Denaturable immobilized enzymatic reaction in a packed-bed reactor with and without substrate inhibitions

Summary An immobilized enzymatic reaction in a packed-bed reactor is investigated in this paper. The thermal denaturation of immobilized enzyme caused by excessive reacting temperature rise is considered. An unsteady state dispersion model is employed to examine the dynamic behaviors of the substrate concentration, temperature and enzyme activity along the reactor. Also included in the present paper is the effect of substrate inhibition which occurs rather frequently in many enzymatic reactions. Comparison of results of the immobilized enzymatic reactions with and without substrate inhibitions are made to show the extent the substrate inhibition affects the enzymatic reaction. Furthermore, the effects of heat reaction and the Peclet number which characterize the reaction and flow behaviors, respectively, on the system considered are analyzed in detail.     

Tannins as gibberellin antagonists

Fourteen chemically defined hydrolyzable tannins and six impure mixtures of either condensed or hydrolyzable tannins were found to inhibit the gibberellin-induced growth of light-grown dwarf pea seedlings. The highest ratio of tannins to gibberellic acid tested (1000: 1 by weight) inhibited from 80 to 95% of the induced growth for all tannins tested except for two monogalloyl glucose tannins which inhibited only 50% of the induced growth. The lowest ratio tested (10: 1) inhibited the induced growth by less than 25% except for the case of terchebin where 50% inhibition was found. The inhibition of gibberellin-induced growth was found to be completely reversed by increasing the amount of gibberellin in three cases tested. Tannins alone did not inhibit endogenous growth of either dwarf or nondwarf pea seedlings. Eight compounds related to tannins, including coumarin, trans-cinnamic acid, and a number of phenolic compounds were also tested as gibberellin antagonists. Most of these compounds showed some inhibition of gibberellin-induced growth, but less than that of the tannins. At the highest ratio (1000: 1) the greatest inhibition was 55%; at the lowest ratio (10: 1) no more than 17% was observed. These compounds did not inhibit endogenous growth, and the inhibition of gibberellin-induced growth could be reversed by increasing the amount of gibberellin in two cases tested.     

Low concentration of condensed tannins from catechu significantly inhibits fatty acid synthase and growth of MCF-7 cells

Tannins exist widely in plants, but because they precipitate proteins, scientists frequently ignore them in search of bioactive components. Catechu, a traditional astringent, is rich in tannins. In this study, we found that condensed tannins from catechu potently inhibited animal fatty acid synthase (FAS). Among them, trimeric condensed tannin showed the most potent inhibition with IC₅₀ of 0.47 μg/ml and it also exhibited strong time-dependent inhibition. Its inhibitory kinetics and reacting sites on FAS were obviously different from the known inhibitors of FAS. Furthermore, condensed tannins were found to suppress the growth of MCF-7 breast cancer cells, and the effect was related to their activity of FAS inhibition. The inhibition of both FAS activity and MCF-7 growth was exhibited by low concentrations of condensed tannins without FAS being precipitated. These results suggest tannins would be a valuable resource of bioactive substances.     

The condensed tannins (proanthocyanidins) in seagrasses

In a survey of 29 species in the 12 seagrass genera, those in the Potamogetonaceae that characteristically have tannin cells in the leaves (Posidonioideae: Posidonia; Cymodoceoideae: Halodule, Syringodium, Cymodocea, Thalassodendron, Amphibolis) contained compounds with the R_f values and color reactions typical of condensed tannins. Species in the Potamogetonaceae that are not characterized by tannin cells in the leaves (Zosteroideae: Zostera, Phyllospadix, Heterozostera) contained compounds with the R_f values associated with condensed tannins but without the typical staining reactions. Two of the three genera in the Hydrocharitaceae (Enhalus, Thalassia) are characterized by tannin cells in the leaves and contain compounds with the R_f values of condensed tannins but only some of the typical staining reactions. The third genus, Halophila, lacks tannin cells in the leaves and contains compounds with the R_f values of condensed tannins without the typical staining reactions. The role of condensed tannins as feeding deterrents because of their protein-binding properties has been well established for many land plants, but their role in seagrass biology has not been assessed fully.     

Chemoenzymatic and microbial dynamic kinetic resolutions

This review tracks a decade of dynamic kinetic resolution developments with a biocatalytic inclination using enzymatic/microbial means for the resolution part followed by the racemization reactions either by means of enzymatic or chemocatalyst. These fast developments are due to the ability of the biocatalysts to significantly reduce the number of synthetic steps which are common for conventional synthesis. Future developments in novel reactions and products of dynamic kinetic resolutions should consider factors that are needed to be extracted at the early synthetic stage to avoid inhibition at scale-up stage have been highlighted.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes

Achievement of efficient enzymatic degradation of cellulose to glucose is one of the main prerequisites and one of the main challenges in the biological conversion of lignocellulosic biomass to liquid fuels and other valuable products. The specific inhibitory interferences by cellobiose and glucose on enzyme-catalyzed cellulose hydrolysis reactions impose significant limitations on the efficiency of lignocellulose conversion — especially at high-biomass dry matter conditions. To provide the base for selecting the optimal reactor conditions, this paper reviews the reaction kinetics, mechanisms, and significance of this product inhibition, notably the cellobiose and glucose inhibition, on enzymatic cellulose hydrolysis. Particular emphasis is put on the distinct complexity of cellulose as a substrate, the multi-enzymatic nature of the cellulolytic degradation, and the particular features of cellulase inhibition mechanisms and kinetics. The data show that new strategies that place the bioreactor design at the center stage are required to alleviate the product inhibition and in turn to enhance the efficiency of enzymatic cellulose hydrolysis. Accomplishment of the enzymatic hydrolysis at medium substrate concentration in separate hydrolysis reactors that allow continuous glucose removal is proposed to be the way forward for obtaining feasible enzymatic degradation in lignocellulose processing.     

Inhibition of the growth of Candida utilis by certain heavy metals

The effect of Zn2+, Mn2+, Cd2+ and Hg2+ ions on the kinetics of growth was studied with Candida utilis. The inhibition of Candida utilis growth by Zn2+ and Mn2+ ions is described by the equation for noncompetitive inhibition of enzymatic reactions which is not the case with Cd2+ and Hg2+ ions. The inhibition constants (Ki) for these metals have been determined.     

短枝木麻黄小枝单宁对其幼苗生长及单宁含量的效应

以短枝木麻黄(Casuarina equisetifolia)小枝中提取的单宁处理其幼苗,探讨其化感作用及其对幼苗单宁含量的影响.结果表明,单宁对木麻黄幼苗的生长有显著抑制作用,且随着处理浓度的升高,抑制作用逐渐增强,处理15 d后,单宁的化感作用对芽长的抑制程度显著高于根长.单宁处理后,幼苗体内单宁含量也发生显著变化,随着处理浓度的升高而逐渐降低,尤其是总酚和可溶性缩合单宁的含量,单宁溶液的浓度与被处理幼苗体内单宁含量间有显著的线性负相关.因此,短枝木麻黄体内的单宁会对同种的幼苗产生化感作用,这种化感作用不仅影响幼苗的生长和发育,也会通过影响幼苗体内次生代谢物质的形成而影响幼苗对食草动物和其他不利因素的抵抗能力.     

The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi-irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.     

Aqueous Leaf Extract of Jatropha gossypiifolia L. (Euphorbiaceae) Inhibits Enzymatic and Biological Actions of Bothrops jararaca Snake Venom

Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A₂ (Lys49 PLA₂). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.     

Microbial degradation of tannins – A current perspective

Tannins are water-soluble polyphenolic compounds having wide prevalence in plants. Hydrolysable and condensed tannins are the two major classes of tannins. These compounds have a range of effects on various organisms – from toxic effects on animals to growth inhibition of microorganisms. Some microbes are, however, resistant to tannins, and have developed various mechanisms and pathways for tannin degradation in their natural milieu. The microbial degradation of condensed tannins is, however, less than hydrolysable tannins in both aerobic and anaerobic environments. A number of microbes have also been isolated from the gastrointestinal tract of animals, which have the ability to break tannin-protein complexes and degrade tannins, especially hydrolysable tannins. Tannase, a key enzyme in the degradation of hydrolysable tannins, is present in a diverse group of microorganisms, including rumen bacteria. This enzyme is being increasingly used in a number of processes. Presently, there is a need for increased understanding of the biodegradation of condensed tannins, particularly in ruminants.     

INHIBITION OF NITRIFICATION BY CLIMAX ECOSYSTEMS. II. ADDITIONAL EVIDENCE AND POSSIBLE ROLE OF TANNINS

The same nine plots were used in this study as in our previous study on inhibition of nitrification (Rice and Pancholy, 1972). These consisted of three stands representing two stages of old field succession and the climax in each of three vegetation types in Oklahoma: tall grass prairie, post oak-blackjack oak forest, and oak-pine forest. Soil samples were analyzed three times during the growing season of 1972 for exchangeable ammonium nitrogen, nitrate, and numbers of Nitrosomonas and Nitrobacter. Results were similar to those obtained during the entire year of 1971. The amount of ammonium nitrogen was lowest in the first successional stage, intermediate in the intermediate successional stage, and highest in the climax. The amount of nitrate was highest in the first successional stage, intermediate in the intermediate successional stage, and lowest in the climax. The numbers of nitrifiers were highest in the first successional stage usually and decreased to a very low number in the climax. These data furnish additional evidence that the nitrifiers are inhibited in the climax so that ammonium nitrogen is not oxidized to nitrate as readily in the climax as in the successional stages. This would aid in the conservation of nitrogen and energy in the climax ecosystem. Some inhibition of nitrification occurred in the intermediate stage of succession also. Previous studies of tannins indicated that these are inhibitory to nitrification, so all important plant species in the intermediate successional stage and the climax were analyzed for total tannin content. A method for extracting and quantifying condensed tannins from soils was developed and the amounts of tannins were determined in each 15-cm level down to 60 cm in the same two plots in each vegetation type. Gallic and ellagic acids, which result from the digestion of hydrolyzable tannins in oak species, were also extracted and quantified in the climax oak-pine forest. All the important herbaceous species, including the grasses, were found to have considerable amounts of condensed tannins. The highest amounts of tannins occurred in the oaks and pine, however. Condensed tannins, hydrolyzable tannins, ellagic acid, gallic acid, digallic acid, and commercial tannic acid (hydrolyzable tannin), in very small concentrations, were all found to completely inhibit nitrification by Nitrosomonas in soil suspensions for 3 weeks, the duration of the tests. Slightly larger concentrations were required to inhibit nitrification by Nitrobacter under similar conditions. The concentrations of tannins, gallic acid, and ellagic acid found in the soil of the research plots were several times higher than the minimum concentrations necessary to completely inhibit nitrification. The inhibition of nitrification was always greater in the climax stand than in the intermediate successional stage in each vegetation type, and the concentration of tannins in the top 15 cm of soil was always higher in the climax stand than in the intermediate successional stage. Moreover, the amounts of tannins calculated to be added to each plot each year are much less than the amounts found in the soil, indicating that the tannins accumulate over a period of time. Thus, it appears that the tannins and tannin derivatives may play a continuous and rather prominent role in the inhibition of nitrification by vegetation.     

Inhibitory action of five tannins on growth induced by several gibberellins

The following tannins, Chinese gallotannin, 1,2,3,4,6-pentagalloyl glucose, chebulinic acid, procyanidin dimers, and procyanidin trimers were tested and found to be antagonists of seven gibberellins (GAs). Each tannin inhibited the growth induced by any of the gibberellins GA(1), GA(3), GA(4), GA(7), GA(9), GA(13), and GA(14) in the dwarf pea assay. Endogenous growth was not affected. The highest ratio of tannin to gibberellin tested (1000:1 by weight) inhibited from 60 to 95% of the induced growth for all tannins and all gibberellins tested. The tannins were particularly inhibitory against GA(4) and GA(14) where a ratio of 10:1 (tannins: GA by weight) resulted in up to 85% growth reduction. Inhibition could be completely reversed by increasing the amount of gibberellin in all combinations studied. The procyanidin dimers and trimers were the first purified components of condensed tannins to be tested in this system and were potent inhibitors particularly against growth induced by GA(4) and GA(14). Inhibition by these compounds along with similar inhibition by previously tested hydrolyzable tannins demonstrates that the effect is general to tannins of all classes.     

A method of isolating nick-translated DNA by subsequent separation on low-melting-temperature agarose

The use of nick translation of cloned DNA segments followed by separation on low-melting-temperature agarose has been used to obtain multiple radiolabeled DNA probes from a single nick-translation procedure. This technique avoids gel matrix inhibition of enzymatic reactions. Examples of the utility of this procedure are presented and the advantages and drawbacks discussed.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack.

Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results in loss of enzymatic activity. Comparative studies with a range of peroxides have shown that this inhibition is concentration, peroxide, and time dependent, with H2O2 less efficient than some peptide peroxides. Enzyme inhibition correlates with loss of both the peroxide and enzyme thiol residues, with a stoichiometry of two thiols lost per peroxide consumed. Blocking the thiol residues prevents reaction with the peroxide. This stoichiometry, the lack of metal-ion dependence, and the absence of electron paramagnetic resonance (EPR)-detectable species, is consistent with a molecular (nonradical) reaction between the active-site thiol of the enzyme and the peroxide. A number of low-molecular-mass compounds including thiols and ascorbate, but not Trolox C, can prevent inhibition by removing the initial peroxide, or species derived from it. In contrast, glutathione reductase and lactate dehydrogenase are poorly inhibited by these peroxides in the absence of added Fe2+-EDTA. The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions. Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target protein can result in selective subsequent damage to other proteins, as evidenced by loss of enzymatic activity, via the formation and subsequent reactions of protein peroxides. These reactions may be important in the development of cellular dysfunction as a result of photo-oxidation.     

Effects of chemically defined tannins on poly (ADP-ribose) glycohydrolase activity.

Three classes of chemically defined tannins, gallotannins, ellagitannins and condensed tannins were examined for their inhibitory activities against purified poly (ADP-ribose) glycohydrolase. Ellagitannins showed higher inhibitory activities than gallotannins. In contrast, condensed tannins, which consist of an epicathechin gallate (ECG) oligomer without a glucose core were not appreciably inhibitory. Kinetic analysis revealed that the inhibition of ellagitannins was competitive with respect to the substrate poly(ADP-ribose), whereas gallotannins exhibited mixed-type inhibition. These results suggest that conjugation with glucose of hexahydroxy-diphenoyl (HHDP) group, which is a unique component of ellagitannins, potentiated the inhibitory activity, and that the structure of ellagitannins may have a functional domain which competes with poly(ADP-ribose) on the poly(ADP-ribose) glycohydrolase molecule.     

Production of glucose syrups in highly concentrated systems

We have investigated the hydrolysis of maltodextrins in a high concentration (up to 70%), by means of enzymatic and acid catalysis. The study revealed that the equilibrium compositions of the catalyzed reactions were kinetically determined by the selectivity of the catalyst, the substrate concentration and the reaction time. A model comprising a set of two kinetic equations was used to describe the hydrolysis and condensation reactions of glucoamylase-catalyzed reactions, even to highly concentrated systems. Increased substrate concentration resulted in the formation of more condensation products. The enzyme inhibition was low and was found to be independent of the substrate concentration.     

Antihypertensive effects of tannins isolated from traditional Chinese herbs as non-specific inhibitors of angiontensin converting enzyme

The tannins are natural polyphenols, able to precipitate water-soluble alkaloids and possess an inhibitory action on the angiotensin converting enzyme (ACE). We identified 18 polyphenolic compounds (tannins) from Chinese herbs and examined the in vitro effects of these tannins on ACE activity, including determination of the 50% inhibitory concentrations (IC50), specificity and mode of inhibition. We also assessed the in vivo inhibitory effect of the tannins on angiotensin I-induced blood pressure elevation in spontaneously hypertensive rats (SHR). Nine tannins with an IC50 <200 microM for ACE inhibitors were identified belonging to three tannin classes: caffeoylquinates, flavan-3-ols and gallotannins. In vitro, we found caffeoylquinates chelate the ACE zinc cofactor. Two of the flavan-3-ols: epigallocatechin-3-O-gallate and epigallocatechin-3-O-methylgallate, and one of gallotannin: 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose were non-specific inhibitors because also reduced the activity of trypsin and chymotrypsin. The ACE inhibition of 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose was also reduced after addition of bovine serum albumin, suggesting a non-specific mode of action. In vivo, 1,2,3,6-tetra-O-galloyl-beta-D-glucose and epigallocatechin-3-O-methylgallate had a strong dose-dependent hypotensive effect reducing the blood pressure significantly in the SHR with infusion of the angiotensin I. These findings indicate that some of the tannins isolated from herbs inhibit ACE activity non-specifically. The ACE inhibitory effect of these tannins may explain the hypotensive effects of some traditional Chinese herbs.     

Effect of tannic acid on Lactobacillus hilgardii analysed by a proteomic approach

AIMS: A contribution towards the elucidation of the mechanisms of tannins on bacteria growth inhibition, with particular focus on the interaction between tannins and bacterial proteins. METHODS AND RESULTS: The interaction between tannic acid (TA) and Lactobacillus hilgardii, a wine spoilage bacterium, was investigated by a combination of physiologic and proteomic approaches. Growing tests were performed on medium supplemented with TA at concentrations ranging from 100 to 1000 mg l(-1) demonstrating the inhibitory effect of TA on the growth rate. Total proteins extracted from cells unexposed and exposed to TA were then analysed by 2D-electrophoresis and significant quantitative variations with a marked decrease of protein intensity upon TA exposure were observed. Most of the proteins, identified by ESI tandem Mass Spectrometry, were metabolic enzymes of different pathways, located in cytoplasm and membrane. CONCLUSIONS: The effects of TA on cells are deduced by the involvement of metabolic enzymes, and functional proteins on the tannin-protein interaction. These results might be related to the altered functions of the cell metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: The possible role of tannins in the inhibition of the bacterial survival and growth in a natural environment such as wine. A similar approach could be applied for evaluating the effects of tannins on food borne and pathogenic bacteria.