土壤碳水化合物的测定方法及其指示作用

碳水化合物是土壤有机质的重要组成成分,也是土壤中易降解的有机成分之一,对土壤有机质的转化和土壤团聚体的形成有着重要影响.土壤碳水化合物的水解方法主要有硫酸、盐酸和三氟乙酸水解法,其检测方法主要有比色分析法、气相色谱法、液相色谱法和高效阴离子交换色谱 脉冲电流检测法.文中论述了土壤碳水化合物的水解、纯化与检测方法,并重点介绍了气相色谱的衍生方法及各方法的优缺点,简要概述了碳水化合物对土壤有机质变化的指示作用.     

Lactone-ring-cleaving enzymes of microorganisms: their diversity and applications

Microbial lactonohydrolases (lactone-ring-cleaving enzymes) with unique characteristics were found. The Fusarium oxysporum enzyme catalyzes the reversible and stereospecific hydrolysis of aldonate lactones and D-pantolactone (D-PL), and is useful for the optical resolution of racemic PL. The Agrobacterium tumefaciens enzyme hydrolyzes several aromatic lactones, and catalyzes the stereospecific hydrolysis of PL like the Fusarium enzyme, but its selectivity is opposite. The Acinetobacter calcoaceticus enzyme catalyzing the specific hydrolysis of dihydrocoumarin belongs to serine-enzyme family, and is useful for enantioselective hydrolysis of methyl DL-beta-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate. This enzyme also catalyzes the bromination of monochlorodimedon when incubated with H(2)O(2) and dihydrocoumarin.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Mechanistic studies of reaction coupling in Glu-tRNAGln amidotransferase

Organisms lacking Gln-tRNA synthetase produce Gln-tRNA(Gln) from misacylated Glu-tRNA(Gln) through the transamidation activity of Glu-tRNA(Gln) amidotransferase (Glu-AdT). Glu-AdT hydrolyzes Gln to Glu and NH(3), using the latter product to transamidate Glu-tRNA(Gln) in concert with ATP hydrolysis. In the absence of the amido acceptor, Glu-tRNA(Gln), the enzyme has basal glutaminase activity that is unaffected by ATP. However, Glu-tRNA(Gln) activates the glutaminase activity of the enzyme about 10-fold; addition of ATP elicits a further 7-fold increase. These enhanced activities mainly result from increases in k(cat) without significant effects on the K(m) for Gln. To determine if ATP binding is sufficient to induce full activation, we tested a variety of ATP analogues for their ability to stimulate tRNA-dependent glutaminase activity. Despite their binding to Glu-AdT, none of the ATP analogues induced glutaminase activation except ATP-gammaS, which stimulates glutaminase activity to the same level as ATP, but without formation of Gln-tRNA(Gln). ATP-gammaS hydrolysis by Glu-AdT is very low in the absence or presence of Glu-tRNA(Gln) and Gln. In contrast, Glu-tRNA(Gln) stimulates basal ATP hydrolysis slightly, but full activation of ATP hydrolysis requires both Gln and Glu-tRNA(Gln). Simultaneous monitoring of ATP or ATP-gammaS hydrolysis and glutaminase and transamidase activities reveals tight coupling among these activities in the presence of ATP, with all three activities waning in concert when Glu-tRNA(Gln) levels become exhausted. ATP-gammaS stimulates the glutaminase activity to an extent similar to that with ATP, but without concomitant transamidase activity and with a very low level of ATP-gammaS hydrolysis. This uncoupling between ATP-gammaS hydrolysis and glutaminase activities suggests that the activation of glutaminase activity by ATP or ATP-gammaS, together with Glu-tRNA(Gln), results either from an allosteric effect due simply to binding of these analogues to the enzyme or from some structural changes that attend ATP or ATP-gammaS hydrolysis.     

Analysis of DNA damage at the dinucleoside monophosphate level: application to the formamido lesion.

The dinucleoside monophosphates d(TpG), d(TpC), and d(TpT) were X-irradiated in oxygenated solution. In each case the modification of the dinucleoside in which the thymine base is degraded to a formamido remnant was observed as a principal product. The hydrolysis of the phosphoester bond of formamido-modified dinucleosides is much slower than that of the corresponding unmodified dinucleosides. This effect is also observable in the hydrolysis of irradiated DNA, where hydrolysis by nuclease P1 (plus acid phosphatase) generates the modified dinucleosides d(TFpN), TF being the modified thymidine. The total yield of the formamido lesion in all its forms, d(TFpN), exceeds the yield of any other base modification.     

The catalytic serine of meta-cleavage product hydrolases is activated differently for C-O bond cleavage than for C-C bond cleavage

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k(cat) value (6.3 ± 0.5 s(-1)) similar to that of HOPDA (6.5 ± 0.5 s(-1)). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k(cat)/K(m) values were independent of methanol concentration, while both k(cat) and K(m) values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k(obs) values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.     

The effect of microwave irradiation on enzymatic hydrolysis of rice straw

A series of experiments involving microwave irradiation were carried out to evaluate the effect of microwave irradiation on enzymatic hydrolysis of rice straw. Compared with microwave irradiation free hydrolysis, rice straw pretreated by combining microwave irradiation with alkali could increase the initial hydrolysis rate but the hydrolysis yield remained unchanged. When the enzyme solution was treated by microwave irradiation, the initial hydrolysis rate increased slightly, but the yield was decreased remarkably. Its optimal hydrolysis conditions were temperature (45 degrees C), pH (4.8) and enzyme loading (20 mg g(-1) substrate), which was determined by an orthogonal experiment. When intermittent microwave irradiation was used, initial hydrolysis rate was greatly accelerated but the yield was decreased slightly. Its optimal hydrolysis conditions were temperature (50 degrees C), pH (4.8) and enzyme loading (20 mg g(-1) substrate), which was determined by another orthogonal experiment.     

CP/MAS 13C NMR analysis of cellulase treated bleached softwood kraft pulp

Fully bleached softwood kraft pulps were hydrolyzed with cellulase (1,4-(1,3:1,4)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) from Trichoderma reesei. Supra-molecular structural features of cellulose during enzymatic hydrolysis were examined by using CP/MAS 13C NMR spectra in combination with line-fitting analysis. Different types of cellulose allomorphs (cellulose I(alpha), cellulose I(beta), para-crystalline) and amorphous regions were hydrolyzed to a different extent by the enzyme used. Also observed was a rapid initial phase for hydrolysis of regions followed by a slow hydrolysis phase. Cellulose I(alpha), para-crystalline, and non-crystalline regions of cellulose are more susceptible to enzymatic hydrolysis than cellulose I(beta) during the initial phase. After the initial phase, all the regions are then similarly susceptible to enzymatic hydrolysis.     

Structural and mutational studies of organophosphorus hydrolase reveal a cryptic and functional allosteric-binding site

Organophosphorus hydrolase detoxifies a broad range of organophosphate pesticides and the chemical warfare agents (CWAs) sarin and VX. Previously, rational genetic engineering produced OPH variants with 30-fold enhancements in the hydrolysis of CWA and their analogs. One interesting variant (H254R) in which the histidine at position 254 was changed to an arginine showed a 4-fold increase in the hydrolysis of demetonS (VX analog), a 14-fold decrease with paraoxon (an insecticide), and a 183-fold decrease with DFP (sarin analog). The three-dimensional structure of this enzyme at 1.9A resolution with the inhibitor, diethyl 4-methylbenzylphosphonate (EBP), revealed that the inhibitor did not bind at the active site, but bound exclusively into a well-defined surface pocket 12 A away from the active site. This structural feature was accompanied by non-competitive inhibition of paraoxon hydrolysis by EBP with H254R, in contrast to the native enzyme, which showed competitive inhibition. These parallel structure-function characteristics identify a functional, allosteric site on the surface of this enzyme.     

Intracellular hydrolysis of EGTA-esters

It has been established that the hydrolysis of EGTA-acetoxymethylester (AME) by red blood cells is about the tenth of the hydrolysis of acetylthiocholine (Ac-S-Ch). This splitting of AME could be inhibited by about 50% by prostigmine at a concentration of 0.75 X 10(-5) mol/l, while the splitting of Ac-S-Ch was totally inhibited by the same prostigmine concentration. The hydrolysis of AME by the so-called white-ghost preparation was considerably inhibited by prostigmine (KI = 5 X 10(-8) mol/l), and this inhibition proved to be a competitive one. The splitting of AME by membrane-free cytosol fraction could not be inhibited by prostigmine. Human red blood cells do not hydrolyse EGTA-ethylester (EE). This compound decomposes spontaneously at room-temperature, its reaction-product formed in the Hestrin-reaction is unstable, the developed colour gradually turns pale. On the other hand, AME does not hydrolyse spontaneously at room-temperature and the colour-intensity of its Hestrin-reaction does not decrease with time. Using chelator-and dye-indicator esters to reach different concentrations of free chelators and dye-indicators intracellulary (IC), the extracellular hydrolysis of esters has to be taken into account or this external breakdown has to be inhibited.     

Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases

In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.     

Sugar determination in ulvans by a chemical-enzymatic method coupled to high performance anion exchange chromatography

The sugar determination of ulvans, the water-soluble polysaccharides from Ulva sp. and Enteromorpha sp., was optimized by combining partial acid prehydrolysis (2 mol L-1 trifluoroacetic acid, 120°C) with enzymic hydrolysis (with β-D-glucuronidase). The different constitutive sugars (rhamnose, galactose, glucose, xylose, glucuronic acid), released after hydrolysis, were separated by high performance anion-exchange chromatography and determined by pulsed amperometric detection. The ulvanobiouronic acid, β-D-GlcA-(1,4)-L-Rha, which is the main constituent of ulvans was always present after 3 h of trifluoroacetic acid hydrolysis (approx. 2% D.M.) when acid hydrolysis was performed alone but the xylose amount fell to 75% of its maximum value at this time. The optimal duration of 2 mol L⁻¹ trifluoroacetic acid hydrolysis of ulvans (i.e. without any degradation of xylose, rhamnose and glucuronic acid) was 45 min. Additionnal treatment of the partial acid hydrolysate by purified β-D-glucuronidase allowed the hydrolysis of the residual ulvanobiouronic acid in rhamnose and glucuronic acid. High performance anion exchange chromatography coupled to this chemical-enzymic hydrolysis revealed to be a high resolution chromatographic technique for monitoring the hydrolysis of the aldobiouronic acid by β-D-glucuronidase. This method allowed the simultaneous quantitative determination of neutral and acidic sugars and revealed the presence of iduronic acid inulvans. This revised version was published online in September 2006 with corrections to the Cover Date.     

Study of a specific lignin model: γ-oxidation and how it influences the hydrolysis efficiency of alcohol-aldehyde dehydrogenation copolymers

Six coniferyl alcohol-coniferaldehyde dehydrogenation copolymers (DHcoPs) were synthesized in order to determine the influence of an increased number of aldehyde functions on hydrolysis. After heterogeneous hydrolysis using acidic Montmorillonite K10 clay, the DHcoPs were thioacidolyzed and analyzed by gel permeation chromatography (GPC). Comparison of the thioacidolyzed products, with and without the hydrolysis step, showed that there was a greater proportion of condensation reaction in the absence of aldehyde. When the coniferaldehyde content in the initial synthetic mixture was more than 30% (w/w), only a low fraction of condensed products was generated during the K10 clay hydrolysis step. This suggests that condensation pathways are mainly due to the alcohol present in the γ-position in the DHcoPs. Investigation of the reactivity and the potential condensation of aldehyde and alcohol monomers under hydrolysis conditions showed the important conversion of coniferyl alcohol and conversely the stability of coniferaldehyde.     

3-Phosphono-2-imino-4-oxoimidazoline--a competitive substrate of AMP-dephosphorylating enzymes from the cytosol fraction of the rat heart

3-Phosphono-2-imino-1-methyl-4-oxoimidazolidine (PIMOI), AMP and p-nitrophenyl phosphate (pNPP) were dephosphorylated in the presence of rat heart cytosol at 37 degrees C pH 6.3 at the rates of 0.71, 0.45 and 1.07 mumol/mg X h, respectively. When mixed together, these compounds inhibited the hydrolysis of each other, which points to the participation of common enzyme(s) in this process. The inhibitor of 5'-nucleotidase (alpha,beta-methylene)-ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by 30-50%), thus suggesting that acidic phosphatases are responsible for PIMOI and AMP hydrolysis under these conditions (pH 6.3). Phosphocreatine (PCr) and phosphocyclocreatine (PcCr) were stable to hydrolysis by the cytosolic fraction. However, addition of AMP to the medium containing PCr or PcCr resulted in AMP phosphorylation down to ATP due to the effects of these phosphagens and, probably, of microcontaminations of ATP. This was followed by gradual disappearance of PCr or PcCr and by accumulation of Pi as a result of the "ATPase" activity in the cytosol. The hydrolysis of AMP, PIMOI and p-NPP was sensitive to sulfhydryl reagents [5,5'-dithio-bis-(2-nitrobenzoate) and, in part, 2,4-dinitro-fluorobenzene] and fluoride ion. Thus, PIMOI is a competitive substrate of acidic phosphatases in heart cytosol with respect to AMP and p-NPP. This may partly explain the protective effect of PIMOI on ischemic myocardium.     

Apolipoprotein E enhances hepatic lipase-mediated hydrolysis of reconstituted high-density lipoprotein phospholipid and triacylglycerol in an isoform-dependent manner

This study compares the kinetics of hepatic lipase (HL)-mediated phospholipid and triacylglycerol hydrolysis in spherical, reconstituted high-density lipoproteins (rHDL) that contain either apolipoprotein E2 (apoE2), apoE3, apoE4, or apoA-I as the sole apolipoprotein. HL-mediated phospholipid hydrolysis was assessed by incubating various concentrations of rHDL that contained only cholesteryl esters (CE) in their core, (E2/CE)rHDL, (E3/CE)rHDL, (E4/CE)rHDL, and (A-I/CE)rHDL, with a constant amount of HL. The rate of phospholipid hydrolysis was determined as the formation of nonesterified fatty acid mass. HL-mediated triacylglycerol hydrolysis was assessed in rHDL containing CE, unlabeled triacylglycerol, and [(3)H]triacylglycerol in their core, (E2/TG)rHDL, (E3/TG)rHDL, (E4/TG)rHDL, and (A-I/TG)rHDL. Triacylglycerol hydrolysis was determined as the ratio of (3)H-labeled hydrolysis products to (3)H-labeled unhydrolyzed triacylglycerol. The rates of phospholipid hydrolysis in the (E2/CE)rHDL, (E3/CE)rHDL, and (E4/CE)rHDL were significantly greater than that in the (A-I/CE)rHDL. The rates of triacylglycerol hydrolysis were also greater in the (E2/TG)rHDL, (E3/TG)rHDL, and (E4/TG)rHDL compared to the (A-I/TG)rHDL, although to a lesser degree than observed with phospholipid hydrolysis. Furthermore, the rates of both phospholipid and triacylglycerol hydrolyses were greater in the (E2)rHDL than in either the (E3)rHDL or the (E4)rHDL. These results show that apoE increases the rate of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL and that this influence is isoform dependent.     

MALD-MS in the quantitative analysis of peptides and proteins

A modified method of isotope dilution was applied to the quantitative determination of peptides and proteins by MALDI MS at subpicomolar level. The essence of the method consists in the quantitative analysis of the enzymic hydrolysis products rather than the starting compounds. This allows the measurements to be performed at a higher resolution and makes the method independent of the molecular mass of oligopeptides and proteins examined. Fragments obtained by hydrolysis of the same oligopeptide or protein in a known concentration by the same enzyme and labeled with the stable 18O isotope are used as internal standards. The label is introduced by carrying out the hydrolysis in H(2)18O, and the oligopeptide concentration is calculated from the isotope distribution between the labeled and unlabeled hydrolysis products in the mass spectrum. This method was tested in the determination of concentrations of the angiotensinogen (1-14) fragment (oligopeptide), extracellular RNAase from Bacillus amyloliquefaciens (protein) and its protein inhibitor, barstar M. Usefulness of this method in kinetic studies was also demonstrated.     

Changes in activity coefficient gamma(w) of water and the foaming capacity of protein during hydrolysis

The changes in the interaction between food proteins and water and in their surface functional property during enzymatic hydrolysis were investigated. Ovalbumin, a soy protein isolate (SPI), and casein were hydrolyzed with trypsin, and the degree of hydrolysis, water activity a(w), and foaming capacity of each hydrolysate were measured. Ovalbumin showed the minimum value for a(w), and the values for SPI and casein progressively decreased during hydrolysis. Therefore, the activity coefficient of water, gamma(w) (=a(w)/x(w), where x(w) is the mole fraction of water) was obtained to remove the influence of mole change and to examine the interaction of protein hydrolysates with water. In order to calculate x(w) in a sample during protein hydrolysis, a method for roughly estimating the number of moles of the protein hydrolysate in a solution was developed. The strategy was to modify the TNBS (2,4,6-trinitrobenzenesulfonic acid) method and to combine this method with the modified Ellman method and the determination of lysine by an amino acid analyzer. During enzymatic hydrolysis, each protein sample showed a minimum gamma(w) value and maximum foaming capacity.     

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.     

Role of Phosphoinositide Hydrolysis in Astrocyte Volume Regulation

Abstract: Astrocytes exposed to hypoosmotic stress swell and subsequently reduce their size to almost their original volume, a phenomenon called regulatory volume decrease (RVD). We found that during hypoosmotic swelling there was a twofold increase in phosphatidylinositol (PI) hydrolysis. This increase was inhibited by the phosphdipase C inhibitor, U-73122 (10 μM ). Inhibition of PI hydrolysis resulted in blockage of RVD. We also examined whether agents that stimulate PI hydrolysis would enhance RVD. These agents significantly accelerated RVD. The rank order of potency was endothelin (20 n M ) ≥ norepinephrine (100 μM) > endothelin-3 (7 n M ) > thrombin (1 U/ml) ≥ ATP (500 μ M ) > bradykinin (20 μ M ) ≥ carbachol (500 μ M ), as indicated by RVD rate constants. The extent of PI hydrolysis induced by these agents at the beginning of RVD exhibited a logarithmic relationship with the magnitude of RVD enhancement. Also, there was a linear relationship between the rate of PI hydrolysis and RVD rate constants. Our results suggest that stimulated PI hydrolysis is involved in the regulation of cell volume in astrocytes.