Enzymes in Bast Fibrous Plant Processing

The program COST Action 847 Textile Quality and Biotechnology (2000–2005) has given an excellent chance to review the possibilities of the research, aiming at development of the industrial application of enzymes for bast fibrous plant degumming and primary processing. The recent advancements in enzymatic processing of bast fibrous plants (flax, hemp, jute, ramie and alike plants) and related textiles are given. The performance of enzymes in degumming, modification of bast fibres, roving, yarn, related fabrics as well as enzymatic bonding of lignocellulosic composites is provided.     

Stereospecificity for nicotinamide nucleotides in enzymatic and chemical hydride transfer reactions

The pyridine nucleotide (NAD and NADP)-linked enzymes are a large class of enzymes constituting approximately 17% of all classified enzymes. When these enzymes catalyze their reactions, the hydride transfer between the substrate and the reaction site (i.e., C-4 of the nicotinamide/dihydronicotinamide ring) of the coenzyme takes place in a stereospecific manner. Thus, in the reaction of oxidation of the reduced coenzyme, one group of enzymes catalyzes the extraction of only the hydrogen having the R configuration at the No. 4 carbon, while the other group catalyzes the removal of only that with the S configuration. Because this aspect of enzyme stereospecificity provides essential information for a given enzyme's reaction mechanism, active site structure, and evolutionary relationship with other enzymes, intensive effort has been made to establish the stereospecificities of as many enzymes as possible. This review presents the compilation of the stereospecificities of these enzymes. Some empirical rules, which are useful but not definitive, in predicting a given enzyme's stereospecificity are also described. In addition, the stereospecificity in enzymatic reactions is compared to the stereo-preference in chemical oxidoreduction of the coenzyme. In order to elucidate the mechanism for the enzyme stereospecificity, the conformations of the coenzyme in free-state and enzyme-bound state are extensively discussed here.     

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.     

Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn's disease in mice

Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing LAB were evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohn's disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing LAB showed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered LAB that produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies.     

Detergents as selective inhibitors and inactivators of enzymes

In order to study the detergent-enzyme interaction and to clarify whether such an interaction produces specific or non-specific effects, we investigated the action of natural and synthetic detergents on enzymatic systems of different levels of complexity (crystalline enzymes, crude homogenates, organ preparations, organisms in toto i.e. rats and germinating seeds). The enzyme-detergent interaction was examined both as a time-independent phenomenon (inhibition) and as a time-dependent phenomenon (inactivation). In in vitro experiments a clear inhibition of pyridine-dependent dehydrogenases by long-chain anionic detergents was found. Cationic detergents have their greatest effect on lipase, LDH, MDH and ICDH from rat liver homogenates. At low concentrations SDS inactivates all the dehydrogenase enzymes studied. With high concentrations (10 mM) of SDS and dodecyltrimethylammonium bromide (C12), there was a sharp and non-specific decrease of enzymatic activities. In the in vivo studies, rats were given detergents to drink; the cationic detergent (C12) was far more effective than SDS with enzymes from both intestine and liver homogenates. SDS and C12 do not seem to interfere with enzyme activities at the beginning of the germination of Pinus pinea and Triticum durum seeds. However a marked reduction of activities does occur at the respective maximum germination times of these seeds. The nonionic detergent is ineffective both as inhibitor and as inactivator.     

Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT

Escherichia coli outer membrane protease OmpT has been characterised as a serine protease based on its inhibitor profile, but serine protease consensus sequences are absent. By site-directed mutagenesis we substituted all conserved serines and histidines. Substitution of His(101) and His(212) by Ala, Asn or Gln resulted in variant enzymes with 0.01 and 9-20% residual enzymatic activity towards a fluorogenic pentapeptide substrate, respectively. The mutations S140A and S201A did not decrease activity, while variants S40A and S99A yielded 0.5 and 0.2% residual activities, respectively. When measured with a dipeptide substrate the variant S40A demonstrated full activity, whereas variant S99A displayed at least 500-fold reduced activity. We conclude that Ser(99) and His(212) are essential active site residues. We propose that OmpT is a novel serine protease with Ser(99) as the active site nucleophile and His(212) as general base.     

糖苷合成酶——— 一类新型的寡糖高效合成工具

寡糖是哺乳动物细胞表面糖蛋白和糖脂以及微生物来源的生理活性物质的要素之一,其应用于医药的巨大潜能至今还没有得到充分体现,主要原因是合成足够于临床使用的寡糖非常困难.传统的化学法和酶法在大规模合成寡糖方面都有一定局限性.近年来,分子生物学技术大大推动了糖苷酶合成寡糖的研究,将糖苷酶催化中心亲核体氨基酸定点突变为非亲核体氨基酸,导致酶的原有水解活性丧失,只催化糖苷键合成反应,寡糖产量最高可达99%,人工产生了一类新酶——糖苷合成酶(glycosynthases),随后又产生了硫代糖苷酶(thioglycoligases)和硫代糖苷合成酶(thioglycosynthases).糖苷合成酶的高通量筛选可用双质粒系统和酵母三杂交系统进行,其活性的进一步改进可通过亲核体氨基酸位点不同氨基酸取代、其他位点氨基酸突变、反应条件优化等方法进行,其区域选择性的改变或增强可通过改变糖基受体分子达到.糖苷合成酶作为一种新型高效的生物催化剂,对寡糖的工业化合成有着重要意义,它的出现对糖生物学的发展必将起到巨大的推动作用.     

R-Factor-Mediated β-Lactamases That Hydrolyze Oxacillin: Evidence for Two Distinct Groups

The enzymatic and molecular properties of 14 oxacillin-hydrolyzing beta-lactamases, all of them R-factor-specified, were studied, and two distinct groups were found. Four of the enzymes had a molecular weight of 24,000, were active against methicillin, and had an electrophoretic mobility of -0.1 cm/h. Eight enzymes had a molecular weight of 45,000, low activity against methicillin, and an electrophoretic mobility of +0.5 cm/h. The remaining two enzymes were similar to those of the second group in being relatively inactive against methicillin, but their molecular weight was lower (42,000) and their electrophoretic mobility was different (-0.1 cm/h). All the enzymes of both groups were sensitive to inhibition by sodium chloride. The two groups were not completely homogeneous in their enzymatic properties; seven possible subtypes could be recognized.     

A generic rate law for surface-active enzymes

Many biochemical reactions are confined to interfaces, such as membranes or cell walls. Despite their importance, no canonical rate laws describing the kinetics of surface-active enzymes exist. Combining the approach chosen by Michaelis and Menten 100 years ago with concepts from surface chemical physics, we here present an approach to derive generic rate laws of enzymatic processes at surfaces. We illustrate this by a simple reversible conversion on a surface to stress key differences to the classical case in solution. The available area function, a concept from surface physics which enters the rate law, covers different models of adsorption and presents a unifying perspective on saturation effects and competition between enzymes. A remarkable implication is the direct dependence of the rate of a given enzyme on all other enzymatic species able to bind at the surface. The generic approach highlights general principles of the kinetics of surface-active enzymes and allows to build consistent mathematical models of more complex pathways involving reactions at interfaces.     

Glycosyl fluorides in enzymatic reactions

Glycosyl fluorides have considerable importance as substrates and inhibitors in enzymatic reactions. Their good combination of stability and reactivity has enabled their use as glycosyl donors with a variety of carbohydrate processing enzymes. Moreover, the installation of fluorine elsewhere on the carbohydrate scaffold commonly modifies the properties of the glycosyl fluoride such that the resultant compounds act as slow substrates or even inhibitors of enzyme action. This review covers the use of glycosyl fluorides as substrates for wild-type and mutant glycosidases and other enzymes that catalyze glycosyl transfer. The use of substituted glycosyl fluorides as inhibitors of enzymes that catalyze glycosyl transfer and as tools for investigation of their mechanism is discussed, including the labeling of active site residues. Synthetic applications in which glycosyl fluorides are used as glycosyl donors in enzymatic transglycosylation reactions for the synthesis of oligo- and polysaccharides are then covered, including the use of mutant glycosidases, the so-called glycosynthases, which are able to catalyze the formation of glycosides without competing hydrolysis. Finally, a short overview of the use of glycosyl fluorides as substrates and inhibitors of phosphorylases and phosphoglucomutase is given.     

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of K_d values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.     

(R)-oxynitrilase-catalysed synthesis of chiral silicon-containing aliphatic (R)-ketone-cyanohydrins

Optically active 2-trimethylsilyl-2-hydroxyl-ethylcyanide was prepared by enzymatic enantioselective transcyanation of acetyltrimethylsilane with acetone cyanohydrin in a biphasic system at 35°C and pH 5. (R)-Oxynitrilase from apple seed meal was the best among all the enzymes explored and diisopropyl ether was the most suitable organic phase. Acetyltrimethylsilane was a better substrate of the enzyme than its carbon analogue. The substrate conversion and product enantiomeric excess of 2-trimethylsilyl-2-hydroxyl-ethylcyanide were >99% and >99%, respectively.     

Temperature-converting factors for enzymes: comparison of methods

Temperature-converting factors for six enzymes, which are determined very often in the clinical laboratory, are given. The determinations were carried out according to the recommendations of the German Society for Clinical Chemistry and of the Committee on Enzymes of the Dutch Society for Clinical Chemistry. These factors are described not only for human sera but also for some batches of commercial test sera and for the pure enzymes. There are two main conclusions. (1) Factors determined according to the German recommendations cannot be used in any simple way to convert enzymatic activities which have been determined according to the Dutch recommendations. (2) Factors determined using commercial test sera cannot be used for converting enzymatic activities of human origin.     

Micellar enzymology

Experimental approaches to modelling the enzymatic function of biological membranes are discussed. Emphasis is given to pseudohomogeneous systems such as proteolipid complexes and enzymes in organic solvents; the latter are solubilized with phospholipids or synthetic surfactants. Methods for producing and studying such micellar systems are considered. The key research problems of micellar enzymology are formulated and its relation to enzyme membranology is discussed. Finally, the new potentialities are noted of applied enzymology (biotechnology) offered by application of a colloidal solution of water in organic solvents as a microheterogeneous medium for enzymatic reactions.     

Development of chequerboard pattern in human foetal skeletal muscle.

The study was undertaken to race the histochemical and electron microscopic development patterns of human myogenesis from the 9th to the 26th week of foetal life. Particular attention was paid to the possibility of appearance of metabolic or structural differences between individual skeletal muscle fibres in early periods of myogenesis. The 9th week of foetal life is the period when primitive myotubes are formed. Irregular distribution of the ATPase (pH 9-4) activity observed at this time is due to differences in the structure of fusing myoblasts. The early myotubes show a high activity of the oxidative enzymes and lack of phosphorylase. Conversion of immature muscle cells into structurally mature fibres begins between the 20th and the 24th week. The latter fibres exhibit a uniform activity of all the studied enzymes and thus resemble the intermediate type of fibres of mature muscles. From about the 26th week on the typical mosaic pattern of the enzymatic activity is observed. All the differences in enzymatic activity which appear in fibers prior to their full morphologicaal maturity result from differences in developmental stages of the fibres at the given moment. The present study also suggests that there are no morphological or metabolic differences between individual humanskeletal muscle fibres prior to their metabolic differentiation into types which occurs after their innervation.     

An enzymatic method for inosine 5'-monophosphate in the femtomole range

A method for measuring inosine 5'-monophosphate (IMP) by enzymatic generation of NADPH is described. Procedures are given for direct fluorometric assay in the nanomole range and indirect measurement with amplification by enzymatic cycling in the pico- and femtomole ranges. The most sensitive procedure represents a nearly 50,000-fold increase in sensitivity over enzymatic methods now available. Specificity of the assay was greatly enhanced by the use of the antibiotic coformycin, a potent inhibitor of adenosine deaminase (EC 3.5.4.4). This enzyme was found to be a major contaminant of one of the necessary enzymes, phosphoglucomutase (EC 2.7.5.1). The use of the method is illustrated by measurements of IMP in single stimulated and control rat muscle fibers.     

Enzymatic Resolution of α‐Methyleneparaconic Acids and Evaluation of their Biological Activity

Both enantiomers of three biologically relevant paraconic acids—MB‐3, methylenolactocin, and C75—were obtained with enantioselectivities up to 99% by kinetic enzymatic resolutions. Good enantiomeric excesses were obtained for MB‐3 and methylenolactocin, using α‐chymotrypsin and aminoacylase as enantiocomplementary enzymes, while C75 was resolved with aminoacylase. They all were evaluated for their antiproliferative, antibacterial, and antifungal activities, showing weak effects and practically no difference between enantiomers in each case. At high concentrations (16–64 µg/mL), (–)‐ C75 acted as an antimicrobial agent against Gram‐positive bacteria. Chirality 27:239–246, 2015. © 2015 Wiley Periodicals, Inc.     

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.     

Pathogen-induced plant proteins

Pathogen-induced plant proteins are classified by their functional characteristics: (a) involvement in plant cell signaling; (b) inhibition of enzymes excreted by the pathogens; (c) stabilization of plant cell walls or ability to trigger apoptosis; (d) enzymatic activity producing lysis of cell walls of pathogenic fungi and bacteria; (e) enzymatic activity in metabolic pathways of phenylpropane and terpene phytoalexins; and (f) ability to affect the pathogens directly, by disturbing the function of their cell membranes or by inactivating their ribosomes. Examples of transgenic plants with increased immunity against pathogens are also provided.     

Inhibition of enzymatic hydrolysis by residual lignins from softwood--study of enzyme binding and inactivation on lignin-rich surface

Lignin-derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin-rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin-bound enzymes. Severe inactivation of the lignin-bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS-PAGE analysis of the lignin-bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat-induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature.