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Journal articles on the topic 'OYE Enzymes'
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Williams, Richard E., Deborah A. Rathbone, Nigel S. Scrutton, and Neil C. Bruce. "Biotransformation of Explosives by the Old Yellow Enzyme Family of Flavoproteins." Applied and Environmental Microbiology 70, no. 6 (June 2004): 3566–74. http://dx.doi.org/10.1128/aem.70.6.3566-3574.2004.
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ABSTRACT Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.
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van Dillewijn, Pieter, Rolf-Michael Wittich, Antonio Caballero, and Juan-Luis Ramos. "Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida." Applied and Environmental Microbiology 74, no. 21 (September 12, 2008): 6703–8. http://dx.doi.org/10.1128/aem.00386-08.
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ABSTRACT To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.
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García-Huertas, Paola, Ana María Mejía-Jaramillo, Carlos Renato Machado, Anna Cláudia Guimarães, and Omar Triana-Chávez. "Prostaglandin F2α synthase in Trypanosoma cruzi plays critical roles in oxidative stress and susceptibility to benznidazole." Royal Society Open Science 4, no. 9 (September 2017): 170773. http://dx.doi.org/10.1098/rsos.170773.
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Nifurtimox (Nfx) and benznidazole (Bz) are the current drugs used for the treatment of Chagas disease. The mechanisms of action and resistance to these drugs in this parasite are poorly known. Prostaglandin F2α synthase or old yellow enzyme (OYE), an NAD(P)H flavin oxidoreductase, has been involved in the activation pathway of other trypanocidal drugs such as Nfx; however, its role in the mechanism of action of Bz is uncertain. In this paper, we performed some experiments of functional genomics in the parasite Trypanosoma cruzi with the aim to test the role of this gene in the resistance to Bz. For this, we overexpressed this gene in sensitive parasites and evaluated the resistance level to the drug and other chemical compounds such as hydrogen peroxide, methyl methanesulfonate and gamma radiation. Interestingly, parasites overexpressing OYE showed alteration of enzymes associated with oxidative stress protection such as superoxide dismutase A and trypanothione reductase. Furthermore, transfected parasites were more sensitive to drugs, genetic damage and oxidative stress. Additionally, transfected parasites were less infective than wild-type parasites and they showed higher alteration in mitochondrial membrane potential and cell cycle after treatment with Bz. These results supply essential information to help further the understanding of the mechanism of action of Bz in T. cruzi .
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Tentori, Francesca, Teodora Bavaro, Elisabetta Brenna, Danilo Colombo, Daniela Monti, Riccardo Semproli, and Daniela Ubiali. "Immobilization of Old Yellow Enzymes via Covalent or Coordination Bonds." Catalysts 10, no. 2 (February 20, 2020): 260. http://dx.doi.org/10.3390/catal10020260.
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Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziGTM particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziGTM, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziGTM Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions.
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Hall, Mélanie, Clemens Stueckler, Bernhard Hauer, Rainer Stuermer, Thomas Friedrich, Michael Breuer, Wolfgang Kroutil, and Kurt Faber. "Asymmetric Bioreduction of Activated C=C Bonds UsingZymomonas mobilis NCR Enoate Reductase and Old Yellow Enzymes OYE 1–3 from Yeasts." European Journal of Organic Chemistry 2008, no. 9 (March 2008): 1511–16. http://dx.doi.org/10.1002/ejoc.200701208.
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Odat, Osama, Samer Matta, Hadi Khalil, Sotirios C. Kampranis, Raymond Pfau, Philip N. Tsichlis, and Antonios M. Makris. "Old Yellow Enzymes, Highly Homologous FMN Oxidoreductases with Modulating Roles in Oxidative Stress and Programmed Cell Death in Yeast." Journal of Biological Chemistry 282, no. 49 (September 26, 2007): 36010–23. http://dx.doi.org/10.1074/jbc.m704058200.
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In a genetic screen to identify modifiers of Bax-dependent lethality in yeast, the C terminus of OYE2 was isolated based on its capacity to restore sensitivity to a Bax-resistant yeast mutant strain. Overexpression of full-length OYE2 suppresses Bax lethality in yeast, lowers endogenous reactive oxygen species (ROS), increases resistance to H2O2-induced programmed cell death (PCD), and significantly lowers ROS levels generated by organic prooxidants. Reciprocally, Δoye2 yeast strains are sensitive to prooxidant-induced PCD. Overexpression and knock-out analysis indicate these OYE2 antioxidant activities are opposed by OYE3, a highly homologous heterodimerizing protein, which functions as a prooxidant promoting H2O2-induced PCD in wild type yeast. To exert its effect OYE3 requires the presence of OYE2. Deletion of the 12 C-terminal amino acids and catalytic inactivation of OYE2 by a Y197F mutation enhance significantly survival upon H2O2-induced PCD in wild type cells, but accelerate PCD in Δoye3 cells, implicating the oye2p-oye3p heterodimer for promoting cell death upon oxidative stress. Unexpectedly, a strain with a double knock-out of these genes (Δoye2 oye3) is highly resistant to H2O2-induced PCD, exhibits increased respiratory capacity, and undergoes less cell death during the adaptive response in chronological aging. Simultaneous deletion of OYE2 and other antioxidant genes hyperinduces endogenous levels of ROS, promoting H2O2-induced cell death: in Δoye2 glr1 yeast high levels of oxidized glutathione elicited gross morphological aberrations involving the actin cytoskeleton and defects in organelle partitioning. Altering the ratio of reduced to oxidized glutathione by exogenous addition of GSH fully reversed these alterations. Based on this work, OYE proteins are firmly placed in the signaling network connecting ROS generation, PCD modulation, and cytoskeletal dynamics in yeast.
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Hall, Mélanie, Clemens Stueckler, Bernhard Hauer, Rainer Stuermer, Thomas Friedrich, Michael Breuer, Wolfgang Kroutil, and Kurt Faber. "Asymmetric Bioreduction of Activated C=C Bonds UsingZymomonas mobilis NCR Enoate Reductase and Old Yellow Enzymes OYE 1–3 from Yeasts (Eur. J. Org. Chem. 9/2008)." European Journal of Organic Chemistry 2008, no. 9 (March 2008): 1479. http://dx.doi.org/10.1002/ejoc.200890018.
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Nizam, Shadab, Rajesh Kumar Gazara, Sandhya Verma, Kunal Singh, and Praveen Kumar Verma. "Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding, Organization of Active Site Residues and Stereopreferences." PLoS ONE 9, no. 4 (April 28, 2014): e95989. http://dx.doi.org/10.1371/journal.pone.0095989.
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Memon, Safyan Aman, Kinaan Aamir Khan, and Hammad Naveed. "HECNet: a hierarchical approach to enzyme function classification using a Siamese Triplet Network." Bioinformatics 36, no. 17 (May 25, 2020): 4583–89. http://dx.doi.org/10.1093/bioinformatics/btaa536.
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Abstract Motivation Understanding an enzyme’s function is one of the most crucial problem domains in computational biology. Enzymes are a key component in all organisms and many industrial processes as they help in fighting diseases and speed up essential chemical reactions. They have wide applications and therefore, the discovery of new enzymatic proteins can accelerate biological research and commercial productivity. Biological experiments, to determine an enzyme’s function, are time-consuming and resource expensive. Results In this study, we propose a novel computational approach to predict an enzyme’s function up to the fourth level of the Enzyme Commission (EC) Number. Many studies have attempted to predict an enzyme’s function. Yet, no approach has properly tackled the fourth and final level of the EC number. The fourth level holds great significance as it gives us the most specific information of how an enzyme performs its function. Our method uses innovative deep learning approaches along with an efficient hierarchical classification scheme to predict an enzyme’s precise function. On a dataset of 11 353 enzymes and 402 classes, we achieved a hierarchical accuracy and Macro-F1 score of 91.2% and 81.9%, respectively, on the 4th level. Moreover, our method can be used to predict the function of enzyme isoforms with considerable success. This methodology is broadly applicable for genome-wide prediction that can subsequently lead to automated annotation of enzyme databases and the identification of better/cheaper enzymes for commercial activities. Availability and implementation The web-server can be freely accessed at http://hecnet.cbrlab.org/. Supplementary information Supplementary data are available at Bioinformatics online.
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Cieśla, Joanna. "Metabolic enzymes that bind RNA: yet another level of cellular regulatory network?" Acta Biochimica Polonica 53, no. 1 (January 12, 2006): 11–32. http://dx.doi.org/10.18388/abp.2006_3360.
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Several enzymes that were originally characterized to have one defined function in intermediatory metabolism are now shown to participate in a number of other cellular processes. Multifunctional proteins may be crucial for building of the highly complex networks that maintain the function and structure in the eukaryotic cell possessing a relatively low number of protein-encoding genes. One facet of this phenomenon, on which I will focus in this review, is the interaction of metabolic enzymes with RNA. The list of such enzymes known to be associated with RNA is constantly expanding, but the most intriguing question remains unanswered: are the metabolic enzyme-RNA interactions relevant in the regulation of cell metabolism? It has been proposed that metabolic RNA-binding enzymes participate in general regulatory circuits linking a metabolic function to a regulatory mechanism, similar to the situation of the metabolic enzyme aconitase, which also functions as iron-responsive RNA-binding regulatory element. However, some authors have cautioned that some of such enzymes may merely represent "molecular fossils" of the transition from an RNA to a protein world and that the RNA-binding properties may not have a functional significance. Here I will describe enzymes that have been shown to interact with RNA (in several cases a newly discovered RNA-binding protein has been identified as a well-known metabolic enzyme) and particularly point out those whose ability to interact with RNA seems to have a proven physiological significance. I will also try to depict the molecular switch between an enzyme's metabolic and regulatory functions in cases where such a mechanism has been elucidated. For most of these enzymes relations between their enzymatic functions and RNA metabolism are unclear or seem not to exist. All these enzymes are ancient, as judged by their wide distribution, and participate in fundamental biochemical pathways.
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Molina, M. Asunción, Victoria Gascón-Pérez, Manuel Sánchez-Sánchez, and Rosa M. Blanco. "Sustainable One-Pot Immobilization of Enzymes in/on Metal-Organic Framework Materials." Catalysts 11, no. 8 (August 20, 2021): 1002. http://dx.doi.org/10.3390/catal11081002.
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The industrial use of enzymes generally necessitates their immobilization onto solid supports. The well-known high affinity of enzymes for metal-organic framework (MOF) materials, together with the great versatility of MOFs in terms of structure, composition, functionalization and synthetic approaches, has led the scientific community to develop very different strategies for the immobilization of enzymes in/on MOFs. This review focuses on one of these strategies, namely, the one-pot enzyme immobilization within sustainable MOFs, which is particularly enticing as the resultant biocomposite Enzyme@MOFs have the potential to be: (i) prepared in situ, that is, in just one step; (ii) may be synthesized under sustainable conditions: with water as the sole solvent at room temperature with moderate pHs, etc.; (iii) are able to retain high enzyme loading; (iv) have negligible protein leaching; and (v) give enzymatic activities approaching that given by the corresponding free enzymes. Moreover, this methodology seems to be near-universal, as success has been achieved with different MOFs, with different enzymes and for different applications. So far, the metal ions forming the MOF materials have been chosen according to their low price, low toxicity and, of course, their possibility for generating MOFs at room temperature in water, in order to close the cycle of economic, environmental and energy sustainability in the synthesis, application and disposal life cycle.
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Rinaldo, Serena, Giorgio Giardina, Nicoletta Castiglione, Valentina Stelitano, and Francesca Cutruzzolà. "The catalytic mechanism of Pseudomonas aeruginosa cd1 nitrite reductase." Biochemical Society Transactions 39, no. 1 (January 19, 2011): 195–200. http://dx.doi.org/10.1042/bst0390195.
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The cd1 NiRs (nitrite reductases) are enzymes catalysing the reduction of nitrite to NO (nitric oxide) in the bacterial energy conversion denitrification process. These enzymes contain two distinct redox centres: one covalently bound c-haem, which is reduced by external electron donors, and another peculiar porphyrin, the d1-haem (3,8-dioxo-17-acrylate-porphyrindione), where nitrite is reduced to NO. In the present paper, we summarize the most recent results on the mechanism of nitrite reduction by the cd1 NiR from Pseudomonas aeruginosa. We discuss the essential catalytic features of this enzyme, with special attention to the allosteric regulation of the enzyme's activity and to the mechanism employed to avoid product inhibition, i.e. trapping of the active-site reduced haem by the product NO. These results shed light on the reactivity of cd1 NiRs and assign a central role to the unique d1-haem, present only in this class of enzymes.
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Dhindwal, Sonali, Leticia Gomez-Gil, David B. Neau, Thi Thanh My Pham, Michel Sylvestre, Lindsay D. Eltis, Jeffrey T. Bolin, and Pravindra Kumar. "Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase." Journal of Bacteriology 198, no. 10 (March 7, 2016): 1499–512. http://dx.doi.org/10.1128/jb.00952-15.
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ABSTRACTBiphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400in which a seven-residue segment,335TFNNIRI341, has been replaced by the corresponding segment of BphAEB356,333GINTIRT339, transforms a broader range of PCB congeners than does either BphAELB400or BphAEB356, including 2,6-dichlorobiphenyl, 3,3′-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,3,4′-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9transformed 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl more efficiently than did BphAELB400and BphAEB356. BphAEII9also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparentkcat/Kmof 2.2 ± 0.5 mM−1s−1, versus 0.9 ± 0.5 mM−1s−1for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes.IMPORTANCEBiphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.
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Antoun, G. R., and D. G. Williamson. "Age-dependent changes in the multiple forms of the soluble 17 β-hydroxysteroid dehydrogenase of female rabbit liver." Biochemical Journal 225, no. 2 (January 15, 1985): 391–98. http://dx.doi.org/10.1042/bj2250391.
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The soluble NADP-dependent 17 beta-hydroxysteroid dehydrogenase activity of female rabbit liver increases with the age of the animal, the specific activity of the enzyme in the 56-day-old rabbit being 3 times that of the 28-day-old animal. The increase in activity is accompanied by a change in the molecular heterogeneity of the enzyme. Three forms (enzymes I, II and III) were identified in the liver cytosol of the 56-day-old female rabbit, whereas only one major form (enzyme IIIY) was present in the 28-day-old animal. Peptide maps of the four purified enzymes showed that there were minor differences in structure. The enzyme present in the liver of the 28-day-old rabbit was distinct from the three enzymes of the 56-day-old animal. All of the enzymes exhibited bifunctional activity, having 17 beta-hydroxysteroid dehydrogenase activity towards androgen and oestrogen substrates and 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. The differences in substrate specificity of the enzymes paralleled their differences in structure. The data suggest that one enzyme (enzyme III) may have a special role in steroid metabolism during development in the female rabbit.
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Jovov, B., N. K. Wills, P. J. Donaldson, and S. A. Lewis. "Vectorial secretion of a kallikrein-like enzyme by cultured renal cells. I. General properties." American Journal of Physiology-Cell Physiology 259, no. 6 (December 1, 1990): C869—C882. http://dx.doi.org/10.1152/ajpcell.1990.259.6.c869.
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Urinary kallikreins are proteolytic enzymes known to be secreted by distal nephron tubules. In this study, we demonstrate (using the chromogenic tripeptide substrate S 2266) that the renal cell line A6 from Xenopus laevis secretes a kallikrein-like enzyme. Secretion is present only when the cells are grown on filters, and enzyme is secreted only into the apical membrane bathing solution. Enzyme secretion consists of two components, one soybean trypsin inhibitor (SBTI) sensitive (SSBTI) and the other insensitive to SBTI (ISBTI). Both enzymes were inhibited by aprotinin, a kallikrein-like enzyme inhibitor. Using a bioassay, only the ISBTI enzyme produced a hypotensive effect on blood pressure and is thus a kallikrein-like enzyme. The apical membrane of cells grown on filters contains both enzyme species, whereas the basolateral membrane contains only the ISBTI (kallikrein-like) enzyme. Both enzymes were present in the apical membrane of cells grown on plastic. Initiation of enzyme secretion occurred after the cells formed electrically tight monolayers and the increase in membrane activity always preceded enzyme secretion. Using an irreversible inhibitor of the apical membrane-bound enzymes, the turnover rate for the SSBTI and ISBTI enzymes (cells on filters) was 3 and 7 h, respectively. Because the recovery of enzyme secretion was proportional to the recovery of membrane-bound enzyme activities, this suggests that enzyme secretion is due to the release of membrane-bound enzyme.
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SAITO, AKINOBU, and RYO HONDO. "Genome Variation among Listeria monocytogenes Isolates Derived from Epidemiologically Related Raw Milk and Other Strains." Journal of Food Protection 59, no. 9 (September 1, 1996): 998–1002. http://dx.doi.org/10.4315/0362-028x-59.9.998.
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Listeria monocytogenes strains were examined by restriction-enzyme analysis of chromosomal DNA using a total of 18 restriction enzymes. Ten of the 6-base restriction enzymes and one 8-base restriction enzyme produced distinguishable fragments among these strains. Six strains (serotype 1/2a) recovered from raw milk suspected of the same contaminant were compared with seven epidemiologically unrelated strains (serotype 1/2a) using 10 of the 6-base restriction enzymes. The restriction enzyme patterns of the six raw milk isolates were identical to each other, but differed from those of the other strains. Restriction-enzyme analysis of the chromosomal DNA of L. monocytogenes by using the 6-base restriction enzymes may be a useful method of epidemiological analysis for listeriosis outbreaks.
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Jimoh, Abdulhameed, and Job Atteh. "Improving the metabolisable energy value of brewers’ dried grains with enzyme cocktails in poultry nutrition." Journal of Agricultural Sciences, Belgrade 63, no. 4 (2018): 409–19. http://dx.doi.org/10.2298/jas1804409j.
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The determination of the positive effects of exogenous enzymes is essential to ensure their inclusion in poultry feed formulation. This study was conducted to determine the effect of enzymes on the apparent metabolisable energy (AME) value of brewers? dried grain (BDG). Xylanase, phytase and multipurpose enzymes were used in a completely randomised design to determine the effects of individual exogenous enzymes and their cocktails on poultry metabolisable energy using adult cockerels. There were eight treatments comprising a control and seven experimental treatments with BDG and one, two or three enzymes. The AME values were determined using the intubation method. Data collected were analysed using the statistical analysis system. Enzymes individually and as a cocktail improved the AME value of BDG compared to the control. An increase in the AME value was 3.48%, 5.39%, 5.92%, 14.29%, 18.13%, 23.21% and 29.58% respectively for phytase, xylanase, cocktail of xylanase and phytase, multipurpose enzyme, cocktail of multipurpose enzyme and phytase, cocktail of xylanase and multipurpose enzyme and cocktail of xylanase, phytase and multipurpose enzyme. Cocktails of enzymes were significantly better (P?0.05) than individual enzymes in their effects on apparent metabolisable energy of BDG. Phytase gave a marginal increase in AME of the studied feedstuff. It has been concluded that the cocktail of enzymes is better than individual enzymes in their effects on AME of BDG. If different enzymes are available, it is recommended that the enzyme with higher units should be used.
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Li, Ling, Zixi Gao, Huige Zhang, Hongying Du, Cuiling Ren, Shengda Qi, and Hongli Chen. "One-pot surface modification of magnetic nanoparticles using phase-transitioned lysozyme for robust immobilization of enzymes." New Journal of Chemistry 45, no. 25 (2021): 11153–59. http://dx.doi.org/10.1039/d1nj00957e.
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Datta, Rahul, Swati Anand, Amitava Moulick, Divyashri Baraniya, Shamina Imran Pathan, Klement Rejsek, Valerie Vranova, et al. "How enzymes are adsorbed on soil solid phase and factors limiting its activity: A Review." International Agrophysics 31, no. 2 (April 25, 2017): 287–302. http://dx.doi.org/10.1515/intag-2016-0049.
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Abstract A majority of biochemical reactions are often catalysed by different types of enzymes. Adsorption of the enzyme is an imperative phenomenon, which protects it from physical or chemical degradation resulting in enzyme reserve in soil. This article summarizes some of the key results from previous studies and provides information about how enzymes are adsorbed on the surface of the soil solid phase and how different factors affect enzymatic activity in soil. Many studies have been done separately on the soil enzymatic activity and adsorption of enzymes on solid surfaces. However, only a few studies discuss enzyme adsorption on soil perspective; hence, we attempted to facilitate the process of enzyme adsorption specifically on soil surfaces. This review is remarkably unmatched, as we have thoroughly reviewed the relevant publications related to protein adsorption and enzymatic activity. Also, the article focuses on two important aspects, adsorption of enzymes and factors limiting the activity of adsorbed enzyme, together in one paper. The first part of this review comprehensively lays emphasis on different interactions between enzymes and the soil solid phase and the kinetics of enzyme adsorption. In the second part, we encircle various factors affecting the enzymatic activity of the adsorbed enzyme in soil.
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Rahman, M. M., U. F. Shahjadee, F. Khanam, A. Z. Rupa, and M. A. K. Azad. "Effect of chemical agents, metallic salts on the stability of α-amylase, protease and comparative analyses of enzyme activity of selected salad vegetables." Food Research 4, no. 4 (March 3, 2020): 1066–70. http://dx.doi.org/10.26656/fr.2017.4(4).259.
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The present investigation was conducted to analyze the activity of enzymes (protease, αamylase, cellulase and urease) of selected salad vegetables (white radish, red radish, beet, carrot, papaya, cucumber and tomato) as well as to determine the effect of chemical agents and metallic salts on the stability of enzymes. Salad vegetables are one of the cheap sources of adequate vitamins, minerals and enzymes. The protease, α-amylase and cellulase activity were highest in papaya (4.11±0.21 U/g, 1.68±0.21 U/g and 0.26±0.13 U/ g) whereas the urease activity was not detected in papaya, cucumber and tomato. The protease, α-amylase and cellulase activity of papaya were increased 24.83%, 42.26% and 57.69% than cucumber while the cellulase activity of carrot was increased 44.45% than tomato. The activity of protease and α-amylase of beet were decreased 52.44%, 33.63% while the cellulase activity of beet was increased 36.36% from radish. The toxicity indicating urease enzyme activity was not detected in papaya, tomato and cucumber but negligible in radish, beet and carrot which can be nullified. The activities of enzymes were increased in the presence of metallic salts such as Ca2+, Mg2+ and Mn2+ while Fe2+, Zn2+ and Cu2+ inhibited the enzyme's activity moderately. Results revealed that in the presence of higher concentrations of urea, EDTA and acetic acid, the activities of all the enzymes were completely inhibited.
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Ismail, Nurul Izzaty, Wan Heng Fong, and Nor Haniza Sarmin. "Computation of splicing languages from DNA splicing system with one palindromic restriction enzyme." Malaysian Journal of Fundamental and Applied Sciences 14, no. 2 (June 3, 2018): 188–92. http://dx.doi.org/10.11113/mjfas.v14n2.879.
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In DNA splicing system, the potential effects of sets of restriction enzymes and a ligase that allow DNA molecules to be cleaved and reassociated to produce further molecules are studied. A splicing language depicts the molecules resulting from a splicing system. In this research, a C++ programming code for DNA splicing system with one palindromic restriction enzyme for one and two (non-overlapping) cutting sites is developed. A graphical user interface, GUI is then designed to allow the user to insert the initial DNA string and restriction enzymes to generate the splicing languages which are the result of the computation of the C++ programming. This interface displays the resulting splicing languages, which depict the results from in vitro experiments of the respective splicing system. The results from this research simplify the lenghty manual computation of the resulting splicing languages of DNA splicing systems with one palindromic restriction enzyme.
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Sparkes, Emily I., Chisom S. Egedeuzu, Billie Lias, Rehana Sung, Stephanie A. Caslin, S. Yasin Tabatabaei Dakhili, Peter G. Taylor, Peter Quayle, and Lu Shin Wong. "Biocatalytic Silylation: The Condensation of Phenols and Alcohols with Triethylsilanol." Catalysts 11, no. 8 (July 22, 2021): 879. http://dx.doi.org/10.3390/catal11080879.
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Silicatein-α (Silα), a hydrolytic enzyme derived from siliceous marine sponges, is one of the few enzymes in nature capable of catalysing the metathesis of silicon–oxygen bonds. It is therefore of interest as a possible biocatalyst for the synthesis of organosiloxanes. To further investigate the substrate scope of this enzyme, a series of condensation reactions with a variety of phenols and aliphatic alcohols were carried out. In general, it was observed that Silα demonstrated a preference for phenols, though the conversions were relatively modest in most cases. In the two pairs of chiral alcohols that were investigated, it was found that the enzyme displayed a preference for the silylation of the S-enantiomers. Additionally, the enzyme’s tolerance to a range of solvents was tested. Silα had the highest level of substrate conversion in the nonpolar solvents n-octane and toluene, although the inclusion of up to 20% of 1,4-dioxane was tolerated. These results suggest that Silα is a potential candidate for directed evolution toward future application as a robust and selective biocatalyst for organosiloxane chemistry.
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Sunaga, H., H. Sugimoto, Y. Nagamachi, and S. Yamashita. "Purification and properties of lysophospholipase isoenzymes from pig gastric mucosa." Biochemical Journal 308, no. 2 (June 1, 1995): 551–57. http://dx.doi.org/10.1042/bj3080551.
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Two lysophospholipases, named gastric lysophospholipases I and II (enzymes I and II), were purified 3730- and 2680-fold from pig gastric mucosa. The preparations showed 22 and 23 kDa single protein bands on SDS/PAGE respectively. Both enzymes lacked transacylase activity and appeared to exist as monomers. Their activities were not affected by Ca2+, Mg2+ or EDTA. Enzyme I was most active at pH 8.5 and hydrolysed a variety of lysophospholipids including acidic lysophospholipids and the acyl analogue of platelet-activating factor, whereas enzyme II was most active at pH 8 and its activity was confined to lysophosphatidylcholine and lysophosphatidylethanolamine. When 1-palmitoylglycerophosphocholine was used as substrate, enzymes I and II showed half-maximal activities at 11 and 12 microM respectively. The enzymes exhibited no phospholipase B, lipase or general esterase activity. Enzyme II was significantly inhibited by lysophosphatidic acid whereas enzyme I was only moderately inhibited. Peptide mapping with V8 protease and papain revealed structural dissimilarity between the two enzymes. Antiserum raised against enzyme I did not recognize enzyme II, but did recognize the small-sized lysophospholipase purified from rat liver. Anti-(enzyme II) consistently did not cross-react with enzyme I or the liver enzyme. These antisera specifically recognized neither the 60 kDa lysophospholipase transacylase purified from liver nor any peritoneal macrophage protein. Thus gastric mucosa contains two different small-sized lysophospholipases: one is closely related to the small-sized lysophospholipase of liver, but the other appears to be a novel isoform.
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Zainol, Norazwina, and Siti Natrah Ismail. "Evaluation of Enzyme Kinetic Parameters to Produce Methanol Using Michaelis-Menten Equation." Bulletin of Chemical Reaction Engineering & Catalysis 14, no. 2 (August 1, 2019): 436. http://dx.doi.org/10.9767/bcrec.14.2.3317.436-442.
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Determination of kinetic parameters of enzymes is important in biotechnology research. It is also one of the most challenging processes in methanol production. The activity of enzyme is determined in term of initial rates at various substrate concentrations. The enzymatic hydrolysis of methanol by pectin methyl esterase (PME) enzyme was investigated at 25 °C and pH 9 over the reaction time range from 0 to 90 min. In this study, the parameters of the enzyme's kinetic, KM and Vmax were directly determined using a modified Michaelis-Menten equation by applying the Lineweaver-Burk plots. Besides, nonlinear regression of Michaelis-Menten equation was calculated based on Euler’s and Runge-Kutta 4th order methods by using Solver supplement application. The result of kinetic constant was tested by comparing the experimental data with model predictions. It was found that Euler and Runge-Kutta method was successful in determining the kinetic parameter rather than Lineweaver-Burk plot. The application of the Michaelis-Menten equation describes the enzyme kinetic very well. From the kinetic analysis, it showed the good agreement between the result obtained and the predictions model in the production of methanol using PME enzyme. Copyright © 2019 BCREC Group. All rights reserved
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Brooks, S. P. J., T. Espinola, and C. H. Suelter. "Erratum: Theory and practical application of coupled enzyme reactions: one and two auxiliary enzymes." Canadian Journal of Biochemistry and Cell Biology 63, no. 5 (May 1, 1985): 387. http://dx.doi.org/10.1139/o85-056.
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Antoun, G. R., I. Brglez, and D. G. Williamson. "A 17 β-hydroxysteroid dehydrogenase of female rabbit liver cytosol. Purification and characterization of multiple forms of the enzyme." Biochemical Journal 225, no. 2 (January 15, 1985): 383–90. http://dx.doi.org/10.1042/bj2250383.
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Multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver were identified. NAD-dependent and NADP-dependent enzyme activities were separated by affinity chromatography on agarose-immobilized Procion Red HE3B, and three forms of the NADP-dependent enzyme activity were purified by chromatofocusing. These three enzyme forms are charge isomers and have no quaternary structure. The enzymes catalysed the C-17 oxidoreduction of oestrogens and androgens; with all enzyme forms the activity towards androgens was higher than that toward oestrogens. The enzymes also exhibited 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. Comparison of the relative activities of the enzymes towards a number of oestrogen and androgen substrates revealed differences among the enzyme forms for both the oxidative and the reductive reactions. In particular, one enzyme form had a significantly lower Km for the 3 alpha-hydroxysteroid substrate and a higher 3 alpha-/17 beta-hydroxysteroid dehydrogenase activity ratio than the other two enzyme forms.
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Lenneman, Eric M., Janet M. Ohlert, Nagendra P. Palani, and Brett M. Barney. "Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway." Applied and Environmental Microbiology 79, no. 22 (September 6, 2013): 7055–62. http://dx.doi.org/10.1128/aem.02420-13.
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ABSTRACTThe biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previousin vitroenzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol inMarinobacter aquaeoleiVT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesisin vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previousin vitrostudies and provide the first glimpse into the role of each enzymein vivoin the native organism.
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Suresh, Harsha Garadi, Aline Xavier da Silveira dos Santos, Wanda Kukulski, Jens Tyedmers, Howard Riezman, Bernd Bukau, and Axel Mogk. "Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae." Molecular Biology of the Cell 26, no. 9 (May 2015): 1601–15. http://dx.doi.org/10.1091/mbc.e14-11-1559.
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Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.
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Zhu, Zheng, Song Ling, Qi-Heng Yang, and Lin Li. "The Difference in the Carboxy-Terminal Sequence Is Responsible for the Difference in the Activity of Chicken and Rat Liver Fructose-2,6-Bisphosphatase." Biological Chemistry 381, no. 12 (December 18, 2000): 1195–202. http://dx.doi.org/10.1515/bc.2000.147.
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Abstract The fructose-2,6-bisphosphatase domain of the bifunctional chicken liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase shares approximately 95% amino acid sequence homology with that of the rat enzyme. However, these two enzymes are significantly different in their phosphatase activities. In this report, we show that the COOH-terminal 25 amino acids of the two enzymes are responsible for the different enzymatic activities. Although these 25 amino acids are not required for the phosphatase activity, their removal diminishes the differences in the activities between the two enzymes. In addition, two chimeric molecules (one consisting of the catalytic core of the chicken bisphosphatase domain and the rat COOH-terminal 25 amino acids, and the other consisting of most of the intact chicken enzyme and the rat COOH-terminal 25 amino acids) showed the same kinetic properties as the rat enzyme. Furthermore, substitution of the residues Pro456pro457Ala458 of the chicken enzyme with GluAlaGlu, the corresponding sequence in the rat liver enzyme, yields a chicken enzyme that behaves like the rat enzyme. These results demonstrate that the different bisphosphatase activities of the chicken and rat liver bifunctional enzymes can be attributed to the differences in their COOH-terminal amino acid sequences, particularly the three residues.
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Rasiah, Indira A., and Bernd H. A. Rehm. "One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli." Applied and Environmental Microbiology 75, no. 7 (February 5, 2009): 2012–16. http://dx.doi.org/10.1128/aem.02782-08.
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ABSTRACT Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable α-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting α-amylase activity. The α-amylase beads were assessed with respect to α-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized α-amylase showed Michaelis-Menten enzyme kinetics exerting a V max of about 506 mU/mg of bead protein with a Km of about 5 μM, consistent with that of free α-amylase. The stability of the enzyme at 85°C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized α-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.
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Ainscough, R. J., J. M. McGree, M. J. Callaghan, and R. E. Speight. "Effective incorporation of xylanase and phytase in lick blocks for grazing livestock." Animal Production Science 59, no. 9 (2019): 1762. http://dx.doi.org/10.1071/an18424.
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The addition of feed enzymes to livestock diets has contributed to significant increases in productivity over recent decades. The use of enzymes has been the most common in systems where enzyme delivery and diets can be easily managed, such as for poultry and pigs. Lick blocks supplement the forage diets of ruminants with nitrogen and minerals but not enzymes, due in part to concerns that block manufacturing temperatures would lead to unacceptable levels of enzyme degradation. The nutritional value of low quality pasture could be improved using enzyme supplemented lick blocks if enzymes remain active at the high lick block manufacturing temperatures. The aim of this study was to determine the extent of xylanase and phytase activity survival when exposed to the production of hot poured lick blocks. Lick block formulations and methods of manufacturing vary, so two enzyme containing molasses-based lick blocks were produced, one at 60°C and another at 100°C. The results showed that both the xylanase and phytase enzymes have high levels of survival at 60°C. In the 100°C lick block, the phytase displayed a half-life of ~10 min, whereas the xylanase retained 90% of the original activity after 30 min of exposure. The inherent thermostability of the enzymes were critical factors for enzyme survival and the enzymes were more stable in the lick blocks than in solution. The results indicate that it should be possible to add enzymes to lick blocks manufactured at elevated temperatures to enhance low quality pasture and thereby aid ruminant digestion and production.
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Morgan, J. Alun W., and Roger W. Pickup. "Activity of microbial peptidases, oxidases, and esterases in lake waters of varying trophic status." Canadian Journal of Microbiology 39, no. 8 (August 1, 1993): 795–803. http://dx.doi.org/10.1139/m93-117.
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The range and activities of microbial enzymes present in lake water were assessed directly in cells concentrated by tangential flow filtration. A total of 108 enzymes were assayed in this study, which included tests for 60 peptidases, 20 oxidases, and 10 esterases, and 18 miscellaneous tests. In general, no trends in the range of enzymes were associated with trophic status of the lakes. However, one lake that was hypereutrophic had a greater range of enzymes than the other lakes tested. An increase in total enzyme activity (activity/mL) was recorded with an increase in trophic status of the water. The relationship between the physical and chemical attributes of each lake and microbial enzyme activities was investigated by principal component analysis. Quantitative changes between lakes in 11 of the 21 variables were shown to be closely related to changes in the enzyme activities of the lakes; total organic carbon, particulate carbon, particulate nitrogen, pH, and chlorophyll a showed the closest relationships.Key words: lake water, enzyme activity, trophic status.
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Riegert, Ulrich, Sibylle Bürger, and Andreas Stolz. "Altering Catalytic Properties of 3-Chlorocatechol-Oxidizing Extradiol Dioxygenase fromSphingomonas xenophaga BN6 by Random Mutagenesis." Journal of Bacteriology 183, no. 7 (April 1, 2001): 2322–30. http://dx.doi.org/10.1128/jb.183.7.2322-2330.2001.
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ABSTRACT The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.
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Jee, Ah-Young, Tsvi Tlusty, and Steve Granick. "Master curve of boosted diffusion for 10 catalytic enzymes." Proceedings of the National Academy of Sciences 117, no. 47 (November 9, 2020): 29435–41. http://dx.doi.org/10.1073/pnas.2019810117.
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Molecular agitation more rapid than thermal Brownian motion is reported for cellular environments, motor proteins, synthetic molecular motors, enzymes, and common chemical reactions, yet that chemical activity coupled to molecular motion contrasts with generations of accumulated knowledge about diffusion at equilibrium. To test the limits of this idea, a critical testbed is the mobility of catalytically active enzymes. Sentiment is divided about the reality of enhanced enzyme diffusion, with evidence for and against. Here a master curve shows that the enzyme diffusion coefficient increases in proportion to the energy release rate—the product of Michaelis-Menten reaction rate and Gibbs free energy change (ΔG)—with a highly satisfactory correlation coefficient of 0.97. For 10 catalytic enzymes (urease, acetylcholinesterase, seven enzymes from the glucose cascade cycle, and one other), our measurements span from a roughly 40% enhanced diffusion coefficient at a high turnover rate and negativeΔGto no enhancement at a slow turnover rate and positiveΔG. Moreover, two independent measures of mobility show consistency, provided that one avoids undesirable fluorescence photophysics. The master curve presented here quantifies the limits of both ideas, that enzymes display enhanced diffusion and that they do not within instrumental resolution, and has possible implications for understanding enzyme mobility in cellular environments. The striking linear dependence of ΔGfor the exergonic enzymes (ΔG<0), together with the vanishing effect for endergonic enzyme (ΔG>0), are consistent with a physical picture in which the mechanism boosting the diffusion is an active one, utilizing the available work from the chemical reaction.
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Rydzy, Małgorzata, Michał Tracz, Andrzej Szczepaniak, and Joanna Grzyb. "Insights into the Structure of Rubisco from Dinoflagellates-in Silico Studies." International Journal of Molecular Sciences 22, no. 16 (August 7, 2021): 8524. http://dx.doi.org/10.3390/ijms22168524.
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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the best studied enzymes. It is crucial for photosynthesis, and thus for all of biosphere’s productivity. There are four isoforms of this enzyme, differing by amino acid sequence composition and quaternary structure. However, there is still a group of organisms, dinoflagellates, single-cell eukaryotes, that are confirmed to possess Rubisco, but no successful purification of the enzyme of such origin, and hence a generation of a crystal structure was reported to date. Here, we are using in silico tools to generate the possible structure of Rubisco from a dinoflagellate representative, Symbiodinium sp. We selected two templates: Rubisco from Rhodospirillum rubrum and Rhodopseudomonas palustris. Both enzymes are the so-called form II Rubiscos, but the first is exclusively a homodimer, while the second one forms homo-hexamers. Obtained models show no differences in amino acids crucial for Rubisco activity. The variation was found at two closely located inserts in the C-terminal domain, of which one extends a helix and the other forms a loop. These inserts most probably do not play a direct role in the enzyme’s activity, but may be responsible for interaction with an unknown protein partner, possibly a regulator or a chaperone. Analysis of the possible oligomerization interface indicated that Symbiodinium sp. Rubisco most likely forms a trimer of homodimers, not just a homodimer. This hypothesis was empowered by calculation of binding energies. Additionally, we found that the protein of study is significantly richer in cysteine residues, which may be the cause for its activity loss shortly after cell lysis. Furthermore, we evaluated the influence of the loop insert, identified exclusively in the Symbiodinium sp. protein, on the functionality of the recombinantly expressed R. rubrum Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme.
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Hu, Chong, Yunxiu Bai, Miao Hou, Yisu Wang, Licheng Wang, Xun Cao, Chiu-Wing Chan, et al. "Defect-induced activity enhancement of enzyme-encapsulated metal-organic frameworks revealed in microfluidic gradient mixing synthesis." Science Advances 6, no. 5 (January 2020): eaax5785. http://dx.doi.org/10.1126/sciadv.aax5785.
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Mimicking the cellular environment, metal-organic frameworks (MOFs) are promising for encapsulating enzymes for general applications in environments often unfavorable for native enzymes. Markedly different from previous researches based on bulk solution synthesis, here, we report the synthesis of enzyme-embedded MOFs in a microfluidic laminar flow. The continuously changed concentrations of MOF precursors in the gradient mixing on-chip resulted in structural defects in products. This defect-generating phenomenon enables multimodal pore size distribution in MOFs and therefore allows improved access of substrates to encapsulated enzymes while maintaining the protection to the enzymes. Thus, the as-produced enzyme-MOF composites showed much higher (~one order of magnitude) biological activity than those from conventional bulk solution synthesis. This work suggests that while microfluidic flow synthesis is currently underexplored, it is a promising strategy in producing highly active enzyme-MOF composites.
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Abell, Lynn M. "Biochemical Approaches to Herbicide Discovery: Advances in Enzyme Target Identification and Inhibitor Design." Weed Science 44, no. 3 (September 1996): 734–42. http://dx.doi.org/10.1017/s0043174500094613.
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This paper focuses primarily on the means by which biochemical information can be used to identify enzymes which, upon inhibition, produce lethal phenotypes and the enzyme inhibitor design strategies that have the highest probability of not only inhibiting the enzyme but also translating that inhibition into herbicidal efficacy. The identification of an exquisitely lethal target site is the key initial component to this approach and has often been one of the most difficult steps because the attributes of a lethal site have, at best, been ill-defined. An examination of the characteristics of known targets provides some insight as to the definition of a lethal target. Recently, antisense RNA suppression of enzyme translation has been used to determine the extent of inhibition required for toxicity and offers potential as a strategy for identifying lethal target sites. After identification of a lethal target, detailed knowledge of the enzyme's chemical and kinetic mechanism as well as the protein's structure may be used to design potent inhibitors. Various types of inhibitors may be designed for a given enzyme. The advantages and disadvantages of a given type with respect to in vivo efficacy as well as the probability of herbicide resistance development will be discussed.
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Taylor, I. N., R. C. Brown, M. Bycroft, G. King, J. A. Littlechild, M. C. Lloyd, C. Praquin, H. S. Toogood, and S. J. C. Taylor. "Application of thermophilic enzymes in commercial biotransformation processes." Biochemical Society Transactions 32, no. 2 (April 1, 2004): 290–92. http://dx.doi.org/10.1042/bst0320290.
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Biocatalysis is a useful tool in the provision of chiral technology and extremophilic enzymes are just one component in that toolbox. Their role is not always attributable to their extremophilic properties; as with any biocatalyst certain other criteria should be satisfied. Those requirements for a useful biocatalyst will be discussed including issues of selectivity, volume efficiency, security of supply, technology integration, intellectual property and regulatory compliance. Here we discuss the discovery and commercialization of an l-aminoacylase from Thermococcus litoralis, the product of a LINK project between Chirotech Technology and the University of Exeter. The enzyme was cloned into Escherichia coli to aid production via established mesophilic fermentation protocols. A simple downstream process was then developed to assist in the production of the enzyme as a genetically modified-organism-free reagent. The fermentation and downstream processes are operated at the 500 litre scale. Characterization of the enzyme demonstrated a substrate preference for N-benzoyl groups over N-acetyl groups. The operational parameters have been defined in part by substrate-concentration tolerances and also thermostabilty. Several examples of commercial biotransformations will be discussed including a process that is successful by virtue of the enzyme's thermotolerance.
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Chasanah, Ekowati, Mahrus Ali, and Miftahul Ilmi. "IDENTIFICATION AND PARTIAL CHARACTERIZATION OF CRUDE EXTRACELLULAR ENZYMES FROM BACTERIA ISOLATED FROM SHRIMP WASTE PROCESSING." Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology 7, no. 1 (May 23, 2013): 11. http://dx.doi.org/10.15578/squalen.v7i1.11.
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Attention on chitin degrading enzymes has been growing since their ability to reduce the waste of shrimp/other crustaceans processing industries and converting them into value added products such as biologically active chitin and chitosan oligomer. Previous experiment found that KLU 11.16 isolate was one of the potential bacteria isolated from shrimp waste producing chitinolytic enzymes including chitosanases. A study on the identification of KLU 11.16 extracellularcrude enzyme was carried out by cultivating the bacteria on chitin medium. Due to the wide application of chitosanase, the characterization of the crude chitosanase was carried out after an identification of the enzymes secreted. Based on assessment using zymogram technique, this bacteria secreted a mixed extracellular chitinolytic enzyme and other hydrolytic enzyme. The crudechitinolytic enzyme degraded 85% deacetylated (DA) better than 100% DA chitosan, and slightly degraded glycol chitin, indicating that KLU 11.16 secreted chitosanases and chitinases enzyme. In addition to the chitinolytic enzyme, the bacteria also secreted protein and carbohydrate degrading enzymes when running at SDS-PAGE enriched with casein, soluble starch and CMC substrates.Crude chitosanases enzyme was performed well at pH 6 and temperature of 300C, and the activity can be increased by addition of 1 mM Fe 2+ in form of chloride salt. Addition of detergent, i.e1mM of Triton X-100 and SDS slightly decreased the activity. Future application of the crude chitosanase from KLU 11.16 was on producing chitosan derivative such as chitosan oligomer using substrateof 85% DA chitosan, which is more digestable by other enzymes secreted by KLU 11.16
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Ebert, D. L., K. B. Jordan, and R. L. Dimond. "Lysosomal enzyme secretory mutants of Dictyostelium discoideum." Journal of Cell Science 96, no. 3 (July 1, 1990): 491–500. http://dx.doi.org/10.1242/jcs.96.3.491.
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Dictyostelium discoideum secretes a number of lysosomal enzymes during axenic growth and upon suspension in a low ionic strength, non-nutrient buffer (standard secretion conditions). These secretory characteristics have allowed us to identify 74 lysosomal enzyme secretory mutants generated by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The majority of these mutants fell into one of four classes, on the basis of their secretory characteristics in non-nutrient buffer. The four mutant classes indicate that a minimum of three distinct sets of genes are necessary for proper secretion of lysosomal enzymes from D. discoideum cells under standard secretion conditions: one set of genes that is involved in general lysosomal enzyme secretion, one that is involved in glycosidase type secretion, and a third that is involved in acid phosphatase type secretion. These three classes likely reflect heterogeneity in the intracellular destination of lysosomal enzymes, the secretory mechanism, or both. A fourth set of genes may be necessary for proper secretion during growth, but plays no role under standard secretion conditions. These are likely altered in the regulation of secretion or in lysosomal enzyme targeting. Of the 74 secretory mutants, 36 were also modification mutants resulting in decreased pI, thermolability, or in vivo instability of lysosomal enzyme activities. The high frequency of modification mutants indicates an integral relationship between lysosomal enzyme modification, and lysosomal enzyme targeting and secretion in D. discoideum.
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Møller, Peter L., Flemming Jørgensen, Ole C. Hansen, Søren M. Madsen, and Peter Stougaard. "Intra- and Extracellular β-Galactosidases fromBifidobacterium bifidum and B. infantis: Molecular Cloning, Heterologous Expression, and Comparative Characterization." Applied and Environmental Microbiology 67, no. 5 (May 1, 2001): 2276–83. http://dx.doi.org/10.1128/aem.67.5.2276-2283.2001.
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ABSTRACT Three β-galactosidase genes from Bifidobacterium bifidum DSM20215 and one β-galactosidase gene fromBifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum β-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published β-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantisβ-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 β-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal β-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidumβ-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four β-galactosidases were highly specific for hydrolysis of β-d-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.
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Dai, Yong, Pieter C. Wensink, and Robert H. Abeles. "One Protein, Two Enzymes." Journal of Biological Chemistry 274, no. 3 (January 15, 1999): 1193–95. http://dx.doi.org/10.1074/jbc.274.3.1193.
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Jang, Ho Hee, Kyun Oh Lee, Yong Hun Chi, Bae Gyo Jung, Soo Kwon Park, Jin Ho Park, Jung Ro Lee, et al. "Two Enzymes in One." Cell 117, no. 5 (May 2004): 625–35. http://dx.doi.org/10.1016/j.cell.2004.05.002.
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Lemonidis, Kimon, Martin W. Werno, Jennifer Greaves, Cinta Diez-Ardanuy, Maria C. Sanchez-Perez, Christine Salaun, David M. Thomson, and Luke H. Chamberlain. "The zDHHC family of S-acyltransferases." Biochemical Society Transactions 43, no. 2 (April 1, 2015): 217–21. http://dx.doi.org/10.1042/bst20140270.
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The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are 24 ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzyme-substrate specificity and unravelling the importance of this enzyme family for cellular physiology and pathology.
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HART, PETER W. "Differences in bleaching responses from fungal- versus bacterial-derived enzymes." March 2012 11, no. 3 (April 1, 2012): 21–27. http://dx.doi.org/10.32964/tj11.3.21.
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Several mills in North America have been successful in using xylanase enzymes expressed from Trichoderma reesei (a fungus) as part of their bleaching sequence for many years. These mills process hardwood and softwood species, with and without oxygen delignification. These mills also use three-, four-, and five-stage bleaching sequences. North American mills tend to report increased pulp brightness ceilings and decreased bleaching costs as benefits associated with the application of enzymes in the bleaching process. Laboratory testing suggests that eucalyptus pulp is highly susceptible to fungal- and bacterial-derived enzyme bleaching and should result in significant cost savings in South American mills. At least four different mills in South America have attempted to perform enzyme bleaching trials using bacterial-derived enzymes. Each of these mill trials resulted in significantly increased operating costs and/or unsustainable operating conditions. More recently, one of these South American mills performed a short trial using a commercially available, fungal-derived enzyme. This trial was technically successful. This report attempts to determine why the South American mill experiences with bacterial-derived enzymes have been poor, while North American mills and the one South American mill trial have had good results with fungal-derived enzymes. Operating conditions and trial goals for the North and South American mills also were examined.
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Ratnaningsih, Enny, Sulistiya Nirta Sunaryo, Idris Idris, and Rindia Maharani Putri. "Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1." Reaktor 21, no. 2 (August 1, 2021): 59–64. http://dx.doi.org/10.14710/reaktor.21.2.59-64.
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In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification
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Polya, G. M., and M. Haritou. "Purification and characterization of two wheat-embryo protein phosphatases." Biochemical Journal 251, no. 2 (April 15, 1988): 357–63. http://dx.doi.org/10.1042/bj2510357.
Full textAbstract:
Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.
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Fukuda, Wakao, Yulia Sari Ismail, Toshiaki Fukui, Haruyuki Atomi, and Tadayuki Imanaka. "Characterization of an archaeal malic enzyme from the hyperthermophilic archaeonThermococcus kodakaraensisKOD1." Archaea 1, no. 5 (2005): 293–301. http://dx.doi.org/10.1155/2005/250757.
Full textAbstract:
Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeonT hermococcus kodakaraensisKOD1 (Tk-Mae). The amino acid sequence ofTk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure ofTk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km) ofTk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encodingTk-Mae and intracellular malic enzyme activity inT. kodakaraensiswere constitutively weak, regardless of the growth substrates. Possible roles ofTk-Mae are discussed based on these results and the metabolic pathways ofT. kodakaraensisdeduced from the genome sequence.
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Gascón-Pérez, Victoria, Mayra Belen Jiménez, Asunción Molina, Rosa María Blanco, and Manuel Sánchez-Sánchez. "Efficient One-Step Immobilization of CaLB Lipase over MOF Support NH2-MIL-53(Al)." Catalysts 10, no. 8 (August 10, 2020): 918. http://dx.doi.org/10.3390/catal10080918.
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Metal-organic framework (MOF) materials possess the widest versatility in structure, composition, and synthesis procedures amongst the known families of materials. On the other hand, the extraordinary affinity between MOFs and enzymes has led to widely investigating these materials as platforms to support these catalytic proteins in recent years. In this work, the MOF material NH2-MIL-53(Al) has been tested as a support to immobilize by one-step methodology (in situ) the enzyme lipase CaLB from Candida antarctica by employing conditions that are compatible with its enzymatic activity (room temperature, aqueous solution, and moderate pH values). Once the nature of the linker deprotonating agent or the synthesis time were optimized, the MOF material resulted in quite efficient entrapping of the lipase CaLB through this in situ approach (>85% of the present enzyme in the synthesis media) while the supported enzyme retained acceptable activity (29% compared to the free enzyme) and had scarce enzyme leaching. The equivalent post-synthetic method led to biocatalysts with lower enzyme loading values. These results make clear that the formation of MOF support in the presence of the enzyme to be immobilized substantially improves the efficiency of the biocatalysts support for retaining the enzyme and limits their leaching.
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Ishak, Nadiah, Angzzas Sari Mohd Kassim, Ashuvila Mohd Aripin, Dayang Norulfairuz Abang Zaidel, and Muhd Hafeez Zainulabidin. "Identification and Expression of Ligninase Enzymes from Tropical Asia Wood Insect for Agro-Pulp Biodelignification: A Theoretical Framework." Applied Mechanics and Materials 773-774 (July 2015): 1380–83. http://dx.doi.org/10.4028/www.scientific.net/amm.773-774.1380.
Full textAbstract:
Current pulp-processing in pulp and paper based industries are inefficient in removing the lignin as this compound is recalcitrant towards degradation. Transitioning from conventional pulping process into bio-delignification through utilisation of ligninase enzymes is one of the alternatives to improve the ability to fully utilize all components of wood to produce high quality fibres. Extensive research efforts have been focused on increase the production of ligninase enzymes from white rot fungi as a whole organism for industrial applications. However, enzymes activity produced from fungi are rather low as lignin modification is a secondary metabolism in which the enzyme only be expressed under particular conditions. Using genetic manipulations to incorporate genes associate for delignification isolated from different organisms such as tropical Asian wood-feeding insect into bacteria expression system will allow rapid enzyme production. This theoretical framework aims to produce an enzyme with high ligninase activity that will be used for removal of lignin during pulp-processing. These enzymes are thought to be more economically efficient in degrading lignin and involves less use of chemicals thus make this processing more environmentally friendly.Keywords: Biodelignification, Asian wood tropical insect, fungi, ligninase enzyme, bacterial expression system