientific and Technical Information (OSTI.GOV)

RAHIMIAN,KAMYAR; ASSINK,ROGER A.; LOY,DOUGLAS A.

2000-04-04

There has been an increasing amount of interest in silsesquioxanes and polysilsesquioxanes. They have been used as models for silica surfaces and have been shown to have great potential for several industrial applications. Typical synthesis of polysilsesquioxanes involves the hydrolysis of organotricholorosilanes and/or organotrialkoxysilanes in the presence of acid or base catalysts, usually in the presence of organic solvents.

Continuous Reductive Amination of Biomass-Derived Molecules over Carbonized Filter Paper-Supported FeNi Alloy.

PubMed

Chieffi, Gianpaolo; Braun, Max; Esposito, Davide

2015-11-01

This paper reports the continuous reductive amination of different molecules, including biomass-related compounds, over carbon-supported FeNi nanoparticles obtained on the basis of inexpensive and abundant metal precursors and cellulose. A biorefinery case study for the preparation of pyrrolidones via acid-catalyzed hydrolysis of glucose followed by reductive amination of the obtained levulinic acid is described. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acid-catalyzed conversion of mono- and poly-sugars into platform chemicals: effects of molecular structure of sugar substrate.

PubMed

Hu, Xun; Wu, Liping; Wang, Yi; Song, Yao; Mourant, Daniel; Gunawan, Richard; Gholizadeh, Mortaza; Li, Chun-Zhu

2013-04-01

Hydrolysis/pyrolysis of lignocellulosic biomass always produces a mixture of sugars with distinct structures as intermediates or products. This study tried to elucidate the effects of molecular structure of sugars on their acid-catalyzed conversions in ethanol/water. Location of carbonyl group in sugars (fructose versus glucose) and steric configuration of hydroxyl groups (glucose versus galactose) significantly affected yields of levulinic acid/ester (fructose>glucose>galactose). The dehydration of fructose to 5-(hydroxymethyl)furfural produces much less soluble polymer than that from glucose and galactose, which results in high yields of levulinic acid/ester from fructose. Anhydrate sugar such as levoglucosan tends to undergo the undesirable decomposition to form less levulinic acid/ester. Catalytic behaviors of the poly-sugars (sucrose, maltose, raffinose, β-cyclodextrins) were determined much by their basic units. However, their big molecular sizes create the steric hindrance that significantly affects their followed conversion over solid acid catalyst. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis and Biological Evaluation of Non-Hydrolizable 1,2,3-Triazole Linked Sialic Acid Derivatives as Neuraminidase Inhibitors

PubMed Central

Weïwer, Michel; Chen, Chi-Chang; Kemp, Melissa M.; Linhardt, Robert J.

2013-01-01

α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition (“click chemistry”). Our approach is to generate non-natural N-glycosides of sialic acid that are resistant to neuraminidase catalyzed hydrolysis as opposed to the natural O-glycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71-89% yield. A disaccharide mimic of sialic acid has also been prepared using the α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor in 68% yield. A model sialic acid coated dendrimer was also synthesized from a per-propargylated pentaerythritol acceptor. These novel sialic acid derivatives were then evaluated as potential neuraminidase inhibitors using a 96-well plate fluorescence assay; micromolar IC50 values were observed, comparable to the known sialidase inhibitor Neu5Ac2en. PMID:24223493

Structural Analogues of Selfotel.

PubMed

Dziuganowska, Zofia A; Ślepokura, Katarzyna; Volle, Jean-Noël; Virieux, David; Pirat, Jean-Luc; Kafarski, Paweł

2016-06-17

A small library of phosphonopiperidylcarboxylic acids, analogues of NMDA antagonist selfotel (CGS 19755), was synthesized. First, the series of aromatic esters was obtained via a palladium-catalyzed cross-coupling reaction (Hirao coupling) of dialkyl phosphites with bromopyridinecarboxylates, followed by their hydrolysis. Then, hydrogenation of the resulting phosphonopyridylcarboxylic acids over PtO2 yielded the desired phosphonopiperidylcarboxylic acids. NMR studies indicated that the hydrogenation reaction proceeds predominantly by cis addition. Several compounds were obtained as monocrystal structures. Preliminary biological studies performed on cultures of neurons suggest that the obtained compounds possess promising activity toward NMDA receptors.

Enhancing enzymatic hydrolysis of sugarcane bagasse by ferric chloride catalyzed organosolv pretreatment and Tween 80.

PubMed

Zhang, Hongdan; Fan, Meishan; Li, Xin; Zhang, Aiping; Xie, Jun

2018-06-01

In this work, a FeCl 3 -catalyzed organosolv pretreatment was employed at 160 °C to remove hemicellulose and lignin in sugarcane bagasse leaving the cellulose-enriched residue for enzymatic hydrolysis to sugars. The solubilized hemicellulose fractions consisted more monomer xylose than oligomer xylose. The FeCl 3 -catalyzed organosolv pretreatment significantly improved the enzymatic hydrolysis, nearly 100% of cellulose components were converted to glucose after pretreatment with 0.05 M FeCl 3 . Structural analysis was employed to reveal how pretreatment affected the enzymatic hydrolysis. With the addition of Tween 80, the same level of glucose was obtained with 50% reduction of enzyme dosage after 24 h. Furthermore, the influence of Tween 80 on different pretreatment systems was investigated, indicating that the improvement was increased as the lignin content increased, decreased with high enzyme loading and extending hydrolysis time. This work suggested that the addition of Tween 80 could improve the enzymatic hydrolysis, reduce the hydrolysis time and enzyme dosage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ferric Hydrogensulfate [Fe(HSO4)3] As a Reusable Heterogeneous Catalyst for the Synthesis of 5-Substituted-1H-Tetrazoles and Amides

PubMed Central

Eshghi, Hossein; Seyedi, Seyed Mohammad; Zarei, Elaheh Rahimi

2011-01-01

Ferric hydrogensulfate catalyzed the synthesis of 5-substituted 1H-tetrazoles via [2 + 3] cycloaddition of nitriles and sodium azide. This method has the advantages of high yields, simple methodology, and easy workup. The catalyst can be recovered by simple filtration and reused delivering good yields. Also, ferric hydrogensulfate catalyzed the hydrolysis of nitriles to primary amides under aqueous conditions. Various aliphatic and aromatic nitriles converted to the corresponding amides in good yields without any contamination with carboxylic acids. PMID:24052817

Design, synthesis and in vitro kinetic study of tranexamic acid prodrugs for the treatment of bleeding conditions

NASA Astrophysics Data System (ADS)

Karaman, Rafik; Ghareeb, Hiba; Dajani, Khuloud Kamal; Scrano, Laura; Hallak, Hussein; Abu-Lafi, Saleh; Mecca, Gennaro; Bufo, Sabino A.

2013-07-01

Based on density functional theory (DFT) calculations for the acid-catalyzed hydrolysis of several maleamic acid amide derivatives four tranexamic acid prodrugs were designed. The DFT results on the acid catalyzed hydrolysis revealed that the reaction rate-limiting step is determined on the nature of the amine leaving group. When the amine leaving group was a primary amine or tranexamic acid moiety, the tetrahedral intermediate collapse was the rate-limiting step, whereas in the cases by which the amine leaving group was aciclovir or cefuroxime the rate-limiting step was the tetrahedral intermediate formation. The linear correlation between the calculated DFT and experimental rates for N-methylmaleamic acids 1- 7 provided a credible basis for designing tranexamic acid prodrugs that have the potential to release the parent drug in a sustained release fashion. For example, based on the calculated B3LYP/6-31G(d,p) rates the predicted t1/2 (a time needed for 50 % of the prodrug to be converted into drug) values for tranexamic acid prodrugs ProD 1- ProD 4 at pH 2 were 556 h [50.5 h as calculated by B3LYP/311+G(d,p)] and 6.2 h as calculated by GGA: MPW1K), 253 h, 70 s and 1.7 h, respectively. Kinetic study on the interconversion of the newly synthesized tranexamic acid prodrug ProD 1 revealed that the t1/2 for its conversion to the parent drug was largely affected by the pH of the medium. The experimental t1/2 values in 1 N HCl, buffer pH 2 and buffer pH 5 were 54 min, 23.9 and 270 h, respectively.

Supramolecular Complexes Formed by the Self-assembly of Hydrophobic Bis(Zn(2+)-cyclen) Complexes, Copper, and Di- or Triimide Units for the Hydrolysis of Phosphate Mono- and Diesters in Two-Phase Solvent Systems (Cyclen=1,4,7,10-Tetraazacyclododecane).

PubMed

Hisamatsu, Yosuke; Miyazawa, Yuya; Yoneda, Kakeru; Miyauchi, Miki; Zulkefeli, Mohd; Aoki, Shin

2016-01-01

We previously reported on supramolecular complexes 4 and 5, formed by the 4 : 4 : 4 or 2 : 2 : 2 assembly of a dimeric zinc(II) complex (Zn2L(1)) having 2,2'-bipyridyl linker, dianion of cyanuric acid (CA) or 5,5-diethylbarbituric acid (Bar), and copper(II) ion (Cu(2+)) in an aqueous solution. The supermolecule 4 possesses Cu2(μ-OH)2 centers and catalyzes hydrolysis of phosphate monoester dianion, mono(4-nitrophenyl)phosphate (MNP), at neutral pH. In this manuscript, we report on design and synthesis of hydrophobic supermolecules 9 and 10 by 4 : 4 : 4 and 2 : 2 : 2 self-assembly of hydrophobic Zn2L(2) and Zn2L(3) containing long alkyl chains, CA or Bar, and Cu(2+) and their phosphatase activity for the hydrolysis of MNP and bis(4-nitrophenyl)phosphate (BNP) in two-phase solvent systems. We assumed that the Cu2(μ-OH)2 active sites of 9 and 10 would be more stable in organic solvent than in aqueous solution and that product inhibition of the supermolecules might be avoided by the release of HPO4(2-) into the aqueous layer. The findings indicate that 9 and 10 exhibit phosphatase activity in the two-phase solvent system, although catalytic turnover was not observed. Furthermore, the hydrolysis of BNP catalyzed by the hydrophobic 2 : 2 : 2 supermolecules in the two-phase solvent system is described.

Catalytic polymer-clay composite for enhanced removal and degradation of diazinon.

PubMed

Shabtai, Itamar A; Mishael, Yael G

2017-08-05

It is well established that organophosphate pesticides, such as diazinon, pose environmental and health risks. Diazinon is prone to rapid acidic hydrolysis, forming the less toxic compound 2-isopropyl-6-methyl-4-pyrimidinol (IMP). In this study, diazinon surface catalyzed hydrolysis was achieved by its adsorption to a composite, based on protonated poly (4-vinyl-pyridine-co-styrene) (HPVPcoS) and montmorillonite (MMT) clay. The adsorption affinity and kinetics of diazinon to HPVPcoS-MMT were significantly higher than those obtained to the deprotonated PVPcoS-MMT, emphasizing the importance of hydrogen bonding. Correspondingly, diazinon filtration by HPVPcoS-MMT columns was highly efficient (100% for 100 pore volumes), while filtration by columns of PVPcoS-MMT or granular activated carbon (GAC) reached only 55% and 85%, respectively. Regeneration of HPVPcoS-MMT by pH increase was demonstrated and sorbent reuse was successful, whereas regeneration and reuse of GAC and PVPcoS-MMT were inefficient. Proton transfer from HPVPcos-MMT to diazinon, investigated by FTIR analysis, supports the suggested mechanism of surface catalyzed hydrolysis. These findings demonstrate the applicability of such bi-functional sorbents, to adsorb and degrade pollutants, for efficient water treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Reaction pathways and free energy profiles for cholinesterase-catalyzed hydrolysis of 6-monoacetylmorphine

PubMed Central

Qiao, Yan; Han, Keli; Zhan, Chang-Guo

2014-01-01

As the most active metabolite of heroin, 6-monoacetylmorphine (6-MAM) can penetrate into the brain for the rapid onset of heroin effects. The primary enzymes responsible for the metabolism of 6-MAM to the less potent morphine in humans are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The detailed reaction pathways for AChE- and BChE-catalyzed hydrolysis of 6-MAM to morphine have been explored, for the first time, in the present study by performing first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the two enzymatic reaction processes follow the similar catalytic reaction mechanism, and the whole catalytic reaction pathway for each enzyme consists of four reaction steps. According to the calculated results, the second reaction step associated with the transition state TS2a/TS2b should be rate-determining for the AChE/BChE-catalyzed hydrolysis, and the free energy barrier calculated for the AChE-catalyzed hydrolysis (18.3 kcal/mol) is 2.5 kcal/mol lower than that for the BChE-catalyzed hydrolysis (20.8 kcal/mol). The free energy barriers calculated for the AChE- and BChE-catalyzed reactions are in good agreement with the experimentally derived activation free energies (17.5 and 20.7 kcal/mol for the AChE- and BChE-catalyzed reactions, respectively). Further structural analysis reveals that the aromatic residues Phe295 and Phe297 in the acyl pocket of AChE (corresponding to Leu286 and Val288 in BChE) contribute to the lower energy of TS2a relative to TS2b. The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse. PMID:24595354

Characterization of an anti-tuberculosis resin glycoside from the prairie medicinal plant Ipomoea leptophylla.

PubMed

Barnes, Curtis C; Smalley, Mary K; Manfredi, Kirk P; Kindscher, Kelly; Loring, Hillary; Sheeley, Douglas M

2003-11-01

The organic soluble extract from the leaves of the native North American prairie plant Ipomoea leptophylla (big root morning glory) showed in vitro activity against M. tuberculosis. Bioassay-guided fractionation of this extract resulted in the identification of two new resin glycosides (6, 7). Base-catalyzed hydrolysis of these glycosides gave operculinic acid (1) as the glycosidic acid component as well as trans-cinnamic acid, propanoic acid, and lauric acid. The complete structure elucidation was accomplished through derivatization, 1D and 2D NMR spectroscopy (TOCSY, ROESY, HSQC, HMBC), and MS/MS experiments on 6 and 7 as well as the permethylated derivative 8.

Bio-preparation of (R)-DMPM using whole cells of Pseudochrobactrum asaccharolyticum WZZ003 and its application on kilogram-scale synthesis of fungicide (R)-metalaxyl.

PubMed

Zhang, Yinjun; Fan, Yicheng; Zhang, Wei; Wu, Guanzhong; Wang, Jinghong; Cheng, Feng; Zheng, Jianyong; Wang, Zhao

2018-04-25

Methyl (R)-N-(2,6-dimethylphenyl)alaninate ((R)-DMPM) is a key chiral intermediate for the production of (R)-metalaxyl, which is one of the best-selling fungicides. A new strain, Pseudochrobactrum asaccharolyticum WZZ003, was identified as a biocatalyst for the enantioselective hydrolysis of (R,S)-DMPM. The key parameters including pH, temperature, rotation speed and substrate concentrations were optimized in the enantioselective hydrolysis of (R,S)-DMPM. After the 48 h hydrolysis of 256 mM (R,S)-DMPM under the optimized reaction conditions, the enantiomeric excess of product (e.e. p ) was up to 99% and the conversion was nearly 50%. Subsequently, the unhydrolyzed (S)-DMPM was converted to (R,S)-DMPM through the n-butanal-catalyzed racemization. Furthermore, stereoselective hydrolysis of (R,S)-DMPM catalyzed by whole cells of P. asaccharolyticum WZZ003 was scaled up to kilogram-scale, offering (R)-MAP-acid with 98.6% e.e. p and 48.0% yield. Moreover, (R)-metalaxyl was prepared at kilogram scale after subsequent esterification and coupling reactions. Therefore, a practical production process of (R)-DMPM and (R)-metalaxyl with the prospect of industrialization was developed in this study. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

USDA-ARS?s Scientific Manuscript database

Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabi...

Adsorption of acetanilide herbicides on soil and its components. II. Adsorption and catalytic hydrolysis of diethatyl-ethyl on saturated Na(+)-, K(+)-, Ca(2+)-, and Mg(2+)-montmorillonite.

PubMed

Liu, W P; Fang, Z; Liu, H J; Yang, W C

2001-04-01

Adsorption and catalytic hydrolysis of the herbicide diethatyl-ethyl [N-chloroacetyl-N-(2,6-diethylphenyl)glycine ethyl ester] on homoionic Na(+)-, K(+)-, Ca(2+)-, and Mg(2+)-montmorillonite clays were investigated in water solution. The Freundlich adsorption coefficient, Ki, got from isotherms on clay followed the order of Na+ approximately K+ > Mg2+ approximately Ca2+. Analysis of FT-IR spectra of diethatyl-ethyl adsorbed on clay suggests probable bonding at the carboxyl and amide carbonyl groups of the herbicide. The rate of herbicide hydrolysis in homoionic clay suspensions followed the same order as that for adsorption, indicating that adsorption may have preceded and thus caused hydrolysis. Preliminary product identification showed that hydrolysis occurred via nucleophilic substitution at the carboxyl carbon, causing the cleavage of the ester bond and formation of diethatyl and its dechlorinated derivative, and at the amide carbon, yielding an ethyl ester derivative and its acid. These pathways also suggest that hydrolysis of diethatyl-ethyl was catalyzed by adsorption on the clay surface.

THE CHEMISTRY OF TRIBUTYL PHOSPHATE: A REVIEW

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burger, L.L.

1955-10-27

The preparation, purification, and chemical properties of THP have been reviewed with emphasis on the hydrolytic reactions. TBP is chemically a very stable compound as evidenced by its thermal stability and resistance to oxidation. The most important reactions are hydrolytic which cleave the butyl or butoxy group and normally produce butyl alcohol together with dibutyl and monobutyl phosphate (DBP and MBP, respectively), and eventually H/sub 3/PO/sub 4/. Hydrolysis occurs in either the organic phase or the aqueous phase and is first order with respect to the ester. Although the rate in the aqueous phase is much faster than in themore » organic phase, the solubility is so low in aqueous solutions that the organic phase reactions become more important. Acid hydrolysis depends on both the nature of the acid and the concentration. The order with respect to acid concentration is close to one but often less than one. Hydrolysis is catalyzed by both acids and bases. In the latter case, the reaction occurs only in the aqueous phase and normally stops with the formation of dibutyl phosphate. The hydrolysis rate increases greatly as the temperature is raised and an activation energy of the order of 20 kcal is often found. The rates observed in the presence of 5 M acid at 60 and 70 deg C may be high enough to cause some concern in solvent extraction technology, since the product, dibutyl phosphate, has undesirable properties. Impurities produced during manufacture or by thermal degradation during purification such as the pyrophosphates, if present, would yield the same objectionable products as TBP hydrolysis, but at a faster rate. Included in the survey is a selected tabulation of physical properties of TBP. (auth)« less

Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

NASA Technical Reports Server (NTRS)

Degani, CH.; Kawatsuji, M.; Halmann, M.

1975-01-01

It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

Synthesis and acid catalysis of cellulose-derived carbon-based solid acid

NASA Astrophysics Data System (ADS)

Suganuma, Satoshi; Nakajima, Kiyotaka; Kitano, Masaaki; Yamaguchi, Daizo; Kato, Hideki; Hayashi, Shigenobu; Hara, Michikazu

2010-06-01

SO 3H-bearing amorphous carbon, prepared by partial carbonization of cellulose followed by sulfonation in fuming H 2SO 4, was applied as a solid catalyst for the acid-catalyzed hydrolysis of β-1,4 glucan, including cellobiose and crystalline cellulose. Structural analyses revealed that the resulting carbon material consists of graphene sheets with 1.5 mmol g -1 of SO 3H groups, 0.4 mmol g -1 of COOH, and 5.6 mmol g -1 of phenolic OH groups. The carbon catalyst showed high catalytic activity for the hydrolysis of β-1,4 glycosidic bonds in both cellobiose and crystalline cellulose. Pure crystalline cellulose was not hydrolyzed by conventional strong solid Brønsted acid catalysts such as niobic acid, Nafion ® NR-50, and Amberlyst-15, whereas the carbon catalyst efficiently hydrolyzes cellulose into water-soluble saccharides. The catalytic performance of the carbon catalyst is due to the large adsorption capacity for hydrophilic reactants and the adsorption ability of β-1,4 glucan, which is not adsorbed to other solid acids.

Hydrolysis of aspartic acid phosphoramidate nucleotides: a comparative quantum chemical study.

PubMed

Michielssens, Servaas; Tien Trung, Nguyen; Froeyen, Matheus; Herdewijn, Piet; Tho Nguyen, Minh; Ceulemans, Arnout

2009-09-07

L-Aspartic acid has recently been found to be a good leaving group during HIV reverse transcriptase catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. This showed that L-Asp is a good mimic for the pyrophosphate moiety of deoxyadenosine triphosphate. The present work explores the thermochemistry and mechanism for hydrolysis of several models for L-aspartic-dAMP using B3LYP/DGDZVP, MP2/6-311++G** and G3MP2 level of theory. The effect of the new compound is gradually investigated: starting from a simple methyl amine leaving group up to the aspartic acid leaving group. The enzymatic environment was mimicked by involving two Mg(2+) ions and some important active site residues in the reaction. All reactions are compared to the corresponding O-coupled leaving group, which is methanol for methyl amine and malic acid for aspartic acid. With methyl amine as a leaving group a tautomeric associative or tautomeric dissociative mechanism is preferred and the barrier is lower than the comparable mechanism with methanol as a leaving group. The calculations on the aspartic acid in the enzymatic environment show that qualitatively the mechanism is the same as for triphosphate but the barrier for hydrolysis by the associative mechanism is higher for L-aspartic-dAMP than for L-malic-dAMP and pyrophosphate.

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase.

PubMed

Rögner, M; Gräber, P

1986-09-01

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach*

PubMed Central

Kim, Young-Min; Kiso, Yoshiaki; Muraki, Tomoe; Kang, Min-Sun; Nakai, Hiroyuki; Saburi, Wataru; Lang, Weeranuch; Kang, Hee-Kwon; Okuyama, Masayuki; Mori, Haruhide; Suzuki, Ryuichiro; Funane, Kazumi; Suzuki, Nobuhiro; Momma, Mitsuru; Fujimoto, Zui; Oguma, Tetsuya; Kobayashi, Mikihiko; Kim, Doman; Kimura, Atsuo

2012-01-01

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr451–Val1082), a portion of which shares identity (35% at Ala39–Ser1304 of PsDex) with Pro32–Ala755 of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val837–Met932 for PsDex and Tyr404–Tyr492 for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala39–Ser1304) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp189, Asp340, Glu412, and Asp1254 of PsDex) of catalytic candidates. Their amide mutants decreased activity (11,500 to 140,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp340 and Glu412 as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN3. PMID:22461618

Photo-catalyzed surface hydrolysis of iridium(iii) ions on semiconductors: a facile method for the preparation of semiconductor/IrOx composite photoanodes toward oxygen evolution reaction.

PubMed

Wu, Qingyong; Xu, Di; Xue, Ning; Liu, Tengyi; Xiang, Min; Diao, Peng

2016-12-21

We previously reported that the hydrolysis of Ir 3+ in homogeneous solution could be triggered by irradiation with light whose energy was larger than a threshold value. In this work, we demonstrated that, by introducing Fe 2 O 3 particles into solution, the incident light energy-restriction for the photo-catalyzed hydrolysis could be broken and the hydrolysis occurred at the Fe 2 O 3 /solution interface. The photo-generated holes on the Fe 2 O 3 surface played a key role in oxidizing Ir(iii) to Ir(iv) species and triggered the deposition of IrO x . We showed that this photo-catalyzed surface hydrolysis is a universal phenomenon that takes place on the surface of many n-type semiconductors such as Fe 2 O 3 , TiO 2 , and Ag 3 PO 4 . As IrO x is an efficient catalyst for oxygen evolution reaction, surface hydrolysis is a general, facile and efficient strategy to prepare semiconductor/IrO x composites, which can be used as anodic materials for photoelectrochemical water splitting.

Isomerization of 1-O-indol-3-ylacetyl-beta-D-glucose. Enzymatic hydrolysis of 1-O, 4-O, and 6-O-indol-3-ylacetyl-beta-D-glucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex

NASA Technical Reports Server (NTRS)

Kowalczyk, S.; Bandurski, R. S.

1990-01-01

The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-beta-D-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzyme-catalyzed hydrolysis of 4-O and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.

Aminoacyl-nucleotide reactions - Studies related to the origin of the genetic code and protein synthesis

NASA Technical Reports Server (NTRS)

Mullins, D. W., Jr.; Senaratne, N.; Lacey, J. C., Jr.

1984-01-01

In the present paper, a report is presented on the effect of pH and carbonate on the hydrolysis rate constants of N-blocked and free aminoacyl adenylate anhydrides. Whereas the hydrolysis of free aminoacyl adenylates seems principally catalyzed by OH(-), the hydrolysis of the N-blocked species is also catalyzed by H(+), giving this compound a U-shaped hydrolysis vs. pH curve. Furthermore, at pH's less than 8, carbonate has an extreme catalytic effect on the hydrolysis of free aminoacyl-AMP anhydride, but essentially no effect on the hydrolysis of N-blocked aminoacyl-AMP anhydride. Furthermore, the N-blocked aminoacyl-AMP anhydride is a very efficient generator of peptides using free glycine as acceptor. The possible significance of the observations to prebiological peptide synthesis is discussed.

Scaled-up production of poacic acid, a plant-derived antifungal agent

DOE PAGES

Yue, Fengxia; Gao, Ruili; Piotrowski, Jeff S.; ...

2017-09-01

Poacic acid, a decarboxylated product from 8–5-diferulic acid that is commonly found in monocot lignocellulosic hydrolysates, has been identified as a natural antifungal agent against economically significant fungi and oomycete plant pathogens. Starting from commercially available or monocot-derivable ferulic acid, a three-step synthetic procedure has been developed for the production of poacic acid needed for field testing in a controlled agricultural setting. First, ferulic acid was esterified to produce ethyl ferulate in 92% yield. Second, peroxidase-catalyzed free radical dehydrodimerization of ethyl ferulate produced crude diferulates, mainly 8–5-diferulate, in 91% yield. Finally, crystalline poacic acid was obtained in 25% yield viamore » alkaline hydrolysis of the crude diferulates after purification by flash-column chromatography. Thus, this new procedure offers two key improvements relevant to large-scale production: 1) bubbling air through the reaction mixture in the second step to remove acetone greatly improves the recovery efficiency of the crude diferulates; and 2) telescoping minor impurities directly into the alkaline hydrolysis step eliminates the need for additional column purifications, thus reducing the overall cost of production and removing a major impediment to process scale-up.« less

Scaled-up production of poacic acid, a plant-derived antifungal agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yue, Fengxia; Gao, Ruili; Piotrowski, Jeff S.

Poacic acid, a decarboxylated product from 8–5-diferulic acid that is commonly found in monocot lignocellulosic hydrolysates, has been identified as a natural antifungal agent against economically significant fungi and oomycete plant pathogens. Starting from commercially available or monocot-derivable ferulic acid, a three-step synthetic procedure has been developed for the production of poacic acid needed for field testing in a controlled agricultural setting. First, ferulic acid was esterified to produce ethyl ferulate in 92% yield. Second, peroxidase-catalyzed free radical dehydrodimerization of ethyl ferulate produced crude diferulates, mainly 8–5-diferulate, in 91% yield. Finally, crystalline poacic acid was obtained in 25% yield viamore » alkaline hydrolysis of the crude diferulates after purification by flash-column chromatography. Thus, this new procedure offers two key improvements relevant to large-scale production: 1) bubbling air through the reaction mixture in the second step to remove acetone greatly improves the recovery efficiency of the crude diferulates; and 2) telescoping minor impurities directly into the alkaline hydrolysis step eliminates the need for additional column purifications, thus reducing the overall cost of production and removing a major impediment to process scale-up.« less

Crystallization and preliminary X-ray diffraction analysis of a novel sphingomyelinase D from Loxosceles gaucho venom.

PubMed

Ullah, Anwar; Magalhães, Geraldo Santana; Masood, Rehana; Mariutti, Ricardo Barros; Coronado, Monika Aparecida; Murakami, Mário Tyago; Barbaro, Katia Cristina; Arni, Raghuvir Krishnaswamy

2014-10-01

Brown spider envenomation results in dermonecrosis, intravascular coagulation, haemolysis and renal failure, mainly owing to the action of sphingomyelinases D (SMases D), which catalyze the hydrolysis of sphingomyelin to produce ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidylcholine to produce lysophosphatidic acid. Here, the heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of LgRec1, a novel SMase D from Loxosceles gaucho venom, are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 52.98, b = 62.27, c = 84.84 Å and diffracted to a maximum resolution of 2.6 Å.

Crystallization and preliminary X-ray diffraction analysis of a novel sphingomyelinase D from Loxosceles gaucho venom

PubMed Central

Ullah, Anwar; Magalhães, Geraldo Santana; Masood, Rehana; Mariutti, Ricardo Barros; Coronado, Monika Aparecida; Murakami, Mário Tyago; Barbaro, Katia Cristina; Arni, Raghuvir Krishnaswamy

2014-01-01

Brown spider envenomation results in dermonecrosis, intravascular coagulation, haemolysis and renal failure, mainly owing to the action of sphingomyelinases D (SMases D), which catalyze the hydrolysis of sphingomyelin to produce ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidylcholine to produce lysophosphatidic acid. Here, the heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of LgRec1, a novel SMase D from Loxosceles gaucho venom, are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 52.98, b = 62.27, c = 84.84 Å and diffracted to a maximum resolution of 2.6 Å. PMID:25286953

Phospholipase activity in rat liver mitochondria studied by the use of endogenous substrates.

PubMed

Bjornstad, P

1966-09-01

The hydrolysis of endogenous phosphatidyl ethanolamine and lecithin in rat liver mitochondria has been studied by using mitochondria from rats injected with ethanolamine-1,2-(14)C or choline-1,2-(14)C. A phospholipase A-like enzyme has been demonstrated, which catalyzes the hydrolysis of one fatty acid ester linkage in phosphatidyl ethanolamine and lecithin. Phosphatidyl ethanolamine is hydrolyzed in preference to lecithin and the main reaction products are free fatty acids and lysophosphatidyl ethanolamine. The further breakdown of lysophospholipids appears to be limited in mitochondria, which indicates that lysophospholipase activity is mainly located extramitochondrially. The enzymic system is greatly stimulated by calcium ions, and also slightly by magnesium ions, while EDTA inhibits it almost completely. These findings are discussed in relation to previous observations on the effect of calcium and of EDTA on the functions of mitochondria. The possible function of the mitochondrial phospholipase for the formation of phospholipids with special fatty acids at the alpha- and -position is discussed.

Ultrafiltration membrane reactors for enzymatic resolution of amino acids: design model and optimization.

PubMed

Bódalo, A; Gómez, J L.; Gómez, E; Bastida, J; Máximo, M F.; Montiel, M C.

2001-03-08

In this paper the possibility of continuous resolution of DL-phenylalanine, catalyzed by L-aminoacylase in a ultrafiltration membrane reactor (UFMR) is presented. A simple design model, based on previous kinetic studies, has been demonstrated to be capable of describing the behavior of the experimental system. The model has been used to determine the optimal experimental conditions to carry out the asymmetrical hydrolysis of N-acetyl-DL-phenylalanine.

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester.

PubMed

van Loo, Bert; Schober, Markus; Valkov, Eugene; Heberlein, Magdalena; Bornberg-Bauer, Erich; Faber, Kurt; Hyvönen, Marko; Hollfelder, Florian

2018-03-30

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S-O bond cleavage) or carbon (C-O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S-O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C-O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (k cat /K M =4.8×10 3 s -1 M -1 ) as well as arylsulfate 4-nitrophenyl sulfate (k cat /K M =12s -1 M -1 ). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although >70% of the amino acids between protomers align structurally (RMSDs 1.79-1.99Å), the oligomeric structures show distinctly different packing and protomer-protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H 2 18 O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S-O cleavage in alkyl sulfate esters with extreme catalytic proficiency. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification and biochemical characterization of an acid sphingomyelinase-like protein from the bacterial plant pathogen Ralstonia solanacearum that hydrolyzes ATP to AMP but not sphingomyelin to ceramide.

PubMed

Airola, Michael V; Tumolo, Jessica M; Snider, Justin; Hannun, Yusuf A

2014-01-01

Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases.

Hydrolysis of Ketene Catalyzed by Formic Acid: Modification of Reaction Mechanism, Energetics, and Kinetics with Organic Acid Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Louie, Matthew K.; Francisco, Joseph S.; Verdicchio, Marco

2015-05-14

The hydrolysis of ketene (H2C=C=O) to form acetic acid involving two water molecules and also separately in the presence of one to two water molecules and formic acid (FA) was investigated. Our results show that, while the currently accepted indirect mechanism, involving addition of water across the carbonyl C=O bond of ketene to form an ene-diol followed by tautomerization of the ene-diol to form acetic acid, is the preferred pathway when water alone is present, with formic acid as catalyst, addition of water across the ketene C=C double bond to directly produce acetic acid becomes the kinetically favored pathway formore » temperatures below 400 K. We find not only that the overall barrier for ketene hydrolysis involving one water molecule and formic acid (H2C2O + H2O + FA) is significantly lower than that involving two water molecules (H2C2O + 2H(2)O) but also that FA is able to reduce the barrier height for the direct path, involving addition of water across the C=C double bond, so that it is essentially identical with (6.4 kcal/mol) that for the indirect ene-diol formation path involving addition of water across the C=O bond. For the case of ketene hydrolysis involving two water molecules and formic acid (H2C2O + 2H(2)O + FA), the barrier for the direct addition of water across the C=C double bond is reduced even further and is 2.5 kcal/mol lower relative to the ene-diol path involving addition of water across the C=O bond. In fact, the hydrolysis barrier for the H2C2O + 2H(2)O + FA reaction through the direct path is sufficiently low (2.5 kcal/mol) for it to be an energetically accessible pathway for acetic acid formation under atmospheric conditions. Given the structural similarity between acetic and formic acid, our results also have potential implications for aqueous-phase chemistry. Thus, in an aqueous environment, even in the absence of formic acid, though the initial mechanism for ketene hydrolysis is expected to involve addition of water across the carbonyl bond as is currently accepted, the production and accumulation of acetic acid will likely alter the preferred pathway to one involving addition of water across the ketene C=C double bond as the reaction proceeds.« less

Biotechnological Production of Methyl-Branched Aldehydes.

PubMed

Fraatz, Marco Alexander; Goldmann, Michael; Geissler, Torsten; Gross, Egon; Backes, Michael; Hilmer, Jens-Michael; Ley, Jakob; Rost, Johanna; Francke, Alexander; Zorn, Holger

2018-03-14

A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after flavoring compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For the biotechnological production of 12-methyltridecanal, the literature was screened for fungi forming iso-fatty acids. Suitable organisms were identified and successfully grown in submerged cultures. The culture medium was optimized to increase the yields of branched fatty acids. A recombinant carboxylic acid reductase was used to reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell catalysis was compared to that of the purified enzyme preparation. After lipase-catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were converted to the corresponding aldehydes, including 12-methyltridecanal and 12-methyltetradecanal.

Pyrolytic sugars from cellulosic biomass.

PubMed

Kuzhiyil, Najeeb; Dalluge, Dustin; Bai, Xianglan; Kim, Kwang Ho; Brown, Robert C

2012-11-01

Depolymerization of cellulose offers the prospect of inexpensive sugars from biomass. Breaking the glycosidic bonds of cellulose to liberate glucose has usually been pursued by acid or enzymatic hydrolysis although a purely thermal depolymerization route to sugars is also possible. Fast pyrolysis of pure cellulose yields primarily the anhydrosugar levoglucosan (LG) whereas the presence of naturally occurring alkali and alkaline earth metals (AAEMs) in biomass strongly catalyzes ring-breaking reactions that favor formation of light oxygenates. Here, we show a method of significantly increasing the yield of sugars from biomass by purely thermal means through infusion of certain mineral acids (phosphoric and sulfuric acid) into the biomass to convert the AAEMs into thermally stable salts (particularly potassium sulfates and phosphates). These salts not only passivate AAEMs that normally catalyze fragmentation of pyranose rings, but also buffer the system at pH levels that favor glycosidic bond breakage. It appears that AAEM passivation contributes to 80 % of the enhancement in LG yield while the buffering effect of the acid salts contributes to the balance of the enhancement. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The hydrolysis kinetics of monobasic and dibasic aminoalkyl esters of ketorolac.

PubMed

Qandil, Amjad M; Jamhawi, Noor M; Tashtoush, Bassam M; Al-Ajlouni, Ahmad M; Idkaidek, Nasir M; Obaidat, Aiman A

2013-09-01

Six aminoethyl and aminobutyl esters of ketorolac containing 1-methylpiperazine (MPE and MPB), N-acetylpiperazine (APE and APB) or morpholine (ME and MB), were synthesized and their hydrolysis kinetics were studied. The hydrolysis was studied at pH 1 to 9 (for MPE, APE and ME) and pH 1 to 8 (for MPB, APB and MB) in aqueous phosphate buffer (0.16 M) with ionic strength (0.5 M) at 37°C. Calculation of k(obs), construction of the pH-rate profiles and determination of the rate equations were performed using KaleidaGraph® 4.1. The hydrolysis displays pseudo-first order kinetics and the pH-rate profiles shows that the aminobutyl esters, MPE, APB and MB, are the most stable. The hydrolysis of the ethyl esters MPE, APE and ME, depending on the pH, is either fast and catalyzed by the hydroxide anion or slow and uncatalyzed for the diprotonated, monoprotonated and nonprotonated forms. The hydrolysis of the butyl esters showed a similar profile, albeit it was also catalyzed by hydronium cation. In addition, the hydroxide anion is 105 more effective in catalyzing the hydrolysis than the hydronium cation. The hydrolysis pattern of the aminoethyl esters is affected by the number and pKa of its basic nitrogen atoms. The monobasic APE and ME, show a similar hydrolysis pattern that is different than the dibasic MPE. The length of the side chain and the pKa of the basic nitrogen atoms in the aminoethyl moiety affect the mechanism of hydrolysis as the extent of protonation at a given pH is directly related to the pKa.

Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

PubMed Central

2015-01-01

Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance. PMID:25309810

Au NPs immersed in sol-gel matrix: nonlinear optical characterization

NASA Astrophysics Data System (ADS)

Aguilera-Zavala, Angélica; Trejo-Durán, Mónica; Ortiz-Jiménez, Orlando; Cornejo-Monroy, Delfino; Severiano-Carrillo, Israel; Alvarado-Méndez, Edgar

2016-09-01

Physical and optical characterization of thin films doped with Au Nanoparticles onto a silica substrate is presented. Films were prepared through sol-gel process, by using Au nanoparticles immersed in lipoic acid as dopant by means of hydrolysis and acid catalyzed reaction of tetraethyl-orthosilicate. The surface was characterized by SEM and AFM microscopies. Z-scan technique was used to measure nonlinear optical properties as nonlinear absorption and refraction indexes, using two different wavelengths. At 633 nm it was possible to observe nonlinear absorption only but at 514 nm both nonlinear properties were observed.

Acid-functionalized nanoparticles for biomass hydrolysis

NASA Astrophysics Data System (ADS)

Pena Duque, Leidy Eugenia

Cellulosic ethanol is a renewable source of energy. Lignocellulosic biomass is a complex material composed mainly of cellulose, hemicellulose, and lignin. Biomass pretreatment is a required step to make sugar polymers liable to hydrolysis. Mineral acids are commonly used for biomass pretreatment. Using acid catalysts that can be recovered and reused could make the process economically more attractive. The overall goal of this dissertation is the development of a recyclable nanocatalyst for the hydrolysis of biomass sugars. Cobalt iron oxide nanoparticles (CoFe2O4) were synthesized to provide a magnetic core that could be separated from reaction using a magnetic field and modified to carry acid functional groups. X-ray diffraction (XRD) confirmed the crystal structure was that of cobalt spinel ferrite. CoFe2O4 were covered with silica which served as linker for the acid functions. Silica-coated nanoparticles were functionalized with three different acid functions: perfluoropropyl-sulfonic acid, carboxylic acid, and propyl-sulfonic acid. Transmission electron microscope (TEM) images were analyzed to obtain particle size distributions of the nanoparticles. Total carbon, nitrogen, and sulfur were quantified using an elemental analyzer. Fourier transform infra-red spectra confirmed the presence of sulfonic and carboxylic acid functions and ion-exchange titrations accounted for the total amount of catalytic acid sites per nanoparticle mass. These nanoparticles were evaluated for their performance to hydrolyze the beta-1,4 glycosidic bond of the cellobiose molecule. Propyl-sulfonic (PS) and perfluoropropyl-sulfonic (PFS) acid functionalized nanoparticles catalyzed the hydrolysis of cellobiose significantly better than the control. PS and PFS were also evaluated for their capacity to solubilize wheat straw hemicelluloses and performed better than the control. Although PFS nanoparticles were stronger acid catalysts, the acid functions leached out of the nanoparticle during the catalytic reactions. PS nanoparticles were further evaluated for the pretreatment of corn stover in order to increase digestibility of the biomass. The pretreatment was carried out at three different catalyst load and temperature levels. At 180°C, the total glucose yield was linearly correlated to the catalyst load. A maximum glucose yield of 90% and 58% of the hemicellulose sugars were obtained at this temperature.

Strategy to Overcome Effect of Raw Materials on Enzymatic Process of Biodiesel from Non-edible Oils Using Candida sp. 99-125 Lipase.

PubMed

Nie, Kaili; Wang, Fang; Tan, Tianwei; Liu, Luo

2015-11-01

Non-edible oils are preferred raw materials for biodiesel production. However, the properties of raw materials significantly affect the synthesis process, leading to difficulties to design one process suitable for any kind of raw material. In this study, the composition of five typical non-edible oils was analyzed. The major difference was the content of free fatty acids, reflected from their acid values. The influence of different oils was investigated by using lipase from Candida sp. 99-125. At low lipase dosage and low water content, the conversion was found proportional to the acid value. However, by increasing the water content or lipase dosage, we observed that the conversions for all kinds of oils used in this study could exceed 80%. Time course analysis indicates that the lipase used in this study catalyzed hydrolysis followed by esterification, rather than direct transesterification. Accumulation of free fatty acids at the very beginning was necessary. A high water content facilitated the hydrolysis of oils with low acid value. This lipase showed capability to transform all the oils by controlling the water content.

Non-catalytic steam hydrolysis of fats. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deibert, M.C.

1992-08-28

Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steammore » mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.« less

Non-catalytic steam hydrolysis of fats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deibert, M.C.

1992-08-28

Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steammore » mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.« less

Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design.

PubMed

Xu, Li; Liu, Xiaohong; Yin, Zhenhao; Liu, Qian; Lu, Lili; Xiao, Min

2016-12-01

The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp 252 , Asp 257 , Asp 264 , Glu 530 , Arg 548 , His 553 , and Trp 555 ) and may form the hydrophobic pocket in stabilizing donor (Trp 261 , Tyr 302 , Tyr 316 , and Trp 369 ) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp 257 . Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

Conversion of cellulose and cellobiose into sorbitol catalyzed by ruthenium supported on a polyoxometalate/metal-organic framework hybrid.

PubMed

Chen, Jinzhu; Wang, Shengpei; Huang, Jing; Chen, Limin; Ma, Longlong; Huang, Xing

2013-08-01

Cellulose and cellobiose were selectively converted into sorbitol over water-tolerant phosphotungstic acid (PTA)/metal- organic-framework-hybrid-supported ruthenium catalysts, Ru-PTA/MIL-100(Cr), under aqueous hydrogenation conditions. The goal was to investigate the relationship between the acid/metal balance of bifunctional catalysts Ru-PTA/MIL-100(Cr) and their performance in the catalytic conversion of cellulose and cellobiose into sugar alcohols. The control of the amount and strength of acid sites in the supported PTA/MIL-100(Cr) was achieved through the effective control of encapsulated-PTA loading in MIL-100(Cr). This design and preparation method led to an appropriately balanced Ru-PTA/MIL-100(Cr) in terms of Ru dispersion and hydrogenation capacity on the one hand, and acid site density of PTA/MIL-100(Cr) (responsible for acid-catalyzed hydrolysis) on the other hand. The ratio of acid site density to the number of Ru surface atoms (nA /nRu ) of Ru-PTA/MIL-100(Cr) was used to monitor the balance between hydrogenation and hydrolysis functions; the optimum balance between the two catalytic functions, that is, 8.84

Improving a natural enzyme activity through incorporation of unnatural amino acids.

PubMed

Ugwumba, Isaac N; Ozawa, Kiyoshi; Xu, Zhi-Qiang; Ely, Fernanda; Foo, Jee-Loon; Herlt, Anthony J; Coppin, Chris; Brown, Sue; Taylor, Matthew C; Ollis, David L; Mander, Lewis N; Schenk, Gerhard; Dixon, Nicholas E; Otting, Gottfried; Oakeshott, John G; Jackson, Colin J

2011-01-19

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

A new sol-gel synthesis of 45S5 bioactive glass using an organic acid as catalyst.

PubMed

Faure, J; Drevet, R; Lemelle, A; Ben Jaber, N; Tara, A; El Btaouri, H; Benhayoune, H

2015-02-01

In this paper a new sol-gel approach was explored for the synthesis of the 45S5 bioactive glass. We demonstrate that citric acid can be used instead of the usual nitric acid to catalyze the sol-gel reactions. The substitution of nitric acid by citric acid allows to reduce strongly the concentration of the acid solution necessary to catalyze the hydrolysis of silicon and phosphorus alkoxides. Two sol-gel powders with chemical compositions very close to that of the 45S5 were obtained by using either a 2M nitric acid solution or either a 5mM citric acid solution. These powders were characterized and compared to the commercial Bioglass®. The surface properties of the two bioglass powders were assessed by scanning electron microscopy (SEM) and by Brunauer-Emmett-Teller method (BET). The Fourier transformed infrared spectroscopy (FTIR) and the X-ray diffraction (XRD) revealed a partial crystallization associated to the formation of crystalline phases on the two sol-gel powders. The in vitro bioactivity was then studied at the key times during the first hours of immersion into acellular Simulated Body Fluid (SBF). After 4h immersion into SBF we clearly demonstrate that the bioactivity level of the two sol-gel powders is similar and much higher than that of the commercial Bioglass®. This bioactivity improvement is associated to the increase of the porosity and the specific surface area of the powders synthesized by the sol-gel process. Moreover, the nitric acid is efficiently substituted by the citric acid to catalyze the sol-gel reactions without alteration of the bioactivity of the 45S5 bioactive glass. Copyright © 2014 Elsevier B.V. All rights reserved.

Phosphatidic Acid and Lipid Sensing by mTOR

PubMed Central

Foster, David A.

2013-01-01

Mammalian target of rapamycin (mTOR) has been implicated as a sensor of nutrient sufficiency for dividing cells and is activated by essential amino acids and glucose. However, cells also require lipids for membrane biosynthesis. A central metabolite in the synthesis of membrane phospholipids is phosphatidic acid (PA), which is required for the stability and activity of mTOR complexes. While PA is commonly generated by the phospholipase D-catalyzed hydrolysis of phosphatidylcholine, PA is also generated by diacylglycerol kinases and lysophosphatidic acid acyltransferases, which are at the center of phospholipid biosynthesis. It is proposed that the responsiveness of mTOR/TOR to PA evolved as a means for sensing lipid precursors for membrane biosynthesis prior to doubling the mass of a cell and dividing. PMID:23507202

Hydrolysis of cellulose catalyzed by quaternary ammonium perrhenates in 1-allyl-3-methylimidazolium chloride.

PubMed

Wang, Jingyun; Zhou, Mingdong; Yuan, Yuguo; Zhang, Quan; Fang, Xiangchen; Zang, Shuliang

2015-12-01

Quaternary ammonium perrhenates were applied as catalyst to promote the hydrolysis of cellulose in 1-allyl-3-methylimidazolium chloride ([Amim]Cl). The quaternary ammonium perrhenates displayed good catalytic performance for cellulose hydrolysis. Water was also proven to be effective to promote cellulose hydrolysis. Accordingly, 97% of total reduced sugar (TRS) and 42% of glucose yields could be obtained under the condition of using 5mol% of tetramethyl ammonium perrhenate as catalyst, 70μL of water, ca. 0.6mmol of microcrystalline cellulose (MCC) and 2.0g of [Amim]Cl as solvent under microwave irradiation for 30min at 150°C (optimal conditions). The influence of quaternary ammonium cation on the efficiency of cellulose hydrolysis was examined based on different cation structures of perrhenates. The mechanism on perrhenate catalyzed cellulose hydrolysis is also discussed, whereas hydrogen bonding between ReO4 anion and hydroxyl groups of cellulose is assumed to be the key step for depolymerization of cellulose. Copyright © 2015. Published by Elsevier Ltd.

Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA)

PubMed Central

2011-01-01

Background Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity. Results NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates. Conclusion Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses. PMID:22035583

Intact carbohydrate structures as part of the melanoidin skeleton.

PubMed

Cämmerer, Bettina; Jalyschko, Walentina; Kroh, Lothar W

2002-03-27

Model melanoidins from monomeric, oligomeric, and polymeric carbohydrates, and amino acids formed under aqueous as well as water-free reaction conditions, were submitted to acidic catalyzed hydrolysis. Their degradation products were detected qualitatively and quantitatively by HPTLC and HPLC-DAD. A considerable amount of monomer carbohydrates from hydrolysis of model melanoidins formed under water-free reaction conditions was detected. It can be seen clearly that the amount of carbohydrates released increased with increasing degree of polymerization of the carbohydrates used as starting material. In comparison, the hydrolysis of melanoidins formed in aqueous condition resulted in only a small glucose release. It seems that in the Maillard reaction under water-free conditions, a significant amount of di- and oligomer carbohydrates were incorporated into the melanoidin skeleton as complete oligomer with intact glycosidic bond, forming side chains at the melanoidin skeleton. Additional side chains could be formed by transglycosylation reactions. With increasing water content, hydrothermolytic as well as retro-aldol reactions of the starting carbonyl components became significant, and therefore the possibility of forming side chains decreased. The results are consistent with the postulated melanoidin structure being built up mainly from sugar degradation products, probably branched via amino compounds.

Hydrolysis of amphenicol and macrolide antibiotics: Chloramphenicol, florfenicol, spiramycin, and tylosin.

PubMed

Mitchell, Shannon M; Ullman, Jeffrey L; Teel, Amy L; Watts, Richard J

2015-09-01

Antibiotics that enter the environment can present human and ecological health risks. An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence as biologically active contaminants. In this study, hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for two amphenicol (chloramphenicol and florfenicol) and two macrolide (spiramycin and tylosin) antibiotics. Antibiotic hydrolysis rates in pH 4-9 buffer solutions at 25°C, 50°C, and 60°C were quantified, and degradation products were characterized. All of the antibiotics tested remained stable and exhibited no observable hydrolysis under ambient conditions typical of aquatic ecosystems. Acid- and base-catalyzed hydrolysis occurred at elevated temperatures (50-60°C), and hydrolysis rates increased considerably below pH 5 and above pH 8. Hydrolysis rates also increased approximately 1.5- to 2.9-fold for each 10°C increase in temperature. Based on the degradation product masses found, the functional groups that underwent hydrolysis were alkyl fluoride, amide, and cyclic ester (lactone) moieties; some of the resultant degradation products may remain bioactive, but to a lesser extent than the parent compounds. The results of this research demonstrate that amphenicol and macrolide antibiotics persist in aquatic systems under ambient temperature and pH conditions typical of natural waters. Thus, these antibiotics may present a risk in aquatic ecosystems depending on the concentration present. Copyright © 2015. Published by Elsevier Ltd.

Cellulose pretreatment with 1-n-butyl-3-methylimidazolium chloride for solid acid-catalyzed hydrolysis.

PubMed

Kim, Soo-Jin; Dwiatmoko, Adid Adep; Choi, Jae Wook; Suh, Young-Woong; Suh, Dong Jin; Oh, Moonhyun

2010-11-01

This study has been focused on developing a cellulose pretreatment process using 1-n-butyl-3-methylimidazolium chloride ([bmim]Cl) for subsequent hydrolysis over Nafion(R) NR50. Thus, several pretreatment variables such as the pretreatment period and temperature, and the [bmim]Cl amount were varied. Additionally, the [bmim]Cl-treated cellulose samples were characterized by X-ray diffraction analysis, and their crystallinity index values including CI(XD), CI(XD-CI) and CI(XD-CII) were then calculated. When correlated with these values, the concentrations of total reducing sugars (TRS) obtained by the pretreatment of native cellulose (NC) and glucose produced by the hydrolysis reaction were found to show a distinct relationship with the [CI(NC)-CI(XD)] and CI(XD-CII) values, respectively. Consequently, the cellulose pretreatment step with [bmim]Cl is to loosen a crystalline cellulose through partial transformation of cellulose I to cellulose II and, furthermore, the TRS release, while the subsequent hydrolysis of [bmim]Cl-treated cellulose over Nafion(R) NR50 is effective to convert cellulose II to glucose. Copyright 2010 Elsevier Ltd. All rights reserved.

Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

PubMed Central

Diaz-Meco, M T; Dominguez, I; Sanz, L; Municio, M M; Berra, E; Cornet, M E; Garcia de Herreros, A; Johansen, T; Moscat, J

1992-01-01

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1. Images PMID:1309592

Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

PubMed

Hiraishi, Tomohiro

2016-02-01

Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

A Facile Semi-Synthetic Approach towards Halogen-Substituted Aminobenzoic Acid Analogues of Platensimycin.

PubMed

Qiu, Lin; Tian, Kai; Pan, Jian; Jiang, Lin; Yang, Hu; Zhu, Xiangcheng; Shen, Ben; Duan, Yanwen; Huang, Yong

2017-02-09

Platensimycin (PTM), produced by several strains of Streptomyces platensis, is a promising drug lead for infectious diseases and diabetes. The recent pilot-scale production of PTM from S. platensis SB12026 has set the stage for the facile semi-synthesis of a focused library of PTM analogues. In this study, gram-quantity of platensic acid (PTMA) was prepared by the sulfuric acid-catalyzed ethanolysis of PTM, followed by a mild hydrolysis in aqueous lithium hydroxide. Three PTMA esters were also obtained in near quantitative yields in a single step, suggesting a facile route to make PTMA aliphatic esters. 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU)-catalyzed coupling of PTMA and 33 aminobenzoates resulted in the synthesis of 28 substituted aminobenzoate analogues of PTM, among which 26 of them were reported for the first time. Several of the PTM analogues showed weak antibacterial activity against methicillin-resistant Staphylococcus aureus. Our study supported the potential utility to integrate natural product biosynthetic and semi-synthetic approaches for structure diversification.

Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

PubMed

Scollar, M P; Sigal, G; Klibanov, A M

1985-03-01

Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

Facile synthesis of highly efficient and recyclable magnetic solid acid from biomass waste

PubMed Central

Liu, Wu-Jun; Tian, Ke; Jiang, Hong; Yu, Han-Qing

2013-01-01

In this work, sawdust, a biomass waste, is converted into a magnetic porous carbonaceous (MPC) solid acid catalyst by an integrated fast pyrolysis–sulfonation process. The resultant magnetic solid acid has a porous structure with high surface area of 296.4 m2 g−1, which can be attributed to the catalytic effect of Fe. The catalytic activity and recyclability of the solid acid catalyst are evaluated during three typical acid-catalyzed reactions: esterification, dehydration, and hydrolysis. The favorable catalytic performance in all three reactions is attributed to the acid's high strength with 2.57 mmol g−1 of total acid sites. Moreover, the solid acid can be reused five times without a noticeable decrease in catalytic activity, indicating the stability of the porous carbon (PC)–sulfonic acid group structure. The findings in the present work offer effective alternatives for environmentally friendly utilization of abundant biomass waste. PMID:23939253

Click strategy using disodium salts of amino acids improves the water solubility of plinabulin and KPU-300.

PubMed

Yakushiji, Fumika; Muguruma, Kyohei; Hayashi, Yoshiki; Shirasaka, Takuya; Kawamata, Ryosuke; Tanaka, Hironari; Yoshiwaka, Yushi; Taguchi, Akihiro; Takayama, Kentaro; Hayashi, Yoshio

2017-07-15

Plinabulin and KPU-300 are promising anti-microtubule agents; however, the low water solubility of these compounds (<0.1µg/mL) has limited their pharmaceutical advantages. Here, we developed five water-soluble derivatives of plinabulin and KPU-300 with a click strategy using disodium salts of amino acids. The mother skeleton, diketopiperazine (DKP), was transformed into a monolactim-type alkyne and a copper-catalyzed alkyne azide cycloaddition (CuAAC) combined azides that was derived from amino acids as a water-solubilizing moiety. The conversion of carboxyl groups into disodium salts greatly improved the water solubility by 0.8 million times compared to the solubility of the parent molecules. In addition, the α-amino acid side chains of the water-solubilizing moieties affected both the water solubility and the half-lives of the compounds during enzymatic hydrolysis. Our effort to develop a variety of water-soluble derivatives using the click strategy has revealed that the replaceable water-solubilizing moieties can alter molecular solubility and stability under enzymatic hydrolysis. With this flexibility, we are approaching to the in vivo study using water-soluble derivative. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hydrolysis of tannic acid catalyzed by immobilized-stabilized derivatives of Tannase from Lactobacillus plantarum.

PubMed

Curiel, Jose Antonio; Betancor, Lorena; de las Rivas, Blanca; Muñoz, Rosario; Guisan, Jose M; Fernández-Lorente, Gloria

2010-05-26

A recombinant tannase from Lactobacillus plantarum , overexpressed in Escherichia coli , was purified in a single step by metal chelate affinity chromatography on poorly activated nickel supports. It was possible to obtain 0.9 g of a pure enzyme by using only 20 mL of chromatographic support. The pure enzyme was immobilized and stabilized by multipoint covalent immobilization on highly activated glyoxyl agarose. Derivatives obtained by multipoint and multisubunit immobilization were 500- and 1000-fold more stable than both the soluble enzyme and the one-point-immobilized enzyme in experiments of thermal and cosolvent inactivation, respectively. In addition, up to 70 mg of pure enzyme was immobilized on 1 g of wet support. The hydrolysis of tannic acid was optimized by using the new immobilized tannase derivative. The optimal reaction conditions were 30% diglyme at pH 5.0 and 4 degrees C. Under these conditions, it was possible to obtain 47.5 mM gallic acid from 5 mM tannic acid as substrate. The product was pure as proved by HPLC. On the other hand, the immobilized biocatalyst preserved >95% of its initial activity after 1 month of incubation under the optimal reaction conditions.

Efficient and low-cost alternative of lipase concentration aiming at the application in the treatment of waste cooking oils.

PubMed

Preczeski, Karina P; Kamanski, Angela B; Scapini, Thamarys; Camargo, Aline F; Modkoski, Tatiani A; Rossetto, Vanusa; Venturin, Bruno; Mulinari, Jéssica; Golunski, Simone M; Mossi, Altemir J; Treichel, Helen

2018-06-01

In this study, we evaluated the concentration of lipases from Aspergillus niger using efficient and low-cost methods aiming at application in the treatment of waste cooking oils. The change in ionic strength of the medium by the addition of salt and precipitation with ethanol increased the specific activity from 2.90 to 28.50 U/mg, resulting in a purification factor of 9.82-fold. The use of acetone resulted in a specific activity of 33.63 U/mg, resulting in a purification factor of 11.60-fold. After that, the concentrated lipase was used in the hydrolysis of waste cooking oil and 753.07 and 421.60 µmol/mL of free fatty acids were obtained for the enzyme precipitated with ethanol and acetone, respectively. The hydrolysis of waste cooking oil catalyzed by homemade purified lipase in ultrasonic media can be considered a pretreatment of oil by converting a significant amount of triglycerides into free fatty acids.

Catalytic hydrolysis of cellulose into furans

NASA Astrophysics Data System (ADS)

Shi, Chengmei; Tao, Furong; Cui, Yuezhi

2016-12-01

Chromium chloride in 4-(3-methylimidazolium-1-yl)butane-1-sulfonic acid hydrogen sulfate (IL-1) was found to effectively catalyze the hydrolysis of microcrystalline cellulose (MCC) at 150°C for 300 min to achieve 87.8% conversion to a slate of products. With a catalytic amount of CrCl3, the yields of 5-hydroxymethyl furfural (HMF) and furfural were up to 32.4 and 15.2%, respectively, small molecules levulinic acid (LA, 10.8%) and the total reducing sugars (TRS, 10.7%) were also generated. Through LC-MSD analysis and mass spectra, dimer of furan compounds as the main by-products were speculated, and the components of gas products were methane, ethane, CO, CO2, and H2. We suggested that IL-1 and CrCl3 exhibited a coordination interaction; the formation of the intermediate via the hydride shift played a key role in the formation of HMF. The catalyst was recycled and exhibited constant activity for five successive trials.

Fractionation of lignocellulosic biopolymers from sugarcane bagasse using formic acid-catalyzed organosolv process.

PubMed

Suriyachai, Nopparat; Champreda, Verawat; Kraikul, Natthakorn; Techanan, Wikanda; Laosiripojana, Navadol

2018-05-01

A one-step formic acid-catalyzed organosolv process using a low-boiling point acid-solvent system was studied for fractionation of sugarcane bagasse. Compared to H 2 SO 4 , the use of formic acid as a promoter resulted in higher efficiency and selectivity on removals of hemicellulose and lignin with increased enzymatic digestibility of the cellulose-enriched solid fraction. The optimal condition from central composite design analysis was determined as 40 min residence time at 159 °C using water/ethanol/ethyl acetate/formic acid in the respective ratios of 43:20:16:21%v/v. Under this condition, a 94.6% recovery of cellulose was obtained in the solid with 80.2% cellulose content while 91.4 and 80.4% of hemicellulose and lignin were removed to the aqueous-alcohol-acid and ethyl acetate phases, respectively. Enzymatic hydrolysis of the solid yielded 84.5% glucose recovery compared to available glucan in the raw material. Physicochemical analysis revealed intact cellulose fibers with decreased crystallinity while the hemicellulose was partially recovered as mono- and oligomeric sugars. High-purity organosolv lignin with < 1% sugar cross-contamination was obtained with no major structural modification according to Fourier-transform infrared spectroscopy. The work represents an alternative process for efficient fractionation of lignocellulosic biomass in biorefineries.

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

PubMed

Stevenson, Bradley J; Waller, Christopher C; Ma, Paul; Li, Kunkun; Cawley, Adam T; Ollis, David L; McLeod, Malcolm D

2015-10-01

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation. Copyright © 2015 John Wiley & Sons, Ltd.

Towards the design of organocatalysts for nerve agents remediation: The case of the active hydrolysis of DCNP (a Tabun mimic) catalyzed by simple amine-containing derivatives.

PubMed

Barba-Bon, Andrea; Martínez-Máñez, Ramón; Sancenón, Félix; Costero, Ana M; Gil, Salvador; Pérez-Pla, Francisco; Llopis, Elisa

2015-11-15

We report herein a study of the hydrolysis of Tabun mimic DCNP in the presence of different amines, aminoalcohols and glycols as potential suitable organocatalysts for DCNP degradation. Experiments were performed in CD3CN in the presence of 5% D2O, which is a suitable solvent mixture to follow the DCNP hydrolysis. These studies allowed the definition of different DCNP depletion paths, resulting in the formation of diethylphosphoric acid, tetraethylpyrophosphate and phosphoramide species as final products. Without organocatalysts, DCNP hydrolysis occurred mainly via an autocatalysis path. Addition of tertiary amines in sub-stoichiometric amounts largely enhanced DCNP depletion whereas non-tertiary polyamines reacted even faster. Glycols induced very slight increment in the DCNP hydrolysis, whereas DCNP hydrolysis increased sharply in the presence of certain aminoalcohols especially, 2-(2-aminoethylamino)ethanol. For the latter compound, DCNP depletion occurred ca. 80-fold faster than in the absence of organocatalysts. The kinetic studies revealed that DCNP hydrolysis in the presence of 2-(2-aminoethylamino)ethanol occurred via a catalytic process, in which the aminoalcohol was involved. DCNP hydrolysis generally depended strongly on the structure of the amine, and it was found that the presence of the OHCH2CH2N moiety in the organocatalyst structure seems important to induce a fast degradation of DCNP. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.).

PubMed

Van Kerckhoven, Sonia H; de la Torre, Fernando N; Cañas, Rafael A; Avila, Concepción; Cantón, Francisco R; Cánovas, Francisco M

2017-01-01

Asparaginases (ASPG, EC 3.5.1.1) catalyze the hydrolysis of the amide group of L-asparagine producing L-aspartate and ammonium. Three ASPG, PpASPG1, PpASPG2, and PpASPG3, have been identified in the transcriptome of maritime pine ( Pinus pinaster Ait.) that were transiently expressed in Nicotiana benthamiana by agroinfection. The three recombinant proteins were processed in planta to active enzymes and it was found that all mature forms exhibited double activity asparaginase/isoaspartyl dipeptidase but only PpASPG1 was able to catalyze efficiently L-asparagine hydrolysis. PpASPG1 contains a variable region of 77 amino acids that is critical for proteolytic processing of the precursor and is retained in the mature enzyme. Furthermore, the functional analysis of deletion mutants demonstrated that this protein fragment is required for specific recognition of the substrate and favors enzyme stability. Potassium has a limited effect on the activation of maritime pine ASPG what is consistent with the lack of a critical residue essential for interaction of cation. Taken together, the results presented here highlight the specific features of ASPG from conifers when compared to the enzymes from angiosperms.

Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.)

PubMed Central

Van Kerckhoven, Sonia H.; de la Torre, Fernando N.; Cañas, Rafael A.; Avila, Concepción; Cantón, Francisco R.; Cánovas, Francisco M.

2017-01-01

Asparaginases (ASPG, EC 3.5.1.1) catalyze the hydrolysis of the amide group of L-asparagine producing L-aspartate and ammonium. Three ASPG, PpASPG1, PpASPG2, and PpASPG3, have been identified in the transcriptome of maritime pine (Pinus pinaster Ait.) that were transiently expressed in Nicotiana benthamiana by agroinfection. The three recombinant proteins were processed in planta to active enzymes and it was found that all mature forms exhibited double activity asparaginase/isoaspartyl dipeptidase but only PpASPG1 was able to catalyze efficiently L-asparagine hydrolysis. PpASPG1 contains a variable region of 77 amino acids that is critical for proteolytic processing of the precursor and is retained in the mature enzyme. Furthermore, the functional analysis of deletion mutants demonstrated that this protein fragment is required for specific recognition of the substrate and favors enzyme stability. Potassium has a limited effect on the activation of maritime pine ASPG what is consistent with the lack of a critical residue essential for interaction of cation. Taken together, the results presented here highlight the specific features of ASPG from conifers when compared to the enzymes from angiosperms. PMID:28690619

Kinetic mechanism and structural requirements of the amine-catalyzed decarboxylation of oxaloacetic acid.

PubMed

Thalji, Nabil K; Crowe, William E; Waldrop, Grover L

2009-01-02

The kinetic and chemical mechanism of amine-catalyzed decarboxylation of oxaloacetic acid at pH 8.0 has been reevaluated using a new and versatile assay. Amine-catalyzed decarboxylation of oxaloacetic acid proceeds via the formation of an imine intermediate, followed by decarboxylation of the intermediate and hydrolysis to yield pyruvate. The decrease in oxaloacetic acid was coupled to NADH formation by malate dehydrogenase, which allowed the rates of both initial carbinolamine formation (as part of the imination step) and decarboxylation to be determined. By comparing the rates observed for a variety of amines and, in particular, diamines, the structural and electronic requirements for diamine-catalyzed decarboxylation at pH 8.0 were identified. At pH 8.0, monoamines were found to be very poor catalysts, whereas some diamines, most notably ethylenediamine, were excellent catalysts. The results indicate that the second amino group of diamines enhances the rate of imine formation by acting as a proton shuttle during the carbinolamine formation step, which enables diamines to overcome high levels of solvation that would otherwise inhibit carbinolamine, and thus imine, formation. The presence of the second amino group may also enhance the rate of the carbinolamine dehydration step. In contrast to the findings of previous reports, the second amino group participates in the reaction by enhancing the rate of decarboxylation via hydrogen-bonding to the imine nitrogen to either stabilize the negative charge that develops on the imine during decarboxylation or preferentially stabilize the reactive imine over the unreactive enamine tautomer. These results provide insight into the precise catalytic mechanism of several enzymes whose reactions are known to proceed via an imine intermediate.

On-line characterization using ultrasound of pectin hydrolysis catalyzed by the enzyme pectinmethylesterase

NASA Astrophysics Data System (ADS)

Aparicio, C.; Resa, P.; Sierra, C.; Elvira, L.

2012-12-01

The major problem in the fruit juice industry is associated with juice quality deterioration due to the cloud loss of juice concentrates by the enzymatic reaction of pectinmethylesterase enzyme (PME, EC 3.1.1.11). During pectin hydrolysis, pectin and water are transformed into polygalacturonic acid (pectate) and methanol by the action of PME. In this work, a low-intensity ultrasonic technique is used to monitor this enzymatic reaction, with PME both from orange peel and from Aspergillus niger. Changes in sound velocity during pectin hydrolysis (1% concentration of pectin, T = 30°C and pH = 4.5 and 7) with 0.25 ml of enzyme solution (PME) have been measured using a through-transmission technique. Sound velocity decreases as pectin is transformed into pectate and methanol and at the end of the process, the change in sound velocity reaches 0.3 m/s with PME from orange peel and 0.33 m/s with PME from Aspergillus niger.

FeCl3-catalyzed ethanol pretreatment of sugarcane bagasse boosts sugar yields with low enzyme loadings and short hydrolysis time.

PubMed

Zhang, Hongdan; Zhang, Shuaishuai; Yuan, Hongyou; Lyu, Gaojin; Xie, Jun

2018-02-01

An organosolv pretreatment system consisting of 60% ethanol and 0.025 mol·L -1 FeCl 3 under various temperatures was developed in this study. During the pretreatment, the highest xylose yield was 11.4 g/100 g raw material, representing 49.8% of xylose in sugarcane bagasse. Structural features of raw material and pretreated substrates were characterized to better understand how hemicellulose removal and delignification affected subsequent enzymatic hydrolysis. The 160 °C pretreated solid presented a remarkable glucose yield of 93.8% for 72 h. Furthermore, the influence of different additives on the enzymatic hydrolysis of pretreated solid was investigated. The results indicated that the addition of Tween 80 shortened hydrolysis time to 6 h and allowed a 50% reduction of enzyme loading to achieve the same level of glucose yield. This work suggested that FeCl 3 -catalyzed organosolv pretreatment could improve the enzymatic hydrolysis significantly and reduce the hydrolysis time and enzyme dosage with the addition of Tween 80. Copyright © 2017 Elsevier Ltd. All rights reserved.

Detoxification of VX by Chloramine-B. Final report, August 1989-April 1992

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Y.C.; Szafraniec, L.L.; Beaudry, W.T.

1993-07-01

At ambient temperature, the nerve agent O-ethyl S-2(diisopropylamino)ethyl methylphosphonothiolate (VX), can be detoxified in an aqueous solution of Chloramine-B CAB, C6H5SQ2N(Cl)Na only in the presence of sufficient acid (pH 3). The thiolo sulfur is first attacked by the reactive species, benzene chlorosulfonamide, to form a chlorosulfonium ion intermediate followed by hydrolysis and substitution reactions with the sulfonamide anion at the P-S bond. These reactions produce strongly acidic products, which further accelerate the initial reaction. Consequently, one of the acidic hydrolysis products of VX, the toxic S-2-(diisopropylamino)ethyl methylphosphonothioic acid (EA 2192) reacts with CAB instantaneously. This acid-catalyzed mechanism is similar tomore » that reported for bivalent sulfides; direct attack by active chlorine is considered insignificant. A neutral VX analog, O,S-diethyl methylphoshonothiolate, reacts with CAB rapidly in H20 with an initial pH of 8.9 but requires the addition of 0.006 N (H+) for the reaction to occur in D20. By comparison, bivalent sulfides are more reactive than the phosphonothiolates, in general, and can be rapidly oxidized in both H20 and D20, even at high pH values. Chloramine-B, VX, Bivalent sulfide, Benzenechlorosulfonamide, Thiolo sulfur, Phosphonothiolate.« less

Crystal structure of a feruloyl esterase belonging to the tannase family: a disulfide bond near a catalytic triad.

PubMed

Suzuki, Kentaro; Hori, Akane; Kawamoto, Kazusa; Thangudu, Ratna Rajesh; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Yamada, Chihaya; Arakawa, Takatoshi; Wakagi, Takayoshi; Koseki, Takuya; Fushinobu, Shinya

2014-10-01

Feruloyl esterase (FAE) catalyzes the hydrolysis of the ferulic and diferulic acids present in plant cell wall polysaccharides, and tannase catalyzes the hydrolysis of tannins to release gallic acid. The fungal tannase family in the ESTHER database contains various enzymes, including FAEs and tannases. Despite the importance of FAEs and tannases in bioindustrial applications, three-dimensional structures of the fungal tannase family members have been unknown. Here, we determined the crystal structure of FAE B from Aspergillus oryzae (AoFaeB), which belongs to the fungal tannase family, at 1.5 Å resolution. AoFaeB consists of a catalytic α/β-hydrolase fold domain and a large lid domain, and the latter has a novel fold. To estimate probable binding models of substrates in AoFaeB, an automated docking analysis was performed. In the active site pocket of AoFaeB, residues responsible for the substrate specificity of the FAE activity were identified. The catalytic triad of AoFaeB comprises Ser203, Asp417, and His457, and the serine and histidine residues are directly connected by a disulfide bond of the neighboring cysteine residues, Cys202 and Cys458. This structural feature, the "CS-D-HC motif," is unprecedented in serine hydrolases. A mutational analysis indicated that the novel structural motif plays essential roles in the function of the active site. © 2014 Wiley Periodicals, Inc.

Expression and Characterization of a PNPLA3 Protein Isoform (I148M) Associated with Nonalcoholic Fatty Liver Disease*

PubMed Central

Huang, Yongcheng; Cohen, Jonathan C.; Hobbs, Helen H.

2011-01-01

A genetic variant of PNPLA3 (patatin-like phospholipase domain-containing 3; PNPLA3-I148M), a serine protease of unknown function, is associated with accumulation of triacylglycerol (TAG) in the liver. To determine the biological substrates of PNPLA3 and the effect of the I148M substitution on enzymatic activity and substrate specificity, we purified and characterized recombinant human PNPLA3 and PNPLA3-I148M. Maximal hydrolytic activity of PNPLA3 was observed against the three major glycerolipids, TAG, diacylglycerol, and monoacylglycerol, with a strong preference for oleic acid as the acyl moiety. Substitution of methionine for isoleucine at position 148 markedly decreased the Vmax of the enzyme for glycerolipids but had only a modest effect on the Km. Purified PNPLA3 also catalyzed the hydrolysis of oleoyl-CoA, but the Vmax was 100-fold lower for oleoyl-CoA than for triolein. The thioesterase activity required the catalytic serine but was only modestly decreased by the I148M substitution. The enzyme had little or no hydrolytic activity against the other lipid substrates tested, including phospholipids, cholesteryl ester, and retinyl esters. Neither the wild-type nor mutant enzyme catalyzed transfer of oleic acid from oleoyl-CoA to glycerophosphate, lysophosphatidic acid, or diacylglycerol, suggesting that the enzyme does not promote de novo TAG synthesis. Taken together, our results are consistent with the notion that PNPLA3 plays a role in the hydrolysis of glycerolipids and that the I148M substitution causes a loss of function, although we cannot exclude the possibility that the enzyme has additional substrates or activities. PMID:21878620

Reversible formation of intermediates during H/sub 3/O/sup +/-catalyzed hydrolysis of amides. Observation of substantial /sup 18/O exchange accompanying the hydrolysis of acetanilide and N-cyclohexylacetamide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slebocka-Tilk, H.; Brown, R.S.; Olekszyk, J.

1987-07-22

Careful mass spectrometric analysis of the /sup 18/O content of approx. 50% enriched acetanilide (2) and N-cyclohexylacetamide (3) recovered from acidic media during the course of hydrolysis reveals that both species suffer /sup 18/O loss. The percent of /sup 18/O exchange per t/sub 1/2/ of hydrolysis increases as (H/sub 3/O/sup +/) decreases. For 2 at 72/sup 0/C the amount of exchange increases from 0.5 +/- 0.5% (per t/sub 1/2/) in 1 M HCl to 9.4 +/- 0.5% in glycine buffer, (H/sub 3/O/sup +/) = 0.003 M. For 3 at 100/sup 0/C the exchange is 1.05 +/- 0.3% (per t/sub 1/2/)more » at 1 M HCl and 9.0 +/- 0.4% in 0.01 M HCl. When these data are used to compute k/sub ex/ (the exchange rate constant), it shows a first-order dependence on (H/sub 3/O/sup +/) followed by a plateau at high (H/sub 3/O/sup +/) for both 2 and 3.« less

Enzymatic hydrolysis of organic phosphorus in swine manure and soil.

PubMed

He, Zhongqi; Griffin, Timothy S; Honeycutt, C Wayne

2004-01-01

Organic phosphorus (Po) exists in many chemical forms that differ in their susceptibility to hydrolysis and, therefore, bioavailability to plants and microorganisms. Identification and quantification of these forms may significantly contribute to effective agricultural P management. Phosphatases catalyze reactions that release orthophosphate (Pi) from Po compounds. Alkaline phosphatase in tris-HCl buffer (pH 9.0), wheat (Triticum aestivum L.) phytase in potassium acetate buffer (pH 5.0), and nuclease P1 in potassium acetate buffer (pH 5.0) can be used to classify and quantify Po in animal manure. Background error associated with different pH and buffer systems is observed. In this study, we improved the enzymatic hydrolysis approach and tested its applicability for investigating Po in soils, recognizing that soil and manure differ in numerous physicochemical properties. We applied (i) acid phosphatase from potato (Solanum tuberosum L.), (ii) acid phosphatases from both potato and wheat germ, and (iii) both enzymes plus nuclease P1 to identify and quantify simple labile monoester P, phytate (myo-inositol hexakis phosphate)-like P, and DNA-like P, respectively, in a single pH/buffer system (100 mM sodium acetate, pH 5.0). This hydrolysis procedure released Po in sequentially extracted H2O, NaHCO3, and NaOH fractions of swine (Sus scrofa) manure, and of three sandy loam soils. Further refinement of the approach may provide a universal tool for evaluating hydrolyzable Po from a wide range of sources.

Exploring the Degradation of Gallotannins Catalyzed by Tannase Produced by Aspergillus niger GH1 for Ellagic Acid Production in Submerged and Solid-State Fermentation.

PubMed

Chávez-González, Mónica L; Guyot, Sylvain; Rodríguez-Herrera, Raul; Prado-Barragán, Arely; Aguilar, Cristóbal N

2018-06-01

Due to great interest on producing bioactive compounds for functional foods and biopharmaceuticals, it is important to explore the microbial degradation of potential sources of target biomolecules. Gallotannins are polyphenols present in nature, an example of them is tannic acid which is susceptible to enzymatic hydrolysis. This hydrolysis is performed by tannase or tannin acyl hydrolase, releasing in this way, biomolecules with high-added value. In the present study, chemical profiles obtained after fungal degradation of tannic acid under two bioprocesses (submerged fermentation (SmF) and solid state fermentation (SSF)) were determined. In both fermentation systems (SmF and SSF), Aspergillus niger GH1 strain and tannic acid as a sole carbon source and inducer were used (the presence of tannic acid promotes production of enzyme tannase). In case of SSF, polyurethane foam (PUF) was used like as support of fermentation; culture medium only was used in case of submerged fermentation. Fermentation processes were monitored during 72 h; samples were taken kinetically every 8 h; and all extracts obtained were partially purified to obtain polyphenolic fraction and then were analyzed by liquid chromatography-mass spectrometry (LC-MS). Molecules like gallic acid and n-galloyl glucose were identified as intermediates in degradation of tannic acid; during SSF was identified ellagic acid production. The results obtained in this study will contribute to biotechnological production of ellagic acid.

Increased saccharification yields from aspen biomass upon treatment with enzymatically generated peracetic acid.

PubMed

Duncan, Shona; Jing, Qing; Katona, Adrian; Kazlauskas, Romas J; Schilling, Jonathan; Tschirner, Ulrike; Aldajani, Waleed Wafa

2010-03-01

The recalcitrance of lignocellulosic biomass to enzymatic release of sugars (saccharification) currently limits its use as feedstock for biofuels. Enzymatic hydrolysis of untreated aspen wood releases only 21.8% of the available sugars due primarily to the lignin barrier. Nature uses oxidative enzymes to selectively degrade lignin in lignocellulosic biomass, but thus far, natural enzymes have been too slow for industrial use. In this study, oxidative pretreatment with commercial peracetic acid (470 mM) removed 40% of the lignin (from 19.9 to 12.0 wt.% lignin) from aspen and enhanced the sugar yields in subsequent enzymatic hydrolysis to about 90%. Increasing the amount of lignin removed correlated with increasing yields of sugar release. Unfortunately, peracetic acid is expensive, and concentrated forms can be hazardous. To reduce costs and hazards associated with using commercial peracetic acid, we used a hydrolase to catalyze the perhydrolysis of ethyl acetate generating 60-70 mM peracetic acid in situ as a pretreatment to remove lignin from aspen wood. A single pretreatment was insufficient, but multiple cycles (up to eight) removed up to 61.7% of the lignin enabling release of >90% of the sugars during saccharification. This value corresponds to a predicted 581 g of fermentable sugars from 1 kg of aspen wood. Improvements in the enzyme stability are needed before the enzymatically generated peracetic acid is a commercially viable alternative.

Organosolv pretreatment for enzymatic hydrolysis of poplars: I. enzyme hydrolysis of cellulosic residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chum, H.L.; Johnson, D.K.; Black, S.

1988-01-01

Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H/sub 2/SO/sub 4/ and H/sub 3/PO/sub 4/) such that the cooking liquor pH less than or equal to 3 are easily digested by enzymes. The total yields of hydrolysis residues (pulps) are in the 40-60% range; the acid-catalyzed delignification followed by enzyme hydrolysis can generate 70-88% of the original six-carbon sugars contained in the wood. Glucomannan and arabinogalactan are dissolved in to the pulping liquor in the pH range of 2-4.5. Lowermore » pH (less than or equal to 3) leads to additional solubilization of six-carbon sugars. These sugars may be fermented directly. From the insoluble hydrolysis residues, 36-41% conversions of wood into fermentable sugars were obtained after enzyme hydrolysis; the starting feedstocks contain 50.8 and 46.6% hexosans, respectively, for aspen and black cottonwood. The kinetics of enzymatic hydrolysis of cellulose can be formally treated as two simultaneous pseudo-first-order reactions in which fast and slow hydrolysis of cellulose occur. Correlations between the glucan digestibility and the effect of the pretreatment have been made. The higher residual xylan content reduces the amount of the rapidly hydrolyzable glucan fraction and lowers the glucan digestibility. The proposed simple kinetic treatment is very helpful in assessing the effect of the pretreatment on pulp enzyme hydrolyzability.« less

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

DOE PAGES

Shiga, Tânia M.; Xiao, Weihua; Yang, Haibing; ...

2017-12-27

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates. Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 degrees C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability,more » and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl3-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose. Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.« less

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiga, Tânia M.; Xiao, Weihua; Yang, Haibing

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates. Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 degrees C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability,more » and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl3-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose. Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.« less

Enzymatic hydrolysis of 1-monoacyl-SN-glycerol-3-phosphoryl-choline (1-lysolecithin) by phospholipases from peanut seeds.

PubMed

Strauss, H; Leibovitz-Ben Gershon, Z; Heller, M

1976-06-01

Hydrolysis of 1-lysolecithin (1-acyl glycerophosphorylcholine [1-acyl GPC]) by preparations of phospholipase D from peanut seeds was investigated. 1-Lysolecithin was hydrolyzed at a much slower rate than phosphatidylcholine (lecithin). Although Ca+2 ions are required for the cleavage of lecithin by the enzyme, their effect on the hydrolysis of lysolecithin depended upon the concentration of the substrate: at 0.2 mM 1-lysolecithin, Ca+2 ions increased the reaction rates, whereas at concentrations of the substrate lower than 0.1 mM, Ca+2 ions were inhibitory. A broad pH activity curve between 5 and 8 was obtained with higher rates in the alkaline range, both in the absence and presence of Ca+2 ions. The increased hydrolysis of lysolecithin due to Ca+2 was noticed over the entire pH range. Upon storage of the enzyme solutions at 4 C, decreased rates of hydrolysis of lecithin were observed, with t 1/2 values of ca. 50 and 100 days depending on the purity of the preparation. During the same period, no reduction occurred in the activity of these preparations on lysolecithin as substrate. The effects of Ca+2 ions and the analysis of the products of 1-acyl GPC cleavage by the enzyme preparations revealed the presence of more than one enzyme and the formation of the following compounds: lysophosphatidic acids (1 acyl glycerophosphoric acids), free fatty acids, glycerophosphorylcholine, and choline. The possible pathways leading to the degradation of lysolecithin and the formation of these products include reactions catalyzed by lysophospholipase A1 (lysophosphatidylcholine 1-acyl hydrolase, E.C. 3.1.1.5) and a phosphodiesterase (L-3-glycerylphosphorylcholine glycerophosphohydrolase, E.C.3.1.4.2), in addition to phospholipase D (phosphatidyl-choline phosphatidohydrolase, E.C. 3.1.4.4).

A New Synthetic Route to N-Benzyl Carboxamides through the Reverse Reaction of N-Substituted Formamide Deformylase

PubMed Central

Hashimoto, Yoshiteru; Sakashita, Toshihide; Fukatsu, Hiroshi; Sato, Hiroyoshi

2014-01-01

Previously, we isolated a new enzyme, N-substituted formamide deformylase, that catalyzes the hydrolysis of N-substituted formamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi, Proc. Natl. Acad. Sci. U. S. A. 101:13726–13731, 2004, doi:10.1073/pnas.0405082101). Here, we discovered that this enzyme catalyzed the reverse reaction, synthesizing N-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction proceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were almost the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the same catalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamine analogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi) mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent release of NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analysis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids with numbers of carbon atoms ranging from C1 to C3), were active as acid substrates for the reverse reaction. Through this reaction, N-substituted carboxamides, such as NBFA, N-benzylacetamide, and N-benzylpropionamide, were synthesized from benzylamine and the corresponding acid substrates. PMID:24123742

Hydrolysis of triacetin catalyzed by immobilized lipases: effect of the immobilization protocol and experimental conditions on diacetin yield.

PubMed

Hernandez, Karel; Garcia-Verdugo, Eduardo; Porcar, Raul; Fernandez-Lafuente, Roberto

2011-05-06

The effect of the immobilization protocol and some experimental conditions (pH value and presence of acetonitrile) on the regioselective hydrolysis of triacetin to diacetin catalyzed by lipases has been studied. Lipase B from Candida antarctica (CALB) and lipase from Rhizomucor miehei (RML) were immobilized on Sepabeads (commercial available macroporous acrylic supports) activated with glutaraldehyde (covalent immobilization) or octadecyl groups (adsorption via interfacial activation). All the biocatalysts accumulated diacetin. Covalently immobilized RML was more active towards rac-methyl mandelate than the adsorbed RML. However, this covalent RML preparation presented the lowest activity towards triacetin. For this reason, this preparation was discarded as biocatalyst for this reaction. At pH 7, acyl migration occurred giving a mixture of 1,2 and 1,3 diacetin, but at pH 5.5, only 1,2 diacetin was produced. Yields were improved at acidic pH values and in the presence of 20% acetonitrile (to over 95%). RML immobilized on octadecyl Sepabeads was proposed as optimal preparation, mainly due to its higher specific activity. Each enzyme preparation presented very different properties. Moreover, changes in the reaction conditions affected the various immobilized enzymes in a different way. Copyright © 2011 Elsevier Inc. All rights reserved.

Hydrolysis of the quinone methide of butylated hydroxytoluene in aqueous solutions.

PubMed

Willcockson, Maren Gulsrud; Toteva, Maria M; Stella, Valentino J

2013-10-01

Butylated hydroxytoluene or BHT is an antioxidant commonly used in pharmaceutical formulations. BHT upon oxidation forms a quinone methide (QM). QM is a highly reactive electrophilic species that can undergo nucleophilic addition. Here, the kinetic reactivity of QM with water at various apparent pH values in a 50% (v/v) water-acetonitrile solution at constant ionic strength of I = 0.5 (NaCl)4 , was studied. The hydrolysis of QM in the presence of added acid, base, sodium chloride, and phosphate buffer resulted in the formation of only one product--the corresponding 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (BA). The rate of BA formation was catalyzed by the addition of acid and base, but not chloride and phosphate species. Nucleophilic excipients, used in the pharmaceutical formulation, or nucleophilic groups on active pharmaceutical ingredient molecule may form adducts with QM, the immediate oxidative product of BHT degradation, thus having implications for drug product impurity profiles. Because of these considerations, BHT should be used with caution in formulations containing drugs or excipients capable of acting as nucleophiles. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Kinetic hydrogen isotope effects in the concerted mechanism for the hydrolysis of acetals, ketals, and ortho esters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eliason, R.; Kreevoy, M.M.

1978-10-25

The hydrolysis of many ortho esters, and some acetals and ketals, is general acid catalyzed, and in some examples these generate linear Bronsted plots over substantial ranges of catalyst acidity. This suggests that the reaction coordinate is primarily a reorganization of heavy atoms since proton transfer from one oxygen to another has been shown to generate strongly curved Bronsted plots. However, the isotopic fractionation factor for the catalytically active proton in these transition states is substantially less than 1.0; in several examples it is less than 0.5. Such values have been thought to require that the reaction coordinate be largelymore » a motion of the hydrogen giving the low fractionation factor. This dilemma has been resolved by the observation and rationalization of fractionation factors as low as 0.28 for stable, hydrogen bridge-bonded complexes, AHA/sup -/. A similar, bounded coordinate is now suggested for the catalytically active protons in question. This permits the reaction coordinate to be pictured. 3 figures, 2 tables.« less

Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics.

PubMed

Majiduddin, Fahd K; Palzkill, Timothy

2003-03-01

Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring beta-lactamases and extended-spectrum beta-lactamases. Four enzymes from the class A group of beta-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these beta-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of beta-lactam antibiotics.

Amino Acid Sequence Requirements at Residues 69 and 238 for the SME-1 β-Lactamase To Confer Resistance to β-Lactam Antibiotics

PubMed Central

Majiduddin, Fahd K.; Palzkill, Timothy

2003-01-01

Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring β-lactamases and extended-spectrum β-lactamases. Four enzymes from the class A group of β-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these β-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of β-lactam antibiotics. PMID:12604542

A coupled photometric assay for characterization of S-adenosyl-l-homocysteine hydrolases in the physiological hydrolytic direction.

PubMed

Kailing, Lyn L; Bertinetti, Daniela; Herberg, Friedrich W; Pavlidis, Ioannis V

2017-10-25

S-Adenosyl-l-homocysteine hydrolases (SAHases) are important metabolic enzymes and their dysregulation is associated with some severe diseases. In vivo they catalyze the hydrolysis of S-adenosyl-l-homocysteine (SAH), the by-product of methylation reactions in various organisms. SAH is a potent inhibitor of methyltransferases, thus its removal from the equilibrium is an important requirement for methylation reactions. SAH hydrolysis is also the first step in the cellular regeneration process of the methyl donor S-adenosyl-l-methionine (SAM). However, in vitro the equilibrium lies towards the synthetic direction. To enable characterization of SAHases in the physiologically relevant direction, we have developed a coupled photometric assay that shifts the equilibrium towards hydrolysis by removing the product adenosine, using a high affinity adenosine kinase (AK). This converts adenosine to AMP and thereby forms equimolar amounts of ADP, which is phosphorylated by a pyruvate kinase (PK), in turn releasing pyruvate. The readout of the assay is the consumption of NADH during the lactate dehydrogenase (LDH) catalyzed reduction of pyruvate to lactic acid. The applicability of the assay is showcased for the determination of the kinetic constants of an SAHase from Bradyrhizobium elkanii (K M,SAH 41±5μM, v max,SAH 25±1μM/min with 0.13mg/mL enzyme). This assay is a valuable tool for in vitro characterization of SAHases with biotechnological potential, and for monitoring SAHase activity in diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme

PubMed Central

Schoefer, Lilian; Braune, Annett; Blaut, Michael

2004-01-01

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37°C and 7.0, respectively. The Km for phloretin was 13 ± 3 μM and the kcat was 10 ± 2 s−1. The enzyme did not transform phloretin-2′-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl2 to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively. PMID:15466559

ATP hydrolysis is critical for induction of conformational changes in GroEL that expose hydrophobic surfaces.

PubMed

Gorovits, B M; Ybarra, J; Horowitz, P M

1997-03-14

The degree of hydrophobic exposure in the molecular chaperone GroEL during its cycle of ATP hydrolysis was analyzed using 1,1'-bis(4-anilino)naphthalene-5,5'disulfonic acid (bisANS), a hydrophobic probe, whose fluorescence is highly sensitive to the environment. In the presence of 10 mM MgCl2 and 10 mM KCl the addition of ATP, but not ADP or AMP-PNP, resulted in a time-dependent, linear increase in the bisANS fluorescence. The rate of the increase in the bisANS fluorescence depended on the concentrations of both GroEL and the probe. The effect could be substantially inhibited by addition of excess ADP or by converting ATP to ADP using hexokinase, showing that the increase in the bisANS fluorescence was correlated with ATP hydrolysis. The rate of ATP hydrolysis catalyzed by GroEL was uncompetitively inhibited in the presence of bisANS. This uncompetitive inhibition suggests that the probe can interact with the GroEL-ATP complex. The inability of the nonhydrolyzable ATP analog, AMP-PNP, to cause a similar effect is explained by the interaction of bisANS with a transient conformational state of GroEL formed consequent to ATP hydrolysis. It is suggested that this short lived hydrophobic exposure reflects a conformational shift in GroEL that results from electrostatic repulsion between the bound products of ATP hydrolysis, and it plays an important role in the mechanism of the chaperonin cycle.

Selective ultrasound-enhanced enzymatic hydrolysis of oleuropein to its aglycon in olive (Olea europaea L.) leaf extracts.

PubMed

Delgado-Povedano, María Del Mar; Priego-Capote, Feliciano; Luque de Castro, María Dolores

2017-04-01

Hydrolysis of oleuropein, the main phenol in olive (Olea europaea L.) leaf extracts, to oleuropein aglycon and other subsequent products in the hydrolytic pathway can be catalyzed by different enzymes. Three of the most used hydrolases were assayed to catalyze the process, and β-glucosidase from Aspergillus niger was selected. Acceleration of the enzymatic hydrolysis by ultrasound (US) was studied using a Box-Behnken design (duty cycle, amplitude, cycle time) and an oleuropein standard, and the optimum US conditions for achieving maximum yield of oleuropein aglycon were 0.5s/s duty cycle, 50% amplitude and 45s cycle. The method was applied to obtain oleuropein aglycon from commercial and laboratory extracts from olive leaves, which may have a pharmacological use as deduced by its healthy properties. The kinetics of the US-assisted enzymatic hydrolysis was monitored by analysis of the target compounds using liquid chromatography-tandem mass spectrometry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toward antibody-catalyzed hydrolysis of organophosphorus poisons

PubMed Central

Vayron, Philippe; Renard, Pierre-Yves; Taran, Frédéric; Créminon, Christophe; Frobert, Yveline; Grassi, Jacques; Mioskowski, Charles

2000-01-01

We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-α-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-α-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M−1⋅min−1 at pH 7.4 and 25°C, which corresponds to a catalytic proficiency of 14,400 M−1 toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents. PMID:10860971

Enzymatic modification of natural and synthetic polymers using lipases and proteases

NASA Astrophysics Data System (ADS)

Chakraborty, Soma

Enzymatic modification of natural/synthetic polymers [starch nanoparticles, poly (n-alkyl acrylates) and poly(vinyl formamide)] was studied. Enzymes used for catalysis were lipases and proteases. Starch nanoparticles (40nm diameter) were incorporated into AOT-coated reverse micelles. Reactions performed with the acylating agents vinyl stearate, epsilon-caprolactone and maleic anhydride in toluene in presence of Novozyme-435 at 40°C for 36h gave products with degrees of substitution of 0.8, 0.6 and 0.4 respectively. DEPT-135 NMR spectra revealed that the modification occurred regioselectively at the C-6 position of the glucose units. Infrared microspectroscopy showed that the surfactant coated starch nanoparticles diffuse into pores of Novozyme-435 beads, coming in close proximity with CALB to promote modification. The modified products retained nanoscale dimensions. Catalysis of amide bond formation between a low molar mass amine and ester side groups of poly(n-alkyl acrylates)[poly(ethyl acrylate), poly(methyl acrylate) and poly(butyl acrylate)] was also examined. The nucleophiles were mono and diamines. Among the poly(n-alkyl acrylates) and the lipases studied, poly(ethyl acrylate) was the preferred substrate and Novozyme-435 the most active lipase. Poly(ethyl acrylate) in 80% by-volume toluene was reacted with 1 equivalent per repeat unit of hexyl amine at 70°C in presence of Novozyme-435. The product contained 10.6 mol% amide groups. Attempts to increase the amidation beyond 10--11 mol% by increasing the reaction time or use of fresh enzyme were unsuccessful, showing that poly(ethylacrylate-co-10mol%hexylacrylamide) is a poor substrate for further acylation. When chiral amines ([R,S]-alpha-methyl benzylamine, [R,S]-beta-methyl phenyl amine) were used as nucleophiles, Novozyme-435 enantioselectively catalyzed amidation of poly(ethyl acrylate). Poly(vinyl formamide), P(VfAm) by acid or base-catalyzed hydrolysis leads to poly(vinylamine), P(VAm), and corresponding copolymers. As an alternative to chemical hydrolysis a mild and selective enzymatic method was discovered. Fifteen proteases were evaluated for this transformation. Of these, PROT 7 was the most active. Within 24h PROT 7 gave products with 44% hydrolysis. Further hydrolysis was not observed by extending the reaction time because poly(vinylformamide-co-40%vinylamine) is a poor substrate for further hydrolysis. The sequence distribution of copolymers formed by chemical hydrolysis and enzymatic hydrolysis was compared. Chemical hydrolysis gave random copolymer. In contrast, PROT 7 gave block-like arrangement of VAm units.

Aroma Precursors in Grapes and Wine: Flavor Release during Wine Production and Consumption.

PubMed

Parker, Mango; Capone, Dimitra L; Francis, I Leigh; Herderich, Markus J

2018-03-14

Pioneering investigations into precursors of fruity and floral flavors established the importance of terpenoid and C 13 -norisoprenoid glycosides to the flavor of aromatic wines. Nowadays flavor precursors in grapes and wine are known to be structurally diverse, encompassing glycosides, amino acid conjugates, odorless volatiles, hydroxycinnamic acids, and many others. Flavor precursors mainly originate in the grape berry but also from oak or other materials involved in winemaking. Flavors are released from precursors during crushing and subsequent production steps by enzymatic and nonenzymatic transformations, via microbial glycosidases, esterases, C-S lyases, and decarboxylases, and through acid-catalyzed hydrolysis and chemical rearrangements. Flavors can also be liberated from glycosides and amino acid conjugates by oral microbiota. Hence, it is increasingly likely that flavor precursors contribute to retronasal aroma formation through in-mouth release during consumption, prompting a shift in focus from identifying aroma precursors in grapes to understanding aroma precursors present in bottled wine.

Crystallization and preliminary X-ray analysis of recombinant human acid beta-glucocerebrosidase, a treatment for Gaucher's disease

NASA Technical Reports Server (NTRS)

Roeber, Dana; Achari, Aniruddha; Manavalan, Partha; Edmunds, Tim; Scott, David L.

2003-01-01

Acid beta-glucocerebrosidase (N-acylsphingosyl-1-O-beta-D-glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme (Genzyme Corporation, Cambridge, MA, USA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is used in the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. Here, the crystallization and the initial diffraction analysis of Cerezyme are reported. The crystals are C-centered orthorhombic, with unit-cell parameters a = 285.0, b = 110.2, c = 91.7 A. A 99.9% complete data set has been collected to 2.75 A with an R(sym) of 8.8%.

Crystallization and Preliminary X-ray analysis of Human Recombinant Acid beta-glucocerebrosidase, a treatment for Gaucher's Disease

NASA Technical Reports Server (NTRS)

Roeber, Dana F.; Achari, Aniruddha; Manavalan, Partha; Edmunds, Tim; Scott, David L.; Curreri, Peter A. (Technical Monitor)

2002-01-01

Acid beta-glucocerebrosidase (N-acylsphingosyl - O - beta-D - glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme(R) (Genzyme Corporation, Cambridge, MA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is commercially available for the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. We report the crystallization and the initial diffraction analysis of Cerezyme(R). The crystals are C-centered orthorhombic, with unit-cell parameters of a = 285.0 A, b = 110.2 A, and c = 91.7 A. A 99.9 A complete data set has been collected to 2.75 A with an R(sub sym) of 8.8 %.

Characterization of a tannase from Emericella nidulans immobilized on ionic and covalent supports for propyl gallate synthesis.

PubMed

Gonçalves, Heloísa Bressan; Jorge, João Atílio; Pessela, Benevides Costa; Lorente, Glória Fernandez; Guisán, José Manuel; Guimarães, Luis Henrique Souza

2013-04-01

The extracellular tannase from Emericela nidulans was immobilized on different ionic and covalent supports. The derivatives obtained using DEAE-Sepharose and Q-Sepharose were thermally stable from 60 to 75 °C, with a half life (t50) >24 h at 80 °C at pH 5.0. The glyoxyl-agarose and amino-glyoxyl derivatives showed a thermal stability which was lower than that observed for ionic supports. However, when the stability to pH was considered, the derivatives obtained from covalent supports were more stable than those obtained from ionic supports. DEAE-Sepharose and Q-Sepharose derivatives as well as the free enzyme were stable in 30 and 50 % (v/v) 1-propanol. The CNBr-agarose derivative catalyzed complete tannic acid hydrolysis, whereas the Q-Sepharose derivative catalyzed the transesterification reaction to produce propyl gallate (88 % recovery), which is an important antioxidant.

Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases.

PubMed

Hajighasemi, Mahbod; Nocek, Boguslaw P; Tchigvintsev, Anatoli; Brown, Greg; Flick, Robert; Xu, Xiaohui; Cui, Hong; Hai, Tran; Joachimiak, Andrzej; Golyshin, Peter N; Savchenko, Alexei; Edwards, Elizabeth A; Yakunin, Alexander F

2016-06-13

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

Reaction Kinetic Model of Dilute Acid-Catalyzed Hemicellulose Hydrolysis of Corn Stover under High-Solid Conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Suan; Guan, Wenjian; Kang, Li

High solid conditions are desirable in pretreatment of lignocellulosic biomass. An advanced dilute-acid pretreatment reactor has been developed at National Renewable Energy Laboratory (NREL). It is a continuous auger-driven reactor that can be operated with high-solid charge at high temperature and with short residence time resulting high productivity and high sugar concentration. Here, we investigated the kinetics of the reactions associated with dilute-acid pretreatment of corn stover, covering the reaction conditions of the NREL reactor operation: 155-185 C, 1-2 wt% sulfuric acid concentration, and 1:2 solid to liquid ratio. The experimental data were fitted to a first-order biphasic model whichmore » assumes that xylan is comprised of two different fragments: fast and slow reacting fractions. Due to the high solid loading condition, significant amount of xylose oligomers was observed during the pretreatment. We also included the oligomers as an intermediate entity in the kinetic model. The effect of acid concentration was incorporated into the pre-exponential factor of Arrhenius equation. The kinetic model with bestfit kinetic parameters has shown good agreement with experimental data. The kinetic parameter values of the proposed model were noticeably different from those previously reported. The activation energies of xylan hydrolysis are lower and the acid exponents are higher than the average of literature values. The proposed model can serve as a useful tool for design and operation of pretreatment system pertaining to corn stover.« less

Reaction Kinetic Model of Dilute Acid-Catalyzed Hemicellulose Hydrolysis of Corn Stover under High-Solid Conditions

DOE PAGES

Shi, Suan; Guan, Wenjian; Kang, Li; ...

2017-09-13

High solid conditions are desirable in pretreatment of lignocellulosic biomass. An advanced dilute-acid pretreatment reactor has been developed at National Renewable Energy Laboratory (NREL). It is a continuous auger-driven reactor that can be operated with high-solid charge at high temperature and with short residence time resulting high productivity and high sugar concentration. Here, we investigated the kinetics of the reactions associated with dilute-acid pretreatment of corn stover, covering the reaction conditions of the NREL reactor operation: 155-185 C, 1-2 wt% sulfuric acid concentration, and 1:2 solid to liquid ratio. The experimental data were fitted to a first-order biphasic model whichmore » assumes that xylan is comprised of two different fragments: fast and slow reacting fractions. Due to the high solid loading condition, significant amount of xylose oligomers was observed during the pretreatment. We also included the oligomers as an intermediate entity in the kinetic model. The effect of acid concentration was incorporated into the pre-exponential factor of Arrhenius equation. The kinetic model with bestfit kinetic parameters has shown good agreement with experimental data. The kinetic parameter values of the proposed model were noticeably different from those previously reported. The activation energies of xylan hydrolysis are lower and the acid exponents are higher than the average of literature values. The proposed model can serve as a useful tool for design and operation of pretreatment system pertaining to corn stover.« less

Structure of the Cell Wall of Bacillus stearothermophilus: Mode of Action of a Thermophilic Bacteriophage Lytic Enzyme

PubMed Central

Welker, N. E.

1971-01-01

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings. PMID:4255338

Rubber muscle actuation with pressurized CO2 from enzyme-catalyzed urea hydrolysis

NASA Astrophysics Data System (ADS)

Sutter, Thomas M.; Dickerson, Matthew B.; Creasy, Terry S.; Justice, Ryan S.

2013-09-01

A biologically inspired pneumatic pressure source was designed and sized to supply high pressure CO2(g) to power a rubber muscle actuator. The enzyme urease served to catalyze the hydrolysis of urea, producing CO2(g) that flowed into the actuator. The actuator’s power envelope was quantified by testing actuator response on a custom-built linear-motion rig. Reaction kinetics and available work density were determined by replacing the actuator with a double-action piston and measuring volumetric gas generation against a fixed pressure on the opposing piston. Under the conditions investigated, urease catalyzed the generation of up to 0.81 MPa (117 psi) of CO2(g) in the reactor headspace within 18 min, and the evolved gas produced a maximum work density of 0.65 J ml-1.

Acid monolayer functionalized iron oxide nanoparticle catalysts

NASA Astrophysics Data System (ADS)

Ikenberry, Myles

Superparamagnetic iron oxide nanoparticle functionalization is an area of intensely active research, with applications across disciplines such as biomedical science and heterogeneous catalysis. This work demonstrates the functionalization of iron oxide nanoparticles with a quasi-monolayer of 11-sulfoundecanoic acid, 10-phosphono-1-decanesulfonic acid, and 11-aminoundecanoic acid. The carboxylic and phosphonic moieties form bonds to the iron oxide particle core, while the sulfonic acid groups face outward where they are available for catalysis. The particles were characterized by thermogravimetric analysis (TGA), transmission electron microscopy (TEM), potentiometric titration, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), inductively coupled plasma optical emission spectrometry (ICP-OES), X-ray photoelectron spectrometry (XPS), and dynamic light scattering (DLS). The sulfonic acid functionalized particles were used to catalyze the hydrolysis of sucrose at 80° and starch at 130°, showing a higher activity per acid site than the traditional solid acid catalyst Amberlyst-15, and comparing well against results reported in the literature for sulfonic acid functionalized mesoporous silicas. In sucrose catalysis reactions, the phosphonic-sulfonic nanoparticles (PSNPs) were seen to be incompletely recovered by an external magnetic field, while the carboxylic-sulfonic nanoparticles (CSNPs) showed a trend of increasing activity over the first four recycle runs. Between the two sulfonic ligands, the phosphonates produced a more tightly packed monolayer, which corresponded to a higher sulfonic acid loading, lower agglomeration, lower recoverability through application of an external magnetic field, and higher activity per acid site for the hydrolysis of starch. Functionalizations with 11-aminoundecanoic acid resulted in some amine groups binding to the surfaces of iron oxide nanoparticles. This amine binding is commonly ignored in iron oxide nanoparticle syntheses and functionalizations for biomedical and catalytic applications, affecting understandings of surface charge and other material properties.

The Acid-Base Properties, Hydrolytic Mechanism, and Susceptibility to O2 Oxidation of Fe4S4(SR)4-2 Clusters

PubMed Central

Bruice, Thomas C.; Maskiewicz, Richard; Job, Robert

1975-01-01

The iron-sulfur cluster compounds Fe4S4(SR)4-2 [where —SR = —SCH3, —S—C(CH3)3, and —S— CH2—CH(CH3)2] have been found to represent the base species of weak acids of pKa comparable to that of carboxylic acids. The acid species Fe4S4(SR)4H- is most subject to reaction with O2 and to acid-catalyzed solvolysis, while the base species Fe4S4(SR)4-2 most readily undergoes ligand exchange. The kinetics for hydrolysis of the isobutyl mercaptide cluster salt has been investigated in detail and a mechanism involving the stepwise process [Formula: see text] has been proposed. The importance of the acid-base equilibria in determining the reactivity of the iron-sulfur clusters and its possible importance as a factor in the determination of the potentials of ferredoxins and high potential iron protein are discussed. PMID:16592211

Study of the kinetic parameters for synthesis and hydrolysis of pharmacologically active salicin isomer catalyzed by baker's yeast maltase

NASA Astrophysics Data System (ADS)

Veličković, D. V.; Dimitrijević, A. S.; Bihelović, F. J.; Jankov, R. M.; Milosavić, N.

2011-12-01

One of the key elements for understanding enzyme reactions is determination of its kinetic parameters. Since transglucosylation is kinetically controlled reaction, besides the reaction of synthesis, very important is the reaction of enzymatic hydrolysis of created product. Therefore, in this study, kinetic parameters for synthesis and secondary hydrolysis of pharmacologically active α isosalicin by baker's yeast maltase were calculated, and it was shown that specifity of maltase for hydrolysis is approximately 150 times higher then for synthesis.

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

PubMed

Rival, S; Besson, C; Saulnier, J; Wallach, J

1999-02-01

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.

Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods.

PubMed

Tang, Pei-Ling; Hassan, Osman; Maskat, Mohamad Yusof; Badri, Khairiah

2015-01-01

In this study, oil palm empty fruit bunch (OPEFBF) was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen) and enzymatic [cutinase versus manganese peroxidase (MnP)] approaches was investigated. The effects of sodium hydroxide concentration (2, 5, and 10% wt.) and reaction time (30, 90, and 180 minutes) on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield (333.17 ± 49.44 ppm hydroxybenzoic acid, 5.67 ± 0.25 ppm p-hydroxybenzaldehyde, 25.57 ± 1.64 ppm vanillic acid, 168.68 ± 23.23 ppm vanillin, 75.44 ± 6.71 ppm syringic acid, 815.26 ± 41.77 ppm syringaldehyde, 15.21 ± 2.19 ppm p-coumaric acid, and 44.75 ± 3.40 ppm ferulic acid), among the tested methods. High sodium hydroxide concentration (10% wt.) was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid). Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g(-1) lignin at pH 8 and 55°C for 24 hours, about 642.83 ± 14.45 ppm hydroxybenzoic acid, 70.19 ± 3.31 ppm syringaldehyde, 22.80 ± 1.04 ppm vanillin, 27.06 ± 1.20 ppm p-coumaric acid, and 50.19 ± 2.23 ppm ferulic acid were produced.

PROCESSING ALTERNATIVES FOR DESTRUCTION OF TETRAPHENYLBORATE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lambert, D; Thomas Peters, T; Samuel Fink, S

Two processes were chosen in the 1980's at the Savannah River Site (SRS) to decontaminate the soluble High Level Waste (HLW). The In Tank Precipitation (ITP) process (1,2) was developed at SRS for the removal of radioactive cesium and actinides from the soluble HLW. Sodium tetraphenylborate was added to the waste to precipitate cesium and monosodium titanate (MST) was added to adsorb actinides, primarily uranium and plutonium. Two products of this process were a low activity waste stream and a concentrated organic stream containing cesium tetraphenylborate and actinides adsorbed on monosodium titanate (MST). A copper catalyzed acid hydrolysis process wasmore » built to process (3, 4) the Tank 48H cesium tetraphenylborate waste in the SRS's Defense Waste Processing Facility (DWPF). Operation of the DWPF would have resulted in the production of benzene for incineration in SRS's Consolidated Incineration Facility. This process was abandoned together with the ITP process in 1998 due to high benzene in ITP caused by decomposition of excess sodium tetraphenylborate. Processing in ITP resulted in the production of approximately 1.0 million liters of HLW. SRS has chosen a solvent extraction process combined with adsorption of the actinides to decontaminate the soluble HLW stream (5). However, the waste in Tank 48H is incompatible with existing waste processing facilities. As a result, a processing facility is needed to disposition the HLW in Tank 48H. This paper will describe the process for searching for processing options by SRS task teams for the disposition of the waste in Tank 48H. In addition, attempts to develop a caustic hydrolysis process for in tank destruction of tetraphenylborate will be presented. Lastly, the development of both a caustic and acidic copper catalyzed peroxide oxidation process will be discussed.« less

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries.

PubMed

Cobucci-Ponzano, Beatrice; Strazzulli, Andrea; Iacono, Roberta; Masturzo, Giuseppe; Giglio, Rosa; Rossi, Mosè; Moracci, Marco

2015-10-01

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2h vs 12h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5'-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP).

PubMed

Guranowski, Andrzej; Wojdyła, Anna Maria; Zimny, Jarosław; Wypijewska, Anna; Kowalska, Joanna; Jemielity, Jacek; Davis, Richard E; Bieganowski, Paweł

2010-01-04

Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5'-phosphosulfate (APS or SO(4)-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed.

Acetone and Butanone Metabolism of the Denitrifying Bacterium “Aromatoleum aromaticum” Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

PubMed Central

Schühle, Karola

2012-01-01

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium “Aromatoleum aromaticum” is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)2 composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 Pi. The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content. PMID:22020645

Human mitochondrial pyrophosphatase: cDNA cloning and analysis of the gene in patients with mtDNA depletion syndromes.

PubMed

Curbo, Sophie; Lagier-Tourenne, Clotilde; Carrozzo, Rosalba; Palenzuela, Lluis; Lucioli, Simona; Hirano, Michio; Santorelli, Filippo; Arenas, Joaquin; Karlsson, Anna; Johansson, Magnus

2006-03-01

Pyrophosphatases (PPases) catalyze the hydrolysis of inorganic pyrophosphate generated in several cellular enzymatic reactions. A novel human pyrophosphatase cDNA encoding a 334-amino-acid protein approximately 60% identical to the previously identified human cytosolic PPase was cloned and characterized. The novel enzyme, named PPase-2, was enzymatically active and catalyzed hydrolysis of pyrophosphate at a rate similar to that of the previously identified PPase-1. A functional mitochondrial import signal sequence was identified in the N-terminus of PPase-2, which targeted the enzyme to the mitochondrial matrix. The human pyrophosphatase 2 gene (PPase-2) was mapped to chromosome 4q25 and the 1.4-kb mRNA was ubiquitously expressed in human tissues, with highest levels in muscle, liver, and kidney. The yeast homologue of the mitochondrial PPase-2 is required for mitochondrial DNA maintenance and yeast cells lacking the enzyme exhibit mitochondrial DNA depletion. We sequenced the PPA2 gene in 13 patients with mitochondrial DNA depletion syndromes (MDS) of unknown cause to determine if mutations in the PPA2 gene of these patients were associated with this disease. No pathogenic mutations were identified in the PPA2 gene of these patients and we found no evidence that PPA2 gene mutations are a common cause of MDS in humans.

Hydrolysis of Methylal Catalyzed by Ion Exchange Resins in Aqueous Media

NASA Astrophysics Data System (ADS)

He, Gaoyin; Dai, Fangfang; Shi, Midong; Li, Qingsong; Yu, Yingmin

2018-05-01

In the present work, the chemical equilibrium and kinetics of methylal (PODE1) hydrolysis catalyzed by ion-exchange resin in aqueous solutions were investigated. The study covers temperatures between 333.15 and 363.15 K at various starting compositions covering (PODE1 + MeOH)/water molar ratio ranges from 0.5 to 1.5 in a time scale. On the basis of the experimental results, a mole fraction-based model of the chemical equilibrium and a pseudohomogeneous model are proposed to fit data based on true amount of monomeric formaldehyde. It has been demonstrated that the hydrolysis of PODE1 is slightly endothermic with the enthalpy 8.19 kJ/mol and the rate determining step. Finally, a feed-forward artificial neural networks (ANN) model is developed to model the concentration change of methanol in aqueous solutions. The results showed that the predicted data from designed ANN model were in good agreement with the experimental data with the coefficient ( R 2) of 0.98. Designed ANN provides a reliable method for modeling the hydrolysis reaction of methylal (PODE1).

Involvement of cytosolic NAD+ glycohydrolase in cyclic ADP-ribose metabolism.

PubMed

Matsumura, N; Tanuma, S

1998-12-18

The NAD+ glycohydrolase homogeneously purified from bovine brain cytosol was found to catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). Although the formation of cADPR from NAD+ does not exceed about 2% of the reaction products, the cyclase activity is clearly evidenced by its conversion of NGD+ to cyclic GDP-ribose (cGDPR), which cannot be hydrolyzed to GDPR. Importantly, a steep increase in cADPR hydrolytic activity was observed at cADPR concentrations above 60 microM, which could be reproduced on a Hill curve with a Hill coefficient of 2. Thus, the allosteric binding of cADPR to the NAD+ glycohydrolase (E) molecule promotes the hydrolysis of cADPR. These results suggest that NAD+ hydrolysis to ADPR and nicotinamide catalyzed by the NAD+ glycohydrolase occurs through the formation of a cADPR. E. cADP-ribosyl complex. The low production of cADPR by NAD+ glycohydrolase compared with invertebrate ADP-ribosyl cyclase is believed to be attributable to the fast hydrolysis of cADPR by the allosteric effect of cADPR bound to the same enzyme that produces it. Copyright 1998 Academic Press.

Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds Along Two Different Reaction Pathways: Implica-tions for Engineering Bioscavengers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wymore, Troy W; Langan, Paul; Smith, Jeremy C

Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ( bioscavengers ) is an emerging prophylactic approach to diminishing their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical (QM/MM) umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalentmore » intermediate. P F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily due to the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed -propeller folds for more efficient degradation of OP compounds.« less

Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds along Two Different Reaction Pathways: Implications for Engineering Bioscavengers

PubMed Central

2015-01-01

Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents (“bioscavengers”) is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P–F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds. PMID:24720808

Influence of thermal processing on hydrolysis and stability of folate poly-gamma-glutamates in broccoli (Brassica oleracea var. italica), carrot (Daucus carota) and tomato (Lycopersicon esculentum).

PubMed

Munyaka, Ann Wambui; Verlinde, Philippe; Mukisa, Ivan Muzira; Oey, Indrawati; Van Loey, Ann; Hendrickx, Marc

2010-04-14

The folate poly-gamma-glutamate profile, their concentrations, and hydrolysis by endogenous gamma-glutamyl hydrolase (GGH) were evaluated in broccoli, carrot and tomato. Further studies on the effect of time and temperature on folate poly-gamma-glutamate hydrolysis and stability were carried out in broccoli since this vegetable showed the highest long-chain and total folate poly-gamma-glutamate concentration. The evolution of l-ascorbic acid, total phenols and Trolox equivalent antioxidant capacity (TEAC) values was evaluated in parallel. Upon thermal inactivation of GGH prior to crushing, it was observed that broccoli, carrot and tomato contained poly-gamma-glutamates with one to seven glutamate residues but differed in the predominant poly-gamma-glutamates. Crushing of raw broccoli, carrot and tomato resulted in significant poly-gamma-glutamate profile changes in broccoli and carrot (indicating GGH-catalyzed hydrolysis) but not in tomato. In this study, the actual crushing of raw broccoli matrix had a greater effect on folate poly-gamma-glutamate hydrolysis than incubation conditions (0-30 min at 25-55 degrees C). During treatments at 25-140 degrees C, folate retention was higher at 80 and 100 degrees C than at the other temperatures. A similar trend in thermal stability was observed for folates, vitamin C, total phenols and TEAC value, an indication that conditions that result in endogenous antioxidants degradation might also result in folate degradation.

The metabolism of isocytidine in Escherichia coli

PubMed Central

Doskočil, J.; Holý, A.; Filip, J.

1974-01-01

Intact cells and cell-free extracts of E. coli convert isocytidine to isocytosine and uracil. The radioactive label of 5-[3H]isocytidine is incorporated into RNA and, DNA of growing bacteria at a rate equal to about 1.4% of that of cytidine under similar conditions; the radioactivity is found in uridylic, cytidylic and 2′-deoxythymidylic acids, while less than 0.4% of incorporated radioactive material might be due to possible incorporation of intact isocytidine. Uridine phosphorylase and cytidine deaminase apparently do not participate in the metabolic conversion of isocytidine. 5-[3H]isocytidine was prepared by platinum-catalyzed dehalogenation of 5-bromoisocytidine in the presence of tritium. The 5-bromo derivative was obtained from 2′,3′-0- -isopropylideneisocytidine by N-bromsuccinimide bromination followed by acidic hydrolysis. PMID:10793683

Metal-Catalyzed Cleavage of tRNA[superscript Phe

ERIC Educational Resources Information Center

Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.

2008-01-01

This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which students assay metal-catalyzed hydrolysis of tRNA[superscript Phe]…

Methods and catalysts for making biodiesel from the transesterification and esterification of unrefined oils

DOEpatents

Yan, Shuli [Detroit, MI; Salley, Steven O [Grosse Pointe Park, MI; Ng, K Y. Simon [West Bloomfield, MI

2012-04-24

A method of forming a biodiesel product and a heterogeneous catalyst system used to form said product that has a high tolerance for the presence of water and free fatty acids (FFA) in the oil feedstock is disclosed. This catalyst system may simultaneously catalyze both the esterification of FAA and the transesterification of triglycerides present in the oil feedstock. The catalyst system according to one aspect of the present disclosure represents a class of zinc and lanthanum oxide heterogeneous catalysts that include different ratios of zinc oxide to lanthanum oxides (Zn:La ratio) ranging from about 10:0 to 0:10. The Zn:La ratio in the catalyst is believed to have an effect on the number and reactivity of Lewis acid and base sites, as well as the transesterification of glycerides, the esterification of fatty acids, and the hydrolysis of glycerides and biodiesel.

Peptide and protein quantitation by acid-catalyzed 18O-labeling of carboxyl groups.

PubMed

Haaf, Erik; Schlosser, Andreas

2012-01-03

We have developed a new method that applies acidic catalysis with hydrochloric acid for (18)O-labeling of peptides at their carboxyl groups. With this method, peptides get labeled at their C-terminus, at Asp and Glu residues, and at carboxymethylated cysteine residues. Oxygen atoms at phosphate groups of phosphopeptide are not exchanged. Our elaborated labeling protocol is easy to perform, fast (5 h and 30 min), and results in 95-97 atom % incorporation of (18)O at carboxyl groups. Undesired side reactions, such as deamidation or peptide hydrolysis, occur only at a very low level under the conditions applied. In addition, data analysis can be performed automatically using common software tools, such as Mascot Distiller. We have demonstrated the capability of this method for the quantitation of peptides as well as for phosphopeptides. © 2011 American Chemical Society

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II. Directing Influence of RNA in the Reaction

PubMed Central

Leis, Jonathan P.; Hurwitz, Jerard

1972-01-01

The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg2+, and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qβ; and synthetic homopolymers such as polyadenylate·polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3′ hydroxyl ends of primer strands. The product is an RNA·DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S. PMID:4333539

Oligonucleotide synthesis catalyzed by the Zn/2+/ ion

NASA Technical Reports Server (NTRS)

Sawai, H.; Orgel, L. E.

1975-01-01

Results of experiments are reported in which Zn(2+) ion catalyzed the formation of oligonucleotides from nucleoside phosphorimidazolides in aqueous solution, even in the absence of a template. Specifically, the imidazolides (ImpU or ImpA) polymerized to form ImpApA, and pApA, pApApA, and pApApApA, or the analogous uracil compounds. In addition, the expected hydrolysis products of the hydrolysis of ImpA were formed (pA, imidazole). Judging from the ratio of pA(n) over pA (with and without zinc ion), this ion increased the efficiency of phosphodiester-bond formation by up to 10 times. Possible mechanisms for the reaction are tentatively proposed.

Regio-selective lipase catalyzed hydrolysis of oxanorbornane-based sugar-like amphiphiles at air-water interface: A polarized FT-IRRAS study.

PubMed

Sarangi, Nirod Kumar; Ganesan, M; Muraleedharan, K M; Patnaik, Archita

2017-04-01

Interfacial hydrolysis of oxanorbornane-based amphiphile (Triol C16) by Candida rugosa lipase was investigated using real-time polarized Fourier transform-infrared reflection absorption spectroscopy (FT-IRRAS). The kinetics of hydrolysis was studied by analyzing the ester carbonyl ν(CO) stretching vibration band across the two dimensional (2D) array of molecules at the confined interface. In particular, we demonstrate Triol C16 to form Michaelis-Menten type complex, like that of lipid-substrate analogues, where the Triol C16 head group remained accessible to the catalytic triad of the lipase. The enzyme-induced selective cleavage of the ester bond was spectroscopically monitored by the disappearance of the intense ν(CO) resonance at 1736cm -1 . Consequently, the in situ spectroscopic measurements evidenced selective ester hydrolysis of Triol C16 yielding Tetrol C 2 OH and Palmitic acid, which remained predominantly in the undissociated form at the interface. The conformation sensitive amide I (majorly ν(CO)) and the interfacial water reorganization suggested 2D ordering of the enzyme molecules following which interfacial reactions were employed towards probing the enzyme kinetics at the air/water interface. The investigation demonstrated further the potential of IRRAS spectroscopy for real-time monitoring the hydrolytic product formation and selectivity at biomimetic interfaces. Copyright © 2017 Elsevier B.V. All rights reserved.

Arginase Inhibitor in the Pharmacological Correction of Endothelial Dysfunction

PubMed Central

Pokrovskiy, Mihail V.; Korokin, Mihail V.; Tsepeleva, Svetlana A.; Pokrovskaya, Tatyana G.; Gureev, Vladimir V.; Konovalova, Elena A.; Gudyrev, Oleg S.; Kochkarov, Vladimir I.; Korokina, Liliya V.; Dudina, Eleonora N.; Babko, Anna V.; Terehova, Elena G.

2011-01-01

This paper is about a way of correction of endothelial dysfunction with the inhibitor of arginase: L-norvaline. There is an imbalance between vasoconstriction and vasodilatation factors of endothelium on the basis of endothelial dysfunction. Among vasodilatation agents, nitrogen oxide plays the basic role. Amino acid L-arginine serves as a source of molecules of nitrogen oxide in an organism. Because of the high activity of arginase enzyme which catalyzes the hydrolysis of L-arginine into ornithine and urea, the bioavailability of nitrogen oxide decreases. The inhibitors of arginase suppress the activity of the given enzyme, raising and production of nitrogen oxide, preventing the development of endothelial dysfunction. PMID:21747978

Recent research progress on microbial L-asparaginases.

PubMed

Zuo, Shaohua; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2015-02-01

L-Asparaginases (EC 3.5.1.1) are enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and found in a variety of organisms from microorganisms to mammals. However, they are mainly expressed and produced by microorganisms. Microbial L-asparaginases have received sustained attention due to their irreplaceable role in the therapy of acute lymphoblastic leukemia and for their inhibition of acrylamide formation during food processing. In this article, we review the application of microbial L-asparaginases in medical treatments and acrylamide mitigation. In addition, we describe in detail recent advances in the existing sources, purification, production, properties, molecular modification, and immobilization of L-asparaginase.

Selective deuteration for molecular insights into the digestion of medium chain triglycerides.

PubMed

Salentinig, Stefan; Yepuri, Nageshwar Rao; Hawley, Adrian; Boyd, Ben J; Gilbert, Elliot; Darwish, Tamim A

2015-09-01

Medium chain triglycerides (MCTs) are a unique form of dietary fat that have a wide range of health benefits. They are molecules with a glycerol backbone esterified with medium chain (6-12 carbon atoms) fatty acids on the two outer (sn-1 and sn-3) and the middle (sn-2) positions. During lipid digestion in the gastrointestinal tract, pancreatic lipase stereoselectively hydrolyses the ester bonds of these triglycerides on the sn-1 and sn-3 positions resulting in sn-2 monoglyceride and fatty acids as major products. However, the sn-2 monoglycerides are thermodynamically less stable than their sn-1/3 counterparts. Isomerization or fatty acid migration from the sn-2 monoglyceride to sn-1/3 monoglyceride may occur spontaneously and would lead to glycerol and fatty acid as final products. Here, tricaprin (C10) with selectively deuterated fatty acid chains was used for the first time to monitor chain migration and the stereoselectivity of the pancreatic lipase-catalyzed hydrolysis of ester bonds. The intermediate and final digestion products were studied using NMR and mass spectrometry under biologically relevant conditions. The hydrolysis of the sn-2 monocaprin to glycerol and capric acid did not occur within biologically relevant timescales and fatty acid migration occurs only in limited amounts as a result of the presence of undigested diglyceride species over long periods of time in the digestion medium. The slow kinetics for the exchange of the sn-2 fatty acid chain and the stereoselectivity of pancreatic lipase on MCTs is relevant for industrial processes that involve enzymatic interesterification and the production of high-value products such as specific structured triacylglycerols, confectionery fats and nutritional products. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Study of stability of methotrexate in acidic solution spectrofluorimetric determination of methotrexate in pharmaceutical preparations through acid-catalyzed degradation reaction.

PubMed

Sabry, Suzy M; Abdel-Hady, M; Elsayed, M; Fahmy, Osama T; Maher, Hadir M

2003-07-14

Study of the degradation reaction of methotrexate (MTX) in acidic solution was carried out. Optimization of the experimental parameters of MTX acid hydrolysis was investigated. Spectrofluorimetric method for determination of MTX through measurement of its acid-degradation product, 4-amino-4-deoxy-10-methylpteroic acid (AMP), was developed. Stability of the standard solution of MTX prepared in sulfuric acid was discussed in the view of accelerated stability analysis. Two other comparative spectroflourimetric methods based on measuring the fluorescence intensities from either a condensation reaction with acetylacetone-formaldehyde (Hantzsch reaction) or a reaction with fluorescamine were also described. Beer's law validation, accuracy, precision, limits of detection, limits of quantification, and other aspects of analytical merit are presented in the text. The proposed methods were successfully applied for the analysis of MTX in pure drug and tablets dosage form. The sensitivity of the developed methods was favorable, so it was possible to be adopted for determination of MTX in plasma samples for routine use in high-dose MTX therapy.

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Brian C.; Anderson, Mark A.; Hoadley, Kelly A.

2012-05-08

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD{sup +} salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction in which ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogues acting as inhibitors or substrates were examined, revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehydemore » and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary {sup 15}N and {sup 13}C kinetic isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed. These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.« less

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine β-lactamases and DD-peptidases.

PubMed

Pelto, Ryan B; Pratt, R F

2012-09-28

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl 5 of may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

Structural Investigation of a Phosphorylation-Catalyzed, Isoaspartate-Free, Protein Succinimide: Crystallographic Structure of Post-Succinimide His15Asp Histidine-Containing Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Napper, Scott; Prasad, Lata; Delbaere, Louis T.J.

2008-09-08

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through themore » high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.« less

Fractionation for further conversion: from raw corn stover to lactic acid

NASA Astrophysics Data System (ADS)

He, Ting; Jiang, Zhicheng; Wu, Ping; Yi, Jian; Li, Jianmei; Hu, Changwei

2016-12-01

Fractionation is considered to be one promising strategy to utilize raw biomass to its fullest and produce chemicals with high selectivity. Herein, ethanol/H2O (1/1, v/v) co-solvent with 0.050 M oxalic acid is used to simultaneously fractionate 88.0 wt% of hemicellulose and 89.2 wt% of lignin in corn stover, while cellulose is not obviously degraded. H2O dissolves hemicellulose, G unit and those with β-O-4 linkage of lignin; whereas ethanol extracts G and S units as well as the skeleton with β-5 and β-β linkages of lignin. Oxalic acid effectively catalyzes the hydrolysis of hemicellulose and breaks the intermolecular linkages between hemicellulose and lignin, therefore further promotes the release of lignin. The dissolved hemicelluloses derivatives are reprocessed to produce lactic acid obtaining a high yield of 79.6 wt% with 90% selectivity by the catalysis of MgO. The remained cellulose and recovered lignin can be used further as feedstock to produce chemicals.

Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures

PubMed Central

Rodas-Junco, Beatriz A; Cab-Guillen, Yahaira; Muñoz-Sanchez, J Armando; Vázquez-Flota, Felipe; Monforte-Gonzalez, Miriam; Hérnandez-Sotomayor, S M Teresa

2013-01-01

Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

Fractionation for further conversion: from raw corn stover to lactic acid

PubMed Central

He, Ting; Jiang, Zhicheng; Wu, Ping; Yi, Jian; Li, Jianmei; Hu, Changwei

2016-01-01

Fractionation is considered to be one promising strategy to utilize raw biomass to its fullest and produce chemicals with high selectivity. Herein, ethanol/H2O (1/1, v/v) co-solvent with 0.050 M oxalic acid is used to simultaneously fractionate 88.0 wt% of hemicellulose and 89.2 wt% of lignin in corn stover, while cellulose is not obviously degraded. H2O dissolves hemicellulose, G unit and those with β-O-4 linkage of lignin; whereas ethanol extracts G and S units as well as the skeleton with β-5 and β-β linkages of lignin. Oxalic acid effectively catalyzes the hydrolysis of hemicellulose and breaks the intermolecular linkages between hemicellulose and lignin, therefore further promotes the release of lignin. The dissolved hemicelluloses derivatives are reprocessed to produce lactic acid obtaining a high yield of 79.6 wt% with 90% selectivity by the catalysis of MgO. The remained cellulose and recovered lignin can be used further as feedstock to produce chemicals. PMID:27917955

Detoxification of acidic catalyzed hydrolysate of Kappaphycus alvarezii (cottonii).

PubMed

Meinita, Maria Dyah Nur; Hong, Yong-Ki; Jeong, Gwi-Taek

2012-01-01

Red seaweed, Kappaphycus alvarezii, holds great promise for use in biofuel production due to its high carbohydrate content. In this study, we investigated the effect of fermentation inhibitors to the K. alvarezii hydrolysate on cell growth and ethanol fermentation. In addition, detoxification of fermentation inhibitors was performed to decrease the fermentation inhibitory effect. 5-Hydroxymethylfurfural and levulinic acid, which are liberated from acidic hydrolysis, was also observed in the hydrolysate of K. alvarezii. These compounds inhibited ethanol fermentation. In order to remove these inhibitors, activated charcoal and calcium hydroxide were introduced. The efficiency of activated charcoals was examined and over-liming was used to remove the inhibitors. Activated charcoal was found to be more effective than calcium hydroxide to remove the inhibitors. Detoxification by activated charcoal strongly improved the fermentability of dilute acid hydrolysate in the production of bioethanol from K. alvarezii with Saccharomyces cerevisiae. The optimal detoxifying conditions were found to be below an activated charcoal concentration of 5%.

Theoretical studies of the transition state structures and free energy barriers for base-catalyzed hydrolysis of amides

PubMed Central

Xiong, Ying; Zhan, Chang-Guo

2010-01-01

The transition state structures and free energy barriers for the rate-determining step (i.e. the formation of a tetrahedral intermediate) of base-catalyzed hydrolysis of a series of amides in aqueous solution have been studied by performing first-principle electronic structure calculations using a hybrid supermolecule-polarizable continuum approach. The calculated results and a revisit of recently reported experimental proton inventory data reveal that the favorable transition state structure optimized for the tetrahedral intermediate formation of hydroxide ion-catalyzed hydrolysis of formamide may have three solvating water molecules remaining on the attacking hydroxide oxygen and two additional water molecules attached to the carbonyl oxygen of formamide. The calculated results have also demonstrated interesting substituent effects on the optimized transition state geometries, on the transition-state stabilization, and on the calculated free energy barriers for the base-catalyzed hydrolysis of amides. When some or all of the hydrogen atoms of formamide are replaced by methyl groups, the total number of water molecules hydrogen-bonding with the attacking hydroxide in the transition state decreases from three for formamide to two for N-methylacetamide, N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMA). The larger substituents of the amide hinder the solvent water molecules approaching the attacking hydroxide oxygen in the transition state and, therefore, destabilize the transition state structure and increase the free energy barrier. By using the optimized most favorable transition state structures, the calculated free energy barriers, i.e. 21.6 (or 21.7), 22.7, 23.1, and 26.0 kcal/mol for formamide, N-methylacetamide, DMF, and DMA, respectively, are in good agreement with the available experimental free energy barriers, i.e. 21.2, 21.5, 22.6, and 24.1 kcal/mol for formamide, N-methylacetamide, DMF, and DMA, respectively. PMID:17107116

Low frequency ultrasonic-assisted hydrolysis of starch in the presence of α-amylase.

PubMed

Gaquere-Parker, Anne; Taylor, Tamera; Hutson, Raihannah; Rizzo, Ashley; Folds, Aubrey; Crittenden, Shastina; Zahoor, Neelam; Hussein, Bilal; Arruda, Aaron

2018-03-01

Hydrolysis of starch is an important process in the food industry and in the production of bioethanol or smaller carbohydrate molecules that can be used as starting blocks for chemical synthesis. Such hydrolysis can be enhanced by lowering the pH, heating the reaction mixture or catalyzing the reaction with enzymes. This study reports the effect of sonication on the reaction rate of starch hydrolysis at different temperatures, in the presence or absence of alpha-amylase. Starch Azure, a commercially available potato starch covalently linked with Remazol Brilliant Blue, has been chosen since its hydrolysis releases a blue dye, which concentration can be monitored by UV Vis spectroscopy. Ultrasounds, regardless of experimental conditions, provide the highest reaction rate for such hydrolysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Helicases as molecular motors: An insight

NASA Astrophysics Data System (ADS)

Tuteja, Narendra; Tuteja, Renu

2006-12-01

Helicases are one of the smallest motors of biological system, which harness the chemical free energy of ATP hydrolysis to catalyze the opening of energetically stable duplex nucleic acids and thereby are involved in almost all aspect of nucleic acid metabolism including replication, repair, recombination, transcription, translation, and ribosome biogenesis. Basically, they break the hydrogen bonding between the duplex helix and translocate unidirectionally along the bound strand. Mostly all the helicases contain some conserved signature motifs, which act as an engine to power the unwinding. After the discovery of the first prokaryotic DNA helicase from Escherichia coli bacteria in 1976 and the first eukaryotic one from the lily plant in 1978, many more (>100) have been isolated. All the helicases share some common properties, including nucleic acid binding, NTP hydrolysis and unwinding of the duplex. Many helicases have been crystallized and their structures have revealed an underlying common structural fold for their function. The defects in helicases gene have also been reported to be responsible for variety of human genetic disorders, which can lead to cancer, premature aging or mental retardation. Recently, a new role of a helicase in abiotic stress signaling in plant has been discovered. Overall, helicases act as essential molecular tools for cellular machinery and help in maintaining the integrity of genome. Here an overview of helicases has been covered which includes history, biochemical assay, properties, classification, role in human disease and mechanism of unwinding and translocation.

Carbohydrates as a source of energy and matter for the origin of life

NASA Technical Reports Server (NTRS)

Weber, A. L.

1991-01-01

Recently, we proposed a new model of early glycolysis in which the oxidation of glyceraldehyde self-hemiacetals yielded energy rich polyglyceric acid instead of energy rich thioesters. In this model, polyglyceric acid not only acts as an energy source for phosphoanhydride synthesis, but also as an autocatalyst that can replicate the sequence of D and L residues in its structure. We began our investigation of this new hypothesis - the triose model - by developing a thermal method for the racemization-free synthesis of polyglyceric acid. The hydrolytic stability and the role of chirality in interactions of polyglyceric acid were studied using this thermal polymer. Next, we established that the 2- and 3-glycerol esters of polyglyceric acid are energy rich by measuring the Gibbs free energy change of hydrolysis of the 2- and 3-glycerol esters of 2 and 3-O-L glyceroyl-glyceric acid methyl ester - a model of polyglyceric acid. Recently, we discovered that glyceraldehyde is bound and oxidized to glyceric acid on the surface of ferric hydroxide and that soluble ferric ion catalyzes the rearrangement of glyceraldehyde to lactic acid. We are exploring the possibility that these reactions could yield polyglyceric acid and polylactic acid under plausible prebiotic conditions.

Practical large-scale production of dihydrocapsiate, a nonpungent capsaicinoid-like substance.

PubMed

Kurosawa, Wataru; Nakano, Takashi; Amino, Yusuke

2017-02-01

Capsinoids represent a novel group of capsaicinoid-like substances found in a nonpungent cultivar, Capsicum annuum "CH-19 Sweet." They have capsaicinoid-like physiological and biological properties while lacking the harmful stimuli of capsaicinoids. A large-scale synthesis of dihydrocapsiate (DCT) is established in this work. 8-Methynonanoic acid (MNA) was synthesized by copper-catalyzed cross-coupling of ethyl 6-bromohexanoate with isobutylmagnesium bromide and subsequent hydrolysis. Lipase-catalyzed chemoselective esterification of vanillyl alcohol and MNA was performed at 50 °C under reduced pressure to remove water without solvents or drying agents. A slightly larger stoichiometric amount of MNA was used and the purification in the final stage was simplified to leave a small amount of MNA in the product, because we found that the presence of a small amount of MNA is necessary to stabilize DCT. DCT was synthesized according to the production, and stabilization methods described here has been filed as a new dietary ingredient.

Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in α-Amanitin Biosynthesis

DOE PAGES

Luo, Hong; Hong, Sung-Yong; Sgambelluri, R.  Michael; ...

2014-12-04

Amatoxins are ribosomally encoded and posttranslationally modified peptides that account for the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic octapeptide α-amanitin, formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases have been proposed to play a role in α-amanitin posttranslational processing, the exact mechanistic details are not known. In this paper, we show that a specific POP (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPBmore » catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25-mer from the 35-mer propeptide and transpeptidation at the second Pro to produce the cyclic octamer. Finally on the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP.« less

Applicability of PM3 to transphosphorylation reaction path: Toward designing a minimal ribozyme

NASA Technical Reports Server (NTRS)

Manchester, John I.; Shibata, Masayuki; Setlik, Robert F.; Ornstein, Rick L.; Rein, Robert

1993-01-01

A growing body of evidence shows that RNA can catalyze many of the reactions necessary both for replication of genetic material and the possible transition into the modern protein-based world. However, contemporary ribozymes are too large to have self-assembled from a prebiotic oligonucleotide pool. Still, it is likely that the major features of the earliest ribozymes have been preserved as molecular fossils in the catalytic RNA of today. Therefore, the search for a minimal ribozyme has been aimed at finding the necessary structural features of a modern ribozyme (Beaudry and Joyce, 1990). Both a three-dimensional model and quantum chemical calculations are required to quantitatively determine the effects of structural features of the ribozyme on the reaction it catalyzes. Using this model, quantum chemical calculations must be performed to determine quantitatively the effects of structural features on catalysis. Previous studies of the reaction path have been conducted at the ab initio level, but these methods are limited to small models due to enormous computational requirements. Semiempirical methods have been applied to large systems in the past; however, the accuracy of these methods depends largely on a simple model of the ribozyme-catalyzed reaction, or hydrolysis of phosphoric acid. We find that the results are qualitatively similar to ab initio results using large basis sets. Therefore, PM3 is suitable for studying the reaction path of the ribozyme-catalyzed reaction.

Optimization of biodiesel synthesis by esterification using a fermented solid produced by Rhizopus microsporus on sugarcane bagasse.

PubMed

Botton, Vanderleia; Piovan, Leandro; Meier, Henry França; Mitchell, David Alexander; Cordova, Jesús; Krieger, Nadia

2018-04-01

A fermented solid containing lipases was produced by solid-state fermentation of Rhizopus microsporus on sugarcane bagasse enriched with urea, soybean oil, and a mineral solution. The dry fermented solid produced using R. microsporus (RMFS) was used to catalyze the synthesis of alkyl-esters by esterification in a solvent-free system containing ethanol and oleic acid (as a model system) or a mixture of fatty acids obtained from the physical hydrolysis of soybean soapstock acid oil (FA-SSAO) in subcritical water. The conversions were 93.5 and 84.1%, for oleic acid and FA-SSAO, respectively, at 48 h and 40 °C, at a molar ratio (MR) of ethanol to fatty acid of 5:1. A further increase in the MR to 10:1 improved the production of ethylic-esters, giving conversions at 48 h of 98 and 86% for oleic acid and FA-SSAO, respectively. The results obtained in this work foster further studies on scaling-up of an environmentally friendly process to produce biofuels.

Kinetic analysis of glucoamylase-catalyzed hydrolysis of starch granules from various botanical sources.

PubMed

Tatsumi, Hirosuke; Katano, Hajime; Ikeda, Tokuji

2007-04-01

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k(0)) were determined to be 23.3+/-4.4, 14.8+/-6.0, 6.2+/-1.8, 7.1+/-4.1, 4.6+/-3.0, and 1.6+/-0.6 s(-1) for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k(0) was largely influenced by the type of starch granules. A comparison of the k(0)-values in relation to the crystalline structure of the starch granules suggested that k(0) increases as the crystalline structure becomes dense.

Vapor-Phase Hydrodeoxygenation of Guaiacol to Aromatics over Pt/HBeta: Identification of the Role of Acid Sites and Metal Sites on the Reaction Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nie, Lei; Peng, Bo; Zhu, Xinli

Hydrodeoxygenation of guaiacol, a phenolic compound derived from lignin fraction of biomass, over a Pt/HBeta catalyst at 350 °C and atmospheric pressure produces benzene, toluene, xylenes, and C9+ aromatics with yield of 42%, 29%, 12%, and 5%, respectively. Reaction pathways for conversion of two functional groups (hydroxyl and methoxyl) over the bifunctional catalyst were studied. Both guaiacol and intermediate products (catechol and cyclopentanone) were fed onto zeolite HBeta and Pt/SiO2 to identify the individual role of acid site and metal site. Acid sites (mainly Brønsted acid site, BAS) catalyze transalkylation and dehydroxylation reactions in sequence, producing phenol, cresols and xylenolsmore » as the major products at high conversion. Pt sites catalyze demethylation reaction resulting in catechol as the primary product, which can either be deoxygenated to phenol followed by phenol to benzene, or decarbonylated to cyclopentanone and further to butane. The close proximity of Pt and BAS in bifunctional Pt/HBeta enables both transalkylation and deoxygenation reactions with inhibited demethylation and decarbonylation reactions, producing aromatics as major final products with a total yield > 85%. Both activity and stability of bifunctional Pt/HBeta during hydrodeoxygenation of guaiacol is improved compared to HBeta and Pt/SiO2. The addition of water to the feed further improves the activity and stability via hydrolysis of O-CH3 bond of guaiacol on BAS and removing coke around Pt.« less

Preparation and characterization of silica xerogels as carriers for drugs.

PubMed

Czarnobaj, K

2008-11-01

The aim of the present study was to utilize the sol-gel method to synthesize different forms of xerogel matrices for drugs and to investigate how the synthesis conditions and solubility of drugs influence the change of the profile of drug release and the structure of the matrices. Silica xerogels doped with drugs were prepared by the sol-gel method from a hydrolyzed tetraethoxysilane (TEOS) solution containing two model compounds: diclofenac diethylamine, (DD)--a water-soluble drug or ibuprofen, (IB)--a water insoluble drug. Two procedures were used for the synthesis of sol-gel derived materials: one-step procedure (the sol-gel reaction was carried out under acidic or basic conditions) and the two-step procedure (first, hydrolysis of TEOS was carried out under acidic conditions, and then condensation of silanol groups was carried out under basic conditions) in order to obtain samples with altered microstructures. In vitro release studies of drugs revealed a similar release profile in two steps: an initial diffusion-controlled release followed by a slower release rate. In all the cases studied, the released amount of DD was higher and the released time was shorter compared with IB for the same type of matrices. The released amount of drugs from two-step prepared xerogels was always lower than that from one-step base-catalyzed xerogels. One-step acid-catalyzed xerogels proved unsuitable as the carriers for the examined drugs.

Purification and Characterization of OleA from Xanthomonas campestris and Demonstration of a Non-decarboxylative Claisen Condensation Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frias, JA; Richman, JE; Erickson, JS

2011-03-25

OleA catalyzes the condensation of fatty acyl groups in the first step of bacterial long-chain olefin biosynthesis, but the mechanism of the condensation reaction is controversial. In this study, OleA from Xanthomonas campestris was expressed in Escherichia coli and purified to homogeneity. The purified protein was shown to be active with fatty acyl-CoA substrates that ranged from C(8) to C(16) in length. With limiting myristoyl-CoA (C(14)), 1 mol of the free coenzyme A was released/mol of myristoyl-CoA consumed. Using [(14)C] myristoyl-CoA, the other products were identified as myristic acid, 2-myristoylmyristic acid, and 14-heptacosanone. 2-Myristoylmyristic acid was indicated to be themore » physiologically relevant product of OleA in several ways. First, 2-myristoylmyristic acid was the major condensed product in short incubations, but over time, it decreased with the concomitant increase of 14-heptacosanone. Second, synthetic 2-myristoylmyristic acid showed similar decarboxylation kinetics in the absence of OleA. Third, 2-myristoylmyristic acid was shown to be reactive with purified OleC and OleD to generate the olefin 14-heptacosene, a product seen in previous in vivo studies. The decarboxylation product, 14-heptacosanone, did not react with OleC and OleD to produce any demonstrable product. Substantial hydrolysis of fatty acyl-CoA substrates to the corresponding fatty acids was observed, but it is currently unclear if this occurs in vivo. In total, these data are consistent with OleA catalyzing a non-decarboxylative Claisen condensation reaction in the first step of the olefin biosynthetic pathway previously found to be present in at least 70 different bacterial strains.« less

Unravelling the reaction mechanism of matrix metalloproteinase 3 using QM/MM calculations

NASA Astrophysics Data System (ADS)

Feliciano, Gustavo Troiano; da Silva, Antônio José Roque

2015-07-01

The matrix metalloproteinase family (MMP) constitutes a family of zinc (Zn) proteases that catalyze the breaking of peptide bonds in proteins. These enzymes are very promising drug targets, since they are involved in remodeling and degradation of the extracellular matrix, which is a key process required for cancer metastasis, and thus, their reaction mechanism has been an area of intensive research. Early proposal based on acid base catalyzed hydrolysis, suggested that a conserved zinc bound water molecule acted as the nucleophile attacking the peptide bond carbon, after being activated by essential glutamate. The possibility of a direct nucleophilic attack by the enzyme, performed by the glutamate was also suggested. These are the key yet unsolved issues about MMP reaction mechanism. In the present work, we used hybrid quantum/classical calculations to analyze the structure and energetics of different possible hydrolysis reaction paths. The results support a water mediated mechanism, where both the nucleophile water molecule and the carbonyl oxygen of the scissile peptide bond are coordinated to zinc in the reactive configuration, while the essential glutamate acts as the base accepting the proton from the nucleophilic water. Formation of the carbon-oxygen bond and breaking of carbon-nitrogen bond were found to be concerted events, with a computed barrier of 14.8 kcal/mol. Substrate polarization was found to be important for the observed reaction mechanism, and a substantial change in the metal coordination environment was observed, particularly, regarding the zinc-histidine coordination.

The Hydrolysis of Carbonyl Sulfide at Low Temperature: A Review

PubMed Central

Zhao, Shunzheng; Yi, Honghong; Tang, Xiaolong; Jiang, Shanxue; Gao, Fengyu; Zhang, Bowen; Zuo, Yanran; Wang, Zhixiang

2013-01-01

Catalytic hydrolysis technology of carbonyl sulfide (COS) at low temperature was reviewed, including the development of catalysts, reaction kinetics, and reaction mechanism of COS hydrolysis. It was indicated that the catalysts are mainly involved metal oxide and activated carbon. The active ingredients which can load on COS hydrolysis catalyst include alkali metal, alkaline earth metal, transition metal oxides, rare earth metal oxides, mixed metal oxides, and nanometal oxides. The catalytic hydrolysis of COS is a first-order reaction with respect to carbonyl sulfide, while the reaction order of water changes as the reaction conditions change. The controlling steps are also different because the reaction conditions such as concentration of carbonyl sulfide, reaction temperature, water-air ratio, and reaction atmosphere are different. The hydrolysis of carbonyl sulfide is base-catalyzed reaction, and the force of the base site has an important effect on the hydrolysis of carbonyl sulfide. PMID:23956697

Hydrolytic Glycosidic Bond Cleavage in RNA Nucleosides: Effects of the 2'-Hydroxy Group and Acid-Base Catalysis.

PubMed

Lenz, Stefan A P; Kohout, Johnathan D; Wetmore, Stacey D

2016-12-22

Despite the inherent stability of glycosidic linkages in nucleic acids that connect the nucleobases to sugar-phosphate backbones, cleavage of these bonds is often essential for organism survival. The current study uses DFT (B3LYP) to provide a fundamental understanding of the hydrolytic deglycosylation of the natural RNA nucleosides (A, C, G, and U), offers a comparison to DNA hydrolysis, and examines the effects of acid, base, or simultaneous acid-base catalysis on RNA deglycosylation. By initially examining HCOO - ···H 2 O mediated deglycosylation, the barriers for RNA hydrolysis were determined to be 30-38 kJ mol -1 higher than the corresponding DNA barriers, indicating that the 2'-OH group stabilizes the glycosidic bond. Although the presence of HCOO - as the base (i.e., to activate the water nucleophile) reduces the barrier for uncatalyzed RNA hydrolysis (i.e., unactivated H 2 O nucleophile) by ∼15-20 kJ mol -1 , the extreme of base catalysis as modeled using a fully deprotonated water molecule (i.e., OH - nucleophile) decreases the uncatalyzed barriers by up to 65 kJ mol -1 . Acid catalysis was subsequently examined by selectively protonating the hydrogen-bond acceptor sites of the RNA nucleobases, which results in an up to ∼80 kJ mol -1 barrier reduction relative to the corresponding uncatalyzed pathway. Interestingly, the nucleobase proton acceptor sites that result in the greatest barrier reductions match sites typically targeted in enzyme-catalyzed reactions. Nevertheless, simultaneous acid and base catalysis is the most beneficial way to enhance the reactivity of the glycosidic bonds in RNA, with the individual effects of each catalytic approach being weakened, additive, or synergistic depending on the strength of the base (i.e., degree of water nucleophile activation), the nucleobase, and the hydrogen-bonding acceptor site on the nucleobase. Together, the current contribution provides a greater understanding of the reactivity of the glycosidic bond in natural RNA nucleosides, and has fundamental implications for the function of RNA-targeting enzymes.

Coal liquefaction by base-catalyzed hydrolysis with CO.sub.2 capture

DOEpatents

Xiao, Xin

2014-03-18

The one-step hydrolysis of diverse biomaterials including coal, cellulose materials such as lumber and forestry waste, non-food crop waste, lignin, vegetable oils, animal fats and other source materials used for biofuels under mild processing conditions which results in the formation of a liquid fuel product along with the recovery of a high purity CO.sub.2 product is provided.

By-Product Carrying Humidified Hydrogen: An Underestimated Issue in the Hydrolysis of Sodium Borohydride.

PubMed

Petit, Eddy; Miele, Philippe; Demirci, Umit B

2016-07-21

Catalyzed hydrolysis of sodium borohydride generates up to four molecules of hydrogen, but contrary to what has been reported so far, the humidified evolved gas is not pure hydrogen. Elemental and spectroscopic analyses show, for the first time, that borate by-products pollute the stream as well as the vessel. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alkalizing Reactions Streamline Cellular Metabolism in Acidogenic Microorganisms

PubMed Central

Arioli, Stefania; Ragg, Enzio; Scaglioni, Leonardo; Fessas, Dimitrios; Signorelli, Marco; Karp, Matti; Daffonchio, Daniele; De Noni, Ivano; Mulas, Laura; Oggioni, Marco; Guglielmetti, Simone; Mora, Diego

2010-01-01

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms. PMID:21152088

The effect of phosphomonoesterases on the oxygen isotope composition of phosphate

NASA Astrophysics Data System (ADS)

von Sperber, Christian; Kries, Hajo; Tamburini, Federica; Bernasconi, Stefano M.; Frossard, Emmanuel

2014-01-01

Plants and microorganisms under phosphorus (P) stress release extracellular phosphatases as a strategy to acquire inorganic phosphate (Pi). These enzymes catalyze the hydrolysis of phosphoesters leading to a release of Pi. During the enzymatic hydrolysis an isotopic fractionation (ε) occurs leaving an imprint on the oxygen isotope composition of the released Pi which might be used to trace phosphorus in the environment. Therefore, enzymatic assays with acid phosphatases from wheat germ and potato tuber and alkaline phosphatase from Escherichia coli were prepared in order to determine the oxygen isotope fractionation caused by these enzymes. Adenosine 5‧ monophosphate and glycerol phosphate were used as substrates. The oxygen isotope fractionation caused by acid phosphatases is 20-30‰ smaller than for alkaline phosphatases, resulting in a difference of 5-7.5‰ in δ18O of Pi depending on the enzyme. We attribute the enzyme dependence of the isotopic fractionation to distinct reaction mechanisms of the two types of phosphatases. The observed difference is large enough to distinguish between the two enzymatic processes in environmental samples. These findings show that the oxygen isotope composition of Pi can be used to trace different enzymatic processes, offering an analytical tool that might contribute to a better understanding of the P-cycle in the environment.

Reversible Hydrolysis Reaction with the Spore Photoproduct under Alkaline Conditions.

PubMed

Adhikari, Surya; Lin, Gengjie; Li, Lei

2016-09-16

DNA lesions may reduce the electron density at the nucleobases, making them prone to further modifications upon the alkaline treatment. The dominant DNA photolesion found in UV-irradiated bacterial endospores is a thymine dimer, 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP). Here we report a stepwise addition/elimination reaction in the SP hydrolysis product under strong basic conditions where a ureido group is added to the carboxyl moiety to form a cyclic amide, regenerating SP after eliminating a hydroxide ion. Direct amidation of carboxylic acids by reaction with amines in the presence of a catalyst is well documented; however, it is very rare for an amidation reaction to occur without activation. This uncatalyzed SP reverse reaction in aqueous solution is even more surprising because the carboxyl moiety is not a good electrophile due to the negative charge it carries. Examination of the base-catalyzed hydrolyses of two other saturated pyrimidine lesions, 5,6-dihydro-2'-deoxyuridine and pyrimidine (6-4) pyrimidone photoproduct, reveals that neither reaction is reversible even though all three hydrolysis reactions may share the same gem-diol intermediate. Therefore, the SP structure where the two thymine residues maintain a stacked conformation likely provides the needed framework enabling this highly unusual carboxyl addition/elimination reaction.

An improved purification method for the lysosomal storage disease protein β-glucuronidase produced in CHO cells.

PubMed

Fratz-Berilla, Erica J; Ketcham, Stephanie A; Parhiz, Hamideh; Ashraf, Muhammad; Madhavarao, Chikkathur N

2017-12-01

Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a CHO cell line grown in suspension in a 15 L perfused bioreactor and developed a three step purification procedure that yields ∼99% pure enzyme with a recovery of more than 40%. The method can be completed in two days and has the potential to be integrated into a continuous manufacturing scheme. Published by Elsevier Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skarzewski, J.; Aoki, M.; Sekiquchi, S.

The alkaline hydrolyses of anilides have been studied extensively, but those of anilides bearing ortho substituents seem to be a rather unexplored field. As was shown for p-nitroacetanilide (1), the reaction mechanism of the base-catalyzed hydrolyses of anilides with strongly electron-withdrawing substituents on the phenyl moiety differs somewhat from the general one. The difference is caused by higher acidity of leaving-group nitroanilines. The results obtained in the case of the alkaline hydrolysis of N-ethyl-2,4-dinitroacetanilide are expected to conform to the reaction mechanism proposed for all other anilides, but the rate-determining step may change for such sterically hindered derivatives. That thismore » prediction is fulfilled will be developed in latter sections.« less

Crystallographic and mutational analyses of tannase from Lactobacillus plantarum.

PubMed

Matoba, Yasuyuki; Tanaka, Naomi; Noda, Masafumi; Higashikawa, Fumiko; Kumagai, Takanori; Sugiyama, Masanori

2013-11-01

Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of α/β-hydrolase superfamily with an additional "lid" domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate. Copyright © 2013 Wiley Periodicals, Inc.

Selected soil enzyme activities in an oak-hickory forest following long-term prescribed burning

Treesearch

M. R. Bayan; F. Eivazi

1993-01-01

The biochemical reactions within the soil are mediated by soil flora and fauna, and are catalyzed by enzymes. Therefore, enzymes play a significant role in nutrient cycling. Enzymes are specific for the type of chemical reactions in which they participate. Arylsulfatase is the enzyme that catalyzes the hydrolysis of an arylsulfate anion by fission of the oxygen-sulfur...

Impact of Zeolite Aging in Hot Liquid Water on Activity for Acid-Catalyzed Dehydration of Alcohols.

PubMed

Vjunov, Aleksei; Derewinski, Miroslaw A; Fulton, John L; Camaioni, Donald M; Lercher, Johannes A

2015-08-19

The location and stability of Brønsted acid sites catalytically active in zeolites during aqueous phase dehydration of alcohols were studied on the example of cyclohexanol. The catalytically active hydronium ions originate from Brønsted acid sites (BAS) of the zeolite that are formed by framework tetrahedral Si atom substitution by Al. Al K-edge extended X-ray absorption fine structure (EXAFS) and (27)Al magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopies in combination with density functional theory (DFT) calculations are used to determine the distribution of tetrahedral Al sites (Al T-sites) both qualitatively and quantitatively for both parent and HBEA catalysts aged in water prior to catalytic testing. The aging procedure leads to partial degradation of the zeolite framework evidenced from the decrease of material crystallinity (XRD) as well as sorption capacity (BET). With the exception of one commercial zeolite sample, which had the highest concentration of framework silanol-defects, there is no evidence of Al coordination modification after aging in water. The catalyst weight-normalized dehydration rate correlated best with the sum of strong and weak Brønsted acidic protons both able to generate the hydrated hydronium ions. All hydronium ions were equally active for the acid-catalyzed reactions in water. Zeolite aging in hot water prior to catalysis decreased the weight normalized dehydration reaction rate compared to that of the parent HBEA, which is attributed to the reduced concentration of accessible Brønsted acid sites. Sites are hypothesized to be blocked due to reprecipitation of silica dissolved during framework hydrolysis in the aging procedure.

CHARACTERIZATION OF THE IN VITRO METABOLISM OF SELECTIVE ANDROGEN RECEPTOR MODULATOR USING HUMAN, RAT, AND DOG LIVER ENZYME PREPARATIONS

PubMed Central

Gao, Wenqing; Wu, Zengru; Bohl, Casey E.; Yang, Jun; Miller, Duane D.; Dalton, James T.

2007-01-01

Compound S4 [S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide] is a novel nonsteroidal selective androgen receptor modulator that demonstrates tissue-selective androgenic and anabolic effects. The purpose of this in vitro study was to identify the phase I metabolites, potential species differences in metabolism, and the cytochromes P450 (P450s) involved in the phase I metabolism of S4 using 14C-S4, recombinant P450s, and other liver enzyme preparations from human, rat, and dog. The major phase I metabolism pathways of S4 in humans were identified as deacetylation of the B-ring acetamide group, hydrolysis of the amide bond, reduction of the A-ring nitro group, and oxidation of the aromatic rings, with deacetylation being the predominant pathway observed with most of the enzyme preparations tested. Among the major human P450 enzymes tested, CYP3A4 appeared to be one of the major phase I enzymes that could be responsible for the phase I metabolism of S4 [Km = 16.1 μM, Vmax = 1.6 pmol/(pmol · min)] in humans and mainly catalyzed the deacetylation, hydrolysis, and oxidation of S4. In humans, the cytosolic enzymes mainly catalyzed the hydrolysis reaction, whereas the microsomal enzymes primarily catalyzed the deacetylation reactions. Similar phase I metabolic profiles were observed in rats and dogs as well, except that the amide bond hydrolysis seemed to occur more rapidly in rats. In summary, these results showed that the major phase I reaction of S4 in human, rat, and dog is acetamide group deacetylation. PMID:16272404

Mechanistic Basis for High Stereoselectivity and Broad Substrate Scope in the (salen)Co(III)-Catalyzed Hydrolytic Kinetic Resolution

PubMed Central

Ford, David D.; Nielsen, Lars P. C.; Zuend, Stephan J.; Jacobsen, Eric N.

2013-01-01

In the (salen)Co(III)-catalyzed hydrolytic kinetic resolution (HKR) of terminal epoxides, the rate- and stereoselectivity-determining epoxide ring-opening step occurs by a cooperative bimetallic mechanism with one Co(III) complex acting as a Lewis acid and another serving to deliver the hydroxide nucleophile. In this paper, we analyze the basis for the extraordinarily high stereoselectivity and broad substrate scope observed in the HKR. We demonstrate that the stereochemistry of each of the two (salen)Co(III) complexes in the rate-determining transition structure is important for productive catalysis: a measurable rate of hydrolysis occurs only if the absolute stereochemistry of each of these (salen)Co(III) complexes is the same. Experimental and computational studies provide strong evidence that stereochemical communication in the HKR is mediated by the stepped conformation of the salen ligand, and not the shape of the chiral diamine backbone of the ligand. A detailed computational analysis reveals that the epoxide binds the Lewis acidic Co(III) complex in a well-defined geometry imposed by stereoelectronic, rather than steric effects. This insight serves as the basis of a complete stereochemical and transition structure model that sheds light on the reasons for the broad substrate generality of the HKR. PMID:24041239

Mechanistic basis for high stereoselectivity and broad substrate scope in the (salen)Co(III)-catalyzed hydrolytic kinetic resolution.

PubMed

Ford, David D; Nielsen, Lars P C; Zuend, Stephan J; Musgrave, Charles B; Jacobsen, Eric N

2013-10-16

In the (salen)Co(III)-catalyzed hydrolytic kinetic resolution (HKR) of terminal epoxides, the rate- and stereoselectivity-determining epoxide ring-opening step occurs by a cooperative bimetallic mechanism with one Co(III) complex acting as a Lewis acid and another serving to deliver the hydroxide nucleophile. In this paper, we analyze the basis for the extraordinarily high stereoselectivity and broad substrate scope observed in the HKR. We demonstrate that the stereochemistry of each of the two (salen)Co(III) complexes in the rate-determining transition structure is important for productive catalysis: a measurable rate of hydrolysis occurs only if the absolute stereochemistry of each of these (salen)Co(III) complexes is the same. Experimental and computational studies provide strong evidence that stereochemical communication in the HKR is mediated by the stepped conformation of the salen ligand, and not the shape of the chiral diamine backbone of the ligand. A detailed computational analysis reveals that the epoxide binds the Lewis acidic Co(III) complex in a well-defined geometry imposed by stereoelectronic rather than steric effects. This insight serves as the basis of a complete stereochemical and transition structure model that sheds light on the reasons for the broad substrate generality of the HKR.

Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods

PubMed Central

Tang, Pei-Ling; Hassan, Osman; Maskat, Mohamad Yusof; Badri, Khairiah

2015-01-01

In this study, oil palm empty fruit bunch (OPEFBF) was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen) and enzymatic [cutinase versus manganese peroxidase (MnP)] approaches was investigated. The effects of sodium hydroxide concentration (2, 5, and 10% wt.) and reaction time (30, 90, and 180 minutes) on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield (333.17 ± 49.44 ppm hydroxybenzoic acid, 5.67 ± 0.25 ppm p-hydroxybenzaldehyde, 25.57 ± 1.64 ppm vanillic acid, 168.68 ± 23.23 ppm vanillin, 75.44 ± 6.71 ppm syringic acid, 815.26 ± 41.77 ppm syringaldehyde, 15.21 ± 2.19 ppm p-coumaric acid, and 44.75 ± 3.40 ppm ferulic acid), among the tested methods. High sodium hydroxide concentration (10% wt.) was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid). Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g−1 lignin at pH 8 and 55°C for 24 hours, about 642.83 ± 14.45 ppm hydroxybenzoic acid, 70.19 ± 3.31 ppm syringaldehyde, 22.80 ± 1.04 ppm vanillin, 27.06 ± 1.20 ppm p-coumaric acid, and 50.19 ± 2.23 ppm ferulic acid were produced. PMID:26798644

Identification of fermentation inhibitors in wood hydrolyzates and removal of inhibitors by ion exchange and liquid-liquid extraction

NASA Astrophysics Data System (ADS)

Luo, Caidian

1998-12-01

Common methods employed in the ethanol production from biomass consist of chemical or enzymatic degradation of biomass into sugars and then fermentation of sugars into ethanol or other chemicals. However, some degradation products severely inhibit the fermentation processes and substantially reduce the efficiency of ethanol production. How to remove inhibitors from the reaction product mixture and increase the production efficiency are critical in the commercialization of any processes of energy from biomass. The present study has investigated anion exchange and liquid-liquid extraction as potential methods for inhibitor removal. An analytical method has been developed to identify the fermentation inhibitors in a hydrolyzate. The majority of inhibitors present in hybrid poplar hydrolyzate have positively been identified. Ion exchange with weak basic Dowex-MWA-1 resin has been proved to be an effective mean to remove fermentation inhibitors from hybrid poplar hydrolyzate and significantly increase the fermentation productivity. Extraction with n-butanol might be a preferred way to remove inhibitors from wood hydrolyzates and improve the fermentability of sugars in the hydrolyzates. n-Butanol also removes some glucose, mannose and xylose from the hydrolyzate. Inhibitor identification reveals that lignin and sugar degradation compounds including both aromatic and aliphatic aldehydes and carboxylic acids formed in hydrolysis, plus fatty acids and other components from wood extractives are major fermentation inhibitors in Sacchromyces cerevisiae fermentation. There are 35 components identified as fermentation inhibitors. Among them, 4-hydroxy benzoic acid, 3,4-dihydroxy benzoic acid, syringic acid, syringaldehyde, and ferulic acid are among the most abundant aromatic inhibitors in hybrid poplar hydrolyzate. The conversion of aldehyde groups into carboxylic acid groups in the nitric acid catalyzed hydrolysis reduces the toxicity of the hydrolyzate. A wide spectrum of aliphatic acids has been identified in the wood hydrolyzate studied. They are potential fermentation inhibitors probably similar to acetic acid. Ethyl acetate extraction has also been demonstrated to be a possible method to remove fermentation inhibitors from hydrolyzates. (Abstract shortened by UMI.)

Advances in quantum simulations of ATPase catalysis in the myosin motor.

PubMed

Kiani, Farooq Ahmad; Fischer, Stefan

2015-04-01

During its contraction cycle, the myosin motor catalyzes the hydrolysis of ATP. Several combined quantum/classical mechanics (QM/MM) studies of this step have been published, which substantially contributed to our thinking about the catalytic mechanism. The methodological difficulties encountered over the years in the simulation of this complex reaction are now understood: (a) Polarization of the protein peptide groups surrounding the highly charged ATP(4-) cannot be neglected. (b) Some unsuspected protein groups need to be treated QM. (c) Interactions with the γ-phosphate versus the β-phosphate favor a concurrent versus a sequential mechanism, respectively. Thus, these practical aspects strongly influence the computed mechanism, and should be considered when studying other catalyzed phosphor-ester hydrolysis reactions, such as in ATPases or GTPases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mechanistic Basis for High Reactivity of (salen)Co–OTs in the Hydrolytic Kinetic Resolution of Terminal Epoxides

PubMed Central

Nielsen, Lars P. C.; Zuend, Stephan J.; Ford, David D.; Jacobsen, Eric N.

2012-01-01

The (salen)Co(III)-catalyzed hydrolytic kinetic resolution (HKR) of terminal epoxides is a bimetallic process with a rate controlled by partitioning between a nucleophilic (salen)Co–OH catalyst and a Lewis acidic (salen)Co–X catalyst. The commonly used (salen)Co–OAc and (salen)Co–Cl precatalysts undergo complete and irreversible counterion addition to epoxide during the course of the epoxide hydrolysis reaction, resulting in quantitative formation of weakly Lewis acidic (salen)Co–OH, and severely diminished reaction rates in the late stages of HKR reactions. In contrast, (salen)Co–OTs maintains high reactivity over the entire course of HKR reactions. We describe here an investigation of catalyst partitioning with different (salen)Co–X precatalysts, and demonstrate that counterion addition to epoxide is reversible in the case of the (salen)Co–OTs. This reversible counterion addition results in stable partitioning between nucleophilic and Lewis acidic catalyst species, allowing highly efficient catalysis throughout the course of the HKR reaction. PMID:22292515

Mechanistic basis for high reactivity of (salen)Co-OTs in the hydrolytic kinetic resolution of terminal epoxides.

PubMed

Nielsen, Lars P C; Zuend, Stephan J; Ford, David D; Jacobsen, Eric N

2012-03-02

The (salen)Co(III)-catalyzed hydrolytic kinetic resolution (HKR) of terminal epoxides is a bimetallic process with a rate controlled by partitioning between a nucleophilic (salen)Co-OH catalyst and a Lewis acidic (salen)Co-X catalyst. The commonly used (salen)Co-OAc and (salen)Co-Cl precatalysts undergo complete and irreversible counterion addition to epoxide during the course of the epoxide hydrolysis reaction, resulting in quantitative formation of weakly Lewis acidic (salen)Co-OH and severely diminished reaction rates in the late stages of HKR reactions. In contrast, (salen)Co-OTs maintains high reactivity over the entire course of HKR reactions. We describe here an investigation of catalyst partitioning with different (salen)Co-X precatalysts and demonstrate that counterion addition to epoxide is reversible in the case of the (salen)Co-OTs. This reversible counterion addition results in stable partitioning between nucleophilic and Lewis acidic catalyst species, allowing highly efficient catalysis throughout the course of the HKR reaction.

Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters.

PubMed

Miyamoto, Naoki; Yoshimura, Miho; Okubo, Yuji; Suzuki-Nagata, Kayo; Tsumuraya, Takeshi; Ito, Nobutoshi; Fujii, Ikuo

2018-05-01

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (Asn H33 , Ser H95 , and Arg L96 ) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (Asn H33 Ala, Ser H95 Ala, and Arg L96 Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ultrasound assisted enzyme catalyzed hydrolysis of waste cooking oil under solvent free condition.

PubMed

Waghmare, Govind V; Rathod, Virendra K

2016-09-01

The present work demonstrates the hydrolysis of waste cooking oil (WCO) under solvent free condition using commercial available immobilized lipase (Novozyme 435) under the influence of ultrasound irradiation. The process parameters were optimized using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound intensity. It has been observed that ultrasound-assisted lipase-catalyzed hydrolysis of WCO would be a promising alternative for conventional methods. A maximum conversion of 75.19% was obtained at mild operating parameters: molar ratio of oil to water (buffer pH 7) 3:1, catalyst loading of 1.25% (w/w), lower ultrasound power 100W (ultrasound intensity - 7356.68Wm(-2)), duty cycle 50% and temperature (50°C) in a relatively short reaction time (2h). The activation energy and thermodynamic study shows that the hydrolysis reaction is more feasible when ultrasound is combined with mechanical agitation as compared with the ultrasound alone and simple conventional stirring technique. Application of ultrasound considerably reduced the reaction time as compared to conventional reaction. The successive use of the catalyst for repetitive cycles under the optimum experimental conditions resulted in a loss of enzymatic activity and also minimized the product conversion. Copyright © 2016. Published by Elsevier B.V.

Automated protein hydrolysis delivering sample to a solid acid catalyst for amino acid analysis.

PubMed

Masuda, Akiko; Dohmae, Naoshi

2010-11-01

In this study, we developed an automatic protein hydrolysis system using strong cation-exchange resins as solid acid catalysts. Examining several kinds of inorganic solid acids and cation-exchange resins, we found that a few cation-exchange resins worked as acid catalysts for protein hydrolysis when heated in the presence of water. The most efficient resin yielded amounts of amino acids that were over 70% of those recovered after conventional hydrolysis with hydrochloric acid and resulted in amino acid compositions matching the theoretical values. The solid-acid hydrolysis was automated by packing the resin into columns, combining the columns with a high-performance liquid chromatography system, and heating them. The amino acids that constitute a protein can thereby be determined, minimizing contamination from the environment.

Hydrolysis of dilute acid-pretreated cellulose under mild hydrothermal conditions.

PubMed

Chimentão, R J; Lorente, E; Gispert-Guirado, F; Medina, F; López, F

2014-10-13

The hydrolysis of dilute acid-pretreated cellulose was investigated in a conventional oven and under microwave heating. Two acids--sulfuric and oxalic--were studied. For both hydrothermal conditions (oven and microwave) the resultant total organic carbon (TOC) values obtained by the hydrolysis of the cellulose pretreated with sulfuric acid were higher than those obtained by the hydrolysis of the cellulose pretreated with oxalic acid. However, the dicarboxylic acid exhibited higher hydrolytic efficiency towards glucose. The hydrolysis of cellulose was greatly promoted by microwave heating. The Rietveld method was applied to fit the X-ray patterns of the resultant cellulose after hydrolysis. Oxalic acid preferentially removed the amorphous region of the cellulose and left the crystalline region untouched. On the other hand, sulfuric acid treatment decreased the ordering of the cellulose by partially disrupting its crystalline structure. Copyright © 2014 Elsevier Ltd. All rights reserved.

A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening.

PubMed

De Bank, Paul A; Kendall, David A; Alexander, Stephen P H

2005-04-15

Signalling via the endocannabinoids anandamide and 2-arachidonylglycerol appears to be terminated largely through the action of the enzyme fatty acid amide hydrolase (FAAH). In this report, we describe a simple spectrophotometric assay to detect FAAH activity in vitro using the ability of the enzyme to hydrolyze oleamide and measuring the resultant production of ammonia with a NADH/NAD+-coupled enzyme reaction. This dual-enzyme assay was used to determine Km and Vmax values of 104 microM and 5.7 nmol/min/mgprotein, respectively, for rat liver FAAH-catalyzed oleamide hydrolysis. Inhibitor potency was determined with the resultant rank order of methyl arachidonyl fluorophosphonate>phenylmethylsulphonyl fluoride>anandamide. This assay system was also adapted for use in microtiter plates and its ability to detect a known inhibitor of FAAH demonstrated, highlighting its potential for use in high-throughput screening.

Mediating chemical reactions using polysaccharides

NASA Astrophysics Data System (ADS)

Tyler, Lauren E.

We have studied the NaBH4-mediated hydrogenation of select alkenes catalyzed by polysaccharide-stabilized nanoparticles. We compared the catalytic properties of Ni-based nanoparticles or Au/Co-based nanoparticles on the hydrogenation of cinnamic acid, cinnamide, cinnamyl alcohol, and ethyl cinnamate. We evaluated the possibility that the type of stabilizing polysaccharide surrounding the nanoparticle may affect the selectivity towards the alkene compounds that undergo the hydrogenation reaction. We found that the hydrogenation of cinnamide or ethyl cinnamate proceeded readily to 100% completion independent of the type of polysaccharide stabilizing the nanoparticle. However, the extent of the hydrogenation of cinnamyl alcohol and cinnamic acid varied greatly depending on the type of polysaccharide stabilizing the nanoparticle. In the course of these studies, we observed that some polysaccharides by themselves promoted the hydrolysis of ethyl cinnamate. Thus, we have raised the hypothesis that some polysaccharides may act as "esterases" and explored the interaction between select polysaccharides and a variety of ester compounds.

"Newton's cradle" proton relay with amide-imidic acid tautomerization in inverting cellulase visualized by neutron crystallography.

PubMed

Nakamura, Akihiko; Ishida, Takuya; Kusaka, Katsuhiro; Yamada, Taro; Fushinobu, Shinya; Tanaka, Ichiro; Kaneko, Satoshi; Ohta, Kazunori; Tanaka, Hiroaki; Inaka, Koji; Higuchi, Yoshiki; Niimura, Nobuo; Samejima, Masahiro; Igarashi, Kiyohiko

2015-08-01

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the "Newton's cradle"-like proton relay pathway of the catalytic cycle. Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

Hydrolysis kinetics of secoisolariciresinol diglucoside oligomers from flaxseed.

PubMed

Yuan, Jian-Ping; Li, Xin; Xu, Shi-Ping; Wang, Jiang-Hai; Liu, Xin

2008-11-12

Flaxseed is the richest dietary source of the lignan secoisolariciresinol diglucoside (SDG) and contains the largest amount of SDG oligomers, which are often hydrolyzed to break the ester linkages for the release of SDG and the glycosidic bonds for the release of secoisolariciresinol (SECO). The alkaline hydrolysis reaction kinetics of SDG oligomers from flaxseed and the acid hydrolysis process of SDG and other glucosides were investigated. For the kinetic modeling, a pseudo-first-order reaction was assumed. The results showed that the alkaline hydrolysis of SDG oligomers followed first-order reaction kinetics under mild alkaline hydrolytic conditions and that the concentration of sodium hydroxide had a strong influence on the activation energy of the alkaline hydrolysis of SDG oligomers. The results also indicated that the main acid hydrolysates of SDG included secoisolariciresinol monoglucoside (SMG), SECO, and anhydrosecoisolariciresinol (anhydro-SECO) and that the extent and the main hydrolysates of the acid hydrolysis reaction depended on the acid concentration, hydrolysis temperature, and time. In addition, the production and change of p-coumaric acid glucoside, ferulic acid glucoside and their methyl esters and p-coumaric acid, ferulic acid, and their methyl esters during the process of hydrolysis was also investigated.

Synthesis and properties of the para-trimethylammonium analogues of green fluorescence protein (GFP) chromophore: The mimic of protonated GFP chromophore.

PubMed

Fanjiang, Ming-Wei; Li, Ming-Ju; Sung, Robert; Sung, Kuangsen

2018-04-01

At low pH, protons from the external, bulk solution can protonate the phenoxide group of the p-HBDI chromophore in wild-type green fluorescent protein (wtGFP) and its mutants, and likely continue to tentatively protonate the phenol hydroxyl group of the same chromophores. Because the protonated GFP chromophore is a transient, we prepare the stable p-trimethylammonium analogues (2a and 2b) of the GFP chromophore to mimic it and explore their properties. What we found is that the p-trimethylammonium analogues of the GFP chromophore have the highly electrophilic amidine carbon, blue-shifted electronic absorption, smaller molar absorptivity, smaller fluorescent quantum yield, and faster E-Z thermoisomerization rate. The amidine carbon of the p-trimethylammonium analogue (2b) of the GFP chromophore is the only site that is attacked by very weak nucleophile of water, resulting in ring-opening of the imidazolinone moiety. The half-life of its decay rate in D 2 O is around 33 days. Actually, acid-catalyzed hydrolysis of p-HBDI also results in ring-opening of the imidazolinone moiety. The ratio of the acid-catalyzed hydrolysis rate constants [k obs (p-HBDI)/k obs (1b)] between p-HBDI and 1b (p-dimethylammonium analogue of the GFP chromophore) is dramatically increased from 0.30 at pH = 2 to 0.63 at pH = 0. This is the evidence that more and more phenol hydroxyl groups of p-HBDI are tentatively protonated in a low-pH aqueous solution and that accelerates hydrolysis of p-HBDI in the way similar to the quaternary ammonium derivatives 2a and 2b in water. With this view point, 2a and 2b still can partially mimic the cationic p-HBDI with the protonated phenol hydroxyl group. Implication of the experiment is that the amidine carbon of the chromophore in wtGFP and its mutants at very low pH should be highly electrophilic. Whether ring-opening of the imidazolinone moiety of the GFP chromophore would occur or not depends on if water molecules can reach the amidine carbon of the chromophore inside wtGFP and its mutants. Copyright © 2018 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qasim, Mohammad A., E-mail: qasimm@ipfw.edu; Song, Jikui; Markley, John L.

Research highlights: {yields} Large pK shifts in ionizable groups when buried in the protein interior. {yields} Substrate dependent shifts in pH optimum for serine proteases. {yields} Lys side chain is a stronger acid in serine protease S{sub 1} pocket than Asp side chain. -- Abstract: Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, andmore » {approx}10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S{sub 1} pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.« less

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings.

PubMed

Kim, Donggiun; Lee, Gunsup; Chang, Man; Park, Jongbum; Chung, Youngjae; Lee, Sukchan; Lee, Taek-Kyun

2011-10-26

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.

Off-line solid phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of brivaracetam acid metabolites: Method validation and application to in vitro metabolism assays.

PubMed

Bourgogne, Emmanuel; Culot, Benoit; Dell'Aiera, Sylvie; Chanteux, Hugues; Stockis, Armel; Nicolas, Jean-Marie

2018-06-01

Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x 2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction. Copyright © 2018 Elsevier B.V. All rights reserved.

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources.

PubMed

de Castro, Aline Machado; Carniel, Adriano; Nicomedes Junior, José; da Conceição Gomes, Absai; Valoni, Érika

2017-06-01

Poly(ethylene terephthalate) (PET) is one of the most consumed plastics in the world. The development of efficient technologies for its depolymerization for monomers reuse is highly encouraged, since current recycling rates are still very low. In this study, 16 commercial lipases and cutinases were evaluated for their abilities to catalyze the hydrolysis of two PET samples. Humicola insolens cutinase showed the best performance and was then used in reactions on other PET sources, solely or in combination with the efficient mono(hydroxyethyl terephthalate)-converting lipase from Candida antarctica. Synergy degrees of the final titers of up to 2.2 (i.e., more than double of the concentration when both enzymes were used, as compared to their use alone) were found, with increased terephthalic acid formation rates, reaching a maximum of 59,989 µmol/L (9.36 g/L). These findings open up new possibilities for the conversion of post-consumer PET packages into their minimal monomers, which can be used as drop in at existing industrial facilities.

Mechanistic investigations of the hydrolysis of amides, oxoesters and thioesters via kinetic isotope effects and positional isotope exchange.

PubMed

Robins, Lori I; Fogle, Emily J; Marlier, John F

2015-11-01

The hydrolysis of amides, oxoesters and thioesters is an important reaction in both organic chemistry and biochemistry. Kinetic isotope effects (KIEs) are one of the most important physical organic methods for determining the most likely transition state structure and rate-determining step of these reaction mechanisms. This method induces a very small change in reaction rates, which, in turn, results in a minimum disturbance of the natural mechanism. KIE studies were carried out on both the non-enzymatic and the enzyme-catalyzed reactions in an effort to compare both types of mechanisms. In these studies the amides and esters of formic acid were chosen because this molecular structure allowed development of methodology to determine heavy-atom solvent (nucleophile) KIEs. This type of isotope effect is difficult to measure, but is rich in mechanistic information. Results of these investigations point to transition states with varying degrees of tetrahedral character that fit a classical stepwise mechanism. This article is part of a special issue entitled: Enzyme Transition States from Theory and Experiment. Copyright © 2014 Elsevier B.V. All rights reserved.

α-Ketoglutaramate: An overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle

PubMed Central

Cooper, Arthur J. L.; Kuhara, Tomiko

2013-01-01

Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L-Glutamine + α-keto acid + H2O → α-ketoglutarate + L-amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance. PMID:24234505

Effects of tempering (annealing), acid hydrolysis, low-citric acid substitution on chemical and physicochemical properties of starches of four yam (Dioscorea spp.) cultivars.

PubMed

Falade, Kolawole O; Ayetigbo, Oluwatoyin E

2017-05-01

The effects of tempering (annealing), acid hydrolysis and low-citric acid substitution on chemical and physicochemical properties of starches of four Nigerian yam cultivars were investigated. Crude fat and protein contents of the native starches decreased significantly after the modifications, while nitrogen-free extract increased significantly with acid hydrolysis and citric acid substitution. Acid hydrolysis and low-citric acid substitution reduced the least concentration for gel formation of the starches from 4 to 2% w/v, but tempering had no effect. Swelling power of the starches reduced significantly, and water solubility increased significantly at 75 and 85 °C, especially with acid hydrolysis and low-citric acid substitution. However, tempering significantly reduced starch solubility in the four cultivars. Paste clarity of starches of white (29.17%), water (18.90%), yellow (30.90%) and bitter (10.57%) yams reduced significantly with tempering to 14.43, 11.83, 16.93 and 7.27%, but increased significantly with acid hydrolysis to 41.40, 35.37, 28.77 and 32.33%, and low-citric acid substitution to 36.60, 44.17, 50.67 and 14.33%, respectively. Pasting properties such as peak, trough, breakdown, final, and setback viscosities and peak time of native starches reduced significantly with acid hydrolysis and low-citric acid substitution, however, tempering significantly increased their pasting temperature, peak time, setback and final viscosities.

Extraterrestrial material analysis: loss of amino acids during liquid-phase acid hydrolysis

NASA Astrophysics Data System (ADS)

Buch, Arnaud; Brault, Amaury; Szopa, Cyril; Freissinet, Caroline

2015-04-01

Searching for building blocks of life in extraterrestrial material is a way to learn more about how life could have appeared on Earth. With this aim, liquid-phase acid hydrolysis has been used, since at least 1970 , in order to extract amino acids and other organic molecules from extraterrestrial materials (e.g. meteorites, lunar fines) or Earth analogues (e.g. Atacama desert soil). This procedure involves drastic conditions such as heating samples in 6N HCl for 24 h, either under inert atmosphere/vacuum, or air. Analysis of the hydrolyzed part of the sample should give its total (free plus bound) amino acid content. The present work deals with the influence of the 6N HCl hydrolysis on amino acid degradation. Our experiments have been performed on a standard solution of 17 amino acids. After liquid-phase acid hydrolysis (6N HCl) under argon atmosphere (24 h at 100°C), the liquid phase was evaporated and the dry residue was derivatized with N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF), followed by gas chromatography-mass spectrometry analysis. After comparison with derivatized amino acids from the standard solution, a significant reduction of the chromatographic peak areas was observed for most of the amino acids after liquid-phase acid hydrolysis. Furthermore, the same loss pattern was observed when the amino acids were exposed to cold 6N HCl for a short amount of time. The least affected amino acid, i.e. glycine, was found to be 73,93% percent less abundant compared to the non-hydrolyzed standard, while the most affected, i.e. histidine, was not found in the chromatograms after hydrolysis. Our experiments thereby indicate that liquid-phase acid hydrolysis, even under inert atmosphere, leads to a partial or total loss of all of the 17 amino acids present in the standard solution, and that a quick cold contact with 6N HCl is sufficient to lead to a loss of amino acids. Therefore, in the literature, the reported increase of the total quantity of amino acids after acid hydrolysis, due to the formation/release of amino acids during the whole water extraction / liquid-phase acid hydrolysis, could have hidden a loss of amino acids. Thus, in extraterrestrial material studies involving liquid-phase acid hydrolysis, the quantities of total amino acids may have been underestimated.

Valorization of starchy, cellulosic, and sugary food waste into hydroxymethylfurfural by one-pot catalysis.

PubMed

Yu, Iris K M; Tsang, Daniel C W; Yip, Alex C K; Chen, Season S; Ok, Yong Sik; Poon, Chi Sun

2017-10-01

This study aimed to produce a high-value platform chemical, hydroxymethylfurfural (HMF), from food waste and evaluate the catalytic performance of trivalent and tetravalent metals such as AlCl 3 , CrCl 3 , FeCl 3 , Zr(O)Cl 2 , and SnCl 4 for one-pot conversion. Starchy food waste, e.g., cooked rice and penne produced 4.0-8.1 wt% HMF and 46.0-64.8 wt% glucose over SnCl 4 after microwave heating at 140 °C for 20 min. This indicated that starch hydrolysis was effectively catalyzed but subsequent glucose isomerization was rate-limited during food waste valorization, which could be enhanced by 40-min reaction to achieve 22.7 wt% HMF from cooked rice. Sugary food waste, e.g., kiwifruit and watermelon, yielded up to 13 wt% HMF over Sn catalyst, which mainly resulted from naturally present fructose. Yet, organic acids in fruits may hinder Fe-catalyzed dehydration by competing for the Lewis sites. In contrast, conversion of raw mixed vegetables as cellulosic food waste was limited by marginal hydrolysis at the studied conditions (120-160 °C and 20-40 min). It is interesting to note that tetravalent metals enabled HMF production at a lower temperature and shorter time, while trivalent metals could achieve a higher HMF selectivity at an elevated temperature. Further studies on kinetics, thermodynamics, and reaction pathways of food waste valorization are recommended. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Influence of Monoterpene and Isoprene Nitrates on the Chemistry and Phase State of Secondary Organic Aerosol in a Low-NOx Mixed Deciduous/Coniferous Forest

NASA Astrophysics Data System (ADS)

Slade, J. H., Jr.; Shepson, P. B.; Desrochers, S. J.; Harvey, R. M.; Wallace, W.; Bui, A.; Griffin, R. J.; Kavassalis, S.; Shi, Q.; Murphy, J. G.; Cook, R.; Connor, M.; Ault, A. P.; Pratt, K.; Alwe, H. D.; Millet, D. B.; Bertman, S. B.; Stevens, P. S.; Wennberg, P. O.; Boor, B.; Petrucci, G.

2016-12-01

Particle phase state plays a key role in secondary organic aerosol (SOA) growth, reactive uptake of gas-phase radicals, and condensed phase reactions, influencing atmospheric composition and clouds. While biogenic SOA derived from isoprene and monoterpene oxidation can exhibit glassy or highly viscous semisolid phase states depending on relative humidity, the lifetimes of important SOA precursors including organic nitrates (ON) can depend on the liquid water content (LWC) and particle acidity. Reactions such as acid-catalyzed hydrolysis of particulate ON (pON) are especially sensitive to changes in aerosol LWC. However, the role of particle phase state on the chemistry of pON, and the chemical transformations of pON on the phase of SOA are poorly understood. During the summer of 2016, as part of the PROPHET - Atmospheric Measurements of Oxidants in Summer (AMOS) field study in a northern Michigan mixed deciduous/coniferous forest, we investigated the production of isoprene (IN) and monoterpene nitrates (MTN) at the ground and canopy levels and their role in particle formation, growth, and phase using a combination of high-resolution chemical ionization and aerosol mass spectrometry techniques, size distribution measurements, and particle bounce factors (BF) using two electrical low pressure impactors operating in parallel. Preliminary derived BF suggest the particles may be in a liquid-like state similar to those measured in other high-humidity and isoprene-rich regions such as the Amazon and Southeastern US. However, maxima in BF are observed during periods of high pON, NO, and IN during the day and high NO2 and MTN at night. Some periods were marked with low pON followed by an increase in particulate organic sulfate (pOS), and decrease in BF. Based on these observations, we hypothesize that acid-catalyzed hydrolysis of pON from IN- and MTN-derived SOA may be driving the transition of pON to pOS, leading to a change in the phase state of SOA. Preliminary results also indicate that the smallest particles can be acidic. We will further explore the role of LWC on the phase state of the particles applying ISORROPIA analysis. Although the PROPHET forest is generally NOx-limited, the work presented here highlights the potential importance of NOx and pON chemistry in dictating the phase state of SOA.

Acid hydrolysis of cellulose to yield glucose

DOEpatents

Tsao, George T.; Ladisch, Michael R.; Bose, Arindam

1979-01-01

A process to yield glucose from cellulose through acid hydrolysis. Cellulose is recovered from cellulosic materials, preferably by pretreating the cellulosic materials by dissolving the cellulosic materials in Cadoxen or a chelating metal caustic swelling solvent and then precipitating the cellulose therefrom. Hydrolysis is accomplished using an acid, preferably dilute sulfuric acid, and the glucose is yielded substantially without side products. Lignin may be removed either before or after hydrolysis.

Vectorial Biochemistry

ERIC Educational Resources Information Center

Bowne, S. W., Jr.; Bowne, G. D.

1975-01-01

Describes a biochemical concept called Mitchell's chemiosmotic hypothesis, which proposes a system that catalyzes the hydrolysis of ATP to ADP and inorganic phosphate. The system is reversible and causes a translocation of protons across a physiological membrane. (MLH)

Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan).

PubMed

Balasubramanian, Anuradha; Ponnuraj, Karthe

2008-07-01

Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized and the resulting crystals diffracted to 2.5 A resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 A.

Poly[(ethylene oxide)-co-(methylene ethylene oxide)]: A hydrolytically-degradable poly(ethylene oxide) platform.

PubMed

Lundberg, Pontus; Lee, Bongjae F; van den Berg, Sebastiaan A; Pressly, Eric D; Lee, Annabelle; Hawker, Craig J; Lynd, Nathaniel A

2012-11-20

A facile method for imparting hydrolytic degradability to poly(ethylene oxide) (PEO), compatible with current PEGylation strategies, is presented. By incorporating methylene ethylene oxide (MEO) units into the parent PEO backbone, complete degradation was defined by the molar incorporation of MEO, and the structure of the degradation byproducts was consistent with an acid-catalyzed vinyl-ether hydrolysis mechanism. The hydrolytic degradation of poly[(ethylene oxide)-co-(methylene ethylene oxide)] was pH-sensitive, with degradation at pH 5 being significantly faster than at pH 7.4 at 37 °C in PBS buffer while long-term stability could be obtained in either the solid-state or at pH 7.4 at 6 °C.

Automatic Low-Cost Data Acquisition from Old Polarimetric Instruments

NASA Astrophysics Data System (ADS)

Alibrandi, Giuseppe; D'Aliberti, Santi; Coppolino, Salvatore; Villari, Antonino; Micali, Norberto

2005-03-01

This article describes the design of an apparatus that allows the digital acquisition of polarimetric data from a Lippich polarimeter. This apparatus consists of a low-cost telecamera applied to the ocular of a double-field polarimeter and connected to a PC. The camera is able to reveal with high sensibility the difference in brightness in the two fields allowing more accurate analytical data to be obtained, without need for the analyser to be rotated. This apparatus allows the execution of either single observations or kinetics, because it is able to save previously obtained analytical data. Experimental tests of the apparatus were performed by measuring the rotation angle of solutions of ( )-adrenaline and by following the kinetics of the acid-catalyzed hydrolysis of sucrose.

Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives.

PubMed

Yen, Chih-Chung; Chuang, Yao-Chen; Ko, Chia-Yun; Chen, Long-Fang O; Chen, Sheau-Shyang; Lin, Chia-Jung; Chou, Yi-Li; Shaw, Jei-Fu

2016-07-26

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

PubMed

Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

2012-12-01

A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The hydrolysis of epoxides catalyzed by inorganic ammonium salts in water: kinetic evidence for hydrogen bond catalysis.

PubMed

Nozière, B; Fache, F; Maxut, A; Fenet, B; Baudouin, A; Fine, L; Ferronato, C

2018-01-17

Naturally-occurring inorganic ammonium ions have been recently reported as efficient catalysts for some organic reactions in water, which contributes to the understanding of the chemistry in some natural environments (soils, seawater, atmospheric aerosols, …) and biological systems, and is also potentially interesting for green chemistry as many of their salts are cheap and non-toxic. In this work, the effect of NH 4 + ions on the hydrolysis of small epoxides in water was studied kinetically. The presence of NH 4 + increased the hydrolysis rate by a factor of 6 to 25 compared to pure water and these catalytic effects were shown not to result from other ions, counter-ions or from acid or base catalysis, general or specific. The small amounts of amino alcohols produced in the reactions were identified as the actual catalysts by obtaining a strong acceleration of the reactions when adding these compounds directly to the epoxides in water. Replacing the amino alcohols by other strong hydrogen-bond donors, such as trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) gave the same results, demonstrating that the kinetics of these reactions was driven by hydrogen-bond catalysis. Because of the presence of many hydrogen-bond donors in natural environments (for instance amines and hydroxy-containing compounds), hydrogen-bond catalysis is likely to contribute to many reaction rates in these environments.

Comparison of sulfuric and hydrochloric acids as catalysts in hydrolysis of Kappaphycus alvarezii (cottonii).

PubMed

Meinita, Maria Dyah Nur; Hong, Yong-Ki; Jeong, Gwi-Taek

2012-01-01

In this study, hydrolysis of marine algal biomass Kappaphhycus alvarezii using two different acid catalysts was examined with the goal of identifying optimal reaction conditions for the formation of sugars and by-products. K. alvarezii were hydrolyzed by autoclave using sulfuric acid or hydrochloric acid as catalyst with different acid concentrations (0.1-1.0 M), substrate concentrations (1.0-13.5%), hydrolysis time (10-90 min) and hydrolysis temperatures (100-130 (°)C). A difference in galactose, glucose, reducing sugar and total sugar content was observed under the different hydrolysis conditions. Different by-product compounds such as 5-hydroxymethylfurfural and levulinic acid were also observed under the different reaction conditions. The optimal conditions for hydrolysis were achieved at a sulfuric acid concentration, temperature and reaction time of 0.2 M, 130 °C and 15 min, respectively. These results may provide useful information for the development of more efficient systems for biofuel production from marine biomass.

Chemical evolution. XXI - The amino acids released on hydrolysis of HCN oligomers

NASA Technical Reports Server (NTRS)

Ferris, J. P.; Wos, J. D.; Nooner, D. W.; Oro, J.

1974-01-01

Major amino acids released by hydrolysis of acidic and basic HCN oligomers are identified by chromatography as Gly, Asp, and diaminosuccinic acid. Smaller amounts of Ala, Ile and alpha-aminoisobutyric acid are also detected. The amino acids released did not change appreciably when the hydrolysis medium was changed from neutral to acidic or basic. The presence of both meso and d, l-diaminosuccinic acids was established by paper chromatography and on an amino acid analyzer.

Optimization of cellulose nanocrystal length and surface charge density through phosphoric acid hydrolysis

NASA Astrophysics Data System (ADS)

Vanderfleet, Oriana M.; Osorio, Daniel A.; Cranston, Emily D.

2017-12-01

Cellulose nanocrystals (CNCs) are emerging nanomaterials with a large range of potential applications. CNCs are typically produced through acid hydrolysis with sulfuric acid; however, phosphoric acid has the advantage of generating CNCs with higher thermal stability. This paper presents a design of experiments approach to optimize the hydrolysis of CNCs from cotton with phosphoric acid. Hydrolysis time, temperature and acid concentration were varied across nine experiments and a linear least-squares regression analysis was applied to understand the effects of these parameters on CNC properties. In all but one case, rod-shaped nanoparticles with a high degree of crystallinity and thermal stability were produced. A statistical model was generated to predict CNC length, and trends in phosphate content and zeta potential were elucidated. The CNC length could be tuned over a relatively large range (238-475 nm) and the polydispersity could be narrowed most effectively by increasing the hydrolysis temperature and acid concentration. The CNC phosphate content was most affected by hydrolysis temperature and time; however, the charge density and colloidal stability were considered low compared with sulfuric acid hydrolysed CNCs. This study provides insight into weak acid hydrolysis and proposes `design rules' for CNCs with improved size uniformity and charge density. This article is part of a discussion meeting issue `New horizons for cellulose nanotechnology'.

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

PubMed

Park, Jong-Min; Kim, Jo-Il; Song, Hyun-Woo; Noh, Joo-Yoon; Kang, Min-Jung; Pyun, Jae-Chul

2015-09-15

This work presented a highly sensitive bacterial antibiotic susceptibility test through β-lactamase assay using Parylene-matrix chip. β-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to β-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate β-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/z<500). The β-lactamase assay for the Escherichia coli antibiotic susceptibility test was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a β-lactamase assay with minimal interference of low molecular weight noise peaks. The β-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay. Copyright © 2015 Elsevier B.V. All rights reserved.

"New" Compounds from Old Plastics: Recycling PET Plastics via Depolymerization. An Activity for the Undergraduate Organic Lab

NASA Astrophysics Data System (ADS)

Kaufman, Don; Wright, Geoff; Kroemer, Ryan; Engel, Josh

1999-11-01

This paper describes work done to develop a meaningful undergraduate organic lab activity that illustrates chemistry of the real world while utilizing reactions typically included in the organic lecture and lab. We show how a common plastic can be converted into several compounds using ester hydrolysis and SN2 reactions. Contributing to the critical shortage of landfill space faced by many communities is the large quantity of plastic refuse. Thus, there is a real need to recycle plastic products. One way to recycle plastics such as polyethyleneterephthalate (PET), the polyester from which numerous consumer products such as 2-liter soda bottles are made, is to depolymerize them and then to use the resulting monomers to produce new products. PET is industrially depolymerized via an acid-catalyzed transesterification reaction conducted under conditions of high temperature and pressure that are not feasible in the undergraduate lab. Despite literature reports that PET is remarkably resistant to hydrolysis, we found that PET can be readily hydrolyzed by refluxing with potassium hydroxide or potassium tert-butoxide in amyl alcohol to give terephthalic acid in high yield. It is then possible to readily synthesize terephthalate diesters via SN2 reactions of ammonium terephthalate salts with alkyl halides. Fischer esterification can also be used to prepare the diesters, but yields are significantly lower.

A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157.

PubMed

Preugschat, Frank; Carter, Luke H; Boros, Eric E; Porter, David J T; Stewart, Eugene L; Shewchuk, Lisa M

2014-12-15

hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2'-deoxy-2'-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30s(-1)) is followed by a slow irreversible step (0.1s(-1)) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+). Copyright © 2014 Elsevier Inc. All rights reserved.

Preparation and characterization of directly compactible layer-by-layer nanocoated cellulose.

PubMed

Strydom, Schalk J; Otto, Daniel P; Liebenberg, Wilna; Lvov, Yuri M; de Villiers, Melgardt M

2011-02-14

Microcrystalline cellulose is a commonly used direct compression tablet diluent and binder. It is derived from purified α-cellulose in an environmentally unfriendly process that involves mineral acid catalysed hydrolysis. In this study Kraft softwood fibers was nanocoated using a layer-by-layer self-assembling process. Powder flow and compactibility results showed that the application of nano-thin polymer layers on the fibers turned non-flowing, non-compacting cellulose into powders that can be used in the direct compression of tablets. The powder flow properties and tableting indices of compacts compressed from these nanocoated microfibers were similar or better than that of directly compactible microcrystalline cellulose powders. Cellulose microfibers coated with four PSS/PVP bilayers had the best compaction properties while still producing tablets that were able to absorb water and disintegrate and did not retard the dissolution of a model drug acetaminophen. The advantages of nanocoating rather than traditional pharmaceutical coating are that it add less than 1% to the weight of the fibers and allows control of the molecular properties of the surface and the thickness of the coat to within a few nanometers. This process is potentially friendlier to the environment because of the type and quantity of materials used. Also, it does not involve acid-catalyzed hydrolysis and neutralization of depolymerized cellulose. Copyright © 2010 Elsevier B.V. All rights reserved.

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase.

PubMed

Wickham, Stephanie; Regan, Nicholas; West, Matthew B; Kumar, Vidya Prasanna; Thai, Justin; Li, Pui Kai; Cook, Paul F; Hanigan, Marie H

2012-08-01

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.

Combined effects of π-π stacking and hydrogen bonding on the (N1) acidity of uracil and hydrolysis of 2'-deoxyuridine.

PubMed

Kellie, Jennifer L; Navarro-Whyte, Lex; Carvey, Matthew T; Wetmore, Stacey D

2012-03-01

M06-2X/6-31+G(d,p) is used to study the simultaneous effects of π-π stacking interactions with phenylalanine (modeled as benzene) and hydrogen bonding with small molecules (HF, H(2)O, and NH(3)) on the N1 acidity of uracil and the hydrolytic deglycosylation of 2'-deoxyuridine (dU) (facilitated by fully (OH(-)) or partially (HCOO(-)···H(2)O) activated water). When phenylalanine is complexed with isolated uracil, the proton affinity of all acceptor sites significantly increases (by up to 28 kJ mol(-1)), while the N1 acidity slightly decreases (by ~6 kJ mol(-1)). When small molecules are hydrogen bound to uracil, addition of the phenylalanine ring can increase or decrease the acidity of uracil depending on the number and nature (acidity) of the molecules bound. Furthermore, a strong correlation between the effects of π-π stacking on the acidity of U and the dU deglycosylation reaction energetics is found, where the hydrolysis barrier can increase or decrease depending on the nature and number of small molecules bound, the nucleophile considered (which dictates the negative charge on U in the transition state), and the polarity of the (bulk) environment. These findings emphasize that the catalytic (or anticatalytic) role of the active-site aromatic amino acid residues is highly dependent on the situation under consideration. In the case of uracil-DNA glycosylase (UNG), which catalyzes the hydrolytic excision of uracil from DNA, the type of discrete hydrogen-bonding interactions with U, the nature of the nucleophile, and the anticipated weak, nonpolar environment in the active site suggest that phenylalanine will be slightly anticatalytic in the chemical step, and therefore experimentally observed contributions to catalysis may entirely result from associated structural changes that occur prior to deglycosylation.

Efficiencies of acid catalysts in the hydrolysis of lignocellulosic biomass over a range of combined severity factors

Treesearch

Jae-Won Lee; Thomas W. Jeffries

2011-01-01

Dicarboxylic organic acids have properties that differ from those of sulfuric acid during hydrolysis of lignocellulose. To investigate the effects of different acid catalysts on the hydrolysis and degradation of biomass compounds over a range of thermochemical pretreatments, maleic, oxalic and sulfuric acids were each used at the same combined severity factor (CSF)...

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

PubMed Central

Schenk, Gerhard; Gahan, Lawrence R.; Carrington, Lyle E.; Mitić, Nataša; Valizadeh, Mohsen; Hamilton, Susan E.; de Jersey, John; Guddat, Luke W.

2005-01-01

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 μ-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (kcat/Km) at pH 4.5, whereas its catalytic rate constant (kcat) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pKa of the leaving group. The crystal structure of the phosphate-bound Fe(III)–Mn(II) PAP has been determined to 2.5-Å resolution (final Rfree 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 λ protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis. PMID:15625111

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyunbeom; Doud, Emma H.; Wu, Rui

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

Bioremediation of cooking oil waste using lipases from wastes

PubMed Central

do Prado, Débora Zanoni; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases. PMID:29073166

Bioremediation of cooking oil waste using lipases from wastes.

PubMed

Okino-Delgado, Clarissa Hamaio; Prado, Débora Zanoni do; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando; Fleuri, Luciana Francisco

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

Methyl Ester Production via Heterogeneous Acid-Catalyzed Simultaneous Transesterification and Esterification Reactions

NASA Astrophysics Data System (ADS)

Indrayanah, S.; Erwin; Marsih, I. N.; Suprapto; Murwani, I. K.

2017-05-01

The heterogeneous acid catalysts (MgF2 and ZnF2) have been used to catalyze the simultaneous transesterification and esterification reactions of crude palm oil (CPO) with methanol. Catalysts were synthesized by sol-gel method (combination of fluorolysis and hydrolysis). The physicochemical, structural, textural, thermal stability of the prepared catalysts was investigated by N2 adsorption-desorption, XRD, FT-IR, SEM and TG/DTG. Both MgF2 and ZnF2 have rutile structures with a different phase. The surface area of ZnF2 is smaller than that of MgF2, but the pore size and volume of ZnF2 are larger than those of MgF2. However, these materials are thermally stable. The performance of the catalysts is determined from the yield of catalysts toward the formation of methyl ester determined based on the product of methyl ester obtained from the reaction. The catalytic activity of ZnF2 is higher than MgF2 amounted to 85.21% and 26.82% with the optimum condition. The high activity of ZnF2 could be attributed to its pore diameter and pore volume but was not correlated with its surface area. The yield of methyl ester decreased along with the increase in molar ratio of methanol/CPO from 85.21 to 80.99 for ZnF2, respectively.

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

PubMed

Gerstmeier, Jana; Newcomer, Marcia E; Dennhardt, Sophie; Romp, Erik; Fischer, Jana; Werz, Oliver; Garscha, Ulrike

2016-05-01

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. © FASEB.

Acid-Catalyzed Preparation of Biodiesel from Waste Vegetable Oil: An Experiment for the Undergraduate Organic Chemistry Laboratory

ERIC Educational Resources Information Center

Bladt, Don; Murray, Steve; Gitch, Brittany; Trout, Haylee; Liberko, Charles

2011-01-01

This undergraduate organic laboratory exercise involves the sulfuric acid-catalyzed conversion of waste vegetable oil into biodiesel. The acid-catalyzed method, although inherently slower than the base-catalyzed methods, does not suffer from the loss of product or the creation of emulsion producing soap that plagues the base-catalyzed methods when…

Optimization of dilute acid pretreatment of water hyacinth biomass for enzymatic hydrolysis and ethanol production

PubMed Central

Idrees, Muhammad; Adnan, Ahmad; Sheikh, Shahzad; Qureshic, Fahim Ashraf

2013-01-01

The present study was conducted for the optimization of pretreatment process that was used for enzymatic hydrolysis of lignocellulosic biomass (Water Hyacinth, WH), which is a renewable resource for the production of bioethanol with decentralized availability. Response surface methodology has been employed for the optimization of temperature (oC), time (hr) and different concentrations of maleic acid (MA), sulfuric acid (SA) and phosphoric acid (PA) that seemed to be significant variables with P < 0.05. High F and R2 values and low P-value for hydrolysis yield indicated the model predictability. The pretreated biomass producing 39.96 g/l, 39.86 g/l and 37.9 g/l of reducing sugars during enzymatic hydrolysis with yield 79.93, 78.71 and 75.9 % from PA, MA and SA treated respectively. The order of catalytic effectiveness for hydrolysis yield was found to be phosphoric acid > maleic acid > sulfuric acid. Mixture of sugars was obtained during dilute acid pretreatment with glucose being the most prominent sugar while pure glucose was obtained during enzymatic hydrolysis. The resulting sugars, obtained during enzymatic hydrolysis were finally fermented to ethanol, with yield 0.484 g/g of reducing sugars which is 95 % of theoretical yield (0.51 g/g glucose) by using commercial baker's yeast (Sacchromyces cerveasiae). PMID:26417215

Zinc Enzymes.

ERIC Educational Resources Information Center

Bertini, I.; And Others

1985-01-01

Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline–oxidative pretreatment of hybrid poplar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhalla, Aditya; Fasahati, Peyman; Particka, Chrislyn A.

2018-05-17

When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H2O2) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significant loss inmore » sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified.« less

Platinum-catalyzed hydrolysis etching of SiC in water: A density functional theory study

NASA Astrophysics Data System (ADS)

Van Bui, Pho; Toh, Daisetsu; Isohashi, Ai; Matsuyama, Satoshi; Inagaki, Kouji; Sano, Yasuhisa; Yamauchi, Kazuto; Morikawa, Yoshitada

2018-05-01

A comprehensive study of the physicochemical interactions and the reaction mechanism of SiC etching with water by Pt catalysts can reveal key details about the surface treatment and catalytic phenomena at interfaces. Therefore, density functional theory simulations were performed to study the kinetics of Pt-assisted water dissociation and breaking of a Si–C bond compared to the HF-assisted mechanism. These calculations carefully considered the elastic and chemical interaction energies at the Pt–SiC interface, activation barriers of Si–C bond dissociation, and the catalytic role of Pt. It was found that the Pt-catalyzed etching of SiC in water is initiated via hydrolysis reactions that break the topmost Si–C bonds. The activation barrier strongly depends on the elastic and chemical interactions. However, chemical interactions are a dominant factor and mainly contribute to the lowering of the activation barrier, resulting in an increased rate of reaction.

Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents.

PubMed

Tsai, Shau-Wei; Chen, Chun-Chi; Yang, Hung-Shien; Ng, I-Son; Chen, Teh-Liang

2006-08-01

In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device.

Mechanism of Thioesterase-Catalyzed Chain Release in the Biosynthesis of the Polyether Antibiotic Nanchangmycin

PubMed Central

Liu, Tiangang; Lin, Xin; Zhou, Xiufen; Deng, Zixin; Cane, David E.

2008-01-01

Summary The polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates. PMID:18482697

Occurrence and Characteristics of a Rapid Exchange of Phosphate Oxygens Catalyzed by Sarcoplasmic Reticulum Vesicles

DOE R&D Accomplishments Database

Kanazawa, T.; Boyer, P. D.

1972-01-01

Sarcoplasmic reticulum vesicles isolated from skeletal muscle actively take up Ca{sup ++} from the medium in the presence of Mg{sup ++} and ATP. This transport is coupled to ATP hydrolysis catalyzed by membrane-bound Ca{sup++}, Mg{sup ++}-ATPase which is activated by concurrent presence of Ca{sup ++} and Mg{sup ++}. Considerable informations have accumulated that give insight into the ATPase and its coupling to the calcium transport. The hydrolysis of ATP by this enzyme occurs through a phosphorylated intermediate. Formation and decomposition of the intermediate show vectorial requirements for Ca{sup ++} and Mg{sup ++}, suggesting an intimate involvement of the intermediate in the transport process. ATP synthesis from P{sub i} and ADP coupled to outflow of Ca{sup ++} from sarcoplasmic reticulum vesicles has recently been demonstrated. This indicates the reversibility of the entire process of calcium transport in sarcoplasmic reticulum vesicles.

Marine fungi Aspergillus sydowii and Trichoderma sp. catalyze the hydrolysis of benzyl glycidyl ether.

PubMed

Martins, Mariana Provedel; Mouad, Ana Maria; Boschini, Letícia; Regali Seleghim, Mirna Helena; Sette, Lara Durães; Meleiro Porto, André Luiz

2011-04-01

Whole cells of the marine fungi Aspergillus sydowii Gc12, Penicillium raistrickii Ce16, P. miczynskii Gc5, and Trichoderma sp. Gc1, isolated from marine sponges of the South Atlantic Ocean (Brazil), have been screened for the enzymatic resolution of (±)-2-(benzyloxymethyl)oxirane (benzyl glycidyl ether; 1). Whole cells of A. sydowii Gc12 catalyzed the enzymatic hydrolysis of (R,S)-1 to yield (R)-1 with an enantiomeric excess (ee) of 24-46% and 3-(benzyloxy)propane-1,2-diol (2) with ee values <10%. In contrast, whole cells of Trichoderma sp. Gc1 afforded (S)-1 with ee values up to 60% and yields up to 39%, together with (R)-2 in 25% yield and an ee of 32%. This is the first published example of the hydrolysis of 1 by whole cells of marine fungi isolated from the South Atlantic Ocean. The hydrolases from the two studied fungi exhibited complementary regioselectivity in opening the epoxide ring of racemic 1, with those of A. sydowii Gc12 showing an (S) preference and those of Trichoderma sp. Gc1 presenting an (R) preference for the substrate.

Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates.

PubMed

Hengge, Alvan C; Stein, Ross L

2004-01-27

To probe the mechanistic origins of convex Eyring plots that have been observed for alpha-chymotrypsin (alpha-CT)-catalyzed hydrolysis of specific p-nitroanilide substrates [Case, A., and Stein, R. L. (2003) Biochemistry 42, 3335-3348], we determined the temperature-dependence of (15)N-kinetic isotope effects for the alpha-CT-catalyzed hydrolysis of N-succinyl-Phe p-nitroanilide (Suc-Phe-pNA). To provide an interpretational context for these enzymatic isotope effects, we also determined 15N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30 degrees C), 15N-KIE values are 1.035 and 0.995 (+/-0.001), respectively, and are consistent with the reported [HO-]-dependent change in rate-limiting step from leaving group departure from an anionic tetrahedral intermediate in dilute base, to hydroxide attack in concentrated base. For the alpha-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15N-KIE is on kc/Km and thus reflects structural features of transition states for all reaction steps up to and including acylation of the active site serine. The isotope effect at 35 degrees C is 1.014 (+/-0.001) and suggests that in the transition state for this reaction, departure of leaving group from the tetrahedral intermediate is well advanced. Significantly, 15N-KIE does not vary over the temperature range 5-45 degrees C. This result eliminates one of the competing hypotheses for the convex Eyring plot observed for this reaction, that is, a temperature-dependent change in rate-limiting step within the chemical manifold of acylation, but supports a mechanism in which an isomerization of enzyme conformation is coupled to active site chemistry. We finally suggest that the near absolute temperature-independence of 15N-KIE may point to a unique transition state for this process.

Cu(II)-catalyzed degradation of ampicillin: effect of pH and dissolved oxygen.

PubMed

Guo, Yiming; Tsang, Daniel C W; Zhang, Xinran; Yang, Xin

2018-02-01

Cu(II)-catalyzed hydrolysis of β-lactam antibiotics has been well-identified and recognized as the key mechanism of antibiotic degradation. However, the overlooked Cu(II) oxidation susceptibly also plays an important role comparably with hydrolysis. This study evaluated the roles of hydrolysis and oxidation in Cu(II)-catalyzed degraded ampicillin (AMP), as a typical β-lactam antibiotic, under relevant environmental conditions (pH 5.0, 7.0, and 9.0; oxygen 0.2 and 6.2 mg/L). Under AMP and Cu(II) molar ratio of 1:1, AMP degradation was the fastest at pH 9.0, followed by pH 5.0 and pH 7.0. The facilitation of oxygen on AMP degradation was notable at pH 5.0 and 7.0 rather than pH 9.0. AMP degradation rate increased from 21.8% in 0.2 mg/L O 2 solution to 85.9% in 6.2 mg/L O 2 solution at pH 7.0 after 4-h reaction. AMP oxidation was attributed to both oxygen-derived Cu(I)/Cu(II) cycle and intermediate reactive oxygen species (HO . and O 2 .- ). Several intermediate and final products in AMP degradation were firstly identified by LC-quadrupole time-of-flight-MS analysis. Phenylglycine primary amine on the AMP structure was the essential complexation site to proceed with the oxidation reaction. The oxidation of AMP preferentially occurred on the β-lactam structure. The inherent mechanisms related to pH and oxygen conditions were firstly investigated, which could enhance the understanding of both oxidation and hydrolysis mechanisms in AMP degradation. This study not only has an important implication in predicting β-lactam antibiotic transformation and fate in natural environment but also benefits the developing of strategies of antibiotic control to reduce the environmental risk.

Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight.

PubMed

Wang, Xingchi; Wen, Fanting; Zhang, Shurong; Shen, Ruru; Jiang, Wei; Liu, Jun

2017-03-01

Effect of acid hydrolysis on the morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight was investigated in this study. The hydrolysis degree of C. auriculatum starch rapidly increased to 63.69% after 4days and reached 78.67% at the end of 9days. Morphology observation showed that the starch granules remained intact during the first 4days of hydrolysis. However, serious erosion phenomenon was observed after 5days and starch granules completely fell into pieces after 7days. During acid hydrolysis process, the crystal type of hydrolyzed starch changed from original C B -type to final A-type. Small-angle X-ray scattering patterns showed the semi-crystalline growth rings started to be hydrolyzed after 4days. The proportions of single helix and amorphous components as well as amylose content in starch gradually decreased, whereas the proportion of double helix components continuously increased during acid hydrolysis. However, the contents of rapidly digestible starch, slowly digestible starch and resistant starch were almost constant during acid hydrolysis process, indicating the in vitro digestion property of C. auriculatum starch was not affected by acid hydrolysis. Our results provided novel information on the inner structure of C. auriculatum starch granules. Copyright © 2017 Elsevier B.V. All rights reserved.

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duong-Ly, Krisna C.; Gabelli, Sandra B.; Xu, WenLian

2011-09-06

A Nudix enzyme from Bacillus cereus catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3{prime} {yields} 5{prime} RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-{angstrom} resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an 'enclosed' Nudix pyrophosphatase site and the othermore » with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.« less

Arrhenius analysis of the electrophorus electricus acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

PubMed

Oakes, Jesse; Nguyen, Tina; Britt, B Mark

2003-06-01

Ellman's method was used to determine the Michaelis-Menten parameters for the hydrolysis of acetylthiocholine by Electrophorus electricus acetylcholinesterase from 12 to 37 degrees C. Arrhenius analysis revealed that the activation energy for formation of the enzyme/substrate complex is 22.2 +/- 1.1 kJ/mole. The Arrhenius plot of k(cat) is markedly curved and attributed to comparable rates of acylation and deacylation due to the absence of evidence for a temperature-dependent enzyme conformational change by differential scanning calorimetry.

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

PubMed Central

2016-01-01

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5′-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general. PMID:25616005

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1 S ,3 S )-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyunbeom; Doud, Emma H.; Wu, Rui

gamma-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

Mechanism of inactivation of γ-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115).

PubMed

Lee, Hyunbeom; Doud, Emma H; Wu, Rui; Sanishvili, Ruslan; Juncosa, Jose I; Liu, Dali; Kelleher, Neil L; Silverman, Richard B

2015-02-25

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1 S ,3 S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

DOE PAGES

Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; ...

2015-01-23

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

Characterization of a unique class C acid phosphatase from Clostridium perfringens.

PubMed

Reilly, Thomas J; Chance, Deborah L; Calcutt, Michael J; Tanner, John J; Felts, Richard L; Waller, Stephen C; Henzl, Michael T; Mawhinney, Thomas P; Ganjam, Irene K; Fales, William H

2009-06-01

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens▿

PubMed Central

Reilly, Thomas J.; Chance, Deborah L.; Calcutt, Michael J.; Tanner, John J.; Felts, Richard L.; Waller, Stephen C.; Henzl, Michael T.; Mawhinney, Thomas P.; Ganjam, Irene K.; Fales, William H.

2009-01-01

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated Kms ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of Pi/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class. PMID:19363079

Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella.

PubMed

Wilson, M E; Consigli, R A

1985-06-01

A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella. A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

PubMed

Awate, Sanket; Brosh, Robert M

2017-06-08

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism

PubMed Central

Awate, Sanket; Brosh, Robert M.

2017-01-01

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies. PMID:28594346

Cascade Production of Lactic Acid from Universal Types of Sugars Catalyzed by Lanthanum Triflate.

PubMed

Liu, Dajiang; Kim, Kwang Ho; Sun, Jian; Simmons, Blake A; Singh, Seema

2018-02-09

Lignocellulosic biomass conversion into value-added platform chemicals in the non-toxic, water-tolerant Lewis acid, and water solutions bears the hallmark of green chemistry. Lactic acid derived from biomass is an important chemical building block for biodegradable polymers such as polylactide. Herein, a universal method of converting lignocellulosic sugars into lactic acid using catalytic amount of water-stable Lewis acid La(OTf) 3 is demonstrated. The lignocellulosic sugars studied in this work include 1) pyrolytic sugars from pyrolysis oil, and 2) sugars derived from ionic liquid (IL)-pretreated biomass. Under moderate conditions (250 °C, 1 h), levoglucosan (major pyrolytic sugar), glucose, and xylose were converted into lactic acid with carbon-based molar yields of 75, 74, and 61 %, respectively. Furthermore, roughly 49 mol % (based on levoglucosan) and 74 wt % (relative to pretreated biomass) of lactic acid were obtained from the conversion of pyrolytic sugars and sugar-rich fraction after lignin removal from switchgrass, respectively. To our knowledge, this is the first reported conversion of pyrolytic sugar into lactic acid by chemocatalysis and also lignocellulosic sugars are converted into lactic acid without hydrolysis. This approach could potentially be extended to other lignocellulosic sugars after simple removal of lignin from biomass pretreatment, rendering moderate to high yields of lactic acid. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Degradation of Di(2-Ethylhexyl) Phthalate by a Novel Gordonia alkanivorans Strain YC-RL2.

PubMed

Nahurira, Ruth; Ren, Lei; Song, Jinlong; Jia, Yang; Wang, Junhuan; Fan, Shuanghu; Wang, Haisheng; Yan, Yanchun

2017-03-01

One bacterial strain, YC-RL2, isolated from petroleum-contaminated soil, could utilize environmental hormone Di(2-Ethylhexyl) phthalate (DEHP) as a sole carbon source for growth. Strain YC-RL2 was identified as Gordonia alkanivorans by 16S rRNA gene analysis and Biolog tests. The effects of environmental factors which might affect the degrading process were optimized at 30 °C and pH 8.0. Strain YC-RL2 showed superior halotolerance and could tolerate up to 0-5% NaCl in trace element medium supplemented with DEHP, although the DEHP degradation rates slowed as NaCl concentration increased. It also showed an outstanding performance in a wide range of pH (6.0-11.0). Meanwhile, strain YC-RL2 was able to withstand high concentrations of DEHP (from 100 to 800 mg/L), and the degradation rates were all above 94%. The DEHP intermediates were detected by HPLC-MS, and the degradation pathway was deduced tentatively. DEHP was transformed into phthalic acid (PA) via mono (2-ethylhexyl) phthalate (MEHP), and PA was further utilized for growth via benzoic acid (BA). The enzyme expected to catalyze the hydrolysis of MEHP to PA was identified from strain YC-RL2. Further investigation found that the enzyme could catalyze the transformation of a wide range of monoalkyl phthalates to PA. This study is the first report about species G. alkanivorans which could degrade several kinds of phthalic acid esters (PAEs), and indicates its application potential for bioremediation of PAE-polluted sites.

Snapshots of the maltose transporter during ATP hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oldham, Michael L.; Chen, Jue

2011-12-05

ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

Fundamental Reaction Pathway and Free Energy Profile for Butyrylcholinesterase-Catalyzed Hydrolysis of Heroin

PubMed Central

Qiao, Yan; Han, Keli; Zhan, Chang-Guo

2013-01-01

The pharmacological function of heroin requires an activation process which transforms heroin into 6-monoacetylmorphine (6-MAM) which is the most active form. The primary enzyme responsible for this activation process in human plasma is butyrylcholinesterase (BChE). The detailed reaction pathway of the activation process via BChE-catalyzed hydrolysis has been explored computationally, for the first time, in the present study by performing molecular dynamics simulation and first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the whole reaction process includes acylation and deacylation stages. The acylation consists of two reaction steps, i.e. the nucleophilic attack on the carbonyl carbon of 3-acetyl group of heroin by the hydroxyl oxygen of Ser198 side chain and the dissociation of 6-MAM. The deacylation also consists of two reaction steps, i.e. the nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate by a water molecule and the dissociation of the acetic acid from Ser198. The calculated free energy profile reveals that the second transition state (TS2) should be rate-determining. The structural analysis reveals that the oxyanion hole of BChE plays an important role in the stabilization of the rate-determining transition state TS2. The free energy barrier (15.9±0.2 or 16.1±0.2 kcal/mol) calculated for the rate-determining step is in good agreement with the experimentally-derived activation free energy (~16.2 kcal/mol), suggesting that the mechanistic insights obtained from the present computational study are reliable. The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse. PMID:23992153

Simulating biochemical physics with computers: 1. Enzyme catalysis by phosphotriesterase and phosphodiesterase; 2. Integration-free path-integral method for quantum-statistical calculations

NASA Astrophysics Data System (ADS)

Wong, Kin-Yiu

We have simulated two enzymatic reactions with molecular dynamics (MD) and combined quantum mechanical/molecular mechanical (QM/MM) techniques. One reaction is the hydrolysis of the insecticide paraoxon catalyzed by phosphotriesterase (PTE). PTE is a bioremediation candidate for environments contaminated by toxic nerve gases (e.g., sarin) or pesticides. Based on the potential of mean force (PMF) and the structural changes of the active site during the catalysis, we propose a revised reaction mechanism for PTE. Another reaction is the hydrolysis of the second-messenger cyclic adenosine 3'-5'-monophosphate (cAMP) catalyzed by phosphodiesterase (PDE). Cyclicnucleotide PDE is a vital protein in signal-transduction pathways and thus a popular target for inhibition by drugs (e.g., ViagraRTM). A two-dimensional (2-D) free-energy profile has been generated showing that the catalysis by PDE proceeds in a two-step SN2-type mechanism. Furthermore, to characterize a chemical reaction mechanism in experiment, a direct probe is measuring kinetic isotope effects (KIEs). KIEs primarily arise from internuclear quantum-statistical effects, e.g., quantum tunneling and quantization of vibration. To systematically incorporate the quantum-statistical effects during MD simulations, we have developed an automated integration-free path-integral (AIF-PI) method based on Kleinert's variational perturbation theory for the centroid density of Feynman's path integral. Using this analytic method, we have performed ab initio pathintegral calculations to study the origin of KIEs on several series of proton-transfer reactions from carboxylic acids to aryl substituted alpha-methoxystyrenes in water. In addition, we also demonstrate that the AIF-PI method can be used to systematically compute the exact value of zero-point energy (beyond the harmonic approximation) by simply minimizing the centroid effective potential.

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

PubMed Central

Chow, Jennifer; Kovacic, Filip; Dall Antonia, Yuliya; Krauss, Ulrich; Fersini, Francesco; Schmeisser, Christel; Lauinger, Benjamin; Bongen, Patrick; Pietruszka, Joerg; Schmidt, Marlen; Menyes, Ina; Bornscheuer, Uwe T.; Eckstein, Marrit; Thum, Oliver; Liese, Andreas; Mueller-Dieckmann, Jochen; Jaeger, Karl-Erich; Streit, Wolfgang R.

2012-01-01

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure. PMID:23112831

On the mechanism of aluminum ion-induced neurotoxicity: The effects of aluminum species on G-protein-mediated processes and on drug interactions with the N-methyl-D-aspartate modulated ionophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hubbard, C.M.

1989-01-01

To establish what effects Al{sup 3+} may have on G-protein mediate signal transduction, the effects of Al{sup 3+} on the signal-coupling G-protein from retinal rod outer segments (G{sub t} or transducin) have been investigated as a model for the effects of Al{sup 3+} on signal transduction by G-proteins in general. In this investigation, we have studied the effects of Al{sup 3+} on the isolated, light-dependent rhodopsin catalyzed GTP-GDP exchange on G{sub t}; the light-dependent GTPase activity of G{sub t}; the light-independent cGMP hydrolysis by PDE; and the light activated, rhodopsin catalyzed, cGMP hydrolysis by PDE in vitro. To determine themore » effects of two defined species of aluminum on N-methyl-D-aspartic acid (NMDA) receptor-channel modulation we utilized a specific radioligand binding assay. This allowed us to compare the effects of aluminum to other metal ions on specific ({sup 3}H)MK-801 binding to the NMDA receptor-channel complex. This complex is involved in long-term potentiation, which is currently being investigated as the mechanism by which learning and memory occur and has been implicated in the pathology of Alzheimer's disease. We have investigated the effects of two different species of aluminum, as well as Ca{sup 2+}, Zn{sup 2+}, Mg{sup 2+}, and Li{sup +} on the specific binding of ({sup 3}H)MK-801 to the NMDA receptor-channel complex under depolarized conditions.« less

[Structural characterization of Astragalus polysaccharides using partial acid hydrolysis-hydrophilic interaction liquid chromatography-mass spectrometry].

PubMed

Liang, Tu; Fu, Qing; Xin, Huaxia; Li, Fangbing; Jin, Yu; Liang, Xinmiao

2014-12-01

Water-soluble polysaccharides from traditional Chinese medicine (TCM) have properties of broad-spectrum treatment and low toxicity, making them as important components in natural medicines and health products. In order to solve the problem of polysaccharides characterization caused by their complex structures, a "bottom-up" approach was developed to complete the characterization of polysaccharides from Astragalus. Firstly, Astragalus pieces were extracted with hot water and then were precipitated by ethanol to obtain Astragalus polysaccharides. Secondly, a partial acid hydrolysis method was carried out and the effects of time, acid concentration and temperature on hydrolysis were investigated. The degree of hydrolysis increased along with the increase of hydrolysis time and acid concentration. The temperature played a great role in the hydrolysis process. No hydrolysis of the polysaccharides occurred at low temperature, while the polysaccharides were almost hydrolyzed to monosaccharide at high temperature. Under the optimum hydrolysis conditions (4 h, 1.5 mol/L trifluoroacetic acid, and 80 °C), Astragalus polysaccharides were hydrolyzed to characteristic oligosaccharide fragments. At last, a hydrophilic liquid chromatography-mass spectrometry method was used for the separation and structural characterization of the polysaccharide hydrolysates. The results showed that the resulting polysaccharides were mainly 1--> 4 linear glucan, and gluco-oligosaccharides with the degrees of polymerization (DP) of 4 - 11 were obtained after partial acid hydrolysis. The significance of this study is that it is the guidance for the characterization of other TCM polysaccharides.

Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

PubMed Central

Balasubramanian, Anuradha; Ponnuraj, Karthe

2008-01-01

Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized and the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å. PMID:18607103

Xylanases, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A; Dirmeier, Reinhard

2013-07-16

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.

Xylanases, nucleic acids encoding them and methods for making and using them

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gray, Kevin A.; Dirmeier, Richard

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanasesmore » have increased activity and stability at increased pH and temperature.« less

pH Wave-Front Propagation in the Urea-Urease Reaction

PubMed Central

Wrobel, Magdalena M.; Bánsági, Tamás; Scott, Stephen K.; Taylor, Annette F.; Bounds, Chris O.; Carranza, Arturo; Pojman, John A.

2012-01-01

The urease-catalyzed hydrolysis of urea displays feedback that results in a switch from acid (pH ∼3) to base (pH ∼9) after a controllable period of time (from 10 to >5000 s). Here we show that the spatially distributed reaction can support pH wave fronts propagating with a speed of the order of 0.1−1 mm min−1. The experimental results were reproduced qualitatively in reaction-diffusion simulations including a Michaelis-Menten expression for the urease reaction with a bell-shaped rate-pH dependence. However, this model fails to predict that at lower enzyme concentrations, the unstirred reaction does not always support fronts when the well-stirred reaction still rapidly switches to high pH. PMID:22947878

γ-Glutamyltranspeptidases: sequence, structure, biochemical properties, and biotechnological applications.

PubMed

Castellano, Immacolata; Merlino, Antonello

2012-10-01

γ-Glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are involved in glutathione metabolism and play critical roles in antioxidant defense, detoxification, and inflammation processes. Moreover, γ-GTs have been recently found to be involved in many physiological disorders, such as Parkinson's disease and diabetes. In this review, the main biochemical and structural properties of γ-GTs isolated from different sources, as well as their conformational stability and mechanism of catalysis, are described and examined with the aim of contributing to the discussion on their structure-function relationships. Possible applications of γ-glutamyltranspeptidases in different fields of biotechnology and medicine are also discussed.

Acid-Catalyzed Isomerization of Carvone to Carvacrol

ERIC Educational Resources Information Center

Kjonaas, Richard A.; Mattingly, Shawn P.

2005-01-01

The acid-catalyzed isomerization of carvone to carvacrol, first reported by Ritter and Ginsburg, is especially well suited with a permanent-magnet FT instrument. The acid-catalyzed isomerization of carvone to carvacrol produced a 61% yield after a three hour reflux with 30% aqueous sulfuric acid.

Variability among strains of Aspergillus section Nigri with capacity to degrade tannic acid isolated from extreme environments.

PubMed

Lara-Victoriano, F; Veana, F; Hernández-Castillo, F D; Aguilar, C N; Reyes-Valdés, M H; Rodríguez-Herrera, R

2017-01-01

Tannins are polyphenolic compounds that cause astringent flavor and turbidity in food. Tannase is an enzyme that catalyzes the hydrolysis of tannins and is used in food industry. This study was conducted to determine the genetic variability and the tannase alleles variation in fungal strains isolated from soil and plants at five extreme areas of Coahuila, México. Two screening assays under 1 and 20 % of tannic acid were performed, with the isolations. In these assays, it was possible to identify 756 and 128 fungal strains, respectively. The major fungal variability was observed in "Cuatro Ciénegas" with 26 strains. The microorganisms were distributed in 11 groups, which correspond to Aspergillus section Nigri. AN7 and AN1 groups showed the major number of isolates from "Paila" and "Cuatro Ciénegas" locations, respectively. In the last location, the major diversity and specific richness were found. But in "Ojo Caliente," tannase allele conservations were observed.

L-Asparaginase II Produced by Salmonella Typhimurium Inhibits T Cell Responses and Mediates Virulence

PubMed Central

Kullas, Amy L.; McClelland, Michael; Yang, Hee-Jeong; Tam, Jason W.; Torres, AnnMarie; Porwollik, Steffen; Mena, Patricio; McPhee, Joseph B.; Bogomolnaya, Lydia; Andrews-Polymenis, Helene; van der Velden, Adrianus W.M.

2013-01-01

SUMMARY Salmonella enterica serovar Typhimurium avoids clearance by the host immune system by suppressing T cell responses; however, the mechanisms that mediate this immunosuppression remain unknown. We show that S. Typhimurium inhibit T cell responses by producing L-Asparaginase II, which catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia. L-Asparaginase II is necessary and sufficient to suppress T cell blastogenesis, cytokine production, and proliferation and to downmodulate expression of the T cell receptor. Furthermore, S. Typhimurium-induced inhibition of T cells in vitro is prevented upon addition of L-asparagine. S. Typhimurium lacking the L-Asparaginase II gene (STM3106) are unable to inhibit T cell responses and exhibit attenuated virulence in vivo. L-Asparaginases are used to treat acute lymphoblastic leukemia through mechanisms that likely involve amino acid starvation of leukemic cells, and these findings indicate that pathogens similarly use L-asparagine deprivation to limit T cell responses. PMID:23245323

Structural and functional studies on urease from pigeon pea (Cajanus cajan).

PubMed

Balasubramanian, Anuradha; Durairajpandian, Vishnuprabu; Elumalai, Sagadevan; Mathivanan, Narayanasamy; Munirajan, Arasambattu Kannan; Ponnuraj, Karthe

2013-07-01

Urease is an enzyme that catalyzes the hydrolysis of urea, forming ammonia and carbon dioxide, and is found in plants, microorganisms and invertebrates. Although plant and bacterial ureases are closely related at amino acid and at the structural level, the insecticidal activity is seen only in the plant ureases. In contrast, both plant and bacterial ureases exhibit antifungal activity. These two biological properties are independent of its ureolytic activity. However, till date the mechanism(s) behind the insecticidal and fungicidal activity of ureases are not clearly understood. Here we report the crystal structure of pigeon pea urease (PPU, Cajanus cajan) which is the second structure from the plant source. We have deduced the amino acid sequence of PPU and also report here studies on its stability, insecticidal and antifungal activity. PPU exhibits cellulase activity. Based on the structural analysis of PPU and docking studies with cellopentoase we propose a possible mechanism of antifungal activity of urease. Copyright © 2013 Elsevier B.V. All rights reserved.

Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

PubMed

Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

2017-02-01

5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. Copyright © 2016 Elsevier Ltd. All rights reserved.

Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI.

PubMed

Zhang, Shujun; Ma, Guangcai; Liu, Yongjun; Ling, Baoping

2015-09-01

2-Pyrone-4,6-dicarboxylate lactonase (LigI) is the first identified enzyme from amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. It catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. In this paper, a combined quantum mechanics and molecule mechanics (QM/MM) approach was employed to study the reaction mechanism of LigI from Sphingomonas paucimobilis. According to the results of our calculations, the whole catalytic reaction contains three elementary steps, including the nucleophilic attack, the cleavage of CO of lactone (substrate) and the intramolecular proton transfer. The intermediate has two intramolecular proton transfer pathways, due to which, two final hydrolysis products can be obtained. The energy profile indicates that 4-carboxy-2-hydroxymuconate (CHM) is the main hydrolysis product, therefore, the isomerization between 4-carboxy-2-hydroxymuconate (CHM) and 4-oxalomesaconate (OMA) is suggested to occur in solvent. During the catalytic reaction, residue Asp248 acts as a general base to activate the hydrolytic water molecule. Although His31, His33 and His180 do not directly participate in the chemical process, they play assistant roles by forming electrostatic interactions with the substrate and its involved species in activating the carbonyl group of the substrate and stabilizing the intermediates and transition states. Copyright © 2015 Elsevier Inc. All rights reserved.

The rabbit liver microsomal biotransformation of 1,1-dialkylethylenes: enantioface selection of epoxidation and enantioselectivity of epoxide hydrolysis.

PubMed

Bellucci, G; Chiappe, C; Cordoni, A; Marioni, F

1994-01-01

The rabbit liver microsomal biotransformation of alpha-methylstyrene (1a), 2-methyl-1-hexene (1b), 2,4,4-trimethyl-1-pentene (1c), and 1,3,3-trimethyl-1-butene (1d) has been investigated with the aim at establishing the enantioface selection of the cytochrome P-450-promoted epoxidation of the double bond and the enantioselectivity of microsomal epoxide hydrolase(mEH)-catalyzed hydrolysis of the resulting epoxides. GLC on a Chiraldex G-TA (ASTEC) column was used to determine the enantiomeric composition of the products. The epoxides 2 first produced in incubations carried out in the presence of an NADPH regenerating system were not detected, being rapidly hydrolyzed by mEH to diols 3. The enantiomeric composition of the latter showed that no enantioface selection occurred in the epoxidation of 1c and 1d, and a very low (8%) ee of the (R)-epoxide was formed from 1b. Incubation of racemic epoxides 2b-d with the microsomal fraction showed that the mEH-catalyzed hydrolysis of 2c and 2d was practically nonenantioselective, while that of 2b exhibited a selectivity E = 4.9 favoring the hydrolysis of the (S)-enantiomer. A comparison of these results with those previously obtained for linear and branched chain alkyl monosubstituted oxiranes shows that the introduction of the second alkyl substituent suppresses the selectivity of the mEH reaction of the latter and reverses that of the former substrates.

Enhanced functional properties of tannic acid after thermal hydrolysis

USDA-ARS?s Scientific Manuscript database

Thermal hydrolysis processing of fresh tannic acid was carried out in a closed reactor at four different temperatures (65, 100, 150 and 200°C). Pressures reached in the system were 1.3 and 4.8 MPa at 150 and 200°C, respectively. Hydrolysis products (gallic acid and pyrogallol) were separated and qua...

Pretreatment of wheat straw by nonionic surfactant-assisted dilute acid for enhancing enzymatic hydrolysis and ethanol production.

PubMed

Qi, Benkun; Chen, Xiangrong; Wan, Yinhua

2010-07-01

Pretreating wheat straw (WS) with combined use of varied sulfuric acid concentration (0-3%, w/v) and Tween 20 concentration (0-1%) was investigated in an attempt to enhance the hydrolysis and fermentability of pretreated WS. Enzymatic hydrolysis yield of glucan and xylan and ethanol production by simultaneous saccharification and fermentation (SSF) of water-insoluble solids (WIS) were significantly affected by the amount of Tween 20 added during acid pretreatment. Any further addition of Tween 20 in either hydrolysis stage or fermentation stage only led to small increase in glucan conversion and ethanol production. Determination of adsorption of cellulases during hydrolysis showed that Tween 20-assisted acid treated straw solution contained more free cellulases than individual acid treated straw solution, indicating that modification of lignin surface by Tween 20 added during pretreatment likely occurred. In addition, the effects of pretreatment conditions on overall recovery of glucose and xylose after pretreatment and enzymatic hydrolysis were also investigated. Copyright 2010 Elsevier Ltd. All rights reserved.

Validation of lignocellulosic biomass carbohydrates determination via acid hydrolysis.

PubMed

Zhou, Shengfei; Runge, Troy M

2014-11-04

This work studied the two-step acid hydrolysis for determining carbohydrates in lignocellulosic biomass. Estimation of sugar loss based on acid hydrolyzed sugar standards or analysis of sugar derivatives was investigated. Four model substrates (starch, holocellulose, filter paper and cotton) and three levels of acid/material ratios (7.8, 10.3 and 15.4, v/w) were studied to demonstrate the range of test artifacts. The method for carbohydrates estimation based on acid hydrolyzed sugar standards having the most satisfactory carbohydrate recovery and relative standard deviation. Raw material and the acid/material ratio both had significant effect on carbohydrate hydrolysis, suggesting the acid to have impacts beyond a catalyst in the hydrolysis. Following optimal procedures, we were able to reach a carbohydrate recovery of 96% with a relative standard deviation less than 3%. The carbohydrates recovery lower than 100% was likely due to the incomplete hydrolysis of substrates, which was supported by scanning electron microscope (SEM) images. Copyright © 2014 Elsevier Ltd. All rights reserved.

Preparation and characterization of dialdehyde starch by one-step acid hydrolysis and oxidation.

PubMed

Zuo, Yingfeng; Liu, Wenjie; Xiao, Junhua; Zhao, Xing; Zhu, Ying; Wu, Yiqiang

2017-10-01

Dialdehyde starch was prepared by one-step synthesis of acid hydrolysis and oxidation, using corn starch as the raw material, sodium periodate (NaIO 4 ) as the oxidant, and hydrochloric acid (HCl) as the acid solution. The prepared dialdehyde starch was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and gel permeation chromatography (GPC). The results confirmed that oxidation occurred between the starch and NaIO 4 . The acid hydrolysis reaction reduced the molecular weight of starch and effectively improved the aldehyde group contents (92.7%). Scanning electron microscope (SEM) analysis indicated that the average particle size decreased after acid hydrolysis and oxidation reaction. X-ray diffraction (XRD) and thermal gravimetric analyzer (TGA) analysis demonstrated that the crystallinity of the obtained dialdehyde starch showed a downward trend and a decelerated thermal decomposition rate. The starch after acid hydrolysis and oxidation exhibited lower hot paste viscosity and higher reactivity. Copyright © 2017. Published by Elsevier B.V.

Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

PubMed Central

Lin, Yun-Jian; Le, Guo-Wei; Wang, Jie-Yun; Li, Ya-Xin; Shi, Yong-Hui; Sun, Jin

2010-01-01

This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen. PMID:21151439

Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

PubMed

Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel

2014-11-04

Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar hydrogen bond donors within the active site, dominate in defining this reaction specificity.

Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.

PubMed

Chen, Chao; Cao, Ruikai; Shrestha, Ruben; Ward, Christina; Katz, Benjamin B; Fischer, Christopher J; Tomich, John M; Li, Ping

2015-05-15

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.

Phosphoester hydrolysis: the incoming substrate turns the bridging hydroxido nucleophile into a terminal one.

PubMed

Gouré, Eric; Carboni, Michaël; Troussier, Angélique; Lebrun, Colette; Pécaut, Jacques; Jacquot, Jean-François; Dubourdeaux, Patrick; Clémancey, Martin; Blondin, Geneviève; Latour, Jean-Marc

2015-05-26

Identifying the active nucleophile in hydrolysis reactions catalyzed by binuclear hydrolases is a recurrent problem and a matter of intense debate. We report on the phosphate ester hydrolysis by a Fe(III)Fe(II) complex of a binucleating ligand. This complex presents activities in the range of those observed for similar biomimetic compounds in the literature. The specific electronic properties of the Fe(III)Fe(II) complex allowed us to use (1)H NMR and Mössbauer spectroscopies to investigate the nature of the various species present in the solution in the pH range of 5-10. Both techniques showed that the hydrolysis activity is associated to a μ-hydroxido Fe(III)Fe(II) species. Further (1)H NMR experiments show that binding of anions or the substrate changes this bonding mode suggesting that a terminal hydroxide is the likely nucleophile in these hydrolysis reactions. This view is further supported by the structure determination of the hydrolysis product. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Decomposition of lignin and cellobiose in relation to the enzymatic hydrolysis of cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamanaka, Y.; Carroad, P.A.; Riaz, M.

1977-02-01

Studies are reported on the use of fungal ..beta..-glucosidase in conjunction with Trichoderma viride cellulase and the search for an effective enzyme system for lignin degradation. ..beta..-glucosidase is of potential benefit in cellulose hydrolysis by catalyzing the hydrolysis of cellobiose to glucose thereby reducing product inhibition and producing a higher glucose yield. Removal of lignin from cellulosic material makes the cellulose more accessible to hydrolyzing enzymes. Hydrolysis studies on Solka Floc and newsprint were conducted with T. viride filtrates containing various proportions of B. theobromae filtrates. Significant improvement in hydrolysis rate particularly in glucose content was obtained by thus enrichingmore » the ..beta..-glucosidase content of the cellulase. In the search for a lignin degrading enzyme, major emphasis was given to the fungus Polyporous versicolor. Significant o-diphenol oxidoreductase (catecholase) activity was found in the culture filtrates. Preliminary observations of a surface culture of the fungus in a composting mode suggest that delignification may be obtained in this manner. Work is continuing on this.« less

Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acid

NASA Astrophysics Data System (ADS)

Miranda-Rojas, Sebastián; Toro-Labbé, Alejandro

2015-05-01

Fluoroacetate is a toxic compound whose environmental accumulation may represent an important contamination problem, its elimination is therefore a challenging issue. Fluoroacetate dehalogenase catalyzes its degradation through a two step process initiated by an SN2 reaction in which the aspartate residue performs a nucleophilic attack on the carbon bonded to the fluorine; the second step is hydrolysis that releases the product as glycolate. In this paper, we present a study based on density functional theory calculations of the SN2 initiation reaction modeled through the interaction between the substrate and the propionate anion as the nucleophile. Results are analyzed within the framework of the reaction force and using the reaction electronic flux to identify and characterize the electronic activity that drives the reaction. Our results reveal that the selective protonation of the substrate catalyzes the reaction by decreasing the resistance of the structural and electronic reorganization needed to reach the transition state. Finally, the reaction energy is modulated by the degree of stabilization of the fluoride anion formed after the SN2 reaction. In this way, a site-induced partial protonation acts as a chemical switch in a key process that determines the output of the reaction.

Polysiloxanes derived from the controlled hydrolysis of tetraethoxysilane as precursors to silica for use in ceramic processing

NASA Technical Reports Server (NTRS)

Philipp, Warren H.

1990-01-01

Synthesis, properties, and potential applications in ceramic processing for two polysiloxane silica precursors derived from the controlled hydrolysis of tetraethoxysilane (TEOS) are presented. The higher molecular weight TEOS-A is a thick adhesive liquid of viscosity 8000 to 12,000 c.p. having a SiO2 char yield of about 55 percent. The lower molecular weight TEOS-B is a more fluid liquid of viscosity 150 to 200 c.p. having a SiO2 char yield of about 52 percent. The acid catalyzed hydrolysis of TEOS to hydrated silica gel goes through a series of polysiloxane intermediates. The rate of this transition increases with the quantity of water added to the TEOS; thus, for ease of polymer isolation, the amount of water added must be carefully determined so as to produce the desired polymer in a reasonable time. The water to TEOS mole ratio falls in the narrow range of 1.05 for TEOS-A and 0.99 for TEOS-B. Further polymerization or gelation is prevented by storing at -5 C in a freezer. Both polysiloxanes thermoset to a glassy solid at 115 C. The liquid polymers are organic in nature in that they are miscible with toluene and ethanol, slightly souble in heptane, but immiscible with water. For both polymers, results on viscosity versus time are given at several temperatures and water additions. Based on these results, some examples of practical utilization of the precursors for ceramic fabrication are given.

Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol.

PubMed

Shukor, Hafiza; Abdeshahian, Peyman; Al-Shorgani, Najeeb Kaid Nasser; Hamid, Aidil Abdul; Rahman, Norliza A; Kalil, Mohd Sahaid

2016-10-01

Catalytic depolymerization of mannan composition of palm kernel cake (PKC) by mannanase was optimized to enhance the release of mannan-derived monomeric sugars for further application in acetone-butanol-ethanol (ABE) fermentation. Efficiency of enzymatic hydrolysis of PKC was studied by evaluating effects of PKC concentration, mannanase loading, hydrolysis pH value, reaction temperature and hydrolysis time on production of fermentable sugars using one-way analysis of variance (ANOVA). The ANOVA results revealed that all factors studied had highly significant effects on total sugar liberated (P<0.01). The optimum conditions for PKC hydrolysis were 20% (w/v) PKC concentration, 5% (w/w) mannanase loading, hydrolysis pH 4.5, 45°C temperature and 72h hydrolysis time. Enzymatic experiments in optimum conditions revealed total fermentable sugars of 71.54±2.54g/L were produced including 67.47±2.51g/L mannose and 2.94±0.03g/L glucose. ABE fermentation of sugar hydrolysate by Clostridium saccharoperbutylacetonicum N1-4 resulted in 3.27±1.003g/L biobutanol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anticancer and antiviral effects and inactivation of S-adenosyl-L-homocysteine hydrolase with 5'-carboxaldehydes and oximes synthesized from adenosine and sugar-modified analogues.

PubMed

Wnuk, S F; Yuan, C S; Borchardt, R T; Balzarini, J; De Clercq, E; Robins, M J

1997-05-23

Selectively protected adenine nucleosides were converted into 5'-carboxaldehyde analogues by Moffatt oxidation (dimethyl sulfoxide/dicyclohexylcarbodiimide/dichloroacetic acid) or with the Dess-Martin periodinane reagent. Hydrolysis of a 5'-fluoro-5'-S-methyl-5'-thio (alpha-fluoro thioether) arabinosyl derivative also gave the 5'-carboxaldehyde. Treatment of 5'-carboxaldehydes with hydroxylamine [or O-(methyl, ethyl, and benzyl)hydroxylamine] hydrochloride gave E/Z oximes. Treatment of purified oximes with aqueous trifluoroacetic acid and acetone effected trans-oximation to provide clean samples of 5'-carboxaldehydes. Adenosine (Ado)-5'-carboxaldehyde and its 4'-epimer are potent inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. They bind efficiently to the enzyme and undergo oxidation at C3' to give 3'-keto analogues with concomitant reduction of the NAD+ cofactor to give an inactive, tightly bound NADH-enzyme complex (type I cofactor-depletion inhibition). Potent type I inhibition was observed with 5'-carboxaldehydes that contain a ribo cis-2',3'-glycol. Their oxime derivatives are "proinhibitors" that undergo enzyme-catalyzed hydrolysis to release the inhibitors at the active site. The 2'-deoxy and 2'-epimeric (arabinosyl) analogues were much weaker inhibitors, and the 3'-deoxy compounds bind very weakly. Ado-5'-carboxaldehyde oxime had potent cytotoxicity in tumor cell lines and was toxic to normal human cells. Analogues had weaker cytotoxic and antiviral potencies, and the 3'-deoxy compounds were essentially devoid of cytotoxic and antiviral activity.

Effect of alcohol on skin permeation and metabolism of an ester-type prodrug in Yucatan micropig skin.

PubMed

Fujii, Makiko; Ohara, Rieko; Matsumi, Azusa; Ohura, Kayoko; Koizumi, Naoya; Imai, Teruko; Watanabe, Yoshiteru

2017-11-15

We studied the effect that three alcohols, ethanol (EA), propanol (PA), and isopropanol (IPA), have on the skin permeation of p-hydroxy benzoic acid methyl ester (HBM), a model ester-type prodrug. HBM was applied to Yucatan micropig skin in a saturated phosphate buffered solution with or without 10% alcohol, and HBM and related materials in receptor fluid and skin were determined with HPLC. In the absence of alcohol, p-hydroxy benzoic acid (HBA), a metabolite of HBM, permeated the skin the most. The three alcohols enhanced the penetration of HBM at almost the same extent. The addition of 10% EA or PA to the HBM solution led to trans-esterification into the ethyl ester or propyl ester of HBA, and these esters permeated skin as well as HBA and HBM did. In contrast, the addition of 10% IPA promoted very little trans-esterification. Both hydrolysis and trans-esterification in the skin S9 fraction were inhibited by BNPP, an inhibitor of carboxylesterase (CES). Western blot and native PAGE showed the abundant expression of CES in micropig skin. Both hydrolysis and trans-esterification was simultaneously catalyzed by CES during skin permeation. Our data indicate that the alcohol used in dermal drug preparations should be selected not only for its ability to enhance the solubility and permeation of the drug, but also for the effect on metabolism of the drug in the skin. Copyright © 2017 Elsevier B.V. All rights reserved.

The development of chiral nematic mesoporous materials.

PubMed

Kelly, Joel A; Giese, Michael; Shopsowitz, Kevin E; Hamad, Wadood Y; MacLachlan, Mark J

2014-04-15

Cellulose nanocrystals (CNCs) are obtained from the sulfuric acid-catalyzed hydrolysis of bulk cellulose. The nanocrystals have diameters of ~5-15 nm and lengths of ~100-300 nm (depending on the cellulose source and hydrolysis conditions). This lightweight material has mostly been investigated to reinforce composites and polymers because it has remarkable strength that rivals carbon nanotubes. But CNCs have an additional, less explored property: they organize into a chiral nematic (historically referred to as cholesteric) liquid crystal in water. When dried into a thin solid film, the CNCs retain the helicoidal chiral nematic order and assemble into a layered structure where the CNCs have aligned orientation within each layer, and their orientation rotates through the stack with a characteristic pitch (repeating distance). The cholesteric ordering can act as a 1-D photonic structure, selectively reflecting circularly polarized light that has a wavelength nearly matching the pitch. During CNC self-assembly, it is possible to add sol-gel precursors, such as Si(OMe)4, that undergo hydrolysis and condensation as the solvent evaporates, leading to a chiral nematic silica/CNC composite material. Calcination of the material in air destroys the cellulose template, leaving a high surface area mesoporous silica film that has pore diameters of ~3-10 nm. Importantly, the silica is brilliantly iridescent because the pores in its interior replicate the chiral nematic structure. These films may be useful as optical filters, reflectors, and membranes. In this Account, we describe our recent research into mesoporous films with chiral nematic order. Taking advantage of the chiral nematic order and nanoscale of the CNC templates, new functional materials can be prepared. For example, heating the silica/CNC composites under an inert atmosphere followed by removal of the silica leaves highly ordered, mesoporous carbon films that can be used as supercapacitor electrodes. The composition of the mesoporous films can be varied by using assorted organosilica precursors. After removal of the cellulose by acid-catalyzed hydrolysis, highly porous, iridescent organosilica films are obtained. These materials are flexible and offer the ability to tune the chemical and mechanical properties through variation of the organic spacer. Chiral nematic mesoporous silica and organosilica materials, obtainable as centimeter-scale freestanding films, are interesting hosts for nanomaterials. When noble metal nanoparticles are incorporated into the pores, they show strong circular dichroism signals associated with their surface plasmon resonances that arise from dipolar coupling of the particles within the chiral nematic host. Fluorescent conjugated polymers show induced circular dichroism spectra when encapsulated in the chiral nematic host. The porosity, film structure, and optical properties of these materials could enable their use in sensors. We describe the development of chiral nematic mesoporous silica and organosilica, demonstrate different avenues of host-guest chemistry, and identify future directions that exploit the unique combination of properties present in these materials. The examples covered in this Account demonstrate that there is a rich diversity of composite materials accessible using CNC templating.

Hydrolysis of tRNA(sup Phe) on Suspensions of Amino Acids

NASA Technical Reports Server (NTRS)

Gao, Kui; Orgel, Leslie E.

2001-01-01

RNA is adsorbed strongly on suspensions of many moderately soluble organic solids. In some cases, the hydrolysis of tRNA(sup Phe) is greatly accelerated by adsorption, and the major sites of hydrolysis are changed from those that are important in homogeneous solution. Here we show that the hydrolysis is greatly accelerated by suspensions of aspartic acid and beta-glutamic acid but not by suspensions of alpha-glutamic acid, asparagine, or glutamine. The non-enzymatic hydrolysis of RNA has been studied extensively, especially because of its relevance to the mechanisms of action of ribozymes and to biotechnology and therapy. Many ribonucleases, ribozymes, and non-biological catalysts function via acid-base catalysis of an intramolecular transesterification mechanism in which the 2'-OH group attacks the adjacent phosphate group. The pentacoordinated phosphorane intermediate may collapse back to starting material, or yield isomerized or cleaved products.

Hydrolysis mechanisms for the organopalladium complex [Pd(CNN)P(OMe)3]BF4 in sulfuric acid.

PubMed

García, Begoña; Hoyuelos, Francisco J; Ibeas, Saturnino; Muñoz, María S; Peñacoba, Indalecio; Leal, José M

2009-08-13

The acid-catalyzed hydrolysis of the organopalladium complex [Pd(CNN)P(OMe)3]BF4 species was monitored spectrophotometrically at different sulfuric acid concentrations (3.9 and 11.0 M) in 10% v:v ethanol-water over the 25-45 degrees C temperature range and in 30% and 50% (v/v) ethanol-water at 25 degrees C. Two acidity regions (I and II) could be differentiated. In each of the two regions the kinetic data pairs yielded two different rate constants, k(1obs) and k(2obs), the former being faster. These constants were fitted by an Excess Acidity analysis to different hydrolyses mechanisms: A-1, A-2, and A-SE2. In region I ([H2SO4] < 7.0 M), the k(1obs) values remained constant k(1obs)(av) = 1.6 x 10(-3) s(-1) and the set of k(2obs) values nicely matched an A-SE2 mechanism, yielding a rate-determining constant k(0,ASE2) = 2.4 x 10(-7) M(-1) s(-1). In region II ([H2SO4] > 7.0 M), a switchover was observed from an A-1 mechanism (k(0,A1) = 1.3 x 10(-4) s(-1)) to an A-2 mechanism (k(0,A2) = 3.6 x 10(-3) M(-1) s(-1)). The temperature effect on the rate constants in 10% (v/v) ethanol-water yielded positive DeltaH and negative DeltaS values, except for the A-1 mechanism, where DeltaS adopted positive values throughout. The solvent permittivity effect, epsilonr, revealed that k(1obs)(av) and k(0,A2) dropped with a fall in epsilonr, whereas the k(0,ASE2) value remained unaffected. The set of results deduced is in line with the schemes put forward.

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras

PubMed Central

Warren, Jeremy G.; Lincoln, James E.; Kirkpatrick, Bruce C.

2015-01-01

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date. PMID:26571265

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

PubMed

Warren, Jeremy G; Lincoln, James E; Kirkpatrick, Bruce C

2015-01-01

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.

Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung.

PubMed Central

Lee, J D; Kolattukudy, P E

1995-01-01

Hydrolysis of structural proteins in the lung by extracellular proteinases secreted by Aspergillus fumigatus is thought to play a significant role in invasive aspergillosis. This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase. We report that A. fumigatus also secretes an aspartic proteinase (aspergillopepsin F) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin. The pH optimum for the enzymatic activity was 5.0 with elastin-Congo red as the substrate, and the activity was not significantly inhibited by pepstatin A, diazoacetyl norleucine methylester, and 1,2-epoxy-3-(p-nitrophenoxy) propane. The cDNA and gene encoding this aspartic proteinase were cloned and sequenced. The open reading frame, interrupted by three introns, would encode a protein of 393 amino acids composed of a putative 21-amino-acid signal peptide and a 49-amino-acid propeptide preceding the 323-amino-acid mature protein. The amino acid sequence of A. fumigatus aspartic proteinase has 70, 66, and 67% homology to the sequences of those from Aspergillus oryzae, Aspergillus awamori, and Aspergillus saitoi, respectively. The active-site motif (DTG) and the catalytic aspartic residues characteristic of aspartic proteinases are found in the presently described enzyme, indicating that it belongs to a family of aspartic proteinases. Polyclonal antibodies were produced in rabbits against both the mature and precursor forms of the aspartic proteinase expressed in Escherichia coli. Immunoblotting with both antibodies detected a 39-kDa mature enzyme in the culture supernatant of A. fumigatus. The aspartic proteinase activity was inhibited by the antibodies, suggesting that the aspartic proteinase in the culture supernatant corresponds to the product of the cloned gene. Immunogold electron microscopy showed that the aspartic proteinase was secreted by A. fumigatus invading neutropenic mouse lung and its secretion was directed toward the germ tubes of penetrating hyphae. PMID:7558282

Combined heat treatment and acid hydrolysis of cassava grate waste (CGW) biomass for ethanol production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agu, R.C.; Amadife, A.E.; Ude, C.M.

1997-12-31

The effect of combined heat treatment and acid hydrolysis (various concentrations) on cassava grate waste (CGW) biomass for ethanol production was investigated. At high concentrations of H{sub 2}SO{sub 4} (1--5 M), hydrolysis of the CGW biomass was achieved but with excessive charring or dehydration reaction. At lower acid concentrations, hydrolysis of CGW biomass was also achieved with 0.3--0.5 M H{sub 2}SO{sub 4}, while partial hydrolysis was obtained below 0.3 M H{sub 2}SO{sub 4} (the lowest acid concentration that hydrolyzed CGW biomass) at 120 C and 1 atm pressure for 30 min. A 60% process efficiency was achieved with 0.3 Mmore » H{sub 2}SO{sub 4} in hydrolyzing the cellulose and lignin materials present in the CGW biomass. High acid concentration is therefore not required for CGW biomass hydrolysis. The low acid concentration required for CGW biomass hydrolysis, as well as the minimal cost required for detoxification of CGW biomass because of low hydrogen cyanide content of CGW biomass would seem to make this process very economical. From three liters of the CGW biomass hydrolysate obtained from hydrolysis with 0.3M H{sub 2}SO{sub 4}, ethanol yield was 3.5 (v/v%) after yeast fermentation. However, although the process resulted in gainful utilization of CGW biomass, additional costs would be required to effectively dispose new by-products generated from CGW biomass processing.« less

Poly(N-vinyl-2-pyrrolidone)-stabilized palladium-platinum nanoparticles-catalyzed hydrolysis of ammonia borane for hydrogen generation

NASA Astrophysics Data System (ADS)

Rakap, Murat

2015-02-01

The catalytic use of highly efficient poly(N-vinyl-2-pyrrolidone)-stabilized palladium-platinum nanoparticles (4.2 ± 1.9 nm) in the hydrolysis of ammonia-borane is reported. The catalyst is prepared by co-reduction of two metal ions in ethanol/water mixture by an alcohol reduction method and characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and UV-Vis spectroscopy. They are recyclable and highly active for hydrogen generation from the hydrolysis of ammonia-borane even at very low concentrations and temperature, providing a record numbers of average turnover frequency value (125 mol H2/mol cat.min-1) and maximum hydrogen generation rate (3468 L H2 min-1 (mol cat)-1). They also provide activation energy of 51.7 ± 2 kJ/mol for the hydrolysis of ammonia borane.

Additives enhancing enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Rocha-Martín, Javier; Martinez-Bernal, Claudio; Pérez-Cobas, Yolanda; Reyes-Sosa, Francisco Manuel; García, Bruno Díez

2017-11-01

Linked to the development of cellulolytic enzyme cocktails from Myceliophthora thermophila, we studied the effect of different additives on the enzymatic hydrolysis yield. The hydrolysis of pretreated corn stover (PCS), sugar cane straw (PSCS) and microcrystalline cellulose (Avicel) was performed under industrial conditions using high solid loadings, limited mixing, and low enzyme dosages. The addition of polyethylene glycol (PEG4000) allowed to increase the glucose yields by 10%, 7.5%, and 32%, respectively in the three materials. PEG4000 did not have significant effect on the stability of the main individual enzymes but increased beta-glucosidase and endoglucanase activity by 20% and 60% respectively. Moreover, the presence of PEG4000 accelerated cellulase-catalyzed hydrolysis reducing up to 25% the liquefaction time. However, a preliminary economical assessment concludes that even with these improvements, a lower contribution of PEG4000 to the 2G bioethanol production costs would be needed to reach commercial feasibility. Copyright © 2017. Published by Elsevier Ltd.

Co-production of functional xylooligosaccharides and fermentable sugars from corncob with effective acetic acid prehydrolysis.

PubMed

Zhang, Hongyu; Xu, Yong; Yu, Shiyuan

2017-06-01

A novel and green approach for the coproduction of xylooligosaccharides (XOS), in terms of a series of oligosaccharide components from xylobiose to xylohexose, and fermentable sugars was developed using the prehydrolysis of acetic acid that was fully recyclable and environmentally friendly, followed by enzymatic hydrolysis. Compared to hydrochloric acid and sulfuric acid, acetic acid hydrolysis provided the highest XOS yield of 45.91% and the highest enzymatic hydrolysis yield. More than 91% conversion of cellulose was achieved in a batch-hydrolysis using only a cellulase loading of 20FPU/g cellulose and even a high solid loading of 20% without any special strategies. The acetic acid pretreated corncob should be washed adequately before saccharification to achieve complete hydrolysis. Consequently, a mass balance analysis showed that 139.8g XOS, 328.1g glucose, 25.1g cellobiose, and 147.8g xylose were produced from 1000g oven dried raw corncob. Copyright © 2017. Published by Elsevier Ltd.

Kinetics of Strong Acid Hydrolysis of a Bleached Kraft Pulp for Producing Cellulose Nanocrystals (CNCs)

Treesearch

Qianqian Wang; Xuebing Zhao; J.Y. Zhu

2014-01-01

Cellulose nanocrytals (CNCs) are predominantly produced using the traditional strong acid hydrolysis process. In most reported studies, the typical CNC yield is low (approximately 30%) despite process optimization. This study investigated the hydrolysis of a bleached kraft eucalyptus pulp using sulfuric acid between 50 and 64 wt % at temperatures of 35−80 °C...

Extraction of cellulose nano-crystals from old corrugated container fiber using phosphoric acid and enzymatic hydrolysis followed by sonication.

PubMed

Tang, Yanjun; Shen, Xiaochuang; Zhang, Junhua; Guo, Daliang; Kong, Fangong; Zhang, Nan

2015-07-10

Due to its amazing physicochemical properties and high environmental compatibility, cellulose nano-crystals (CNC) hold great promise for serving as a strategic platform for sustainable development. Now, there has been growing interest in the development of processes using waste or residual biomass as CNC source for addressing economic and environmental concerns. In the present work, a combined process involving phosphoric acid hydrolysis, enzymatic hydrolysis and sonication was proposed aiming to efficiently exact CNC from low-cost old corrugated container (OCC) pulp fiber. The effect of enzymatic hydrolysis on the yield and microstructure of resulting CNC was highlighted. Results showed that the enzymatic hydrolysis was effective in enhancing CNC yield after phosphoric acid hydrolysis. CNC was obtained with a yield of 23.98 wt% via the combined process with phosphoric acid concentration of 60 wt%, cellulase dosage of 2 mL (84 EGU) per 2g fiber and sonication intensity of 200 W. Moreover, the presence of enzymatic hydrolysis imparted the obtained CNC with improved dispersion, increased crystallinity and thermal stability. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of hyper thermal acid hydrolysis of Kappaphycus alvarezii for enhanced bioethanol production.

PubMed

Ra, Chae Hun; Nguyen, Trung Hau; Jeong, Gwi-Taek; Kim, Sung-Koo

2016-06-01

Hyper thermal (HT) acid hydrolysis of Kappaphycus alvarezii, a red seaweed, was optimized to 12% (w/v) seaweed slurry content, 180mM H2SO4 at 140°C for 5min. The maximum monosaccharide concentration of 38.3g/L and 66.7% conversion from total fermentable monosaccharides of 57.6g/L with 120gdw/L K. alvarezii slurry were obtained from HT acid hydrolysis and enzymatic saccharification. HT acid hydrolysis at a severity factor of 0.78 efficiently converted the carbohydrates of seaweed to monosaccharides and produced a low concentration of inhibitory compounds. The levels of ethanol production by separate hydrolysis and fermentation with non-adapted and adapted Kluyveromyces marxianus to high concentration of galactose were 6.1g/L with ethanol yield (YEtOH) of 0.19 at 84h and 16.0g/L with YEtOH of 0.42 at 72h, respectively. Development of the HT acid hydrolysis process and adapted yeast could enhance the overall ethanol fermentation yields of K. alvarezii seaweed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Base-Catalyzed Depolymerization of Solid Lignin-Rich Streams Enables Microbial Conversion

DOE PAGES

Rodriguez, Alberto; Salvachúa, Davinia; Katahira, Rui; ...

2017-08-01

Lignin valorization offers significant potential to enhance the economic viability of lignocellulosic biorefineries. However, because of its heterogeneous and recalcitrant nature, conversion of lignin to value-added coproducts remains a considerable technical challenge. Here, we employ base-catalyzed depolymerization (BCD) using a process-relevant solid lignin stream produced via deacetylation, mechanical refining, and enzymatic hydrolysis to enable biological lignin conversion. BCD was conducted with the solid lignin substrate over a range of temperatures at two NaOH concentrations, and the results demonstrate that the lignin can be partially extracted and saponified at temperatures as low as 60 degrees C. At 120 °C and 2%more » NaOH, the high extent of lignin solubility was accompanied by a considerable decrease in the lignin average molecular weight and the release of lignin-derived monomers including hydroxycinnamic acids. BCD liquors were tested for microbial growth using seven aromatic-catabolizing bacteria and two yeasts. Three organisms (Pseudomonas putida KT2440, Rhodotorula mucilaginosa, and Corynebacterium glutamicum) tolerate high BCD liquor concentrations (up to 90% v/v) and rapidly consume the main lignin-derived monomers, resulting in lignin conversion of up to 15%. Furthermore, as a proof of concept, muconic acid production from a representative lignin BCD liquor was demonstrated with an engineered P. putida KT2440 strain. Our results highlight the potential for a mild lignin depolymerization process to enhance the microbial conversion of solid lignin-rich biorefinery streams.« less

Base-Catalyzed Depolymerization of Solid Lignin-Rich Streams Enables Microbial Conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez, Alberto; Salvachúa, Davinia; Katahira, Rui

Lignin valorization offers significant potential to enhance the economic viability of lignocellulosic biorefineries. However, because of its heterogeneous and recalcitrant nature, conversion of lignin to value-added coproducts remains a considerable technical challenge. Here, we employ base-catalyzed depolymerization (BCD) using a process-relevant solid lignin stream produced via deacetylation, mechanical refining, and enzymatic hydrolysis to enable biological lignin conversion. BCD was conducted with the solid lignin substrate over a range of temperatures at two NaOH concentrations, and the results demonstrate that the lignin can be partially extracted and saponified at temperatures as low as 60 degrees C. At 120 °C and 2%more » NaOH, the high extent of lignin solubility was accompanied by a considerable decrease in the lignin average molecular weight and the release of lignin-derived monomers including hydroxycinnamic acids. BCD liquors were tested for microbial growth using seven aromatic-catabolizing bacteria and two yeasts. Three organisms (Pseudomonas putida KT2440, Rhodotorula mucilaginosa, and Corynebacterium glutamicum) tolerate high BCD liquor concentrations (up to 90% v/v) and rapidly consume the main lignin-derived monomers, resulting in lignin conversion of up to 15%. Furthermore, as a proof of concept, muconic acid production from a representative lignin BCD liquor was demonstrated with an engineered P. putida KT2440 strain. Our results highlight the potential for a mild lignin depolymerization process to enhance the microbial conversion of solid lignin-rich biorefinery streams.« less

Enhancement of lipase catalyzed-fatty acid methyl esters production from waste activated bleaching earth by nullification of lipase inhibitors.

PubMed

Dwiarti, Lies; Ali, Ehsan; Park, Enoch Y

2010-01-01

This study sought to identify inhibitory factors of lipase catalyzed-fatty acid methyl esters (FAME) production from waste activated bleaching earth (wABE). During the vegetable oil refinery process, activated bleaching earth (ABE) is used for removing the impure compounds, but adsorbs vegetable oil up to 35-40% as on a weight basis, and then the wABE is discarded as waste material. The impurities were extracted from the wABE with methanol and evaluated by infra-red (IR) spectroscopy, which revealed that some were chlorophyll-plant pigments. The chlorophylls inhibited the lipase during FAME conversion from wABE. The inhibition by a mixture of chlorophyll a and b was found to be competitive. The inhibition of the enzymatic hydrolysis of waste vegetable oil contained in wABE by chlorophyll a alone was competitive, while the inhibition by chlorophyll b alone was non-competitive. Furthermore, the addition of a small amount of alkali nullified this inhibitory effect and accelerated the FAME production rate. When 0.9% KOH (w/w wABE) was added to the transesterification reaction with only 0.05% lipase (w/w wABE), the maximum FAME production rate improved 120-fold, as compared to that without the addition of KOH. The alkali-combined lipase significantly enhanced the FAME production rate from wABE, in spite of the presence of the plant pigments, and even when a lower amount of lipase was used as a catalyst.

Effect of cooking temperature on the crystallinity of acid hydrolysed-oil palm cellulose

NASA Astrophysics Data System (ADS)

Kuthi, Fatin Afifah Binti Ahmad; Badri, Khairiah Haji

2014-09-01

In this research, we studied the effect of acid hydrolysis temperature on the crystallinity of cellulose produced from empty fruit bunch (EFB). The hydrolysis temperature was studied from 120 to 140 °C at a fixed time and sulfuric acid, H2SO4 concentration which were 1 h and 1% (v/v) respectively. X-ray diffractometry (XRD) was carried out to measure the crystallinity of cellulose produced at varying hydrolysis temperatures. During hydrolysis, the amorphous region of α-cellulose was removed and the crystalline region was obtained. Percentage of crystallinity (CrI) for acid hydrolysed cellulose at 120, 130 and 140 °C were 54.21, 50.59 and 50.55 % respectively. Morphological studies using scanning electron microscope (SEM) showed that acid hydrolysis defibrilised to microfibrils in α-cellulose. The extraction process to produce α-cellulose has also been successfully carried out as the impurities at the outer surface, lignin and hemicellulose were removed. These findings were supported by the disappearance of peaks at 1732, 1512 and 1243 cm-1 on Fourier Transform infrared (FTIR) spectrum of α-cellulose. Similar peaks were identified in both the commercial microcrystalline cellulose (C-MCC) and acid hydrolysed cellulose (H-EFB), indicating the effectiveness of heat-catalysed acid hydrolysis.

Cellulose nanofiber isolation from palm oil Empty Fruit Bunches (EFB) through strong acid hydrolysis

NASA Astrophysics Data System (ADS)

Setyaningsih, Dwi; Uju; Muna, Neli; Isroi; Budi Suryawan, Nyoman; Azid Nurfauzi, Ami

2018-03-01

The palm oil industry produces about 25-26% of palm oil empty fruit bunches. The empty fruit bunch of palm oil contains cellulose up to 36.67%. This is a good opportunity for the synthesis of cellulose nanofiber (CNF). Cellulose nanofiber is a nano-sized cellulose material that has unique physical and mechanical properties. The synthesis was performed using a strong acid method with sulfuric acid. Sulfuric acid removes the amorphous region of cellulose so that the crystalline part can be isolated. CNF yield measurement showed that temperature, time, acid concentration, and interaction between each factor were affecting significantly to CNF yield. The result showed that yield of 14.98 grams, was obtained by hydrolysis at 35°C for 6 hours and 55% acid concentration. The crystallinity measurement showed that the temperature, time, acid concentration, and interaction between each factor during hydrolysis were not affected significantly to percent value of CNF crystallinity. The result showed that 31.1% of crystallinity, was obtained by hydrolysis at 45°C for 3 hours and 55% of acid concentration. The size measurement showed that the temperature, time, acid concentration and interaction between each factor were affected significantly. The result showed 894.25 nm as the best result, obtained by hydrolysis with 35°C and 60% acid concentration for 6 hours. CNF color was white with the best dispersion of hydrolysis at 35°C of 55% for 6 hours.

AN ENZYME MIMIC THAT HYDROLYZES AN UNACTIVATED ESTER WITH CATALYTIC TURNOVER. (R826653)

EPA Science Inventory

Abstract

The Cu(II) complex of a cyclodextrin dimer linked by a bipyridyl unit catalyzes the hydrolysis of an unactivated doubly-bound benzyl ester.

Author Keywords: cyclodextrin dimer; copper

Soil oxidoreductases and FDA hydrolysis

USDA-ARS?s Scientific Manuscript database

The oxidoreductases (E.C. 1.) comprise the largest enzyme group and consist of enzymes that catalyze reactions between two compounds, one of which is oxidized (the donor) while reducing the other (the acceptor) (Dixon and Webb, 1979). In common with all redox reactions, the reaction mechanism involv...

Immobilized lipase from Candida sp. 99-125 on hydrophobic silicate: characterization and applications.

PubMed

Zhao, Bin; Liu, Xinlong; Jiang, Yanjun; Zhou, Liya; He, Ying; Gao, Jing

2014-08-01

Lipase Candida sp. 99-125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99-125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.

Kinetics studies with fruit bromelain (Ananas comosus) in the presence of cysteine and divalent ions.

PubMed

Kaur, Tajwinder; Kaur, Amandeep; Grewal, Ravneet K

2015-09-01

The kinetics of cysteine and divalent ion modulation viz. Ca(2+), Cu(2+), Hg(2+) of fruit bromelain (EC 3.4.22.33) have been investigated in the present study. Kinetic studies revealed that at pH 4.5, cysteine induced V-type activation of bromelain catalyzed gelatin hydrolysis. At pH 3.5, Ca(2+) inhibited the enzyme noncompetitively, whereas, both K-and V-type activations of bromelain were observed in the presence of 0.5 mM Ca(2+) at pH 4.5 and 7.5. Bromelain was inhibited competitively at 0.6 mM Cu(2+) ions at pH 3.5, which changed to an uncompetitive inhibition at pH 4.5 and 7.5. An un-competitive inhibition of bromelain catalyzed gelatin hydrolysis was observed in the presence of 0.6 mM Hg(2+) at pH 3.5 and 4.5. These findings suggest that divalent ions modulation of fruit bromelain is pH dependent.

A thermoresponsive nanorattle containing two different catalysts for controllable one-pot tandem catalysis

NASA Astrophysics Data System (ADS)

Niu, Chengrong; Hu, Jie; Li, Yinfeng; Leng, Jinghang; Li, Songjun

2018-03-01

In the present work, a thermoresponsive nanorattle with a Ag nanoparticle (NP) core (one catalyst in the nanorattle), and a poly(N-isopropylacrylamide) shell was developed. An imidazole group was grafted on the polymer shell by copolymerization as the other catalyst. Owing to the catalytic activities of the imidazole group and Ag NP with regards to hydrolysis and reduction, respectively, this nanorattle exhibited tandem-reaction catalytic abilities. In addition, because of the shrinkage of the poly(N-isopropylacrylamide) shell at high temperatures, the tandem reaction could be controlled to stop at the first reaction step. That is to say, only the hydrolysis reaction was catalyzed by the imidazole group being grafted on the surface of the shell. The reduction step in the tandem reaction catalyzed by the Ag particle, however, was switched off by the shrinkage of the poly(N-isopropylacrylamide) shell. This protocol opens up an opportunity to develop controllable catalysts for complicated chemical processes.

Site-specific hydrolysis of chlorogenic acids by selected Lactobacillus species.

PubMed

Aguirre Santos, Elsa Anaheim; Schieber, Andreas; Weber, Fabian

2018-07-01

Hydroxycinnamic acids are a major group of phenolic compounds widely distributed in plants. Among them, chlorogenic acids and caffeic acid have been in the focus of interest due to their impact on food quality and their putative health benefits. Numerous microorganisms like lactic acid bacteria are able to hydrolyze chlorogenic acids by cinnamoyl esterase enzymes. Data on the specificity of theses enzymes regarding the cleavage of distinct isomers of mono- or dichlorogenic acids is lacking. Lactobacillus reuteri, Lactobacillus helveticus, and Lactobacillus fermentum were screened for their ability to hydrolyze chlorogenic acid isomers in culture medium. Concentrations of chlorogenic acids and the released caffeic acid were determined by UHPLC-ESI-MS. The highest hydrolysis rate (100%) was observed for the hydrolysis of 5-CQA by Lactobacillus helveticus. A so far unknown metabolic pathway for the cleavage of 4-CQA is proposed including isomerization to 5-CQA and 3-CQA followed by hydrolysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Function and application of a non-ester-hydrolyzing carboxylesterase discovered in tulip.

PubMed

Nomura, Taiji

2017-01-01

Plants have evolved secondary metabolite biosynthetic pathways of immense rich diversity. The genes encoding enzymes for secondary metabolite biosynthesis have evolved through gene duplication followed by neofunctionalization, thereby generating functional diversity. Emerging evidence demonstrates that some of those enzymes catalyze reactions entirely different from those usually catalyzed by other members of the same family; e.g. transacylation catalyzed by an enzyme similar to a hydrolytic enzyme. Tuliposide-converting enzyme (TCE), which we recently discovered from tulip, catalyzes the conversion of major defensive secondary metabolites, tuliposides, to antimicrobial tulipalins. The TCEs belong to the carboxylesterase family in the α/β-hydrolase fold superfamily, and specifically catalyze intramolecular transesterification, but not hydrolysis. This non-ester-hydrolyzing carboxylesterase is an example of an enzyme showing catalytic properties that are unpredictable from its primary structure. This review describes the biochemical and physiological aspects of tulipalin biogenesis, and the diverse functions of plant carboxylesterases in the α/β-hydrolase fold superfamily.

Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

PubMed

Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

2016-03-04

Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

Progressing batch hydrolysis process

DOEpatents

Wright, J.D.

1985-01-10

A progressive batch hydrolysis process is disclosed for producing sugar from a lignocellulosic feedstock. It comprises passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with feed stock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feed stock to glucose. The cooled dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, serially fed through a plurality of pre-hydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose. The dilute acid stream containing glucose is cooled after it exits the last prehydrolysis reactor.

Degradation Mechanisms of Poly(ester urethane) Elastomer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edgar, Alexander S.

This report describes literature regarding the degradation mechanisms associated with a poly(ester urethane) block copolymer, Estane® 5703 (Estane), used in conjunction with Nitroplasticizer (NP), and 1,3,5,7-tetranitro-1,3,5,7-tetrazocane, also known as high molecular weight explosive (HMX) to produce polymer bonded explosive PBX 9501. Two principal degradation mechanisms are reported: NO2 oxidative reaction with the urethane linkage resulting in crosslinking and chain scission events, and acid catalyzed hydrolysis of the ester linkage. This report details future work regarding this PBX support system, to be conducted in late 2017 and 2018 at Engineered Materials Group (MST-7), Materials Science and Technology Division, Los Alamos Nationalmore » Laboratory. This is the first of a series of three reports on the degradation processes and trends of the support materials of PBX 9501.« less

HCOOH-induced Controlled-release Hydrolysis of Microalgae (Scenedesmus) to Lactic Acid over Sn-Beta Catalyst.

PubMed

Zan, Yifan; Sun, Yuanyuan; Kong, Lingzhao; Miao, Gai; Bao, Liwei; Wang, Hao; Li, Shenggang; Sun, Yuhan

2018-06-12

Formic acid induced controlled-release hydrolysis of sugar-rich microalgae (Scenedesmus) over the Sn-Beta catalyst was found to be a highly efficient process for producing lactic acid as a platform chemical. One-pot reaction with a very high lactic acid yield of 83.0% was realized in a batch reactor using water as the solvent. Under the attack of formic acid, the cell wall of Scenedesmus was disintegrated, and hydrolysis of the starch inside the cell was strengthened in a controlled-release mode, resulting in a stable and relatively low glucose concentration. Subsequently, the Sn-Beta catalyst was employed for the efficient conversion of glucose into lactic acid with stable catalytic performance through isomerization, retro-aldol and de-/rehydration reactions. Thus, the hydrolysis of polysaccharides and the catalytic conversion of the monosaccharide into lactic acid was realized by the synergy between an organic Brønsted acid and a heterogeneous Lewis acid catalyst. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduction in environmental impact of sulfuric acid hydrolysis of bamboo for production of fuel ethanol.

PubMed

Sun, Zhao-Yong; Tang, Yue-Qin; Morimura, Shigeru; Kida, Kenji

2013-01-01

Fuel ethanol can be produced from bamboo by concentrated sulfuric acid hydrolysis followed by continuous ethanol fermentation. To reduce the environmental impact of this process, treatment of the stillage, reuse of the sulfuric acid and reduction of the process water used were studied. The total organic carbon (TOC) concentration of stillage decreased from 29,688 to 269 mg/l by thermophilic methane fermentation followed by aerobic treatment. Washing the solid residue from acid hydrolysis with effluent from the biological treatment increased the sugar recovery from 69.3% to 79.3%. Sulfuric acid recovered during the acid-sugar separation process was condensed and reused for hydrolysis, resulting in a sugar recovery efficiency of 76.8%, compared to 80.1% when fresh sulfuric acid was used. After acetate removal, the condensate could be reused as elution water in the acid-sugar separation process. As much as 86.3% of the process water and 77.6% of the sulfuric acid could be recycled. Copyright © 2012 Elsevier Ltd. All rights reserved.

Use of an algal hydrolysate to improve enzymatic hydrolysis of anaerobically digested fiber

USDA-ARS?s Scientific Manuscript database

This study investigated the use of acid hydrolyzed algae to enhance the enzymatic hydrolysis of cellulosic biomass. We first characterized wastewater-grown algal samples and determined the optimal conditions (acid concentration, reaction temperature, and reaction time) for algal hydrolysis using di...

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues.

PubMed

Ben Taher, Imen; Fickers, Patrick; Chniti, Sofien; Hassouna, Mnasser

2017-03-01

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L -1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L -1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397-406, 2017. © 2017 American Institute of Chemical Engineers.

Progressing batch hydrolysis process

DOEpatents

Wright, John D.

1986-01-01

A progressive batch hydrolysis process for producing sugar from a lignocellulosic feedstock, comprising passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feedstock to glucose; cooling said dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, then feeding said dilute acid stream serially through a plurality of prehydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose; and cooling the dilute acid stream containing glucose after it exits the last prehydrolysis reactor.

Arrhenius equation for modeling feedyard ammonia emissions using temperature and diet crude protein

USDA-ARS?s Scientific Manuscript database

Temperature controls many processes of ammonia volatilization. For example, urea hydrolysis is an enzymatically catalyzed reaction described by the Arrhenius equation. Diet crude protein (CP) controls ammonia emission by affecting N excretion. Objectives were to use the Arrhenius equation to model a...

Urchin-like TiO₂@C core-shell microspheres: coupled synthesis and lithium-ion battery applications.

PubMed

Liu, Zhenyu; Liu, Jing; Liu, Junfeng; Wang, Li; Zhang, Guoxin; Sun, Xiaoming

2014-05-21

Carbon coated urchin-like TiO2 microspheres were prepared through coupled hydrolysis of titanium tetrachloride and catalyzed carbonization of glucose. Carbon coating endowed the composite with unusual structural stability at high temperature and reasonable Li-ion battery performance.

Acid hydrolysis of cellulosic fibres: Comparison of bleached kraft pulp, dissolving pulps and cotton textile cellulose.

PubMed

Palme, Anna; Theliander, Hans; Brelid, Harald

2016-01-20

The behaviour of different cellulosic fibres during acid hydrolysis has been investigated and the levelling-off degree of polymerisation (LODP) has been determined. The study included a bleached kraft pulp (both never-dried and once-dried) and two dissolving pulps (once-dried). Additionally, cotton cellulose from new cotton sheets and sheets discarded after long-time use was studied. Experimental results from the investigation, together with results found in literature, imply that ultrastructural differences between different fibres affect their susceptibility towards acid hydrolysis. Drying of a bleached kraft pulp was found to enhance the rate of acid hydrolysis and also result in a decrease in LODP. This implies that the susceptibility of cellulosic fibres towards acid hydrolysis is affected by drying-induced stresses in the cellulose chains. In cotton cellulose, it was found that use and laundering gave a substantial loss in the degree of polymerisation (DP), but that the LODP was only marginally affected. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Production of fuel ethanol from bamboo by concentrated sulfuric acid hydrolysis followed by continuous ethanol fermentation.

PubMed

Sun, Zhao-Yong; Tang, Yue-Qin; Iwanaga, Tomohiro; Sho, Tomohiro; Kida, Kenji

2011-12-01

An efficient process for the production of fuel ethanol from bamboo that consisted of hydrolysis with concentrated sulfuric acid, removal of color compounds, separation of acid and sugar, hydrolysis of oligosaccharides and subsequent continuous ethanol fermentation was developed. The highest sugar recovery efficiency was 81.6% when concentrated sulfuric acid hydrolysis was carried out under the optimum conditions. Continuous separation of acid from the saccharified liquid after removal of color compounds with activated carbon was conducted using an improved simulated moving bed (ISMB) system, and 98.4% of sugar and 90.5% of acid were recovered. After oligosaccharide hydrolysis and pH adjustment, the unsterilized saccharified liquid was subjected to continuous ethanol fermentation using Saccharomycescerevisiae strain KF-7. The ethanol concentration, the fermentation yield based on glucose and the ethanol productivity were approximately 27.2 g/l, 92.0% and 8.2 g/l/h, respectively. These results suggest that the process is effective for production of fuel ethanol from bamboo. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tailored and integrated production of carboxylated cellulose nanocrystals (CNC) with nanofibrils (CNF) through maleic acid hydrolysis

Treesearch

Ruibin Wang; Liheng Chen; J.Y. Zhu; Rendang Yang

2017-01-01

This study demonstrates the feasibility of tailored and integrated production of carboxylated cellulose nanocrystals (CNC) with nanofibrils (CNF) from bleached pulp fibers through hydrolysis using a recyclable dicarboxylic acid. Hydrolysis experiments were conducted using ranges of 15–75 wt% maleic acid concentrations, 60–120°C temperatures, and 5–300 min reaction...

Preparation of κ-carra-oligosaccharides with microwave assisted acid hydrolysis method

NASA Astrophysics Data System (ADS)

Li, Guangsheng; Zhao, Xia; Lv, Youjing; Li, Miaomiao; Yu, Guangli

2015-04-01

A rapid method of microwave assisted acid hydrolysis was established to prepare κ-carra-oligosaccharides. The optimal hydrolysis condition was determined by an orthogonal test. The degree of polymerization (DP) of oligosaccharides was detected by high performance thin layer chromatography (HPTLC) and polyacrylamide gel electrophoresis (PAGE). Considering the results of HPTLC and PAGE, the optimum condition of microwave assisted acid hydrolysis was determined. The concentration of κ-carrageenan was 5 mg mL-1; the reaction solution was adjusted to pH 3 with diluted hydrochloric acid; the solution was hydrolyzed under microwave irradiation at 100 for 15 °C min. Oligosaccharides were separated by a Superdex 30 column (2.6 cm × 90 cm) using AKTA Purifier UPC100 and detected with an online refractive index detector. Each fraction was characterized by electrospray ionization mass spectrometry (ESI-MS). The data showed that odd-numbered κ-carra-oligosaccharides with DP ranging from 3 to 21 could be obtained with this method, and the structures of the oligosaccharides were consistent with those obtained by traditional mild acid hydrolysis. The new method was more convenient, efficient and environment-friendly than traditional mild acid hydrolysis. Our results provided a useful reference for the preparation of oligosaccharides from other polysaccharides.

Detection of diastereomer peptides as the intermediates generating D-amino acids during acid hydrolysis of peptides.

PubMed

Miyamoto, Tetsuya; Sekine, Masae; Ogawa, Tetsuhiro; Hidaka, Makoto; Watanabe, Hidenori; Homma, Hiroshi; Masaki, Haruhiko

2016-11-01

In this study, we investigated whether the amino acid residues within peptides were isomerized (and the peptides converted to diastereomers) during the early stages of acid hydrolysis. We demonstrate that the model dipeptides L-Ala-L-Phe and L-Phe-L-Ala are epimerized to produce the corresponding diastereomers at a very early stage, prior to their acid hydrolytic cleavage to amino acids. Furthermore, the sequence-inverted dipeptides were generated via formation of a diketopiperazine during hydrolytic incubation, and these dipeptides were also epimerized. The proportion of diastereomers increased rapidly during incubation for 0.5-2 h. During acid hydrolysis, C-terminal residues of the model dipeptides were isomerized faster than N-terminal residues, consistent with the observation that the D-amino acid values of the C-terminal residues determined by the 0 h-extrapolating method were larger than those of the N-terminal residues. Thus, the artificial D-amino acid contents determined by the 0 h-extrapolating method appear to be products of the isomerization of amino acid residues during acid hydrolysis.

Effect of hydrolysis enzymatic process of corn using protease crude (Rhizopus oligosporus-C1) to produce corn hydrolisate rich folic acid

NASA Astrophysics Data System (ADS)

Maryati, Yati; Susilowati, Agustine; Melanie, Hakiki; Lotulung, Puspa D.

2017-11-01

Corn hydrolyzate (Zea mays L) as a functional food fortificant derived from natural folic acid has been evaluated through a hydrolysis process using protease enzyme Rhizopus oligosporus strain C1. Enzymatic hydrolysis was carried out on two types of corn; yellow and white pearl variety corn, at concentration of protease enzyme (rough) 0.025; 0.125; and 0.225% (v/w of soluble nixtamal corn protein) with a hydrolysis time of 24 h at 30 °C, and pH 5.0. The results showed that the concentration of protease enzymes can increase the folic acid to the optimum condition, from the beginning to the end of the process time. Folic acid optimization of hydrolysis results in each corn was at the concentration of protease enzyme 0.225% (v/w of soluble nixtamal corn protein) in white corn and yellow corn at 24 hours hydrolysis, with folic acid composition, 283.56 µg/mL and 412.52 µg/mL, 1.07 and 1.04 mg/mL of soluble proteins, proteolytic activity 2.09 and 2.06 U/mL, total solids of 21.74 and 17.85%, total sugars of 0.56 and 2.22 mg/mL, and reducing sugar 91.72 and 48.47 mg/mL. In this condition, the increase of optimum folic acid for white corn was 33.57% and for yellow corn was 71.60% after hydrolysis.

Effective, Facile, and Selective Hydrolysis of the Chemical Warfare Agent VX Using Zr6-Based Metal-Organic Frameworks.

PubMed

Moon, Su-Young; Wagner, George W; Mondloch, Joseph E; Peterson, Gregory W; DeCoste, Jared B; Hupp, Joseph T; Farha, Omar K

2015-11-16

The nerve agent VX is among the most toxic chemicals known to mankind, and robust solutions are needed to rapidly and selectively deactivate it. Herein, we demonstrate that three Zr6-based metal-organic frameworks (MOFs), namely, UiO-67, UiO-67-NH2, and UiO-67-N(Me)2, are selective and highly active catalysts for the hydrolysis of VX. Utilizing UiO-67, UiO-67-NH2, and UiO-67-N(Me)2 in a pH 10 buffered solution of N-ethylmorpholine, selective hydrolysis of the P-S bond in VX was observed. In addition, UiO-67-N(Me)2 was found to catalyze VX hydrolysis with an initial half-life of 1.8 min. This half-life is nearly 3 orders of magnitude shorter than that of the only other MOF tested to date for hydrolysis of VX and rivals the activity of the best nonenzymatic materials. Hydrolysis utilizing Zr-based MOFs is also selective and facile in the absence of pH 10 buffer (just water) and for the destruction of the toxic byproduct EA-2192.

Magnesium ion catalyzed P-N bond hydrolysis in imidazolide-activated nucleotides - Relevance to template-directed synthesis of polynucleotides

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia; Bernasconi, Claude F.; Doodokyan, Donald L.; Alberas, Diann J.

1989-01-01

Results are presented from a detailed study of the P-N bond hydrolysis in guanosine 5-prime-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-prime-imidazolide (ImpG) in the presence of 0-0.50 M Mg(2+). Pseudo-first-order rate constants of these compounds were obtained as a function of Mg(2+) concentration, for pH values between 6 and 10 and 37 C. It was found that Mg(2+) catalysis was most effective at pH 10, where a 15-fold increase in hydrolysis was achieved in 0.02 M Mg; at 0.2 M, a 115-fold increase was observed. Implication of these results for the mechanism of template-directed oligomerization is discussed.

Mitigation of Humic Acid Inhibition in Anaerobic Digestion of Cellulose by Addition of Various Salts.

PubMed

Azman, Samet; Khadem, Ahmad F; Zeeman, Grietje; van Lier, Jules B; Plugge, Caroline M

2015-03-25

Humic compounds are inhibitory to the anaerobic hydrolysis of cellulosic biomass. In this study, the impact of salt addition to mitigate the inhibitory effects of humic compounds was investigated. The experiment was conducted using batch tests to monitor the anaerobic hydrolysis of cellulose in the presence of humic acid. Sodium, potassium, calcium, magnesium and iron salts were tested separately for their efficiency to mitigate humic acid inhibition. All experiments were done under mesophilic conditions (30 °C) and at pH 7. Methane production was monitored online, using the Automatic Methane Potential Test System. Methane production, soluble chemical oxygen demand and volatile fatty acid content of the samples were measured to calculate the hydrolysis efficiencies. Addition of magnesium, calcium and iron salts clearly mitigated the inhibitory effects of humic acid and hydrolysis efficiencies reached up to 75%, 65% and 72%, respectively, which were similar to control experiments. Conversely, potassium and sodium salts addition did not mitigate the inhibition and hydrolysis efficiencies were found to be less than 40%. Mitigation of humic acid inhibition via salt addition was also validated by inductively coupled plasma atomic emission spectroscopy analyses, which showed the binding capacity of different cations to humic acid.

Hydrolysis of hemicellulose to produce fermentable monosaccharides by plasma acid.

PubMed

Wang, Ying; Yuan, Bo; Ji, Yingchao; Li, Hong

2013-09-12

In this paper, plasma acid was obtained by treating distilled water with dielectric barrier discharge to hydrolyze hemicellulose. The orthogonal experiment L₂₅(5(6)) was used to optimize such hydrolysis conditions. The total reducing sugar (TRS) was measured by the DNS method. To determine whether the oligosaccharide existed in the hydrolysis products, it was hydrolyzed by sulfuric acid for a second time following the same procedure as reported earlier. The monosaccharide compositions of the hydrolyzed sample were analyzed by high-performance liquid chromatography (HPLC) and Fourier transformed infrared spectroscopy (FTIR). The results showed that pH 2.81 of plasma acid, 100 °C and 50 min were assigned as an optimal hydrolysis condition by plasma acid. Under this condition, the hemicellulose was hydrolyzed completely to produce monosaccharides including xylose, glucose, and galactose with the mole ratio being 17:3:1. The yields of xylose, glucose, and galactose were 38.67%, 9.28% and 3.09%, respectively. Compared with the hemicellulose hydrolysis results by sulfuric acid, it is concluded that plasma acid is an environmental-friendly and efficient method to explore and hydrolyze the hemicellulose existed in biomass. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mitigation of Humic Acid Inhibition in Anaerobic Digestion of Cellulose by Addition of Various Salts

PubMed Central

Azman, Samet; Khadem, Ahmad F.; Zeeman, Grietje; van Lier, Jules B.; Plugge, Caroline M.

2015-01-01

Humic compounds are inhibitory to the anaerobic hydrolysis of cellulosic biomass. In this study, the impact of salt addition to mitigate the inhibitory effects of humic compounds was investigated. The experiment was conducted using batch tests to monitor the anaerobic hydrolysis of cellulose in the presence of humic acid. Sodium, potassium, calcium, magnesium and iron salts were tested separately for their efficiency to mitigate humic acid inhibition. All experiments were done under mesophilic conditions (30 °C) and at pH 7. Methane production was monitored online, using the Automatic Methane Potential Test System. Methane production, soluble chemical oxygen demand and volatile fatty acid content of the samples were measured to calculate the hydrolysis efficiencies. Addition of magnesium, calcium and iron salts clearly mitigated the inhibitory effects of humic acid and hydrolysis efficiencies reached up to 75%, 65% and 72%, respectively, which were similar to control experiments. Conversely, potassium and sodium salts addition did not mitigate the inhibition and hydrolysis efficiencies were found to be less than 40%. Mitigation of humic acid inhibition via salt addition was also validated by inductively coupled plasma atomic emission spectroscopy analyses, which showed the binding capacity of different cations to humic acid. PMID:28955013

Biosynthesis of diphthamide in the yeast Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, J.Y.C.

1985-01-01

Inactivation of EF-2 by diphtheria toxin requires the presence of a posttranslationally synthesized amino acid residue, diphthamide. The present work was undertaken to study the biosynthetic mechanism of diphthamide synthesis in the yeast Saccharomyces cerevisiae in order to gain better understanding of the biological roles of this unique amino acid residue. Thirty-one haploid ADP-ribosylation-negative mutants, comprising 5 complementation groups, were obtained. One of these mutants contains a toxin-resistant form of EF-2 which can be converted to a toxin-sensitive form through the methylation reaction catalyzed by a S-AdoMet:EF-2 methyltransferase enzyme which is present in other yeast strains. The (/sup 3/He)methylated residuemore » in the EF-2 modified by the methyltransferase in the presence of S-Ado-L-(/sup 3/H-methyl)-Met has been analyzed chromatographically following both acid and enzymatic hydrolysis. At the conclusion of the reaction, all of the radiolabel was recovered as diphthine (the unamidated form of diphthamide). The authors conclude that the S-AdoMet:EF-2-methyltransferase is specific for the addition of at least the last two of the three methyl groups present in diphthine.« less

The mechanism of hydrothermal hydrolysis for glycyrrhizic acid into glycyrrhetinic acid and glycyrrhetinic acid 3-O-mono-β-D-glucuronide in subcritical water.

PubMed

Fan, Rui; Li, Nan; Xu, Honggao; Xiang, Jun; Wang, Lei; Gao, Yanxiang

2016-01-01

To improve the bioactivity and sweetness properties of glycyrrhizic acid (GL), the hydrothermal hydrolysis of GL into glycyrrhetinic acid (GA) and glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG) in subcritical water was investigated. The effects of temperature, time and their interaction on the conversion ratios were analyzed and the reactions were elaborated with kinetics and thermodynamics. The results showed that GL hydrothermal hydrolysis was significantly (P < 0.05) affected by reaction time and temperature, as well as their interaction, and could be fitted into first-order kinetics. The thermodynamic analysis indicated that the hydrolysis of GL was endergonic and non-spontaneous. The hydrolytic pathways were composed of complex consecutive and parallel reactions. It was concluded that subcritical water may be a potential medium for producing GAMG and GA. Copyright © 2015 Elsevier Ltd. All rights reserved.

An Integrated Protein Chemistry Laboratory: Chlorophyll and Chlorophyllase

ERIC Educational Resources Information Center

Arkus, Kiani A. J.; Jez, Joseph M.

2008-01-01

Chlorophyll, the most abundant pigment in nature, is degraded during normal plant growth, when leaves change color, and at specific developmental stages. Chlorophyllase catalyzes the first chemical reaction in this process, that is, the hydrolysis of chlorophyll into chlorophyllide. Here, we describe a series of laboratory sessions designed to…

Hydrolysis of Letrozole catalyzed by macrocyclic Rhodium (I) Schiff-base complexes.

PubMed

Reddy, P Muralidhar; Shanker, K; Srinivas, V; Krishna, E Ravi; Rohini, R; Srikanth, G; Hu, Anren; Ravinder, V

2015-03-15

Ten mononuclear Rhodium (I) complexes were synthesized by macrocyclic ligands having N4 and N2O2 donor sites. Square planar geometry is assigned based on the analytical and spectral properties for all complexes. Rh(I) complexes were investigated as catalysts in hydrolysis of Nitrile group containing pharmaceutical drug Letrozole. A comparative study showed that all the complexes are efficient in the catalysis. The percent yields of all the catalytic reaction products viz. drug impurities were determined by spectrophotometric procedures and characterized by spectral studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of Varying Acid Hydrolysis Condition in Gracilaria Sp. Fermentation Using Sasad

NASA Astrophysics Data System (ADS)

Mansuit, H.; Samsuri, M. D. C.; Sipaut, C. S.; Yee, C. F.; Yasir, S. M.; Mansa, R.

2015-04-01

Macroalgae or seaweed is being considered as promising feedstock for bioalcohol production due to high polysaccharides content. Polysaccharides can be converted into fermentable sugar through acid hydrolysis pre-treatment. In this study, the potential of using carbohydrate-rich macroalgae, Gracilaria sp. as feedstock for bioalcohol production via various acid hydrolysis conditions prior to the fermentation process was investigated and evaluated. The seaweed used in this research was from the red algae group, using species of Gracilaria sp. which was collected from Sg. Petani Kedah, Malaysia. Pre-treatment of substrate was done using H2SO4 and HCl with molarity ranging from 0.2M to 0.8M. The pretreatment time were varied in the range of 15 to 30 minutes. Fermentation was conducted using Sasad, a local Sabahan fermentation agent as a starter culture. Alcohol extraction was done using a distillation unit. Reducing sugar analysis was done by Benedict test method. Alcohol content analysis was done using specific gravity test. After hydrolysis, it was found out that acid hydrolysis at 0.2M H2SO4 and pre-treated for 20 minutes at 121°C has shown the highest reducing sugar content which has yield (10.06 mg/g) of reducing sugar. It was followed by other samples hydrolysis using 0.4M HCl with 30 minutes pre-treatment and 0.2M H2SO4, 15 minutes pre-treatment with yield of 8.06 mg/g and 5.75 mg/g reducing sugar content respectively. In conclusion, acid hydrolysis of Gracilaria sp. can produce higher reducing sugar yield and thus it can further enhance the bioalcohol production yield. Hence, acid hydrolysis of Gracilaria sp. should be studied more as it is an important step in the bioalcohol production and upscaling process.

DFT investigations of phosphotriesters hydrolysis in aqueous solution: a model for DNA single strand scission induced by N-nitrosoureas.

PubMed

Liu, Tingting; Zhao, Lijiao; Zhong, Rugang

2013-02-01

DNA phosphotriester adducts are common alkylation products of DNA phosphodiester moiety induced by N-nitrosoureas. The 2-hydroxyethyl phosphotriester was reported to hydrolyze more rapidly than other alkyl phosphotriesters both in neutral and in alkaline conditions, which can cause DNA single strand scission. In this work, DFT calculations have been employed to map out the four lowest activation free-energy profiles for neutral and alkaline hydrolysis of triethyl phosphate (TEP) and diethyl 2-hydroxyethyl phosphate (DEHEP). All the hydrolysis pathways were illuminated to be stepwise involving an acyclic or cyclic phosphorane intermediate for TEP or DEHEP, respectively. The rate-limiting step for all the hydrolysis reactions was found to be the formation of phosphorane intermediate, with the exception of DEHEP hydrolysis in alkaline conditions that the decomposition process turned out to be the rate-limiting step, owing to the extraordinary low formation barrier of cyclic phosphorane intermediate catalyzed by hydroxide. The rate-limiting barriers obtained for the four reactions are all consistent with the available experimental information concerning the corresponding hydrolysis reactions of phosphotriesters. Our calculations performed on the phosphate triesters hydrolysis predict that the lower formation barriers of cyclic phosphorane intermediates compared to its acyclic counter-part should be the dominant factor governing the hydrolysis rate enhancement of DEHEP relative to TEP both in neutral and in alkaline conditions.

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

PubMed

Reiz, Bela; Li, Liang

2010-09-01

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Dilute acid hydrolysis of paper birch : kinetics studies of xylan and acetyl-group hydrolysis

Treesearch

Mark T. Maloney; Thomas W. Chapman; Andrew J. Baker

1985-03-01

Batch hydrolysis kinetics of paper birch (Betula papyrifera) xylan and its associated acetyl groups in dilute sulfuric acid have been measured for acid concentrations of between 0.04 and 0.18 M and temperatures of between 100 and 170°C. Only 5% of the cellulose was hydrolyzed for up to 85% xylan removal. Rate data were correlated well by a parallel reaction model based...

Chemical evolution. XXIX - Pyrimidines from hydrogen cyanide

NASA Technical Reports Server (NTRS)

Ferris, J. P.; Joshi, P. C.; Lawless, J. G.

1978-01-01

Compounds obtained by hydrolysis of HCN oligomers formed by allowing pH 9.2, 0.1 M cyanide to stand at room temperature for 4 to 12 months were analyzed. Hydrolysis of HCN oligomers yielded 4,5-dihydroxypyrimidine and 5-hydroxyuracil; orotic acid was detected after hydrolysis at pH 8.5. A unified pathway from diaminofumaronitrile to the pyrimidines observed is suggested. As purines, pyrimidines and amino acids are released by hydrolysis of HCN oligomers in either acidic or mildly basic aqueous solutions, they could have been formed on the primitive earth in spite of fluctuations in pH. 4,5-dihydroxypyrimidines appear to be likely candidates for incorporation into primitive nucleic acids, as they should undergo Watson-Crick hydrogen bonding with adenine.

Two-stage, dilute sulfuric acid hydrolysis of wood : an investigation of fundamentals

Treesearch

John F. Harris; Andrew J. Baker; Anthony H. Conner; Thomas W. Jeffries; James L. Minor; Roger C. Pettersen; Ralph W. Scott; Edward L Springer; Theodore H. Wegner; John I. Zerbe

1985-01-01

This paper presents a fundamental analysis of the processing steps in the production of methanol from southern red oak (Quercus falcata Michx.) by two-stage dilute sulfuric acid hydrolysis. Data for hemicellulose and cellulose hydrolysis are correlated using models. This information is used to develop and evaluate a process design.

Comparison of cell wall polysaccharide hydrolysis by a dilute acid/enzymatic saccharification process and rumen microorganisms

USDA-ARS?s Scientific Manuscript database

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance of biomass in the bioenergy conversion process. Cell wall polysaccharide hydrolysis by dilute sulfuric acid pretreatment at 121 degrees C followed by cellulase hydrolysis for 72 h (CONV) and in v...

Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC.

PubMed

Dilokpimol, Adiphol; Mäkelä, Miia R; Mansouri, Sadegh; Belova, Olga; Waterstraat, Martin; Bunzel, Mirko; de Vries, Ronald P; Hildén, Kristiina S

2017-07-25

A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Monoglyceride lipase as a drug target: At the crossroads of arachidonic acid metabolism and endocannabinoid signaling.

PubMed

Grabner, Gernot F; Zimmermann, Robert; Schicho, Rudolf; Taschler, Ulrike

2017-07-01

Monoglyerides (MGs) are short-lived, intermediary lipids deriving from the degradation of phospho- and neutral lipids, and monoglyceride lipase (MGL), also designated as monoacylglycerol lipase (MAGL), is the major enzyme catalyzing the hydrolysis of MGs into glycerol and fatty acids. This distinct function enables MGL to regulate a number of physiological and pathophysiological processes since both MGs and fatty acids can act as signaling lipids or precursors thereof. The most prominent MG species acting as signaling lipid is 2-arachidonoyl glycerol (2-AG) which is the most abundant endogenous agonist of cannabinoid receptors in the body. Importantly, recent observations demonstrate that 2-AG represents a quantitatively important source for arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. Accordingly, MGL-mediated 2-AG degradation affects lipid signaling by cannabinoid receptor-dependent and independent mechanisms. Recent genetic and pharmacological studies gave important insights into MGL's role in (patho-)physiological processes, and the enzyme is now considered as a promising drug target for a number of disorders including cancer, neurodegenerative and inflammatory diseases. This review summarizes the basics of MG (2-AG) metabolism and provides an overview on the therapeutic potential of MGL. Copyright © 2017 Elsevier Inc. All rights reserved.

Plant growth regulators induced urease activity in Cucurbita pepo L. cotyledons.

PubMed

El Shora, Hamed M; Ali, Awatif S

2016-03-01

This study is aimed to investigate the activity of urease (EC 3.5.1.5, urea amidohydrolase) that catalyzes the hydrolysis of urea in 5-day-old Cucurbita pepo cotyledons subjected to various concentrations of different growth regulators. The treatment of C. pepo cotyledons with different concentrations (100-600 μmol) of different auxins [indole-3-acetic acid (IAA), indole butyric acid (IBA), indole propionic acid (IPA) and naphthalene acetic acid (NAA)]; or with different concentrations (100-300 μmol) of different cytokinins [kinetin, zeatin and benzyladenine (6-BA)] resulted in a significant increase of urease activity, compared to control. The optimal effects were recorded for each of 500 μmol of IAA and 300 μmol of zeatin treatments. A gradual increase in urease activity was detected in cotyledons treated with various concentrations (0.2-1.0 mM) of 28-homobrassinolide (HBL), in relative to control. A substantial increase in urease activity was observed in cotyledons subjected to different concentrations of triazole (10-60 mg L(-1)), containing either triadimefon (TDM) or hexaconazole (HEX), compared to control. The combination of 300 μmol zeatin with any of protein inhibitors, namely 5-fluorouridine (FUrd), cordycepin and α-amanitin, resulted in the alleviation of their inhibitory effect on the urease activity.

Molecular Docking of Potential Inhibitors of Broccoli Myrosinase.

PubMed

Román, J; Castillo, A; Mahn, A

2018-05-30

Glucosinolates are secondary metabolites occurring in Brassicaceae plants whose hydrolysis may yield isothiocyanates, widely recognized as health-promoting compounds. Myrosinase catalyzes this conversion. The chemical mechanism involves an unstable intermediary (thiohydroxamate- O -sulfonate) that spontaneously decomposes into isothiocyanates or other non-bioactive compounds depending on pH and cofactors. At acidic pH, non-bioactive compounds such as nitriles and thiocyanates are formed, while at neutral pH isothiocyanates are obtained. Broccoli myrosinase has been poorly studied so far. Recently, its amino acidic sequence was elucidated, and a structural model was built. The aim of this work was to study the molecular interaction of broccoli myrosinase with different ligands at acidic pH to propose possible inhibitors that prevent formation of undesirable compounds at acidic pH, and that at neutral pH dissociate from the enzyme, allowing formation of isothiocyanates. The interaction between broccoli myrosinase and 40 ligands was studied by molecular docking simulations. Both the enzyme and each inhibitor were set at pH 3.0. Amygdaline and arbutin showed the highest affinity to broccoli myrosinase in this condition. The residues that stabilize the complexes agree with those that stabilize the substrate (Gln207, Glu429, Tyr352, and Ser433). Accordingly, amygdaline and arbutin would perform as competitive inhibitors of myrosinase at pH 3.0.

Molecular Mechanisms and New Treatment Paradigm for Atrial Fibrillation.

PubMed

Sirish, Padmini; Li, Ning; Timofeyev, Valeriy; Zhang, Xiao-Dong; Wang, Lianguo; Yang, Jun; Lee, Kin Sing Stephen; Bettaieb, Ahmed; Ma, Sin Mei; Lee, Jeong Han; Su, Demetria; Lau, Victor C; Myers, Richard E; Lieu, Deborah K; López, Javier E; Young, J Nilas; Yamoah, Ebenezer N; Haj, Fawaz; Ripplinger, Crystal M; Hammock, Bruce D; Chiamvimonvat, Nipavan

2016-05-01

Atrial fibrillation represents the most common arrhythmia leading to increased morbidity and mortality, yet, current treatment strategies have proven inadequate. Conventional treatment with antiarrhythmic drugs carries a high risk for proarrhythmias. The soluble epoxide hydrolase enzyme catalyzes the hydrolysis of anti-inflammatory epoxy fatty acids, including epoxyeicosatrienoic acids from arachidonic acid to the corresponding proinflammatory diols. Therefore, the goal of the study is to directly test the hypotheses that inhibition of the soluble epoxide hydrolase enzyme can result in an increase in the levels of epoxyeicosatrienoic acids, leading to the attenuation of atrial structural and electric remodeling and the prevention of atrial fibrillation. For the first time, we report findings that inhibition of soluble epoxide hydrolase reduces inflammation, oxidative stress, atrial structural, and electric remodeling. Treatment with soluble epoxide hydrolase inhibitor significantly reduces the activation of key inflammatory signaling molecules, including the transcription factor nuclear factor κ-light-chain-enhancer, mitogen-activated protein kinase, and transforming growth factor-β. This study provides insights into the underlying molecular mechanisms leading to atrial fibrillation by inflammation and represents a paradigm shift from conventional antiarrhythmic drugs, which block downstream events to a novel upstream therapeutic target by counteracting the inflammatory processes in atrial fibrillation. © 2016 American Heart Association, Inc.

Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum.

PubMed

Krstanović, Marina; Brgles, Marija; Halassy, Beata; Frkanec, Ruza; Vrdoljak, Anto; Branović, Karmen; Tomasić, Jelka; Benedetti, Fabio

2006-01-01

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (K(M)=0.6 mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin.

Structural and mutagenetic analyses of a 1,3-1,4-β-glucanase from Paecilomyces thermophila.

PubMed

Cheng, Ya-Shan; Huang, Chun-Hsiang; Chen, Chun-Chi; Huang, Ting-Yung; Ko, Tzu-Ping; Huang, Jian-Wen; Wu, Tzu-Hui; Liu, Je-Ruei; Guo, Rey-Ting

2014-02-01

The thermostable 1,3-1,4-β-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley β-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80Å to 2.25Å resolution. PtLic16A adopts a typical β-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3-1,4-β-glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3-1,4-β-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering. Copyright © 2013 Elsevier B.V. All rights reserved.

Relationships Between Base-Catalyzed Hydrolysis Rates or Glutathione Reactivity for Acrylates and Methacrylates and Their NMR Spectra or Heat of Formation

PubMed Central

Fujisawa, Seiichiro; Kadoma, Yoshinori

2012-01-01

The NMR chemical shift, i.e., the π-electron density of the double bond, of acrylates and methacrylates is related to the reactivity of their monomers. We investigated quantitative structure-property relationships (QSPRs) between the base-catalyzed hydrolysis rate constants (k1) or the rate constant with glutathione (GSH) (log kGSH) for acrylates and methacrylates and the 13C NMR chemical shifts of their α,β-unsaturated carbonyl groups (δCα and δCβ) or heat of formation (Hf) calculated by the semi-empirical MO method. Reported data for the independent variables were employed. A significant linear relationship between k1 and δCβ, but not δCα, was obtained for methacrylates (r2 = 0.93), but not for acrylates. Also, a significant relationship between k1 and Hf was obtained for both acrylates and methacrylates (r2 = 0.89). By contrast, log kGSH for acrylates and methacrylates was linearly related to their δCβ (r2 = 0.99), but not to Hf. These findings indicate that the 13C NMR chemical shifts and calculated Hf values for acrylates and methacrylates could be valuable for estimating the hydrolysis rate constants and GSH reactivity of these compounds. Also, these data for monomers may be an important tool for examining mechanisms of reactivity. PMID:22754331

Influence of ammonium salts on the lipase/esterase activity assay using p-nitrophenyl esters as substrates.

PubMed

De Yan, Hong; Zhang, Yin Jun; Liu, Hong Cai; Zheng, Jian Yong; Wang, Zhao

2013-01-01

p-Nitrophenyl esters with a short-chain carboxylic group, such as p-nitrophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB), could be effectively hydrolyzed by ammonium salts. p-Nitrophenyl esters were usually used as substrates to assay the lipase/esterase activity. Ammonium sulfate precipitation was often used to purify proteins, and some ammonium salts were usually used as nitrogen sources or inorganic salts for the lipase/esterase production. To study the effect of ammonium salts on the assay of the lipase/esterase activity, the contributing factors of hydrolysis of p-NPA/p-NPB catalyzed by ammonium salts were investigated. The lipase activities were compared in the presence and absence of ammonium sulfate. The hydrolysis reaction could be catalyzed under neutral and alkaline circumstances. The hydrolysis rate increased with the increase in the reaction temperature or the concentration of ammonium ion. When p-NPA was employed as the substrate for the analysis of the lipase/esterase activity, the effect of ammonium sulfate on the analysis could be neutralized by setting a control when the concentration of ammonium sulfate was less than 40% saturation. However, when the concentration of ammonium sulfate increased from 40% to 100% saturation, the enzyme activities decreased about 13-40%, which could not be ignored for accurate analysis of the enzyme activity. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Effects of acid impregnated steam explosion process on xylose recovery and enzymatic conversion of cellulose in corncob.

PubMed

Fan, Xiaoguang; Cheng, Gang; Zhang, Hongjia; Li, Menghua; Wang, Shizeng; Yuan, Qipeng

2014-12-19

Corncob residue is a cellulose-rich byproduct obtained from industrial xylose production via dilute acid hydrolysis processes. Enzymatic hydrolysis of cellulose in acid hydrolysis residue of corncob (AHRC) is often less efficient without further pretreatment. In this work, the process characteristics of acid impregnated steam explosion were studied in conjunction with a dilute acid process, and their effects on physiochemical changes and enzymatic saccharification of corncob residue were compared. With the acid impregnated steam explosion process, both higher xylose recovery and higher cellulose conversion were obtained. The maximum conversion of cellulose in acid impregnated steam explosion residue of corncob (ASERC) reached 85.3%, which was 1.6 times higher than that of AHRC. Biomass compositional analysis showed similar cellulose and lignin content in ASERC and AHRC. XRD analysis demonstrated comparable crystallinity of ASERC and AHRC. The improved enzymatic hydrolysis efficiency was attributed to higher porosity in ASERC, measured by mercury porosimetry. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dissecting the role of conformational change and membrane binding by the bacterial cell division regulator MinE in the stimulation of MinD ATPase activity.

PubMed

Ayed, Saud H; Cloutier, Adam D; McLeod, Laura J; Foo, Alexander C Y; Damry, Adam M; Goto, Natalie K

2017-12-15

The bacterial cell division regulators MinD and MinE together with the division inhibitor MinC localize to the membrane in concentrated zones undergoing coordinated pole-to-pole oscillation to help ensure that the cytokinetic division septum forms only at the mid-cell position. This dynamic localization is driven by MinD-catalyzed ATP hydrolysis, stimulated by interactions with MinE's anti-MinCD domain. This domain is buried in the 6-β-stranded MinE "closed" structure, but is liberated for interactions with MinD, giving rise to a 4-β-stranded "open" structure through an unknown mechanism. Here we show that MinE-membrane interactions induce a structural change into a state resembling the open conformation. However, MinE mutants lacking the MinE membrane-targeting sequence stimulated higher ATP hydrolysis rates than the full-length protein, indicating that binding to MinD is sufficient to trigger this conformational transition in MinE. In contrast, conformational change between the open and closed states did not affect stimulation of ATP hydrolysis rates in the absence of membrane binding, although the MinD-binding residue Ile-25 is critical for this conformational transition. We therefore propose an updated model where MinE is brought to the membrane through interactions with MinD. After stimulation of ATP hydrolysis, MinE remains bound to the membrane in a state that does not catalyze additional rounds of ATP hydrolysis. Although the molecular basis for this inhibited state is unknown, previous observations of higher-order MinE self-association may explain this inhibition. Overall, our findings have general implications for Min protein oscillation cycles, including those that regulate cell division in bacterial pathogens. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow-injection chemiluminescence assay.

PubMed

Zhang, Qingfeng; Zhang, Cuiyun; Yang, Meiding; Yu, Donghong; Yu, Cong

2017-11-01

A sensitive and convenient flow-injection chemiluminescence (FI-CL) turn-on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu 2+ can catalyze the luminol-H 2 O 2 CL reaction. Pyrophosphate (PPi) can chelate Cu 2+ and therefore the Cu 2+ -mediated luminol-H 2 O 2 CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu 2+ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained. Copyright © 2017 John Wiley & Sons, Ltd.

Two-stage acid saccharification of fractionated Gelidium amansii minimizing the sugar decomposition.

PubMed

Jeong, Tae Su; Kim, Young Soo; Oh, Kyeong Keun

2011-11-01

Two-stage acid hydrolysis was conducted on easy reacting cellulose and resistant reacting cellulose of fractionated Gelidium amansii (f-GA). Acid hydrolysis of f-GA was performed at between 170 and 200 °C for a period of 0-5 min, and an acid concentration of 2-5% (w/v, H2SO4) to determine the optimal conditions for acid hydrolysis. In the first stage of the acid hydrolysis, an optimum glucose yield of 33.7% was obtained at a reaction temperature of 190 °C, an acid concentration of 3.0%, and a reaction time of 3 min. In the second stage, a glucose yield of 34.2%, on the basis the amount of residual cellulose from the f-GA, was obtained at a temperature of 190 °C, a sulfuric acid concentration of 4.0%, and a reaction time 3.7 min. Finally, 68.58% of the cellulose derived from f-GA was converted into glucose through two-stage acid saccharification under aforementioned conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Acid Hydrolysis of Trioxalatocobaltate (III) Ion

ERIC Educational Resources Information Center

Wiggans, P. W.

1975-01-01

Describes an investigation involving acid hydrolysis and using both volumetric and kinetic techniques. Presents examples of the determination of the rate constant and its variation with temperature. (GS)

In vitro enzymic hydrolysis of chlorogenic acids in coffee.

PubMed

da Encarnação, Joana Amarante; Farrell, Tracy L; Ryder, Alexandra; Kraut, Nicolai U; Williamson, Gary

2015-02-01

Coffee is rich in quinic acid esters of phenolic acids (chlorogenic acids) but also contains some free phenolic acids. A proportion of phenolic acids appear in the blood rapidly after coffee consumption due to absorption in the small intestine. We investigated in vitro whether this appearance could potentially be derived from free phenolic acids in instant coffee or from hydrolysis of chlorogenic acids by pancreatic or brush border enzymes. We quantified six free phenolic acids in instant coffees using HPLC-DAD-mass spectrometry. The highest was caffeic acid, but all were present at low levels compared to the chlorogenic acids. Roasting and decaffeination significantly reduced free phenolic acid content. We estimated, using pharmacokinetic modelling with previously published data, that the contribution of these compounds to small intestinal absorption is minimal. Hydrolysis of certain chlorogenic acids was observed with human-differentiated Caco-2 cell monolayers and with porcine pancreatin, which showed maximal rates on 3- and 5-O-caffeoylquinic acids, respectively. The amounts of certain free phenolic acids in coffee could only minimally account for small intestinal absorption based on modelling. The hydrolysis of caffeoylquinic, but not feruloylquinic acids, by enterocyte and pancreatic esterases is potentially a contributing mechanism to small intestinal absorption. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruzzini, Antonio C.; Bhowmik, Shiva; Ghosh, Subhangi

The meta-cleavage product (MCP) hydrolases utilize a Ser–His–Asp triad to hydrolyze a carbon–carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES red, which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES red decay and product formation showed amore » solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pK a2 (β nuc ~ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His–Asp pair does not play an essential role. The data further suggest that ES red represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.« less

Insight into the Phosphodiesterase Mechanism from Combined QM/MM Free Energy Simulations

PubMed Central

Wong, Kin-Yiu; Gao, Jiali

2011-01-01

Summary Molecular dynamics simulations employing a combined quantum mechanical and molecular mechanical potential have been carried out to elucidate the reaction mechanism of the hydrolysis of a cyclic nucleotide cAMP substrate by phosphodiesterase 4B (PDE4B). PDE4B is a member of the PDE superfamily of enzymes that play crucial roles in cellular signal transduction. We have determined a two-dimensional potential of mean force for the coupled phosphoryl bond cleavage and proton transfer through a general acid catalysis mechanism in PDE4B. The results indicate that the ring-opening process takes place through an SN2 reaction mechanism, followed by a proton transfer to stabilize the leaving group. The computed free energy of activation for the PDE4B-catalyzed cAMP hydrolysis is about 13 kcal/mol and an overall reaction free energy is about −17 kcal/mol, both in accord with experimental results. In comparison with the uncatalyzed reaction in water, the enzyme PDE4B provides a strong stabilization of the transition state, lowering the free energy barrier by 14 kcal/mol. We found that the proton transfer from the general acid residue His234 to the O3' oxyanion of the ribosyl leaving group lags behind the nucleophilic attack, resulting in a shallow minimum on the free energy surface. A key contributing factor to transition state stabilization is the elongation of the distance between the divalent metal ions Zn2+ and Mg2+ in the active site as the reaction proceeds from the Michaelis complex to the transition state. PMID:21595828

Identification of a novel phospholipase D with high transphosphatidylation activity and its application in synthesis of phosphatidylserine and DHA-phosphatidylserine.

PubMed

Mao, Xiangzhao; Liu, Qianqian; Qiu, Yongqian; Fan, Xiaoqin; Han, Qingqing; Liu, Yanjun; Zhang, Lujia; Xue, Changhu

2017-05-10

Phosphatidylserine (PS) and docosahexaenoic acid-phosphatidylserine (DHA-PS) have significant nutritional and biological functions, which are extensively used in functional food industries. Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) or DHA-PC with l-serine, is an effective method for PS and DHA-PS preparation. However, because of the hydrolysis activity of PLD, PC and DHA-PC would be converted to the undesirable byproduct, phosphatidic acid (PA) and DHA-PA. In this study, a novel phospholipase D (PLD a2 ) was firstly cloned from Acinetobacter radioresistens a2 with high transphosphatidylation activity and no hydrolysis activity. In the PLD-catalyzed synthesis process (12h), both the transphosphatidylation conversion rate and selectivity of PS and DHA-PS were about 100%, which is the only PLD enzyme reported with this superiority up till now. In comparison with the majority of other known PLDs, PLD a2 exerted the highest activity at neutral pH, and it was stable from pH 4.0 to pH 9.0. In addition, PLD a2 had excellent thermal stability, with an optimum reaction temperature of 40°C and keeping more than 80% activity from 20°C to 60°C. The high catalytic selectivity mechanism of PLD a2 was explained by utilizing homology modeling, two-step docking, and binding energy and conformation analysis. PLD a2 ensured a stable supply of the biocatalyst with its most preponderant transphosphatidylation activity and PS selectivity, and had great potential in phospholipids industrial production. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolism of endocannabinoids.

PubMed

Biernacki, Michał; Skrzydlewska, Elżbieta

2016-08-11

Endocannabinoids belong to a group of ester, ether and amide derivatives of fatty acids, which are endogenous ligands of receptors CB1, CB2, TRPV1 and GPR55 that are included in the endocannabinoid system of the animal organism. The best known endocannabinoids are: N-arachidonylethanolamide called anandamide (AEA) and 2-arachidonoylglycerol (2-AG). They occur in all organisms, and their highest level is observed in the brain. In this review the mechanisms of synthesis and degradation of both AEA and 2-AG are shown. Endocannabinoids are synthesized from phospholipids (mainly phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) located in the cell membrane. As a result of arachidonic acid transfer from phosphatidylcholine to phosphatidylethanolamine, N-arachidonoyl phosphatidylethanolamine is formed, which is hydrolyzed to AEA by phospholipase D, C and A2. However, 2-AG is formed during the hydrolysis of phosphatidylinositol catalyzed mainly by DAGL. The primary role of endocannabinoids is the activation of cannabinoid receptors. Both AEA and 2-AG are primarily agonists of the CB1 receptor and to a lower degree CB2 and TRPV1r eceptors, but 2-AG has stronger affinity for these receptors. Through activation of receptors, endocannabinoids affect cellular metabolism and participate in the metabolic processes by receptor-independent pathways. Endocannabinoids which are not bound to the receptors are degraded. The main enzymes responsible for the hydrolysis of AEA and 2-AG are FAAH and MAGL, respectively. Apart from hydrolytic degradation, endocannabinoids may also be oxidized by cyclooxygenase-2, lipoxygenases, and cytochrome P450. It has been shown that the metabolites of both endocannabinoids also have biological significance.

The ligand effect on the hydrolytic reactivity of Zn(II) complexes toward phosphate diesters.

PubMed

Bonfá, Lodovico; Gatos, Maddalena; Mancin, Fabrizio; Tecilla, Paolo; Tonellato, Umberto

2003-06-16

The catalytic effects of the Zn(II) complexes of a series of poliaminic ligands in the hydrolysis of the activated phosphodiesters bis-p-nitrophenyl phosphate (BNP) and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) have been investigated. The reactions show first-order rate dependency on both substrate and metal ion complex and a pH dependence which is diagnostic of the acid dissociation of the reactive species. The mechanism of the metal catalyzed transesterification of HPNP has been assessed by solvent isotopic kinetic effect studies and involves the intramolecular nucleophilic attack of the substrate alcoholic group, activated by metal ion coordination. The intrinsic reactivity of the different complexes is controlled by the nature and structure of the ligand: complexes of tridentate ligands, particularly if characterized by a facial coordination mode, are more reactive than those of tetradentate ligands which can hardly allow binding sites for the substrate. In the case of tridentate ligands that form complexes with a facial coordination mode, a linear Brønsted correlation between the reaction rate (log k) and the pK(a) of the active nucleophile is obtained. The beta(nuc) values are 0.75 for the HPNP transesterification and 0.20 for the BNP hydrolysis. These values are indicated as the result of the combination of two opposite Lewis acid effects of the Zn(II) ion: the activation of the substrate and the efficiency of the metal coordinated nucleophile. The latter factor apparently prevails in determining the intrinsic reactivity of the Zn(II) complexes.

Effect of acid hydrolysis and fungal biotreatment on agro-industrial wastes for obtainment of free sugars for bioethanol production.

PubMed

El-Tayeb, T S; Abdelhafez, A A; Ali, S H; Ramadan, E M

2012-10-01

This study was designed to evaluate selected chemical and microbiological treatments for the conversion of certain local agro-industrial wastes (rice straw, corn stalks, sawdust, sugar beet waste and sugarcane bagasse) to ethanol. The chemical composition of these feedstocks was determined. Conversion of wastes to free sugars by acid hydrolysis varied from one treatment to another. In single-stage dilute acid hydrolysis, increasing acid concentration from 1 % (v/v) to 5 % (v/v) decreased the conversion percentage of almost all treated agro-industrial wastes. Lower conversion percentages for some treatments were obtained when increasing the residence time from 90 to 120 min. The two-stage dilute acid hydrolysis by phosphoric acid (1.0 % v/v) followed by sulphuric acid (1.0 % v/v) resulted in the highest conversion percentage (41.3 % w/w) on treated sugar beet waste. This treatment when neutralized, amended with some nutrients and inoculated with baker's yeast, achieved the highest ethanol concentration (1.0 % v/v). Formation of furfural and hydroxymethylfurfural (HMF) were functions of type of acid hydrolysis, acid concentration, residence time and feedstock type. The highest bioconversion of 5 % wastes (37.8 % w/w) was recorded on sugar beet waste by Trichoderma viride EMCC 107. This treatment when followed by baker's yeast fermentation, 0.41 % (v/v) ethanol and 8.2 % (v/w) conversion coefficient were obtained.

Effect of acid hydrolysis and fungal biotreatment on agro-industrial wastes for obtainment of free sugars for bioethanol production

PubMed Central

El-Tayeb, T.S.; Abdelhafez, A.A.; Ali, S.H.; Ramadan, E.M.

2012-01-01

This study was designed to evaluate selected chemical and microbiological treatments for the conversion of certain local agro-industrial wastes (rice straw, corn stalks, sawdust, sugar beet waste and sugarcane bagasse) to ethanol. The chemical composition of these feedstocks was determined. Conversion of wastes to free sugars by acid hydrolysis varied from one treatment to another. In single-stage dilute acid hydrolysis, increasing acid concentration from 1 % (v/v) to 5 % (v/v) decreased the conversion percentage of almost all treated agro-industrial wastes. Lower conversion percentages for some treatments were obtained when increasing the residence time from 90 to 120 min. The two-stage dilute acid hydrolysis by phosphoric acid (1.0 % v/v) followed by sulphuric acid (1.0 % v/v) resulted in the highest conversion percentage (41.3 % w/w) on treated sugar beet waste. This treatment when neutralized, amended with some nutrients and inoculated with baker’s yeast, achieved the highest ethanol concentration (1.0 % v/v). Formation of furfural and hydroxymethylfurfural (HMF) were functions of type of acid hydrolysis, acid concentration, residence time and feedstock type. The highest bioconversion of 5 % wastes (37.8 % w/w) was recorded on sugar beet waste by Trichoderma viride EMCC 107. This treatment when followed by baker’s yeast fermentation, 0.41 % (v/v) ethanol and 8.2 % (v/w) conversion coefficient were obtained. PMID:24031984

An aldonolactonase AltA from Penicillium oxalicum mitigates the inhibition of β-glucosidase during lignocellulose biodegradation.

PubMed

Peng, Shengjuan; Cao, Qing; Qin, Yuqi; Li, Xuezhi; Liu, Guodong; Qu, Yinbo

2017-05-01

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, D-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than D-glucose and D-gluconate on β-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of D-glucono-1,5-lactone to D-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of β-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

Atomic resolution (0.97 Å) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2

PubMed Central

Sekar, K.; Rajakannan, V.; Gayathri, D.; Velmurugan, D.; Poi, M.-J.; Dauter, M.; Dauter, Z.; Tsai, M.-D.

2005-01-01

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 Å). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 Å, β = 101.47°. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (R free = 13.4%) using 63 926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60–70 are ordered and have clear electron density. PMID:16508077

Biodiesel production by combined fatty acids separation and subsequently enzymatic esterification to improve the low temperature properties.

PubMed

Wang, Meng; Nie, Kaili; Cao, Hao; Deng, Li; Wang, Fang; Tan, Tianwei

2014-12-01

The poor low-temperature properties of biodiesel, which provokes easy crystallization at low temperature, can cause fuel line plugging and limits its blending amount with petro-diesel. This work aimed to study the production of biodiesel with a new process of improving the low temperature performance of biodiesel. Waste cooking oil was first hydrolyzed into fatty acids (FAs) by 60g immobilized lipase and 240g RO water in 15h. Then, urea complexation was used to divide the FAs into saturated and unsaturated components. The conditions for complexation were: FA-to-urea ratio 1:2 (w/w), methanol to FA ratio 5:1 (v/v), duration 2h. The saturated and unsaturated FAs were then converted to iso-propyl and methyl esters by lipase, respectively. Finally, the esters were mixed together. The CFPP of this mixture was decreased from 5°C to -3°C. Hydrolysis, urea complexation and enzymic catalyzed esterification processes are discussed in this paper. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hydroxycinnamate Conjugates as Potential Monolignol Replacements: In vitro Lignification and Cell Wall Studies with Rosmarinic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuki, Tobimatsu; Sasikumar, Elumalai; Grabber, John H.

2012-04-01

The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers, such as rosmarinic acid (RA) and analogous catechol derivatives, into cell-wall lignins that are consequently less recalcitrant to biomass processing. In vitro lignin polymerization experiments revealed that RA readily underwent peroxidase-catalyzed copolymerization with monolignols and lignin oligomers to form polymers with new benzodioxane inter-unit linkages. Incorporation of RA permitted extensive depolymerization of synthetic lignins by mild alkaline hydrolysis, presumably by cleavage of ester intra-unit linkages within RA. Copolymerization of RA with monolignols into maize cell walls by in situ peroxidases significantly enhanced alkaline lignin extractability andmore » promoted subsequent cell wall saccharification by fungal enzymes. Incorporating RA also improved cell wall saccharification by fungal enzymes and by rumen microflora even without alkaline pretreatments, possibly by modulating lignin hydrophobicity and/or limiting cell wall cross-linking. Consequently, we anticipate that bioengineering approaches for partial monolignol substitution with RA and analogous plant hydroxycinnamates would permit more efficient utilization of plant fiber for biofuels or livestock production.« less

Enhanced saccharification of sugarcane bagasse using soluble cellulase supplemented with immobilized β-glucosidase.

PubMed

Borges, Diogo Gontijo; Baraldo, Anderson; Farinas, Cristiane Sanchez; Giordano, Raquel de Lima Camargo; Tardioli, Paulo Waldir

2014-09-01

The β-glucosidase (BG) enzyme plays a vital role in the hydrolysis of lignocellulosic biomass. Supplementation of the hydrolysis reaction medium with BG can reduce inhibitory effects, leading to greater conversion. In addition, the inclusion of immobilized BG can be a useful way of increasing enzyme stability and recyclability. BG was adsorbed on polyacrylic resin activated by carboxyl groups (BG-PC) and covalently attached to glyoxyl-agarose (BG-GA). BG-PC exhibited similar behavior to soluble BG in the hydrolysis of cellobiose, while BG-GA hydrolyzed the same substrate at a lower rate. However, the thermal stability of BG-GA was higher than that of free BG. Hydrolysis of pretreated sugarcane bagasse catalyzed by soluble cellulase supplemented with immobilized BG improved the conversion by up to 40% after 96 h of reaction. Both derivatives remained stable up to the third cycle and losses of activity were less than 50% after five cycles. Copyright © 2014 Elsevier Ltd. All rights reserved.

Kinetics study of palm oil hydrolysis using immobilized lipase Candida rugosa in packed bed reactor.

PubMed

Min, C S; Bhatia, S; Kamaruddin, A H

1999-01-01

Continuous hydrolysis of palm oil triglyceride in organic solvent using immobilized Candida rugosa on the Amberlite MB-1 as a source of immobilized lipase was studied in packed bed reactor. The enzymatic kinetics of hydrolysis reaction was studied by changing the substrate concentration, reaction temperature and residence time(tau) in the reactor. At 55 degrees C, the optimum water concentration was found to be 15 % weight per volume of solution (%w/v). The Michaelis-Menten kinetic model was used to obtain the reaction parameters, Km(app) and V max(app). The activation energies were found to be quite low indicating that the lipase-catalyzed process is controlled by diffusion of substrates. The Michaelis-Menten kinetic model was found to be suitable at low water concentration 10-15 %w/v of solution. At higher water concentration, substrate inhibition model was used for data analysis. Reactor operation was found to play an important role in the palm oil hydrolysis kinetic.

Influence of drying of chara cellulose on length/length distribution of microfibrils after acid hydrolysis.

PubMed

Horikawa, Yoshiki; Shimizu, Michiko; Saito, Tsuguyuki; Isogai, Akira; Imai, Tomoya; Sugiyama, Junji

2018-04-01

Chara is a genus of freshwater alga that is evolutionarily observed at the aquatic-terrestrial boundary, whose cellulose microfibrils are similar to those of terrestrial plants regarding the crystallinity and biosynthesis of cellulose. Oven-dried and never-dried celluloses samples were prepared from chara. Terrestrial plant cellulose samples were used as references. The lengths and length distributions of oven-dried and never-dried chara cellulose microfibrils after acid hydrolysis with or without pretreatment by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, which was used for efficient fibrillation of acid-hydrolyzed products, were observed by transmission electron microscopy. All terrestrial plant celluloses and oven-dried chara cellulose had short nanocrystal-like morphologies of 100-300 nm in length after acid hydrolysis. In contrast, the never-dried chara cellulose had much longer microfibrils of ∼970 nm in length after acid hydrolysis. These results indicated that disordered regions present periodically along the cellulose microfibrils, which cause the formation of cellulose nanocrystals after acid hydrolysis, are not present in inherent chara cellulose microfibrils in water, but are formed artificially under drying or dehydration conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Behavior of ellagitannins, gallic acid, and ellagic acid under alkaline conditions

Treesearch

Richard W. Hemingway; W.E. Hillis

1971-01-01

Examination of the rates of hydrolysis of different ellagitannins under conditions comparable with cold soda and alkaline-groundwood pulping processes showed that some ellagitannins are notably resistant to hydrolysis. The rate of hydrolysis was dependent upon the pH and tempemture of the solution and particularly upon the structure of the compound. Decarboxylation of...

Evaluation of abalone β-glucuronidase substitution in current urine hydrolysis procedures.

PubMed

Malik-Wolf, Brittany; Vorce, Shawn; Holler, Justin; Bosy, Thomas

2014-04-01

This study examined the potential of abalone β-glucuronidase as a viable and cost effective alternative to current hydrolysis procedures using acid, Helix pomatia β-glucuronidase and Escherichia coli β-glucuronidase. Abalone β-glucuronidase successfully hydrolyzed oxazepam-glucuronide and lorazepam-glucuronide within 5% of the spiked control concentration. Benzodiazepines present in authentic urine specimens were within 20% of the concentrations obtained with the current hydrolysis procedure using H. pomatia β-glucuronidase. JWH 018 N-(5-hydroxypentyl) β-d-glucuronide was hydrolyzed within 10% of the control concentration. Authentic urine specimens showed improved glucuronide cleavage using abalone β-glucuronidase with up to an 85% increase of drug concentration, compared with the results obtained using E. coli β-glucuronidase. The JWH 018 and JWH 073 carboxylic acid metabolites also showed increased drug concentrations of up to 24%. Abalone β-glucuronidase was able to completely hydrolyze a morphine-3-glucuronide control, but only 82% of total morphine was hydrolyzed in authentic urine specimens compared with acid hydrolysis results. Hydrolysis of codeine and hydromorphone varied between specimens, suggesting that abalone β-glucuronidase may not be as efficient in hydrolyzing the glucuronide linkages in opioid compounds compared with acid hydrolysis. Abalone β-glucuronidase demonstrates effectiveness as a low cost option for enzyme hydrolysis of benzodiazepines and synthetic cannabinoids.

A rapid hydrolysis method and DABS-Cl derivatization for complete amino acid analysis of octreotide acetate by reversed phase HPLC.

PubMed

Akhlaghi, Yousef; Ghaffari, Solmaz; Attar, Hossein; Alamir Hoor, Amir

2015-11-01

Octreotide as a synthetic cyclic octapeptide is a somatostatin analog with longer half-life and more selectivity for inhibition of the growth hormone. The acetate salt of octreotide is currently used for medical treatment of somatostatin-related disorders such as endocrine and carcinoid tumors, acromegaly, and gigantism. Octreotide contains both cysteine and tryptophan residues which make the hydrolysis part of its amino acid analysis procedure very challenging. The current paper introduces a fast and additive-free method which preserves tryptophan and cysteine residues during the hydrolysis. Using only 6 M HCl, this hydrolysis process is completed in 30 min at 150 °C. This fast hydrolysis method followed by pre-column derivatization of the released amino acids with 4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride (DABS-Cl) which takes only 20 min, makes it possible to do the complete amino acid analysis of an octreotide sample in a few hours. The highly stable-colored DABS-Cl derivatives can be detected in 436 nm in a reversed phase chromatographic system, which eliminates spectral interferences to a great extent. The amino acid analysis of octreotide acetate including hydrolysis, derivatization, and reversed phase HPLC determination was validated according to International Conference of Harmonization (ICH) guidelines.

Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction

PubMed Central

Catrina, Irina; O'Brien, Patrick J.; Purcell, Jamie; Nikolic-Hughes, Ivana; Zalatan, Jesse G.; Hengge, Alvan C.; Herschlag, Daniel

2008-01-01

The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are ‘loose’ and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn2+ ion. Furthermore, the KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer. PMID:17411045

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

PubMed

Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

2018-04-09

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The Effect of Phytase on the Oxygen Isotope Composition of Phosphate

NASA Astrophysics Data System (ADS)

von Sperber, C.; Tamburini, F.; Bernasconi, S. M.; Frossard, E.

2013-12-01

Plants and microorganisms under phosphorus (P) stress release extracellular phosphatases as a strategy to acquire inorganic phosphate (Pi) (1-2). These enzymes catalyze the hydrolysis of phosphoesters leading to a release of Pi. The enzymatic hydrolysis leads, via a nucleophilic attack, to the incorporation of one oxygen atom from the water into the newly formed Pi molecule. During the incorporation, an isotopic fractionation occurs, which might be used to identify the origin of Pi in the environment (3-6). While the effect of phosphomonoesterases and phosphodiesterases on the oxygen isotope composition of phosphate has been examined, there are, so far, no studies dealing with the effect of phytases (4-6). Phytases catalyze the hydrolysis of myo-inositol-hexakis-phosphate (IP6), which is an important component of organic P in many ecosystems (7). Enzymatic assays with phytase from wheat germ and Aspergillus niger were prepared under sterile and temperature controlled conditions in order to determine the effect of phytases on the oxygen isotope composition of phosphate, which has been liberated from IP6 via enzymatic hydrolysis. Assays with phytase from wheat germ lead to a turnover of the substrate close to 100%, while assays with phytase from Aspergillus niger lead to a turnover of the substrate close to 80%. In the case of the assays with phytase from wheat germ, our results indicate that one sixth of the total 24 oxygen which are associated to the phosphates in IP6 are exchanged with oxygen from water. From this we conclude that the incorporation of one oxygen atom from water occurs only at four phosphate molecules of IP6, while two phosphate molecules do not experience an incorporation of oxygen. This suggests that during the enzymatic hydrolysis, four P-O bonds and two C-O bonds are broken. Provided that, the isotopic fractionation can be calculated with an isotopic mass balance resulting in -8.4‰ (×3.6 SD). This is a value very similar to those reported for acid phosphatases (6). In contrast, the results from assays with phytase from Aspergillus niger indicate that the exchange of oxygen occurs at more than one third of the total 24 oxygen which are associated to the phosphates in IP6. In addition, we observe a change in the oxygen isotope composition of Pi when using myo-inositol and potassium-dihydrogen-phosphate as sole substrates in the enzymatic assays with phytase from Aspergillus niger. These observations suggest that the reformation of IP6 from the two products of the reaction (myo-inositol and Pi) is taking place at a rate, which is within the time scale of the experiment. In this case, the isotopic fractionation caused by phytase from Aspergillus niger will be determined by the equilibrium of the reaction. Further experiments are in process to verify these findings.

Recent Development in Chemical Depolymerization of Lignin: A Review

DOE PAGES

Wang, Hai; Tucker, Melvin; Ji, Yun

2013-01-01

This article reviewed recent development of chemical depolymerization of lignins. There were five types of treatment discussed, including base-catalyzed, acid-catalyzed, metallic catalyzed, ionic liquids-assisted, and supercritical fluids-assisted lignin depolymerizations. The methods employed in this research were described, and the important results were marked. Generally, base-catalyzed and acid-catalyzed methods were straightforward, but the selectivity was low. The severe reaction conditions (high pressure, high temperature, and extreme pH) resulted in requirement of specially designed reactors, which led to high costs of facility and handling. Ionic liquids, and supercritical fluids-assisted lignin depolymerizations had high selectivity, but the high costs of ionic liquids recyclingmore » and supercritical fluid facility limited their applications on commercial scale biomass treatment. Metallic catalyzed depolymerization had great advantages because of its high selectivity to certain monomeric compounds and much milder reaction condition than base-catalyzed or acid-catalyzed depolymerizations. It would be a great contribution to lignin conversion if appropriate catalysts were synthesized.« less

Catalytic conversion of carbohydrates to 5-hydroxymethylfurfural from the waste liquid of acid hydrolysis NCC.

PubMed

Sun, Yonghui; Liu, Pengtao; Liu, Zhong

2016-05-20

The principal goal of this work was to reuse the carbohydrates and recycle sulfuric acid in the waste liquid of acid hydrolysis nanocrystalline cellulose (NCC). Therefore, in this work, the optimizations of further hydrolysis of waste liquid of acid hydrolysis NCC and catalytic conversion of L4 to 5-hydroxymethylfurfural (5-HMF) were studied. Sulfuric acid was separated by spiral wound diffusion dialysis (SWDD). The results revealed that cellulose can be hydrolyze to glucose absolutely under the condition of temperature 35 °C, 3 h, and sulfuric acid's concentration 62 wt%. And 78.3% sulfuric acid was recovered by SWDD. The yield of 5-HMF was highest in aqueous solution under the optimal condition was as follows, temperature 160 °C, 3 h, and sulfuric acid's concentration 12 wt%. Then the effect of biphasic solvent systems catalytic conversion and inorganic salt as additives were still examined. The results showed that both of them contributed to prepare 5-HMF. The yield and selectivity of 5-HMF was up to 21.0% and 31.4%, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

The integration of dilute acid hydrolysis of xylan and fast pyrolysis of glucan to obtain fermentable sugars.

PubMed

Jiang, Liqun; Wu, Nannan; Zheng, Anqing; Zhao, Zengli; He, Fang; Li, Haibin

2016-01-01

Fermentable sugars are important intermediates in the biological conversion of biomass. Hemicellulose and amorphous cellulose are easily hydrolyzed to fermentable sugars in dilute acid, whereas crystalline cellulose is more difficult to be hydrolyzed. Cellulose fast pyrolysis is an alternative method to liberate valuable fermentable sugars from biomass. The amount of levoglucosan generated from lignocellulose by fast pyrolysis is usually lower than the theoretical yield based on the cellulose fraction. Pretreatment is a promising route to improve the yield of levoglucosan from lignocellulose. The integration of dilute sulfuric acid hydrolysis and fast pyrolysis to obtain fermentable sugars was evaluated in this study. Dilute sulfuric acid hydrolysis could remove more than 95.1 and 93.4 % of xylan (the main component of hemicellulose) from sugarcane bagasse and corncob with high yield of xylose. On the other hand, dilute sulfuric acid hydrolysis was also an effective pretreatment to enhance levoglucosan yield from lignocellulose. Dilute acid hydrolysis could accumulate glucan (the component of cellulose) and remove most of the alkali and alkaline earth metals which were powerful catalysts during fast pyrolysis. Further increase in dilute acid concentration (from 0 to 2 %) in pretreatment could promote the yield of levoglucosan in fast pyrolysis. The acid pretreated sugarcane bagasse and corncob gave levoglucosan yields of 43.8 and 35.2 % which were obvious higher than those of raw sugarcane bagasse (12.0 %) and corncob (7.0 %). Obtaining fermentable sugars by combination dilute acid hydrolysis of xylan and fast pyrolysis of glucan could make full utilization of biomass, and get fermentable sugars economically from biomass for bio-refinery.

Enhancement of hydrolysis of Chlorella vulgaris by hydrochloric acid.

PubMed

Park, Charnho; Lee, Ja Hyun; Yang, Xiaoguang; Yoo, Hah Young; Lee, Ju Hun; Lee, Soo Kweon; Kim, Seung Wook

2016-06-01

Chlorella vulgaris is considered as one of the potential sources of biomass for bio-based products because it consists of large amounts of carbohydrates. In this study, hydrothermal acid hydrolysis with five different acids (hydrochloric acid, nitric acid, peracetic acid, phosphoric acid, and sulfuric acid) was carried out to produce fermentable sugars (glucose, galactose). The hydrothermal acid hydrolysis by hydrochloric acid showed the highest sugar production. C. vulgaris was hydrolyzed with various concentrations of hydrochloric acid [0.5-10 % (w/w)] and microalgal biomass [20-140 g/L (w/v)] at 121 °C for 20 min. Among the concentrations examined, 2 % hydrochloric acid with 100 g/L biomass yielded the highest conversion of carbohydrates (92.5 %) into reducing sugars. The hydrolysate thus produced from C. vulgaris was fermented using the yeast Brettanomyces custersii H1-603 and obtained bioethanol yield of 0.37 g/g of algal sugars.

Hydrolysis products generated by lipoprotein lipase and endothelial lipase differentially impact THP-1 macrophage cell signalling pathways.

PubMed

Essaji, Yasmin; Yang, Yanbo; Albert, Carolyn J; Ford, David A; Brown, Robert J

2013-08-01

Macrophages express lipoprotein lipase (LPL) and endothelial lipase (EL) within atherosclerotic plaques; however, little is known about how lipoprotein hydrolysis products generated by these lipases might affect macrophage cell signalling pathways. We hypothesized that hydrolysis products affect macrophage cell signalling pathways associated with atherosclerosis. To test our hypothesis, we incubated differentiated THP-1 macrophages with products from total lipoprotein hydrolysis by recombinant LPL or EL. Using antibody arrays, we found that the phosphorylation of six receptor tyrosine kinases and three signalling nodes--most associated with atherosclerotic processes--was increased by LPL derived hydrolysis products. EL derived hydrolysis products only increased the phosphorylation of tropomyosin-related kinase A, which is also implicated in playing a role in atherosclerosis. Using electrospray ionization-mass spectrometry, we identified the species of triacylglycerols and phosphatidylcholines that were hydrolyzed by LPL and EL, and we identified the fatty acids liberated by gas chromatography-mass spectrometry. To determine if the total liberated fatty acids influenced signalling pathways, we incubated differentiated THP-1 macrophages with a mixture of the fatty acids that matched the concentrations of liberated fatty acids from total lipoproteins by LPL, and we subjected cell lysates to antibody array analyses. The analyses showed that only the phosphorylation of Akt was significantly increased in response to fatty acid treatment. Overall, our study shows that macrophages display potentially pro-atherogenic signalling responses following acute treatments with LPL and EL lipoprotein hydrolysis products.

Thermus thermophilus as a Source of Thermostable Lipolytic Enzymes

PubMed Central

López-López, Olalla; Cerdán, María-Esperanza; González-Siso, María-Isabel

2015-01-01

Lipolytic enzymes, esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), catalyze the hydrolysis of ester bonds between alcohols and carboxylic acids, and its formation in organic media. At present, they represent about 20% of commercialized enzymes for industrial use. Lipolytic enzymes from thermophilic microorganisms are preferred for industrial use to their mesophilic counterparts, mainly due to higher thermostability and resistance to several denaturing agents. However, the production at an industrial scale from the native organisms is technically complicated and expensive. The thermophilic bacterium Thermus thermophilus (T. thermophilus) has high levels of lipolytic activity, and its whole genome has been sequenced. One esterase from the T. thermophilus strain HB27 has been widely characterized, both in its native form and in recombinant forms, being expressed in mesophilic microorganisms. Other putative lipases/esterases annotated in the T. thermophilus genome have been explored and will also be reviewed in this paper. PMID:27682117

Optimization of Lipase production from a novel strain Thalassospira permensis M35-15 using Response Surface Methodology

PubMed Central

Kai, Wang; Peisheng, Yan

2016-01-01

ABSTRACT Lipases can catalyze the hydrolysis of glycerol, esters and long chain fatty acids. A lipase producing isolate M35-15 was screened and identified as Thalassospira permensis using 16S rRNA gene sequence analysis. To our knowledge this is the first report on Thalassospira permensis producing lipases. In this paper the optimization of medium composition for the increase in bacterial lipase was achieved using statistical methods. Firstly the key ingredients were selected by Plackett-Burman experimental design, then the levels of the ingredients were optimized using central composite design of Response Surface Methodology. The predicted optimal lipase activity was 11.49 U under the conditions that medium composition were 5.15 g/l glucose, 11.74 g/l peptone, 6.74 g/l yeast powder and 22.90 g/l olive oil emulsifier. PMID:27285376

Optimization of Lipase production from a novel strain Thalassospira permensis M35-15 using Response Surface Methodology.

PubMed

Kai, Wang; Peisheng, Yan

2016-09-02

Lipases can catalyze the hydrolysis of glycerol, esters and long chain fatty acids. A lipase producing isolate M35-15 was screened and identified as Thalassospira permensis using 16S rRNA gene sequence analysis. To our knowledge this is the first report on Thalassospira permensis producing lipases. In this paper the optimization of medium composition for the increase in bacterial lipase was achieved using statistical methods. Firstly the key ingredients were selected by Plackett-Burman experimental design, then the levels of the ingredients were optimized using central composite design of Response Surface Methodology. The predicted optimal lipase activity was 11.49 U under the conditions that medium composition were 5.15 g/l glucose, 11.74 g/l peptone, 6.74 g/l yeast powder and 22.90 g/l olive oil emulsifier.

Optical enhancing durable anti-reflective coating

DOEpatents

Maghsoodi, Sina; Varadarajan, Aravamuthan; Movassat, Meisam

2016-07-05

Disclosed herein are polysilsesquioxane based anti-reflective coating (ARC) compositions, methods of preparation, and methods of deposition on a substrate. In embodiments, the polysilsesquioxane of this disclosure is prepared in a two-step process of acid catalyzed hydrolysis of organoalkoxysilane followed by addition of tetralkoxysilane that generates silicone polymers with >40 mol % silanol based on Si-NMR. These high silanol siloxane polymers are stable and have a long shelf-life in the polar organic solvents at room temperature. Also disclosed are low refractive index ARC made from these compositions with and without additives such as porogens, templates, Si--OH condensation catalyst and/or nanofillers. Also disclosed are methods and apparatus for applying coatings to flat substrates including substrate pre-treatment processes, coating processes including flow coating and roll coating, and coating curing processes including skin-curing using hot-air knives. Also disclosed are coating compositions and formulations for highly tunable, durable, highly abrasion-resistant functionalized anti-reflective coatings.

High gain durable anti-reflective coating

DOEpatents

Maghsoodi, Sina; Brophy, Brenor L.; Colson, Thomas E.; Gonsalves, Peter R.; Abrams, Ze'ev R.

2016-07-26

Disclosed herein are polysilsesquioxane-based anti-reflective coating (ARC) compositions, methods of preparation, and methods of deposition on a substrate. In one embodiment, the polysilsesquioxane of this disclosure is prepared in a two-step process of acid catalyzed hydrolysis of organoalkoxysilane followed by addition of tetralkoxysilane that generates silicone polymers with >40 mol % silanol based on Si-NMR. These high silanol siloxane polymers are stable and have a long shelf-life in polar organic solvents at room temperature. Also disclosed are low refractive index ARC made from these compositions with and without additives such as porogens, templates, thermal radical initiator, photo radical initiators, crosslinkers, Si--OH condensation catalyst and nano-fillers. Also disclosed are methods and apparatus for applying coatings to flat substrates including substrate pre-treatment processes, coating processes and coating curing processes including skin-curing using hot-air knives. Also disclosed are coating compositions and formulations for highly tunable, durable, highly abrasion-resistant functionalized anti-reflective coatings.

The Chemistry of Paper Preservation: Part 1. The Aging of Paper and Conservation Techniques

NASA Astrophysics Data System (ADS)

Carter, Henry A.

1996-05-01

This study provides an introduction to the problem of the aging of paper and the conservation techniques that are currently being employed in paper preservation. The chemical reactions that are responsible for the aging of paper are discussed with the conclusion that acid-catalyzed hydrolysis is the predominant mechanism for cellulose degradation and strength loss. A description and the chemistry of a number of mass deacidification methods are presented. The more viable deacidification methods include the DEZ, Wei T'o, FMC, Bookkeeper, Viennese, Book Preservation Associates, Sable and the Batelle processes. A summary of the literature on the evaluations of these processes is presented, and the benefits and limitations of the mass deacidification methods are discussed. Other conservation techniques such as paper strengthening, conservation bleaching, and pest control are briefly introduced, followed by an introduction to alkaline papermaking.

Engineering of isoamylase: improvement of protein stability and catalytic efficiency through semi-rational design.

PubMed

Li, Youran; Zhang, Liang; Ding, Zhongyang; Gu, Zhenghua; Shi, Guiyang

2016-01-01

Isoamylase catalyzes the hydrolysis of α-1,6-glycosidic linkages in glycogen, amylopectin and α/β-limit dextrins. A semi-rational design strategy was performed to improve catalytic properties of isoamylase from Bacillus lentus. Three residues in vicinity of the essential residues, Arg505, Asn513, and Gly608, were chosen as the mutation sites and were substituted by Ala, Pro, Glu, and Lys, respectively. Thermal stability of the mutant R505P and acidic stability of the mutant R505E were enhanced. The k cat /K m values of the mutant G608V have been promoted by 49%, and the specific activity increased by 33%. This work provides an effective strategy for improving the catalytic activity and stability of isoamylase, and the results obtained here may be useful for the improvement of catalytic properties of other α/β barrel enzymes.

Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balasubramanian, Anuradha; Ponnuraj, Karthe, E-mail: pkarthe@hotmail.com

Urease from pigeon pea was purified and crystallized and X-ray diffraction data were collected at 2.5 Å resolution. Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized andmore » the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å.« less

Cloning and overexpression of beta-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide.

PubMed

Matsuo, Ichiro; Kim, Sunhwa; Yamamoto, Yuichi; Ajisaka, Katsumi; Maruyama, Jun-ich; Nakajima, Harushi; Kitamoto, Katsuhiko

2003-03-01

We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.

Process development of short-chain polyols synthesis from corn stover by combination of enzymatic hydrolysis and catalytic hydrogenolysis.

PubMed

Fang, Zhen-Hong; Zhang, Jian; Lu, Qi-Ming; Bao, Jie

2014-09-01

Currently short-chain polyols such as ethanediol, propanediol, and butanediol are produced either from the petroleum feedstock or from the starch-based food crop feedstock. In this study, a combinational process of enzymatic hydrolysis with catalytic hydrogenolysis for short-chain polyols production using corn stover as feedstock was developed. The enzymatic hydrolysis of the pretreated corn stover was optimized to produce stover sugars at the minimum cost. Then the stover sugars were purified and hydrogenolyzed into polyols products catalyzed by Raney nickel catalyst. The results show that the yield of short-chain polyols from the stover sugars was comparable to that of the corn-based glucose. The present study provided an important prototype for polyols production from lignocellulose to replace the petroleum- or corn-based polyols for future industrial applications.

and applications of microbial ß-D-xylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium

USDA-ARS?s Scientific Manuscript database

Xylan 1,4-beta-D xylosidase catalyzes hydrolysis of nonreducing end xylose residues from xylooligosaccharides. The enzyme is currently used in several industrial-scale processes for food and materials, and on a grander scale, the enzyme could find employment along side cellulases and other hemicell...

High Surface Area, Thermally Stable, Hydrophobic, Microporous, Rigid Gels Generated at Ambient from MeSi(OEt)3 /(EtO)3 SiCH2 CH2 Si(OEt)3 Mixtures by F- -Catalyzed Hydrolysis.

PubMed

Furgal, Joseph C; Yamane, Honami; Odykirk, Timothy R; Yi, Eongyu; Chujo, Yoshiki; Laine, Richard M

2018-01-02

High surface area materials are of considerable interest for gas storage/capture, molecular sieving, catalyst supports, as well as for slow-release drug-delivery systems. We report here a very simple and fast route to very high surface area, mechanically robust, hydrophobic polymer gels prepared by fluoride-catalyzed hydrolysis of mixtures of MeSi(OEt) 3 and bis-triethoxysilylethane (BTSE) at room temperature. These materials offer specific surface areas up to 1300 m 2  g -1 , peak pore sizes of 0.8 nm and thermal stabilities above 200 °C. The gelation times and surface areas can be controlled by adjusting the solvent volume (dichloromethane), percent fluoride (as nBu 4 NF or TBAF) and the BTSE contents. Polymers with other corners and linkers were also explored. These materials will further expand the materials databank for use in vacuum insulation panels and as thermally stable release and capture media. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mathematical model for Trametes versicolor growth in submerged cultivation.

PubMed

Tisma, Marina; Sudar, Martina; Vasić-Racki, Durda; Zelić, Bruno

2010-08-01

Trametes versicolor is a white-rot fungus known as a producer of extracellular enzymes such as laccase, manganese-peroxidase, and lignin-peroxidase. The production of these enzymes requires detailed knowledge of the growth characteristics and physiology of the fungus. Submerged cultivations of T. versicolor on glucose, fructose, and sucrose as sole carbon sources were performed in shake flasks. Sucrose hydrolysis catalyzed by the whole cells of T. versicolor was considered as one-step enzymatic reaction described with Michaelis-Menten kinetics. Kinetic parameters of invertase-catalyzed sucrose hydrolysis were estimated (K (m) = 7.99 g dm(-3) and V (m) = 0.304 h(-1)). Monod model was used for description of kinetics of T. versicolor growth on glucose and fructose as sole carbon sources. Growth associated model parameters were estimated from the experimental results obtained by independent experiments (mu(G)(max) = 0.14 h(-1), K(G)(S) = 8.06 g dm(-3), mu(F)(max) = 0.37 h(-1) and K(F)(S) = 54.8 g dm(-3)). Developed mathematical model is in good agreement with the experimental results.

Accelerated hydrolysis of substituted cellulose for potential biofuel production: kinetic study and modeling.

PubMed

Mu, Bingnan; Xu, Helan; Yang, Yiqi

2015-11-01

In this work, kinetics of substitution accelerated cellulose hydrolysis with multiple reaction stages was investigated to lay foundation for mechanism study and molecular design of substituting compounds. High-efficiency hydrolysis of cellulose is critical for cellulose-based bioethanol production. It is known that, substitution could substantially decrease activation energy and increase reaction rate of acidic hydrolysis of glycosidic bonds in cellulose. However, reaction kinetics and mechanism of the accelerated hydrolysis were not fully revealed. In this research, it was proved that substitution therefore accelerated hydrolysis only occurred in amorphous regions of cellulose fibers, and was a process with multiple reaction stages. With molar ratio of substitution less than 1%, the overall hydrolysis rate could be increased for around 10 times. We also quantified the relationship between the hydrolysis rate of individual reaction stage and its major influences, including molar ratio of substitution, activation energy of acidic hydrolysis, pH and temperature. Copyright © 2015 Elsevier Ltd. All rights reserved.

Quantitative predictions of bioconversion of aspen by dilute acid and SPORL pretreatments using a unified combined hydrolysis factor (CHF)

Treesearch

W. Zhu; Carl J. Houtman; J.Y. Zhu; Roland Gleisner; K.F. Chen

2012-01-01

A combined hydrolysis factor (CHF) was developed to predict xylan hydrolysis during pretreatments of native aspen (Populus tremuloides) wood chips. A natural extension of previously developed kinetic models allowed us to account for the effect of catalysts by dilute acid and two sulfite pretreatments at different pH values....

Hydrolysis of virgin coconut oil using immobilized lipase in a batch reactor.

PubMed

Chua, Lee Suan; Alitabarimansor, Meisam; Lee, Chew Tin; Mat, Ramli

2012-01-01

Hydrolysis of virgin coconut oil (VCO) had been carried out by using an immobilised lipase from Mucor miehei (Lipozyme) in a water-jacketed batch reactor. The kinetic of the hydrolysis was investigated by varying the parameters such as VCO concentration, enzyme loading, water content, and reaction temperature. It was found that VCO exhibited substrate inhibition at the concentration more than 40% (v/v). Lipozyme also achieved the highest production of free fatty acids, 4.56 mM at 1% (w/v) of enzyme loading. The optimum water content for VCO hydrolysis was 7% (v/v). A relatively high content of water was required because water was one of the reactants in the hydrolysis. The progress curve and the temperature profile of the enzymatic hydrolysis also showed that Lipozyme could be used for free fatty acid production at the temperature up to 50°C. However, the highest initial reaction rate and the highest yield of free fatty acid production were at 45 and 40°C, respectively. A 100 hours of initial reaction time has to be compensated in order to obtain the highest yield of free fatty acid production at 40°C.

Hydrolysis of Virgin Coconut Oil Using Immobilized Lipase in a Batch Reactor

PubMed Central

Chua, Lee Suan; Alitabarimansor, Meisam; Lee, Chew Tin; Mat, Ramli

2012-01-01

Hydrolysis of virgin coconut oil (VCO) had been carried out by using an immobilised lipase from Mucor miehei (Lipozyme) in a water-jacketed batch reactor. The kinetic of the hydrolysis was investigated by varying the parameters such as VCO concentration, enzyme loading, water content, and reaction temperature. It was found that VCO exhibited substrate inhibition at the concentration more than 40% (v/v). Lipozyme also achieved the highest production of free fatty acids, 4.56 mM at 1% (w/v) of enzyme loading. The optimum water content for VCO hydrolysis was 7% (v/v). A relatively high content of water was required because water was one of the reactants in the hydrolysis. The progress curve and the temperature profile of the enzymatic hydrolysis also showed that Lipozyme could be used for free fatty acid production at the temperature up to 50°C. However, the highest initial reaction rate and the highest yield of free fatty acid production were at 45 and 40°C, respectively. A 100 hours of initial reaction time has to be compensated in order to obtain the highest yield of free fatty acid production at 40°C. PMID:22953055

Partial Purification and Properties of an Alkaline α-Galactosidase from Mature Leaves of Cucurbita pepo1

PubMed Central

Gaudreault, Pierre-Richard; Webb, John A.

1983-01-01

A fourth molecular from of α-galactosidase, designated LIV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. Km determinations in McIlvaine buffer (200 millimolar Na2-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. LIV was partially inhibited by Ca2+, Mg2+, and Mn2+, more so by Ni2+, Zn2+, and Co2+, and highly so by Cu2+, Ag2+, Hg2+ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, LIV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C1, C2, and C4, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C6). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of LIV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of LIVin vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore LIV activity through coordination with some toxic cation introduced as a buffer contaminant. Images Fig. 1 PMID:16662884

The lid domain of the MCP hydrolase DxnB2 contributes to the reactivity towards recalcitrant PCB metabolites

PubMed Central

Yam, Katherine C.; Ghosh, Subhangi; Bolin, Jeffrey T.; Eltis, Lindsay D.

2013-01-01

DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically-shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ESred, an intermediate possessing a bathochromically-shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/β-hydrolase core domain, as a determinant in oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme’s ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/β-hydrolases. PMID:23879719

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

PubMed Central

Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

2013-01-01

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

Enhancement of the Rate of Pyrophosphate Hydrolysis by Nonenzymatic Catalysts and by Inorganic Pyrophosphatase*

PubMed Central

Stockbridge, Randy B.; Wolfenden, Richard

2011-01-01

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, 31P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PPi) in the presence and absence of Mg2+ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPPi2− proceeds with a rate constant of 2.8 × 10−10 s−1, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s−1 under the same conditions. The resulting rate enhancement (2 × 1012-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH‡ = −16.6 kcal/mol). The presence of Mg2+ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼106-fold. Transfer to dimethyl sulfoxide accelerated PPi hydrolysis by reducing the enthalpy of activation. Mg2+ increased the rate of PPi hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation. PMID:21460215

A Kinetic Study of DDGS Hemicellulose Acid Hydrolysis and NMR Characterization of DDGS Hydrolysate.

PubMed

Chen, Hanchi; Liu, Shijie

2015-09-01

Liquid hot water (LHW) extraction was used as a pretreatment method to separate the hemicellulose fraction from dried distiller's grain with solubles (DDGS) into liquid phase. Acid hydrolysis using 3.264 % w/w sulfuric acid at 130 °C was performed to convert polysaccharides in LHW extract to monosaccharides. The structure characterization of DDGS in anomeric carbon region based on proton NMR and heteronuclear single quantum coherence (HSQC) during acid hydrolysis was studied in this work. It reveals that the sugar units in DDGS hemicelluloses are constructed with (1-4)-β-D-xylopyranose and α-L-arabinofuranosyl residues. A kinetic model is included to explain the changing concentration of monomer, oligomer, and sugar units. The model was further tested based on the changing concentration of five carbon sugar units during hydrolysis.

Development of melamine modified urea formaldehyde resins based o nstrong acidic pH catalyzed urea formaldehyde polymer

Treesearch

Chung-Yun Hse

2009-01-01

To upgrade the performance of urea-formaldehyde (UF) resin bonded particleboards, melamine modified urea-formaldehyde (MUF) resins based on strong acidic pH catalyzed UF polymers were investigated. The study was conducted in a series of two experiments: 1) formulation of MUF resins based on a UF polymer catalyzed with strong acidic pH and 2) determination of the...

Acid hydrolysis of Jerusalem artichoke for ethanol fermentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, K.; Hamdy, M.K.

1986-01-01

An excellent substrate for ethanol production is the Jerusalem artichoke (JA) tuber (Helianthus tuberosus). This crop contains a high level of inulin that can be hydrolyzed mainly to D-fructose and has several distinct advantages as an energy source compared to others. The potential ethanol yield of ca. 4678 L/ha on good agricultural land is equivalent to that obtained from sugar beets and twice that of corn. When JA is to be used for ethanol fermentation by conventional yeast, it is first converted to fermentable sugars by enzymes or acids although various strains of yeast were used for the direct fermentationmore » of JA extracts. Fleming and GrootWassink compared various acids (hydrochloric, sulfuric, citric, and phosphoric) and strong cation exchange resin for their effectiveness on inulin hydrolysis and reported that no differences were noted among the acids or resin in their influence on inulin hydrolysis. Undesirable side reactions were noted during acid hydrolysis leading to the formation of HMF and 2-(2-hydroxy acetyl) furan. The HMF at a level of 0.1% is known to inhibit growth and ethanol fermentation by yeast. In this study the authors established optimal conditions for complete acid-hydrolysis of JA with minimum side reactions and maximum sugar-ethanol production. A material balance for the ethanol production was also determined.« less

Hydrolysis of glyoxal in water-restricted environments: formation of organic aerosol precursors through formic acid catalysis.

PubMed

Hazra, Montu K; Francisco, Joseph S; Sinha, Amitabha

2014-06-12

The hydrolysis of glyoxal involving one to three water molecules and also in the presence of a water molecule and formic acid has been investigated. Our results show that glyoxal-diol is the major product of the hydrolysis and that formic acid, through its ability to facilitate intermolecular hydrogen atom transfer, is considerably more efficient than water as a catalyst in the hydrolysis process. Additionally, once the glyoxal-diol is formed, the barrier for further hydrolysis to form the glyoxal-tetrol is effectively reduced to zero in the presence of a single water and formic acid molecule. There are two important implications arising from these findings. First, the results suggest that under the catalytic influence of formic acid, glyoxal hydrolysis can impact the growth of atmospheric aerosols. As a result of enhanced hydrogen bonding, mediated through their polar OH functional groups, the diol and tetrol products are expected to have significantly lower vapor pressure than the parent glyoxal molecule; hence they can more readily partition into the particle phase and contribute to the growth of secondary organic aerosols. In addition, our findings provide insight into how glyoxal-diol and glyoxal-tetrol might be formed under atmospheric conditions associated with water-restricted environments and strongly suggest that the formation of these precursors for secondary organic aerosol growth is not likely restricted solely to the bulk aqueous phase as is currently assumed.

Growth condition and bacterial community for maximum hydrolysis of suspended organic materials in anaerobic digestion of food waste-recycling wastewater.

PubMed

Kim, Man Deok; Song, Minkyung; Jo, Minho; Shin, Seung Gu; Khim, Jee Hyeong; Hwang, Seokhwan

2010-02-01

This paper reports the effects of changing pH (5-7) and temperature (T, 40-60 degrees C) on the efficiencies of bacterial hydrolysis of suspended organic matter (SOM) in wastewater from food waste recycling (FWR) and the changes in the bacterial community responsible for this hydrolysis. Maximum hydrolysis efficiency (i.e., 50.5% reduction of volatile suspended solids) was predicted to occur at pH 5.7 and T = 44.5 degrees C. Changes in short-chain volatile organic acid profiles and in acidogenic bacterial communities were investigated under these conditions. Propionic and butyric acids concentrations increased rapidly during the first 2 days of incubation. Several band sequences consistent with Clostridium spp. were detected using denaturing gel gradient electrophoresis. Clostridium thermopalmarium and Clostridium novyi seemed to contribute to butyric acid production during the first 1.5 days of acidification of FWR wastewater, and C. thermopalmarium was a major butyric acid producer afterward. C. novyi was an important propionic acid producer. These two species appear to be important contributors to hydrolysis of SOM in the wastewater. Other acidogenic anaerobes, Aeromonas sharmana, Bacillus coagulans, and Pseudomonas plecoglossicida, were also indentified.

Comparison of Enzymatic Hydrolysis and Acid Hydrolysis of Sterol Glycosides from Foods Rich in Δ(7)-Sterols.

PubMed

Münger, Linda H; Jutzi, Sabrina; Lampi, Anna-Maija; Nyström, Laura

2015-08-01

In this study, we present the difference in sterol composition of extracted steryl glycosides (SG) hydrolyzed by either enzymatic or acid hydrolysis. SG were analyzed from foods belonging to the plant families Cucurbitaceae (melon and pumpkin seeds) and Amaranthaceae (amaranth and beetroot), both of which are dominated by Δ(7)-sterols. Released sterols were quantified by gas chromatography with a flame ionization detector (GC-FID) and identified using gas chromatography/mass spectrometry (GC-MS). All Δ(7)-sterols identified (Δ(7)-stigmastenyl, spinasteryl, Δ(7)-campesteryl, Δ(7)-avenasteryl, poriferasta-7,25-dienyl and poriferasta-7,22,25-trienyl glucoside) underwent isomerization under acidic conditions and high temperature. Sterols with an ethylidene or methylidene side chain were found to form multiple artifacts. The artifact sterols coeluted with residues of incompletely isomerized Δ(7)-sterols, or Δ(5)-sterols if present, and could be identified as Δ(8(14))-sterols on the basis of relative retention time, and their MS spectra as trimethylsilyl (TMS) and acetate derivatives. For instance, SG from melon were composed of 66% Δ(7)-stigmastenol when enzymatic hydrolysis was performed, whereas with acid hydrolysis only 8% of Δ(7)-stigmastenol was determined. The artifact of Δ(7)-stigmastenol coeluted with residual non-isomerized spinasterol, demonstrating the high risk of misinterpretation of compositional data obtained after acid hydrolysis. Therefore, the accurate composition of SG from foods containing sterols with a double bond at C-7 can only be obtained by enzymatic hydrolysis or by direct analysis of the intact SG.

Kinetic properties of wild-type and altered recombinant amidases by the use of ion-selective electrode assay method.

PubMed

Martins, S; Karmali, A; Serralheiro, M L

2006-08-15

A novel assay method was investigated for wild-type and recombinant mutant amidases (EC 3.5.1.4) from Pseudomonas aeruginosa by ammonium ion-selective electrode (ISE). The initial velocity is proportional to the enzyme concentration by using the wild-type enzyme. The specific activities of the purified amidase were found to be 88.2 and 104.2 U mg protein(-1) for the linked assay and ISE methods, respectively. The kinetic constants--Vmax, Km, and Kcat--determined by Michaelis-Menten plot were 101.13 U mg protein(-1), 1.12x10(-2) M, and 64.04 s(-1), respectively, for acrylamide as the substrate. On the other hand, the lower limit of detection and range of linearity of enzyme concentration were found to be 10.8 and 10.8 to 500 ng, respectively, for the linked assay method and 15.0 and 15.0 to 15,000 ng, respectively, for the ISE method. Hydroxylamine was found to act as an uncompetitive activator of hydrolysis reaction catalyzed by amidase given that there is an increase in Vmax and Km when acetamide was used as the substrate. However, the effect of hydroxylamine on the hydrolysis reaction was dependent on the type of amidase and substrate involved in the reaction mixture. The degrees of activation (epsilon(a)) of the wild-type and mutant (T103I and C91A) enzymes were found to be 2.54, 12.63, and 4.33, respectively, for acetamide as the substrate. However, hydroxylamine did not activate the reaction catalyzed by wild-type and altered (C91A and W138G) amidases by using acrylamide and acetamide, respectively, as the substrate. The activating effect of hydroxylamine on the hydrolysis of acetamide, acrylamide, and p-nitrophenylacetamide can be explained by the fact that additional formation of ammonium ions occurred due to the transferase activity of amidases. However, the activating effect of hydroxylamine on the hydrolysis of p-nitroacetanilide may be due to a change in conformation of enzyme molecule. Therefore, the use of ISE permitted the study of the kinetic properties of wild-type and mutant amidases because it was possible to measure initial velocity of the enzyme-catalyzed reaction in real time.

Mechanisms of catalytic cleavage of benzyl phenyl ether in aqueous and apolar phases

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Jiayue; Lu, Lu; Zhao, Chen

2014-03-01

Catalytic pathways for the cleavage of ether bonds in benzyl phenyl ether (BPE) in liquid phase using Ni- and zeolite-based catalysts are explored. In the absence of catalysts, the C-O bond is selectively cleaved in water by hydrolysis, forming phenol and benzyl alcohol as intermediates, followed by alkylation. The hydronium ions catalyzing the reactions are provided by the dissociation of water at 523 K. Upon addition of HZSM-5, rates of hydrolysis and alkylation are markedly increased in relation to proton concentrations. In the presence of Ni/SiO 2, the selective hydrogenolysis dominates for cleaving the C aliphatic-O bond. Catalyzed by themore » dual-functional Ni/HZSM-5, hydrogenolysis occurs as the major route rather than hydrolysis (minor route). In apolar undecane, the non-catalytic thermal pyrolysis route dominates. Hydrogenolysis of BPE appears to be the major reaction pathway in undecane in the presence of Ni/SiO 2 or Ni/HZSM-5, almost completely suppressing radical reactions. Density functional theory (DFT) calculations strongly support the proposed C-O bond cleavage mechanisms on BPE in aqueous and apolar phases. These calculations show that BPE is initially protonated and subsequently hydrolyzed in the aqueous phase. Finally, DFT calculations suggest that the radical reactions in non-polar solvents lead to primary benzyl and phenoxy radicals in undecane, which leads to heavier condensation products as long as metals are absent for providing dissociated hydrogen.« less

Fuel alcohol production from agricultural lignocellulosic feedstocks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farina, G.E.; Barrier, J.W.; Forsythe, M.L.

1988-01-01

A two-stage, low-temperature, ambient pressure, acid hydrolysis process that utilizes separate unit operations to convert hemicellulose and cellulose in agricultural residues and crops to fermentable sugars is being developed and tested. Based on the results of the bench-scale tests, an acid hydrolysis experimental plant to demonstrate the concepts of low-temperature acid hydrolysis on a much larger scale was built. Plant tests using corn stover have been conducted for more that a year and conversion efficiences have equaled those achieved in the laboratory. Laboratory tests to determine the potential for low-temperature acid hydrolysis of other feedstocks - including red clover, alfalfa,more » kobe lespedeza, winter rape, and rye grass - are being conducted. Where applicable, process modifications to include extraction before or after hydrolysis also are being studied. This paper describes the experimental plant and process, results obtained in the plant, results of alternative feedstocks testing in the laboratory, and a plan for an integrated system that will produce other fuels, feed, and food from crops grown on marginal land.« less

Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods.

PubMed

Liu, Chao; Li, Bin; Du, Haishun; Lv, Dong; Zhang, Yuedong; Yu, Guang; Mu, Xindong; Peng, Hui

2016-10-20

In this work, nanocellulose was extracted from bleached corncob residue (CCR), an underutilized lignocellulose waste from furfural industry, using four different methods (i.e. sulfuric acid hydrolysis, formic acid (FA) hydrolysis, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and pulp refining, respectively). The self-assembled structure, morphology, dimension, crystallinity, chemical structure and thermal stability of prepared nanocellulose were investigated. FA hydrolysis produced longer cellulose nanocrystals (CNCs) than the one obtained by sulfuric acid hydrolysis, and resulted in high crystallinity and thermal stability due to its preferential degradation of amorphous cellulose and lignin. The cellulose nanofibrils (CNFs) with fine and individualized structure could be isolated by TEMPO-mediated oxidation. In comparison with other nanocellulose products, the intensive pulp refining led to the CNFs with the longest length and the thickest diameter. This comparative study can help to provide an insight into the utilization of CCR as a potential source for nanocellulose production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of Limited Hydrolysis and High-Pressure Homogenization on Functional Properties of Oyster Protein Isolates.

PubMed

Yu, Cuiping; Cha, Yue; Wu, Fan; Xu, Xianbing; Du, Ming

2018-03-22

In this study, the effects of limited hydrolysis and/or high-pressure homogenization (HPH) treatment in acid conditions on the functional properties of oyster protein isolates (OPI) were studied. Protein solubility, surface hydrophobicity, particle size distribution, zeta potential, foaming, and emulsifying properties were evaluated. The results showed that acid treatment led to the dissociation and unfolding of OPI. Subsequent treatment such as limited proteolysis, HPH, and their combination remarkably improved the functional properties of OPI. Acid treatment produced flexible aggregates, as well as reduced particle size and solubility. On the contrary, limited hydrolysis increased the solubility of OPI. Furthermore, HPH enhanced the effectiveness of the above treatments. The emulsifying and foaming properties of acid- or hydrolysis-treated OPI significantly improved. In conclusion, a combination of acid treatment, limited proteolysis, and HPH improved the functional properties of OPI. The improvements in the functional properties of OPI could potentiate the use of oyster protein and its hydrolysates in the food industry.

Molecular characterization and expression analysis of soluble trehalase gene in Aphis glycines, a migratory pest of soybean

USDA-ARS?s Scientific Manuscript database

Trehalase catalyzes the hydrolysis of trehalose, a disaccharide sugar into two glucose molecules. In insects, trehalase plays a crucial role in energy metabolism as well as chitin synthesis. There is no report on the characterization of trehalase gene in aphids which are serious pests of various agr...

Merely two mutations switch a DNA-hydrolyzing deoxyribozyme from heterobimetallic (Zn2+/Mn2+) to monometallic (Zn2+-only) behavior

PubMed Central

Xiao, Ying; Allen, Emily C.

2012-01-01

A deoxyribozyme that hydrolyzes DNA phosphodiester linkages with a requirement for both Zn2+ and Mn2+ is switched by only two nucleotide mutations to require Zn2+ alone, demonstrating that DNA-catalyzed DNA hydrolysis can be achieved using only one metal ion cofactor. PMID:21125108

21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

Code of Federal Regulations, 2014 CFR

2014-04-01

... (EC 3.4.24.28), which catalyze the hydrolysis of peptide bonds in proteins. (b) The ingredient meets... ingredient is used as an enzyme as defined in § 170.3(o)(9) of this chapter to hydrolyze proteins or... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bacterially-derived protease enzyme preparation...

21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

Code of Federal Regulations, 2011 CFR

2011-04-01

...: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this chapter to hydrolyze... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Bacterially-derived protease enzyme preparation... subtilisin (EC 3.4.21.62) and neutral proteinase (EC 3.4.24.28), which catalyze the hydrolysis of peptide...

21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

Code of Federal Regulations, 2013 CFR

2013-04-01

...: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this chapter to hydrolyze... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Bacterially-derived protease enzyme preparation... subtilisin (EC 3.4.21.62) and neutral proteinase (EC 3.4.24.28), which catalyze the hydrolysis of peptide...

21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

Code of Federal Regulations, 2012 CFR

2012-04-01

...: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this chapter to hydrolyze... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Bacterially-derived protease enzyme preparation... subtilisin (EC 3.4.21.62) and neutral proteinase (EC 3.4.24.28), which catalyze the hydrolysis of peptide...

21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

Code of Federal Regulations, 2010 CFR

2010-04-01

...: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this chapter to hydrolyze... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bacterially-derived protease enzyme preparation... subtilisin (EC 3.4.21.62) and neutral proteinase (EC 3.4.24.28), which catalyze the hydrolysis of peptide...

Production of xylitol from corn cob hydrolysate through acid and enzymatic hydrolysis by yeast

NASA Astrophysics Data System (ADS)

Mardawati, Efri; Andoyo, R.; Syukra, K. A.; Kresnowati, MTAP; Bindar, Y.

2018-03-01

The abundance of corn production in Indonesia offers the potential for its application as the raw material for biorefinery process. The hemicellulose content in corn cobs can be considered to be used as a raw material for xylitol production. The purpose of this research was to study the effect of hydrolysis methods for xylitol production and the effect of the hydrolyzed corn cobs to produce xylitol through fermentation. Hydrolysis methods that would be evaluated were acid and enzymatic hydrolysis. The result showed that the xylitol yield of fermented solution using enzymatic hydrolysates was 0.216 g-xylitol/g-xylose, which was higher than the one that used acid hydrolysates, which was 0.100 g-xylitol/g-xylose. Moreover, the specific growth rate of biomass in fermentation using enzymatic hydrolysates was also higher than the one that used acid hydrolysates, 0.039/h compared to 0.0056/h.

Assessment of the hydrolysis process for the determination of okadaic acid-group toxin ester: presence of okadaic acid 7-O-acyl-ester derivates in Spanish shellfish.

PubMed

Villar-González, A; Rodríguez-Velasco, M L; Ben-Gigirey, B; Yasumoto, T; Botana, L M

2008-04-01

The contamination of different types of shellfish by okadaic acid (OA)-group toxin esters is an important problem that presents serious risk for human health. During previous investigations carried out in our laboratory by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), the occurrence of a high percentage of esters in relation to the total OA equivalents has been observed in several shellfish species. The determination of these kinds of toxins using LC/MS or other chemical methods requires a hydrolysis step in order to convert the sterified compounds into the parent toxins, OA, dinophysistoxins-1 (DTX-1) and dinophysistoxins-2 (DTX-2). Most of the hydrolysis procedures are based on an alkaline hydrolysis reaction. However, despite hydrolysis being a critical step within the analysis, it has not been studied in depth up to now. The present paper reports the results obtained after evaluating the hydrolysis process of an esterified form of OA by using a standard of 7-O-acyl ester with palmitoyl as the fatty acid (palOA). Investigations were focused on checking the effectiveness of the hydrolysis for palOA using methanol as solvent standard and matrices matched standards. From the results obtained, no matrix influence on the hydrolysis process was observed and the quantity of palOA converted into OA was always above 80%. The analyses of different Spanish shellfish samples showed percentages of palOA in relation to the total OA esters ranging from 27% to 90%, depending on the shellfish specie.

Part I: RNA hydrolysis catalyzed by imidazole compounds. Part II. Hydrophobic acceleration of reactions and mimics of thiamin-dependent enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kool, E.T.

1988-01-01

Catalysts modeled after the active site groups of the enzyme Ribonuclease A were synthesized and tested for catalysis of the hydrolysis of poly(rU), using a quantitative assay. The most effective of all the catalysts is N,N{prime}-bis-imidazolylmethane, which gave a four-fold rate enhancement as compared to N-methyl-imidazole. The structure/activity relationships are discussed in light of the ribonuclease mechanism. Also examined were reactions catalyzed by the coenzyme thiamine. In an investigation of the effects of restricting conformational freedom, a thiazolium salt was attached in two positions to {beta}-cyclodextrin. Since the catalyst gave about the same rate for tritium exchange from benzaldehyde asmore » singly-attached catalysts, we surmise that any rate enhancement due to the restriction of bond rotations has been lost by forcing the structure into less productive conformations. The benzoin condensation catalyzed by cyanide was also investigated. The reaction was shown to be faster in water than in most organic solvents. Kinetic salt effects and the effects of added {beta}- and {gamma}-cyclodextrin were measured in water; salting-out ions and {gamma}-cyclodextrin increase the rate, while salting-in ions and {beta}-cyclodextrin decrease it. Negative salt effects were observed in formamide, ethylene glycol, and DMSO. All these media effects are discussed in relation to the compact, hydrophobic transition state for the reaction.« less

Preparation of micro-fibrillated cellulose based on sugar palm ijuk (Arenga pinnata) fibres through partial acid hydrolysis

NASA Astrophysics Data System (ADS)

Saputro, A.; Verawati, I.; Ramahdita, G.; Chalid, M.

2017-07-01

The aim of this study was to isolate and characterized micro-fibrillated cellulose (MFC) from sugar palm/ijuk fibre (Arenga pinnata) by partial sulfuric acid hydrolysis. Cellulose fibre was prepared by repeated treatments with 5 wt% sodium hydroxide 2 h at 80°C, followed by bleaching with 1.7 wt% sodium chlorite for 2 h at 80°C in acidic environment under stirring. MFC was prepared by partial hydrolysis with sulfuric acid in various concentrations (30, 40, 50, and 60 % for 45 min at 45 °C) under stirring. Fourier Transform Infrared, Field Emission Scanning Electron Microscope, Thermo Gravimetric Analyzer and X-ray Diffraction characterized cellulose fibre and MFC. FTIR measurements showed that alkaline and bleaching treatments were effective to remove non-cellulosic constituents such as wax, lignin and hemicellulose. FESEM observation revealed conversion into more clear surface and defibrillation of cellulosic fibre after pre-treatments. XRD measurement revealed increase in crystallinity after pre-treatments and acid hydrolysis from 54.4 to 87.8%. Thermal analysis showed that increasing acid concentration reduced thermal stability.

Hydrolysis of proteins with methanesulfonic acid for improved HPLC-ICP-MS determination of seleno-methionine in yeast and nuts.

PubMed

Wrobel, Katarzyna; Kannamkumarath, Sasi S; Wrobel, Kazimierz; Caruso, Joseph A

2003-01-01

In this work, the use of methanesulfonic acid for protein hydrolysis is proposed for evaluation of Se-methionine in yeast, Brazil nuts, and possibly other selenium-rich biological samples. The hydrolysis was carried out by heating the sample with 4 mol L(-1) acid at reflux for 8 h. Two chromatographic techniques (size-exclusion and ion-pairing) coupled with ICP-MS detection were used to compare the release of Se-methionine from proteins by enzymatic (proteinase K, protease XIV) and acid hydrolyses. A more efficient liberation of Se-methionine was observed by acid hydrolysis. For quantification, the sample extracts were introduced onto a C8 Alltima column, and the separation was achieved with a mobile phase containing 5 mmol L(-1) hexanesulfonic acid in citrate buffer (pH 4.5)/methanol (95:5). The results obtained by standard addition showed 816+/-17 micro g g(-1) and 36.2+/-1.5 micro g g(-1) of selenium in the form of Se-methionine in yeast and nuts, respectively (65% and 75% of total selenium).

Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

PubMed

Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

2015-10-07

Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

Hydrofluoric Acid-Based Derivatization Strategy To Profile PARP-1 ADP-Ribosylation by LC-MS/MS.

PubMed

Gagné, Jean-Philippe; Langelier, Marie-France; Pascal, John M; Poirier, Guy G

2018-06-11

Despite significant advances in the development of mass spectrometry-based methods for the identification of protein ADP-ribosylation, current protocols suffer from several drawbacks that preclude their widespread applicability. Given the intrinsic heterogeneous nature of poly(ADP-ribose), a number of strategies have been developed to generate simple derivatives for effective interrogation of protein databases and site-specific localization of the modified residues. Currently, the generation of spectral signatures indicative of ADP-ribosylation rely on chemical or enzymatic conversion of the modification to a single mass increment. Still, limitations arise from the lability of the poly(ADP-ribose) remnant during tandem mass spectrometry, the varying susceptibilities of different ADP-ribose-protein bonds to chemical hydrolysis, or the context dependence of enzyme-catalyzed reactions. Here, we present a chemical-based derivatization method applicable to the confident identification of site-specific ADP-ribosylation by conventional mass spectrometry on any targeted amino acid residue. Using PARP-1 as a model protein, we report that treatment of ADP-ribosylated peptides with hydrofluoric acid generates a specific +132 Da mass signature that corresponds to the decomposition of mono- and poly(ADP-ribosylated) peptides into ribose adducts as a consequence of the cleavage of the phosphorus-oxygen bonds.

Isolation, Solubility, and Characterization of D-Mannitol Esters of 4-Methoxybenzeneboronic Acid.

PubMed

Lopalco, Antonio; Marinaro, William A; Day, Victor W; Stella, Valentino J

2017-02-01

The purpose of this study was to determine the aqueous solubility of a model phenyl boronic acid, 4-methoxybenzeneboronic acid, as a function of pH both in the absence and in the presence of varying D-mannitol concentration. Solid isolated D-mannitol esters were characterized by differential scanning calorimetry, thermogravimetric analysis, powder X-ray diffraction, and single-crystal X-ray studies, and the boronic acid-to-D-mannitol ratio was quantified by HPLC. Hydrolysis of the monoester was studied using UV spectral differences between the monoester and the parent boronic acid. Two D-mannitol esters of 4-methoxybenzeneboronic acid were isolated. The triboronate ester was very insoluble whereas a symmetrical monoboronate monohydrate was also less soluble than the parent. Both esters were crystalline. The monoboronate monohydrate was, however, more soluble than the parent at alkaline pH values due to its lower pKa value (6.53) compared to the parent acid (9.41). Hydrolysis of the monoboronate was extremely fast when even small amount of water was added to dry acetonitrile solutions of the ester. The hydrolysis was buffer concentration dependent and apparent pH sensitive with hydrolysis accelerated by acid. Implications affecting the formulation of future boronic acid drugs are discussed. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Highly thermal-stable and functional cellulose nanocrystals and nanofibrils produced using fully recyclable organic acids

Treesearch

Liheng Chen; Junyong Zhu; Carlos Baez; Peter Kitin; Thomas Elder

2016-01-01

Here we report the production of highly thermal stable and functional cellulose nanocrystals (CNC) and nanofibrils (CNF) by hydrolysis using concentrated organic acids. Due to their low water solubility, these solid organic acids can be easily recovered after hydrolysis reactions through crystallization at a lower or ambient temperature. When dicarboxylic acids were...

Method to upgrade bio-oils to fuel and bio-crude

DOEpatents

Steele, Philip H; Pittman, Jr., Charles U; Ingram, Jr., Leonard L; Gajjela, Sanjeev; Zhang, Zhijun; Bhattacharya, Priyanka

2013-12-10

This invention relates to a method and device to produce esterified, olefinated/esterified, or thermochemolytic reacted bio-oils as fuels. The olefinated/esterified product may be utilized as a biocrude for input to a refinery, either alone or in combination with petroleum crude oils. The bio-oil esterification reaction is catalyzed by addition of alcohol and acid catalyst. The olefination/esterification reaction is catalyzed by addition of resin acid or other heterogeneous catalyst to catalyze olefins added to previously etherified bio-oil; the olefins and alcohol may also be simultaneously combined and catalyzed by addition of resin acid or other heterogeneous catalyst to produce the olefinated/esterified product.

Tuning the acid/metal balance of carbon nanofiber-supported nickel catalysts for hydrolytic hydrogenation of cellulose.

PubMed

Van de Vyver, Stijn; Geboers, Jan; Schutyser, Wouter; Dusselier, Michiel; Eloy, Pierre; Dornez, Emmie; Seo, Jin Won; Courtin, Christophe M; Gaigneaux, Eric M; Jacobs, Pierre A; Sels, Bert F

2012-08-01

Carbon nanofibers (CNFs) are a class of graphitic support materials with considerable potential for catalytic conversion of biomass. Earlier, we demonstrated the hydrolytic hydrogenation of cellulose over reshaped nickel particles attached at the tip of CNFs. The aim of this follow-up study was to find a relationship between the acid/metal balance of the Ni/CNFs and their performance in the catalytic conversion of cellulose. After oxidation and incipient wetness impregnation with Ni, the Ni/CNFs were characterized by various analytical methods. To prepare a selective Ni/CNF catalyst, the influences of the nature of oxidation agent, Ni activation, and Ni loading were investigated. Under the applied reaction conditions, the best result, that is, 76 % yield in hexitols with 69 % sorbitol selectivity at 93 % conversion of cellulose, was obtained on a 7.5 wt % Ni/CNF catalyst prepared by chemical vapor deposition of CH(4) on a Ni/γ-Al(2)O(3) catalyst, followed by oxidation in HNO(3) (twice for 1 h at 383 K), incipient wetness impregnation, and reduction at 773 K under H(2). This preparation method leads to a properly balanced Ni/CNF catalyst in terms of Ni dispersion and hydrogenation capacity on the one hand, and the number of acidic surface-oxygen groups responsible for the acid-catalyzed hydrolysis on the other. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deduced catalytic mechanism of d-amino acid amidase from Ochrobactrum anthropi SV3

PubMed Central

Okazaki, Seiji; Suzuki, Atsuo; Komeda, Hidenobu; Asano, Yasuhisa; Yamane, Takashi

2008-01-01

d-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3 catalyzes d-stereospecific hydrolysis of amino acid amides. DAA has attracted attention as a catalyst for the stereospecific production of d-amino acids, although the mechanism that drives the reaction has not been clear. Previously, the structure of DAA was classified into two types, a substrate-bound state with an ordered Ω loop, and a ground state with a disordered Ω loop. Because the binding of the substrate facilitates ordering, this transition was regarded to be induced fit motion. The angles and distances of hydrogen bonds at Tyr149 Oη, Ser60 Oγ and Lys63 Nζ revealed that Tyr149 Oη donates an H atom to a water molecule in the substrate-bound state, and that Tyr149 Oη donates an H atom to Ser60 Oγ or Lys63 Nζ in the ground state. Taking into consideration the locations of the H atoms of Tyr149 Oη, Ser60 Oγ and Lys63 Nζ, a catalytic mechanism of DAA activity is presented, wherein a shift of an H atom at Tyr149 Oη in the substrate-bound versus the ground state plays a significant role in the reaction. This mechanism explains well why acylation proceeds and deacylation does not proceed in the substrate-bound state. PMID:18421151

Impact of recycled effluent on the hydrolysis during anaerobic digestion of vegetable and flower waste.

PubMed

Lü, F; He, P J; Hao, L P; Shao, L M

2008-01-01

Two trials were established to investigate the effect of recycled effluent on hydrolysis during anaerobic co-digestion of vegetable and flower waste. Trial I evaluated the effect by regulating the flow rate of recycled effluent, while Trial II regulated the ratio of hydrolytic effluent to methanogenic effluent, which were recycled to hydrolysis reactor. Results showed that the recirculation of methanogenic effluent could enhance the buffer capability and operation stability of hydrolysis reactor. Higher recycled flow rate was favourable for microbial anabolism and further promoted hydrolysis. After 9 days of hydrolysis, the cumulative SCOD in the hydrolytic effluent reached 334, 407, 413, 581 mg/g at recycled flow rates of 0.1, 0.5, 1.0, 2.0 m3/(m3 x d), respectively. It was feasible to recycling a mixture of hydrolytic and methanogenic effluent to the hydrolysis reactor. This research showed that partially introducing hydrolytic effluent into the recycled liquid could enhance hydrolysis, while excessive recirculation of hydrolytic effluent will inhibit the hydrolysis. The flow ratio 1:3 of hydrolytic to methanogenic effluent was found to provide the highest hydrolysis efficiency and degradation rate of lignocelluloses-type biomass, among four ratios of 0:1, 1:3, 1:1 and 3:1. Under this regime, after 9 days of hydrolysis, the cumulative TOC and TN in the hydrolytic effluent reached 162 mg/g and 15 mg/g, the removal efficiency of TS, VS, C and cellulose in the solid phase were 60.66%, 62.88%, 58.35% and 49.12%, respectively. The flow ratio affected fermentation pathways, i.e. lower ratio favoured propionic acid fermentation and the generation of lactic acid while higher ratio promoted butyric acid fermentation. IWA Publishing 2008.

Reaction mechanism of the acidic hydrolysis of highly twisted amides: Rate acceleration caused by the twist of the amide bond.

PubMed

Mujika, Jon I; Formoso, Elena; Mercero, Jose M; Lopez, Xabier

2006-08-03

We present an ab initio study of the acid hydrolysis of a highly twisted amide and a planar amide analogue. The aim of these studies is to investigate the effect that the twist of the amide bond has on the reaction barriers and mechanism of acid hydrolysis. Concerted and stepwise mechanisms were investigated using density functional theory and polarizable continuum model calculations. Remarkable differences were observed between the mechanism of twisted and planar amide, due mainly to the preference for N-protonation of the former and O-protonation of the latter. In addition, we were also able to determine that the hydrolytic mechanism of the twisted amide will be pH dependent. Thus, there is a preference for a stepwise mechanism with formation of an intermediate in the acid hydrolysis, whereas the neutral hydrolysis undergoes a concerted-type mechanism. There is a nice agreement between the characterized intermediate and available X-ray data and a good agreement with the kinetically estimated rate acceleration of hydrolysis with respect to analogous undistorted amide compounds. This work, along with previous ab initio calculations, describes a complex and rich chemistry for the hydrolysis of highly twisted amides as a function of pH. The theoretical data provided will allow for a better understanding of the available kinetic data of the rate acceleration of amides upon twisting and the relation of the observed rate acceleration with intrinsic differential reactivity upon loss of amide bond resonance.

Hyper-thermal acid hydrolysis and adsorption treatment of red seaweed, Gelidium amansii for butyric acid production with pH control.

PubMed

Ra, Chae Hun; Jeong, Gwi-Taek; Kim, Sung-Koo

2017-03-01

Optimal hyper-thermal (HT) acid hydrolysis conditions for Gelidium amansii were determined to be 12% (w/v) seaweed slurry content and 144 mM H 2 SO 4 at 150 °C for 10 min. HT acid hydrolysis-treated G. amansii hydrolysates produced low concentrations of inhibitory compounds and adsorption treatment using 3% activated carbon. An adsorption time of 5 min was subsequently used to remove the inhibitory 5-hydroxymethylfurfural from the medium. A final maximum monosaccharide concentration of 44.6 g/L and 79.1% conversion from 56.4 g/L total fermentable monosaccharides with 120 g dw/L G. amansii slurry was obtained from HT acid hydrolysis, enzymatic saccharification, and adsorption treatment. This study demonstrates the potential for butyric acid production from G. amansii hydrolysates under non-pH-controlled as well as pH-controlled fermentation using Clostridium acetobutylicum KCTC 1790. The activated carbon treatment and pH-controlled fermentation showed synergistic effects and produced butyric acid at a concentration of 11.2 g/L after 9 days of fermentation.

Activation of Elongation Factor G by Phosphate Analogues

PubMed Central

Salsi, Enea; Farah, Elie

2016-01-01

EF-G is a universally conserved translational GTPase that promotes the translocation of tRNA and mRNA through the ribosome. EF-G binds to the ribosome in a GTP-bound form and subsequently catalyzes GTP hydrolysis. The contribution of the ribosome-stimulated GTP hydrolysis by EF-G to tRNA/mRNA translocation remains debated. Here, we show that while EF-G•GDP does not stably bind to the ribosome and induce translocation, EFG• GDP in complex with phosphate group analogues BeF3− and AlF4− promotes the translocation of tRNA and mRNA. Furthermore, the rates of mRNA translocation induced by EF-G in the presence of GTP and a non-hydrolysable analogue of GTP, GDP•BeF3−are similar. Our results are consistent with the model suggesting that GTP hydrolysis is not directly coupled to mRNA/tRNA translocation. Hence, GTP binding is required to induce the activated, translocation-competent conformation of EF-G while GTP hydrolysis triggers EF-G release from the ribosome. PMID:27063503

Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery.

PubMed

Tuteja, Narendra; Tuteja, Renu

2004-05-01

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3' to 5' or 5' to 3' direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

Cloning of a Novel Arylamidase Gene from Paracoccus sp. Strain FLN-7 That Hydrolyzes Amide Pesticides

PubMed Central

Zhang, Jun; Yin, Jin-Gang; Hang, Bao-Jian; Cai, Shu; Li, Shun-Peng

2012-01-01

The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with Km and kcat values of 29.5 μM and 49.2 s−1, respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments. PMID:22544249

Insight into the phosphodiesterase mechanism from combined QM/MM free energy simulations.

PubMed

Wong, Kin-Yiu; Gao, Jiali

2011-07-01

Molecular dynamics simulations employing a combined quantum mechanical and molecular mechanical potential have been carried out to elucidate the reaction mechanism of the hydrolysis of a cyclic nucleotide cAMP substrate by phosphodiesterase 4B (PDE4B). PDE4B is a member of the PDE superfamily of enzymes that play crucial roles in cellular signal transduction. We have determined a two-dimensional potential of mean force (PMF) for the coupled phosphoryl bond cleavage and proton transfer through a general acid catalysis mechanism in PDE4B. The results indicate that the ring-opening process takes place through an S(N)2 reaction mechanism, followed by a proton transfer to stabilize the leaving group. The computed free energy of activation for the PDE4B-catalyzed cAMP hydrolysis is about 13 kcal·mol(-1) and an overall reaction free energy is about -17 kcal·mol(-1), both in accord with experimental results. In comparison with the uncatalyzed reaction in water, the enzyme PDE4B provides a strong stabilization of the transition state, lowering the free energy barrier by 14 kcal·mol(-1). We found that the proton transfer from the general acid residue His234 to the O3' oxyanion of the ribosyl leaving group lags behind the nucleophilic attack, resulting in a shallow minimum on the free energy surface. A key contributing factor to transition state stabilization is the elongation of the distance between the divalent metal ions Zn(2+) and Mg(2+) in the active site as the reaction proceeds from the Michaelis complex to the transition state. © 2011 The Authors Journal compilation © 2011 FEBS.

Pretreatment of Sugar Beet Pulp with Dilute Sulfurous Acid is Effective for Multipurpose Usage of Carbohydrates.

PubMed

Kharina, M; Emelyanov, V; Mokshina, N; Ibragimova, N; Gorshkova, T

2016-05-01

Sulfurous acid was used for pretreatment of sugar beet pulp (SBP) in order to achieve high efficiency of both extraction of carbohydrates and subsequent enzymatic hydrolysis of the remaining solids. The main advantage of sulfurous acid usage as pretreatment agent is the possibility of its regeneration. Application of sulfurous acid as hydrolyzing agent in relatively low concentrations (0.6-1.0 %) during a short period of time (10-20 min) and low solid to liquid ratio (1:3, 1:6) allowed effective extraction of carbohydrates from SBP and provided positive effect on subsequent enzymatic hydrolysis. The highest obtained concentration of reducing substances (RS) in hydrolysates was 8.5 %; up to 33.6 % of all carbohydrates present in SBP could be extracted. The major obtained monosaccharides were arabinose and glucose (9.4 and 7.3 g/l, respectively). Pretreatment of SBP with sulfurous acid increased 4.6 times the yield of glucose during subsequent enzymatic hydrolysis of remaining solids with cellulase cocktail, as compared to the untreated SBP. Total yield of glucose during SBP pretreatment and subsequent enzymatic hydrolysis amounted to 89.4 % of the theoretical yield. The approach can be applied directly to the wet SBP. Hydrolysis of sugar beet pulp with sulfurous acid is recommended for obtaining of individual monosaccharides, as well as nutritional media.

Kinetics of acid hydrolysis and reactivity of some antibacterial hydrophilic iron(II) imino-complexes

NASA Astrophysics Data System (ADS)

Shaker, Ali Mohamed; Nassr, Lobna Abdel-Mohsen Ebaid; Adam, Mohamed Shaker Saied; Mohamed, Ibrahim Mohamed Abdelhalim

2015-05-01

Kinetic study of acid hydrolysis of some hydrophilic Fe(II) Schiff base amino acid complexes with antibacterial properties was performed using spectrophotometry. The Schiff base ligands were derived from sodium 2-hydroxybenzaldehyde-5-sulfonate and glycine, L-alanine, L-leucine, L-isoleucine, DL-methionine, DL-serine, or L-phenylalanine. The reaction was studied in aqueous media under conditions of pseudo-first order kinetics. Moreover, the acid hydrolysis was studied at different temperatures and the activation parameters were calculated. The general rate equation was suggested as follows: rate = k obs [Complex], where k obs = k 2 [H+]. The evaluated rate constants and activation parameters are consistent with the hydrophilicity of the investigated complexes.

Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens

PubMed Central

Lassen, Søren F.; Breinholt, Jens; Østergaard, Peter R.; Brugger, Roland; Bischoff, Andrea; Wyss, Markus; Fuglsang, Claus C.

2001-01-01

Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U · mg, of protein−1 and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, 1H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26). PMID:11571175

Current applications and different approaches for microbial l-asparaginase production.

PubMed

Cachumba, Jorge Javier Muso; Antunes, Felipe Antonio Fernandes; Peres, Guilherme Fernando Dias; Brumano, Larissa Pereira; Santos, Júlio César Dos; Da Silva, Silvio Silvério

2016-12-01

l-asparaginase (EC 3.5.1.1) is an enzyme that catalysis mainly the asparagine hydrolysis in l-aspartic acid and ammonium. This enzyme is presented in different organisms, such as microorganisms, vegetal, and some animals, including certain rodent's serum, but not unveiled in humans. It can be used as important chemotherapeutic agent for the treatment of a variety of lymphoproliferative disorders and lymphomas (particularly acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma), and has been a pivotal agent in chemotherapy protocols from around 30 years. Also, other important application is in food industry, by using the properties of this enzyme to reduce acrylamide levels in commercial fried foods, maintaining their characteristics (color, flavor, texture, security, etc.) Actually, l-asparaginase catalyzes the hydrolysis of l-asparagine, not allowing the reaction of reducing sugars with this aminoacid for the generation of acrylamide. Currently, production of l-asparaginase is mainly based in biotechnological production by using some bacteria. However, industrial production also needs research work aiming to obtain better production yields, as well as novel process by applying different microorganisms to increase the range of applications of the produced enzyme. Within this context, this mini-review presents l-asparaginase applications, production by different microorganisms and some limitations, current investigations, as well as some challenges to be achieved for profitable industrial production. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Development of silane-hydrolysate binder for UV-resistant thermal control coatings

NASA Technical Reports Server (NTRS)

Patterson, W. J.

1981-01-01

Detailed characterizaton and formulation studies were performed on a methyltriakoxysilane hydrolysate as a binder for thermal control coatings. The binder was optimized by varying hydrolysis temperature, time, catalyst type, and water concentration. The candidate coating formulations, based on this binder with TiO2 pigment, were optimized via a detailed series of sprayed test panels that included the parameters of binder/pigment ratio, ethanol content, pigment particle size, coating thickness and cure conditions. A typical optimized coating was prepared by acetic acid catalyzed hydrolysis of methyltriethoxysilane with 3.25 mol-equivalents of water over a 24 hour period at room temperature. The resulting hydrolysate was directly mixed with pre-milled TiO2 (12 grams pigment/26 grams binder) to yield a sprayable consistency. Panels were sprayed to result in a nominal cure coating thickness of 2 mils. Cure was affected by air drying for 24 hr at room temperature plus 72 hr at 150 F. These coatings are typically extremely tough and abrasion-resistant, with an absorptance (alpha) of 0.20 and emittance (e) of 0.89. No significant coating damage was observed in the mandrel bend test, even after exposure to thermal cycling from -160 to 160 F. Vacuum exposure of the coatings for 930 hours at 1 equivalent UV sun resulted in no visible degradation and no significant increase in absorptance.

Poly(2-oxazoline)-Antibiotic Conjugates with Penicillins.

PubMed

Schmidt, Martin; Bast, Livia K; Lanfer, Franziska; Richter, Lena; Hennes, Elisabeth; Seymen, Rana; Krumm, Christian; Tiller, Joerg C

2017-09-20

The conjugation of antibiotics with polymers is rarely done, but it might be a promising alternative to low-molecular-weight derivatization. The two penicillins penicillin G (PenG) and penicillin V (PenV) were attached to the end groups of different water-soluble poly(2-oxazoline)s (POx) via their carboxylic acid function. This ester group was shown to be more stable against hydrolysis than the β-lactam ring of the penicillins. The conjugates are still antimicrobially active and up to 20 times more stable against penicillinase catalyzed hydrolysis. The antibiotic activity of the conjugates against Staphylococcus aureus in the presence of penicillinase is up to 350 times higher compared with the free antibiotics. Conjugates with a second antimicrobial function, a dodecyltrimethylammonium group (DDA-X), at the starting end of the PenG and PenV POx conjugates are more antimicrobially active than the conjugates without DDA-X and show high activity in the presence of penicillinase. For example, the conjugates DDA-X-PEtOx-PenG and DDA-X-PEtOx-PenV are 200 to 350 times more active against S. aureus in the presence of penicillinase and almost as effective as the penicillinase stable cloxacollin (Clox) under these conditions. These conjugates show even greater activity compared to cloxacollin without this enzyme present. Further, both conjugates kill Escherichia coli more effectively than PenG and Clox.

Analysis of Nerve Agent Metabolites from Hair for Long-Term Verification of Nerve Agent Exposure

DTIC Science & Technology

2016-05-09

Analysis of Nerve Agent Metabolites from Hair for Long-Term Verification of Nerve Agent Exposure Amanda S. Appel,† John H. McDonough,‡ Joseph D...feasible. In this study, hair was evaluated as a long-term repository of nerve agent hydrolysis products. Pinacolyl methylphosphonic acid (PMPA...hydrolysis product of soman) and isopropyl methylphosphonic acid (IMPA; hydrolysis product of sarin) were extracted from hair samples with N,N

ESTIMATION OF CARBOXYLIC ACID ESTER HYDROLYSIS RATE CONSTANTS

EPA Science Inventory

SPARC chemical reactivity models were extended to calculate hydrolysis rate constants for carboxylic acid esters from molecular structure. The energy differences between the initial state and the transition state for a molecule of interest are factored into internal and external...