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To produce more diversified and more valuable commodities from a biobased economy, I have sought to upgrade ethanol into a wider range of products through cell-free platform technologies. I have built two styles of ethanol valorization system, one through acetaldehyde condensation, another through acetyl-CoA. Also, I have developed methods to power biochemical systems, one that converts electrical power into reducing equivalents and another that can regenerate ATP while co-producing acetone. I developed a 1,3-BDO production system that reached a maximum 0.16 g/L/h productivity, and, with replenishment of feedstock and enzymes, achieved a 1,3-BDO titer of 7.7 g/L. I also developed an isoprenol production system able to reach a maximum 1.0 � 0.05 mM/h productivity and 12.5 � 0.8 mM maximum titer. My ATP generator reached titers of 27 � 6 mM ATP and 59 � 15 mM acetone with maximum ATP synthesis rate of 2.8 � 0.6 mM/hour and acetone of 7.8 � 0.8 mM/hour. These results demonstrate a proof-of-concept blueprint for more diversified bioeconomy for commodity chemical productions.

Background: Chaga mushroom (Inonotus obliquus) is an edible macrofungus used in traditional and folk medicine for the treatment of various gastrointestinal disorders. It has shown potent anti-inflammatory, antioxidant and anticancer effects in several experimental studies, including anti-inflammatory and anticancer effects in colorectal cancer and intestinal inflammation. Whole extract or purified compound ergosterol peroxide from chaga mushroom showed anti-inflammatory mechanism via suppression of NF-κB/iNOS-COX-2 and growth inhibitory mechanism via regulation of apoptosis activation and β-catenin suppression. The diverse inflammatory and carcinogenic agents, like carbon tetrachloride (CCl4), a potent hepatotoxic chemical, cause liver damage by inducing lipid peroxidation and other oxidative damages. Aims: The study aimed to analyze the biochemical, cellular and molecular mechanisms of CCl4 induced chronic liver inflammation and carcinoma, and to analyze the effect of the extract of chaga mushroom on liver inflammation and cancer by virtue of anti-inflammatory mechanisms. Method: Physiological, histological and immunohistochemical analyses of the physiological functions and cellular functions were performed. Biochemical assays were conducted for assessing enzymatic changes in tissues. Molecular simulation and docking studies were performed for the evaluation of the molecular interaction. Results: CCl4-exposed mice exhibited a significant decrease in body weight followed by altered histopathological signatures in the liver. Supplementation of IOAE showed that treatment restored the normal structure of the tissues with large round nuclei in most of the cells. CCl4 caused a steep elevation in the levels of SGOT and SGPT to 2.32- and 1.8-fold as compared to control. The LDH level increased to 447 IU/L in CCl4 treated mice as compared to control (236 IU/L). Analysis of the oxidant enzyme pathway showed that CCl4 reduced the GSH level to 16.5 μM as compared to control (52 μM), and induced the catalase enzyme activity up to 259 U/mL as compared to control (124 U/L). These physiological and biochemical alterations were restored towards normal levels by IOAE administration. Immunohistochemical staining for caspase-3 and p53 showed that CCl4 notably increased their expressions, which were subsequently suppressed by administration of IOAE. The molecular simulation and docking studies using ergosterol peroxide from chaga mushroom with iNOS, COX-2 and TNF-α showed binding energy of -10.5, -8.9 and -9.1 Kcal/mol, respectively. These proteins interacting with ergosterol peroxide exerted an inhibitory effect on these critical proinflammatory signaling proteins. Conclusions: The results point out that IOAE is able to prevent damage of hepatic cells caused by CCl4 in mouse models through anti-inflammatory and growth inhibitory mechanisms, which can be utilized for natural prevention of liver toxicity.

Acetyl xylan esterases are among the key enzymes in the xylan degradation enzyme system. However, acetyl xylan esterases from natural microorganisms have low expression and low enzyme activity and ...

Summary Chiral aromatic alcohols have received much attention due to their widespread use in pharmaceutical industries. In the asymmetric synthesis processes, the excellent performance of alcohol dehydrogenase makes it a good choice for biocatalysts. In this study, a novel and robust medium‐chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyse the asymmetric reduction of aromatic ketones to chiral aromatic alcohols. The reduction of 2‐hydroxyacetophenone (2‐HAP) to (R)‐(‐)‐1‐phenyl‐1,2‐ethanediol ((R)‐PED) was chosen as a template to evaluate its catalytic activity. A specific activity of 110 U mg−1 and a 99% purity of e.e. was achieved in the presence of NADH. An efficient bienzyme‐coupled catalytic system (RhADH and formate dehydrogenase, CpFDH) was established using a two‐phase strategy (dibutyl phthalate and buffer), which highly raised the tolerated substrate concentration (60 g l−1). Besides, a broad range of aromatic ketones were enantioselectively reduced to the corresponding chiral alcohols by this enzyme system with highly enantioselectivity. This system is of the potential to be applied at a commercial scale., A novel and robust medium‐chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyze the asymmetric reduction of aromatic ketones to chiral aromatic alcohols.

The term "single enzyme nanoparticle" (SEN) refers to a chemically or biologically engineered single enzyme molecule. SENs are distinguished from conventional protein nanoparticles in that they can maintain their individual structure and enzymatic activity following modification. Furthermore, SENs exhibit enhanced properties as biopharmaceuticals, such as reduced antigenicity, and increased stability and targetability, which are attributed to the introduction of specific moieties, such as poly(ethylene glycol), carbohydrates, and antibodies. Enzyme replacement therapy (ERT) is a crucial therapeutic option for controlling enzyme-deficiency-related disorders. However, the unfavorable properties of enzymes, including immunogenicity, lack of targetability, and instability, can undermine the clinical significance of ERT. As shown in the cases of Adagen®, Revcovi®, Palynziq®, and Strensiq®, SEN can be an effective technology for overcoming these obstacles. Based on these four licensed products, we expect that additional SENs will be introduced for ERT in the near future. In this article, we review the concepts and features of SENs, as well as their preparation methods. Additionally, we summarize different types of enzyme deficiency disorders and the corresponding therapeutic enzymes. Finally, we focus on the current status of SENs in ERT by reviewing FDA-approved products.

Three β-glucosidases (Pectinex Ultra SP-L, Pectinex Ultra Clear and homemade preparation from Aspergillus niger) were immobilized using different strategies: ionic adsorption on aminated (MANAE)-agarose beads at pH 5, 7, and 9, followed by biocatalysts modification with glutaraldehyde, or on glutaraldehyde pre-activated supports. The pH of the immobilization was altered to allow different enzyme molecule orientations on the support surface. The biocatalysts from Pectinex Ultra SP-L showed the highest thermal and operational stabilities when immobilized on MANAE-agarose-glutaraldehyde at pH 7. The β-glucosidase from Pectinex Ultra Clear and from A. niger produced best results when immobilized on MANAE-agarose beads at pH 5 and 7, respectively, which was later treated with glutaraldehyde. The best immobilization results using pre-activated supports were observed for the enzyme present in Pectinex Ultra SP-L, to which the highest thermal stabilities were obtained. Remarkably, the enzyme from A. niger, immobilized on MANAE-agarose at pH 9 and subsequently treated with glutaraldehyde, produced the highest stabilization (approximately 560 times more stable than soluble enzyme at 60 °C). Results showed that optimal protocol for β-glucosidases immobilizations using the glutaraldehyde chemistry must be individually tested and tailored to each type of enzyme.

Many laboratory courses consist of short and seemingly unconnected individual laboratory exercises. To increase the course consistency, relevance, and student engagement, we have developed a research‐inspired and project‐based module, “From Gene to Structure and Function”. This 2.5‐week full‐day biochemistry and structural biology module covers protein expression, purification, structure solving, and characterization. The module is centered around the flavodoxin‐like protein NrdI, involved in the activation of the bacterial ribonucleotide reductase enzyme system. Through an in‐depth focus on one specific protein, the students will learn the basic laboratory skills needed in order to generate a broader knowledge and breadth within the field. With respect to generic skills, the students report their findings as a scientific article, with the aim to learn to present concise research results and write scientific papers. The current research‐inspired project has the potential of being further developed into a more discovery‐driven project and extended to include other molecular biological techniques or biochemical/biophysical characterizations. In student evaluations, this research‐inspired laboratory course has received very high ratings and been highly appreciated, where the students have gained research experience for more independent future work in the laboratory. © 2019 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 47(3):318–332, 2019.

In this chapter you will be introduced to some of the fundamentals of the process of biological N2 fixation and the nature of the enzyme complex, nitrogenase, which mediates the conversion of N2 to NH3. The process is unique in that, so far as is known, only prokaryotic microbes possess this enzyme system. Despite the cumbersome nature of the nitrogenase enzyme complex, about one half–two thirds of the global supply of fixed N comes through biological N2 fixation. Nitrogen fixation occurs in a wide range of habitats wherever sufficient energy supplies and appropriate environmental conditions exist.

In in vitro experiments, the possibility of using a luminescent system extracted from the luminous fungus Armillaria borealis has been shown to detect and determine the concentration of hispidin. A linear dependence of the luminescent response on the content of hispidin in solutions in the concentration range of 5.4 × 10–5–1.4 × 10–2 µM was detected. The stability of the enzyme system and the high sensitivity of the bioluminescent reaction allows carrying out multiple measurements with the analyte detection limit of 1.3 × 10–11 g. The obtained results show the prospects of creating a rapid bioluminescent method for the analysis of medical substances or extracts from various biological objects for the presence of hispidin.

Methods of proteolytic enzymes isolation from muscle tissue and viscera of pond fish are viewed and their properties are investigated. Efficiency of enzymatic processes is determined by physical and chemical characteristics of the enzymes and, first of all, by their activity and stability. The cathepsins from muscle tissue of most pond fish are acidic and have the highest activity under pH 4.5-5.0. The substrate specificity of this enzyme complex predetermines its possible usage in pickling, particularly for reducing its time and improving organoleptic parameters of the product.

Filamentous fungi are considered as the most efficient producers expressing lignocellulose-degrading enzymes. Penicillium oxalicum strains possess extraordinary fungal lignocellulolytic enzyme systems and can efficiently utilize plant biomass. In recent years, the regulatory aspects of production of hydrolytic enzymes by P. oxalicum have been well established. This review aims to discuss the recent developments for the production of lignocellulolytic enzymes by P. oxalicum. The main cellulolytic transcription factors mediating the complex transcriptional-regulatory network are highlighted. The genome-wide identification of cellulolytic transcription factors, the cascade regulation network for cellulolytic gene expression, and the synergistic and dose-controlled regulation by cellulolytic regulators are discussed. A cellulase regulatory network sensitive to inducers in intracellular environments, the cross-talk of regulation of lignocellulose-degrading enzyme and amylase, and accessory enzymes are also demonstrated. Finally, strategies for the metabolic engineering of P. oxalicum, which show promising applications in the enzymatic hydrolysis for biochemical production, are established.

Most biomanufacturing systems developed for the production of biocommodities are based on whole-cell systems. However, with the advent of innovative technologies, the focus has shifted from whole-cell towards cell-free enzyme system. Since more than a century, researchers are using the cell-free extract containing the required enzymes and their respective cofactors in order to study the fundamental aspects of biological systems, particularly fermentation. Although yeast cell-free enzyme system is known since long ago, it is rarely been studied and characterized in detail. In this review, we hope to describe the major pitfalls encountered by whole-cell system and introduce possible solutions to them using cell-free enzyme systems. We have discussed the glycolytic and fermentative pathways and their regulation at both transcription and translational levels. Moreover, several strategies employed for development of cell-free enzyme system have been described with their potential merits and shortcomings associated with these developmental approaches. We also described in detail the various developmental approaches of synthetic cell-free enzyme system such as compartmentalization, metabolic channeling, protein fusion, and co-immobilization strategies. Additionally, we portrayed the novel cell-free enzyme technologies based on encapsulation and immobilization techniques and their development and commercialization. Through this review, we have presented the basics of cell-free enzyme system, the strategies involved in development and operation, and the advantages over conventional processes. Finally, we have addressed some potential directions for the future development and industrialization of cell-free enzyme system.

The pulp of potato tubers (Solanum tuberosum), topinambur (Helianthus tuberosus) and apples (Malus silvestris) can hydrolyse totally, or almost totally, ester bonds in phenyl, α- and β-naphthyl, benzyl and cinnamyl acetates. In methyl 4-acetoxy-3-metoxybenzoate and methyl 2,5-diacetoxybenzoate as well as testosterone propionate and 16,17-acetonide of 21-acetoxy-6-fluoro-16α,17β,21-trihydroxy-4-pregnen-3,20-dione, the hydrolysis is selective towards the substrate and the bioreagent. In contrast, ethyl benzoate and cinnamate are resistant to hydrolysis.

Monitoring of microwave emission from aqueous solution of horseradish peroxidase (HRP) in the process of the enzyme functioning was carried out. For the monitoring, a system containing HRP, luminol and Н2О2 was employed. Microwave emission measurements were carried out in the 3.4-4.2GHz frequency range using the active and passive modes (active-mode and passive-mode measurements). In the active mode, excitation of the solution in the pulsed electromagnetic field was accomplished. In the passive mode, no excitation was induced. It appears that the passive-mode measurements taken in the course of the peroxidase reaction in the enzyme system have shown a 0.5°С increase of the microwave signal. Upon the active-mode measurements, taken in the same reaction conditions, the forced excitation of the solution has also led to the increase (by 2°С) of the level of the microwave signal – i.e. to its 4-fold enhancement compared to the signal obtained in passive-mode measurements.

Single molecule enzymology provides an opportunity to examine details of enzyme mechanisms that are not distinguishable in biomolecule ensemble studies. Here we report, for the first time, detection of the current produced in an electrocatalytic reaction by a single redox enzyme molecule when it collides with an ultramicroelectrode. The catalytic process provides amplification of the current from electron-transfer events at the catalyst leading to a measurable current. This new methodology monitors turnover of a single enzyme molecule. The methodology might complement existing single molecule techniques, giving further insights into enzymatic mechanisms and filling the gap between fundamental understanding of biocatalytic processes and their potential for bioenergy production.

The interactions between enzyme and nanoparticles (NPs) are governed by the key properties of NPs, such as structure, size, surface chemistry, charge, and surface shape. In this report, we compared the effect of oxidized multiwalled carbon nanotubes (OXWNT) and irradiated multiwalled carbon nanotubes (IRWNT) on the enzymatic activity of PchPipA. Both OXWNT and IRWNT decreased the biocatalytic activity of PchPipA to some extent when they were added in the reaction system, while OXWNT exhibited higher inhibition of the activity of PchPipA than IRWNT. These results suggested that the water solubility may be another property that can affect the interaction of bio-macromolecular products and nanoparticles.

Single enzyme molecule assays were performed on E. coli β-galactosidase using a capillary electrophoresis-based protocol. Assays were performed using double incubations and two substrates, resorufin-β-D-galactoside and DDAO-β-D-galactoside, simultaneously. The variation between individual enzyme molecules in the ratio of product peak areas for the two different substrates used was indistinguishable from the variation in peak areas of the replicate incubations for a given enzyme molecule. This suggests that the enzyme is not heterogeneous with respect to its relative activity with the two different substrates used.

A simple, highly efficient, and economical biphasic cell-free system was developed for biocatalytic reduction of prochiral aromatic ketones to furnish enantiopure alcohols. This system is characterized by using endogenous enzymes of the cell-free extract to form enzyme-coupled NADPH recycling system. Besides, it offered much higher productivity than whole cells and greatly simplified the preparation process of biocatalysts in comparison with isolated enzymes. Various prochiral aromatic ketones, especially a-substituted acetophenone derivatives, were reduced to chiral alcohols with excellent enantiomeric excess (ee) and moderate to good yield by this cell-free system. 2010 Published by Elsevier Ltd.

Summarized recent data on the polymorphism of the xenobiotic metabolism and detoxification enzyme (XMDE) system in humans are presented. The current notions on the molecular mechanisms of metabolic processes and the role of genetic polymorphism are reviewed. The roles of transport proteins and nuclear receptors in the regulation of the activity of the XMDE system are shown. The possibilities of using the polymorphism of the XMDE system as the basis for predicting the risk of oncological diseases are considered. Experimental modeling of different levels of the epoxide synthetase and epoxide hydratase activities revealed their close relationship with the toxic, mutagenic, and carcinogenic actions of polycyclic aromatic hydrocarbons. The data indicating the necessity of considering the physiological factors that could influence xenobiotic metabolism and the development of pathological changes are given.

1 It is probable that the “original” yellow enzyme system of Warburg is responsible for a part of the Michaelis Salomon reaction. 2 There are strong arguments against the functioning of amino-acid oxidase and xanthine oxidase.

The investigations for the determination of NADH-tetrazolium reductase enzyme system activity in ram semen during liquid and cryopreservation in relation with their viability, were carried out. The semen samples with lower motility of spermatozoa after collection, possess lower values of mean cytochemical coefficient for NADH-tetrazolium reductase activity ( р

The hepoxilin pathway was discovered over two decades ago. Products in this pathway are derived through the 12S-lipoxygenase/hepoxilin synthase enzyme system and contain intrinsic biological activity. This activity relates to the reorganization of calcium and potassium ions within the cell, and in inflammation and insulin secretion. Although the natural hepoxilins are chemically unstable, chemical analogues (PBTs) have been synthesized with chemical and biological stability. The PBTs antagonize the natural hepoxilins. The PBTs showed bioavailability, excellent tolerance and stability in vivo. In proof of principle studies in vivo in animal models, the PBTs have shown actions as anti-inflammatory agents, anti-thrombotic agents, anti-cancer agents and anti-diabetic agents. These studies demonstrate the effectiveness of the base structure of the hepoxilin (and PBT) molecule and serve as an excellent framework for the design and preparation of second-generation compounds with improved pharmaceutical properties as therapeutics for the above-mentioned diseases.

Numerous approaches to creating protein multilayers have been described for a variety of applications. In this work, the affinity of the enzyme pectinase towards a natural polysaccharide alginate has been exploited to create affinity directed enzyme multilayers on alginate beads. The biocatalyst has been characterized in terms of catalytic efficiency and kinetic parameters. Atomic force microscopy provided a good insight into multilayer formation at the molecular level. It was seen that enzyme layering was not uniform all over the bead. Thus, achieving high catalytic efficiency was an ‘average performance’ of the enzyme molecule, which was part of different layers. The results show that it is possible to create layered biocatalyst designs without using expensive lectins/antibodies for constructing affinity layers.

The objective of this study was to measure the effects of cysteine addition and heat treatment on plasmin (PL) activity and plasminogen (PG) activation in reconstituted non-fat dry milk (NFDM). Cysteine was added to a final concentration of 0.16 mg·mL−1 to raw, defatted milk and pasteurized at 72 °C for 15 s (low heat, LH) or 100 °C for 30 s (medium heat, MH) prior to spray drying. Both MH fluid milk and NFDM had no PL activity or PG activation, regardless of cysteine addition. For both LH fluid milk and NFDM, there was a significant decrease in PL activity upon cysteine addition (P < 0.0001 for both), as well as in PG activation (P = 0.0003 for fluid milk, P < 0.0001 for NFDM), likely due to the polymerization of PG or binding of PL and PG with other milk proteins. Upon storage of reconstituted powders at 21 °C and 4 °C, PL activity increased with a complimentary decrease in PG-derived PL activity in LH samples, though these changes slowed in samples with added cysteine. Urea-PAGE showed proteolysis of β-casein consistent with PL activities in LH samples. No PL or PG-derived PL activities or proteolysis were observed in stored MH samples. These results suggest that the addition of cysteine prior to the pasteurization and manufacture of LH-NFDM significantly decreases the potential for proteolysis during storage of reconstituted powder caused by the PL enzyme system.

Despite the fact that carbon-phosphorus lyase activity was first documented more than 50 years ago, we are yet to completely understand the details of how this enzyme system functions or what it looks like. In this issue of Structure, Yang et al. (2016) now provide a step forward with a view of how PhnK fits into the bigger picture of carbon-phosphorus lyase.

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ1,4-BQTC-1,4-BQ.

The xylanolytic enzyme system was examined in Bifidobacterium adolescentis , B. infantis and B. bifidum to determine their ability to utilize xylooligosaccharides (XOSs). All these species produced only xylosidase and arabinosidase, and no xylanase, α-glucuronidase and acetyl xylan esterase were found. The optimal activity of β- d -xylosidase from B. adolescentis was at pH 5.6 and 45 °C, and α- l -arabinoside was at pH 5.0 and 40 °C. The degradation products of cell-free extracts and the growth rate of B. adolescentis were tested over XOSs and XOSs de-branched by recombinant α-glucuronidase. The results showed that de-branching α-glucuronidase increased the production of xylose and the total cell density by 10%, and accelerated the growth of B. adolescentis by 20%.

Fatty acid synthase (E.C. 2.3.1.85; FASN) is a multifunctional enzyme system that catalyzes the formation of fatty acids from acetyl-CoA, malonyl-CoA, and NADPH and plays a central role in lipid biosynthesis. Two classes of FASN exist: FASN I in animals and fungi, and FASN II in plants and prokaryotes. Animal FASN I is a homodimeric protein found in the cytosol of lipogenic tissues such as the liver and brain. Many human carcinomas exhibit elevated levels of FASN I, though the benefit to cancer cells is still unclear. Inhibition of FASN I selectively effects apoptosis in cancer cells, and the role of FASN I in chemotherapy is a growing area of research with the use of natural products and small molecule inhibitors.

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

3-Acetylpyridine-adenine dinucleotide, reduced form (AcPdADH) is able to act as an alternative reductant in glutamate-synthase-catalysed glutamate synthesis. In the AcPdADH-dependent reaction, kcat and Km values for the other substrates are fourfold lower than those for the NADH-dependent reaction, and Km for AcPdADH is about 3 μM. AcPdADH acts as a competitive inhibitor with respect to NADH in NADH-dependent glutamate synthesis, with a Ki of 1 fμM. Glutamate synthase catalyses NADH-dependent reduction of AcPdAD+. This appears to proceed by a substituted-enzyme (ping-pong) mechanism, with competitive substrate inhibition by NADH at high levels. The Km value for this reaction are 1.4 μM for NADH and 14 μM for ACPdAD+ and kcat, is 51 s−; K1 for NADH is about 10 μM. The latter findings suggest that NADH is capable of reducing the enzyme molecule in the absence of other substrates and that a reduced form of the enzyme can exist in the absence of bound NADH.

Streptomyces K15 possesses a set of exocellular and cell-bound D-alanyl-D-alanine carboxypeptidases. Four of them have been isolated to the stage where each enzyme preparation contains on single penicillin-binding protein. The exocellular 54000-Mr enzyme is extremely sensitive to benzylpenicillin and performs low transpeptidase activity on the carbonyl-donor/amino-acceptor tetrapeptide ACLLys(Gly)-DAla-DAla. The exocellular 40 000-Mr enzyme and the two lysozyme-releasable 40 000-Mr and 38 000-Mr enzymes are moderately sensitive to benzylpenicillin and have a high propensity to catalyse dimer formation from the aforementioned tetrapeptide monomer.

The Nobel Prize in Chemistry for 2004 is shared by Aaron Ciechanover, Avram Hershko and Irwin Rose, who made fundamental discoveries concerning how cells regulate the breakdown of cellular proteins with extreme specificity. The three biochemists discovered ubiquitin-mediated proteolysis, a process where an enzyme system tags unwanted proteins with many molecules of a small protein called ubiquitin and then sends then to the proteasome where they are broken down.

By use of a capillary electrophoresis-based procedure, it is possible to measure the activity of individual molecules of beta-galactosidase. Molecules from the crystallized enzyme as well as the original enzyme preparation used to grow the crystals both displayed a range of activity of 20-fold or greater. beta-Galactosidase molecules obtained from two different crystals had indistinguishable activity distributions of 31,600 +/- 1100 and 31,800 +/- 1100 reactions min(-1) (enzyme molecule)(-1). This activity was found to be significantly different from that of the enzyme used to grow the crystals, which showed an activity distribution of 38,500 +/- 900 reactions min(-1) (enzyme molecule)(-1).

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.

The determination of the CO2/O2 specificity factor (Ω) is very important to investigate the Rubisco carbon assimilation efficiency. In this paper, it is proved that previous formulae can introduce notable errors into calculating the CO2/O2 specificity factor (Ω) because CO2 and O2 are both the substrates and the mutually competitive inhibitors for Rubisco. A simple integrated equation is proposed to calculate the CO2/O2 specificity factor (Ω). On the other hand, previous multi-step procedures, including the manipulation of radioisotope (14C and/or 3H) and the chromatographic separation of the products, are inconvenient and may cause much random error. An improved assay procedure is presented therefore, which includes the spectrophotometric measurement of 3-phosphoglycerate-dependent NADH oxidation with a coupled enzyme system.

Encapsulation within calcium alginate gel capsules was used to produce a coimmobilized enzyme system. Glucose oxidase (GOD) and catalase (CAT) were chosen as model enzymes. The same values of Vmax and Km app for the GOD encapsulated system and for the GOD-CAT coencapsulated system were calculated. When gel beads and capsules were compared, the same catalyst deactivation sequence for the two enzymes was observed. However, when capsules were employed as immobilization support, GOD efficiencies were higher than for the gel beads. These results were explained in terms of the structure of the capsules.

Three experiments were conducted to determine the effects of a sorghum-specific enzyme system, derived from an Aspergillus niger and Bacillus subtilis fermentation extract (carbohydrase activity of 1,650 α-amylase units and cellulase activity of 30 fibrinolytic units/mL), on growth performance of finishing pigs. In Exp. 1,192 pigs (average initial BW of 46.1 kg) were fed sorghum-based diets without or with 360 mL of enzyme system per ton of sorghum in a 78 d growth assay. For d 0 to 39, gain/feed was improved (p 0.15). For d 39 to 78 and overall (d 0 to 78), ADG, gain/feed, and digestibilities of DM and N were not affected (p>0.13) by enzyme supplementation. Backfat thickness, fat-free lean index, and scores for stomach keratinization and ulcers also were not affected (p>0.15) by the dietary treatments. In Exp. 2,168 pigs (average initial BW of 58.4 kg) were fed diets without or with 150, 300, or 450 mL/ton of the same enzyme system used in Exp. 1. Adding as much as 450 mL enzyme system / ton of sorghum did not affect (p>0.15) ADG or gain/feed for d 0 to 29 of the growth assay. However, during d 29 to 63, ADG increased by 11% (linear effect, p 0.14). Carcass characteristics (dressing percentage, backfat thickness, and fat free lean index) also were not affected (p>0.20) by addition of the enzyme system. In Exp. 3,176 pigs (average initial BW of 46.7 kg) were fed diets without or with 450, 900, or 1,350 mL/ton of the same enzyme system used in Exp. 1 and 2 in a 71 d growth assay. Adding up to 1,350 mL/ton of enzyme had no effects (p>0.15) on ADG, gain/feed, digestibilities of DM and N, and carcass characteristics (dressing percentage, backfat thickness, and fat-free lean index). In conclusion, finishing pigs fed diets with a sorghum-specific enzyme system showed some positive trends for improved growth performance, but those effects were not large and (or) consistent. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 1 : 70-76)

An enzyme system organized in a flow device was used to mimic a reversible Controlled NOT (CNOT) gate with two input and two output signals. Reversible conversion of NAD(+) and NADH cofactors was used to perform a XOR logic operation, while biocatalytic hydrolysis of p-nitrophenyl phosphate resulted in an Identity operation working in parallel. The first biomolecular realization of a CNOT gate is promising for integration into complex biomolecular networks and future biosensor/biomedical applications.

General mechanism of transglycosylation reaction by glycosidases contains branched paths to form and destroy the glycosylated intermediate. The probabilistic model was applied for the simulation and analysis of the transglycosylation mechanism. The model is composed of a single enzyme molecule and finite amounts of substrates and water molecules mimicking the possible smallest enzyme-catalyzed reaction system in a microcompartment. Using random numbers and probabilities, progress of distribution of reactants and products can be simulated and predicted with minimum adjustable parameters. Experimental data of beta-xylosidase and beta-glucosidase reactions were quantitatively analyzed with the simple scheme. Since the algorithm and simulation procedures are simple, the model is applicable to related complicated enzyme mechanisms containing many branched reaction paths.

The study of redox-active systems often requires the maintenance of anaerobic conditions. The glucose oxidase system has often been used to maintain anaerobic conditions, but it has some drawbacks, such as the production of H(2)O(2) and limitations on stability. Protocatechuate dioxygenase from Burkholderia cepacia and the substrate, protocatechuate, constitute an alternate effective oxygen-scrubbing system that can be used in a wide variety of biochemical experiments. We have shown its suitability for maintaining rigorous anaerobic environments in solutions of pH 6-9, at temperatures from 4 to 35 degrees C, and for periods of time up to 15 months. The enzyme system was shown to be stable under these conditions and effective for maintaining anaerobic conditions in titrations of FAD. It is also suitable for scrubbing various types of apparatus such as stopped-flow instruments for anaerobic experiments.

In this paper, derivation of conservation relationships for metabolic networks has been discussed. Using computer algebra technique, all conservation relationships for the closed three enzyme system of creatine kinase-hexokinase-glucose-6-phosphate dehydrogenase are derived:Creatinephosphate+ADP@?creatinekinasecreatine+ATPATP+D-Glucose@?hexokinaseD-Glucose6-phosphate+ADPD-Glucose6-phosphate+NADP^+@?G6P-DHD-Gluconate6-phosphate+NADPHThis method is also effective for more complex enzymatic systems such as branched or cyclic pathways.

TDP-3,6-dideoxy-β- l - arabino- hexopyranose (TDP-β- l -ascarylose) has been produced on a 200 mg-scale from TDP-3-deoxy-α- d -glucose by the l -rhamnose synthesising enzyme system which was isolated from Salmonella entericum LT2.

The present study examined whether rifampin attenuated glutathione (GSH) depletion by acetaminophen reactive metabolites generated in the in vitro P450 enzyme system prepared from mouse liver and the possible mechanism involved in this effect. The results showed that GSH concentration was decreased concentration-dependently by acetaminophen in the in vitro P450 enzyme system. Rifampin significantly attenuated acetaminophen-mediated GSH depletion in a concentration-dependent manner. The concentration-response curve for GSH depletion of acetaminophen was shifted to the right in a parallel fashion in the presence of rifampin at the concentration of 3.2 × 10-5 M, which appeared to result from the competitive binding of rifampin to acetaminophen metabolites. Cytochrome c was markedly reduced by acetaminophen metabolites in this enzyme system, and GSH concentration-dependently increased the cytochrome c reduction by acetaminophen metabolites. These findings suggested that cytochrome c was reduced by the GSH conjugate of acetaminophen metabolites rather than by acetaminophen-derived superoxide anion (02.-) and other unbound free radicals. Rifampin was shown to possess an effect similar to that of GSH. It is concluded that the decrease in GSH depletion by rifampin is most likely attributable to the binding of rifampin to the acetaminophen toxic species, and the increase in cytochrome c reduction by rifampin is attributable to the conjugate formed between rifampin and acetaminophen metabolites.

Dynamic behaviour of urea biosensor at quasi-stationary enzyme kinetics is investigated in this article. Mathematical dependencies and digital simulation of non-linear enzyme system at weak inhibition are presented. Forming of uniform scales is proved to be possible for dynamic information parameters.