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multienzyme system

Katsuaki Hirano, Masahiro Kurosaki, Satoshi Nihei, Hiroki Hasegawa, Suguru Shinoda, Mitsuru Haruki, Nobutaka Hirano
The cellulosome is a supramolecular multienzyme complex comprised of a wide variety of polysaccharide-degrading enzymes and scaffold proteins. The cellulosomal enzymes that bind to the scaffold proteins synergistically degrade crystalline cellulose. Here, we report in vitro reconstitution of the Clostridium thermocellum cellulosome from 40 cellulosomal components and the full-length scaffoldin protein that binds to nine enzyme molecules. These components were each synthesized using a wheat germ cell-free protein synthesis system and purified...
October 19, 2016: Scientific Reports
Hai Yu, Yanhong Li, Jie Zeng, Vireak Thon, Dung M Nguyen, Thao Ly, Hui Yu Kuang, Alice Ngo, Xi Chen
Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various molecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingolipids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available. Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid (Neu5Ac8OMe)...
October 13, 2016: Journal of Organic Chemistry
Vanessa Gonçalves Pereira, Matheus Trovão de Queiroz, Vânia D'Almeida
Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-l-iduronidase, involved in degradation of glycosaminoglycans. Clinical manifestations are widely variable and patients with severe phenotype present developmental delay and cognitive decline, among other systemic alterations. MPS I patients present secondary accumulation of gangliosides in neuronal cells, besides accumulation of undegraded glycosaminoglycans. Reduction of Neu1 expression has been previously observed in the cerebellum of MPS I mice; to be active, neuraminidase 1 forms the lysosomal multienzyme complex (LMC) with two other proteins, β-galactosidase and protective protein/cathepsin A, involved in stepwise degradation of gangliosides in the lysosomes...
October 6, 2016: Gene
Debasis Das, Marthe T C Walvoort, Vinita Lukose, Barbara Imperiali
Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays...
2016: Scientific Reports
Lital Davidi, Sarah Moraïs, Lior Artzi, Doriv Knop, Yitzhak Hadar, Yonathan Arfi, Edward A Bayer
Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products...
September 27, 2016: Proceedings of the National Academy of Sciences of the United States of America
Jaroslav Srp, Martina Nussbaumerová, Martin Horn, Michael Mareš
The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities...
August 15, 2016: Insect Biochemistry and Molecular Biology
Sarah Moraïs, Johanna Stern, Amaranta Kahn, Anastasia P Galanopoulou, Shahar Yoav, Melina Shamshoum, Matthew A Smith, Dimitris G Hatzinikolaou, Frances H Arnold, Edward A Bayer
BACKGROUND: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively...
2016: Biotechnology for Biofuels
Maureen B Quin, Sarah A Perdue, Szu-Yi Hsu, Claudia Schmidt-Dannert
Spatial organization via encapsulation of enzymes within recombinant nanocompartments may increase efficiency in multienzyme cascades. Previously, we reported the encapsulation of single cargo proteins within nanocompartments in the heterologous host Escherichia coli. This was achieved by coexpression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK, with a signal sequence EutC1-19 cargo protein fusion. Optimization of this system, leading to the targeting of more than one cargo protein, requires an understanding of the encapsulation mechanism...
November 2016: Applied Microbiology and Biotechnology
Immacolata Venditto, Ana S Luis, Maja Rydahl, Julia Schückel, Vânia O Fernandes, Silvia Vidal-Melgosa, Pedro Bule, Arun Goyal, Virginia M R Pires, Catarina G Dourado, Luís M A Ferreira, Pedro M Coutinho, Bernard Henrissat, J Paul Knox, Arnaud Baslé, Shabir Najmudin, Harry J Gilbert, William G T Willats, Carlos M G A Fontes
The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome...
June 28, 2016: Proceedings of the National Academy of Sciences of the United States of America
Samira Hezaveh, An-Ping Zeng, Uwe Jandt
Targeted manipulation and exploitation of beneficial properties of multienzyme complexes, especially for the design of novel and efficiently structured enzymatic reaction cascades, require a solid model understanding of mechanistic principles governing the structure and functionality of the complexes. This type of system-level and quantitative knowledge has been very scarce thus far. We utilize the human pyruvate dehydrogenase complex (hPDC) as a versatile template to conduct corresponding studies. Here we present new homology models of the core subunits of the hPDC, namely E2 and E3BP, as the first time effort to elucidate the assembly of hPDC core based on molecular dynamic simulation...
May 19, 2016: Journal of Physical Chemistry. B
Ponnuswamy Vijayaraghavan, Arumugaperumal Arun, Naif Abdullah Al-Dhabi, Samuel Gnana Prakash Vincent, Mariadhas Valan Arasu, Ki Choon Choi
BACKGROUND: Hydrolytic enzymes, such as cellulases and proteases, have various applications, including bioethanol production, extraction of fruit and vegetable juice, detergent formulation, and leather processing. Solid-substrate fermentation has been an emerging method to utilize low-cost agricultural residues for the production of these enzymes. Although the production of carboxy methyl cellulase (CMCase) and protease in solid state fermentation (SSF) have been studied extensively, research investigating multienzyme production in a single fermentation process is limited...
2016: Biotechnology for Biofuels
Nidhi C Dubey, Bijay P Tripathi, Martin Müller, Manfred Stamm, Leonid Ionov
We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels...
May 9, 2016: Biomacromolecules
Tobias Elbinger, Markus Gahn, Maria Neuss-Radu, Falk M Hante, Lars M Voll, Günter Leugering, Peter Knabner
Mathematical modeling of biochemical pathways is an important resource in Synthetic Biology, as the predictive power of simulating synthetic pathways represents an important step in the design of synthetic metabolons. In this paper, we are concerned with the mathematical modeling, simulation, and optimization of metabolic processes in biochemical microreactors able to carry out enzymatic reactions and to exchange metabolites with their surrounding medium. The results of the reported modeling approach are incorporated in the design of the first microreactor prototypes that are under construction...
2016: Frontiers in Bioengineering and Biotechnology
Hai Yu, Xi Chen
Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with glycosyltransferases in one pot for efficient production of the target glycans from simple monosaccharides and acceptors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitated the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes...
March 14, 2016: Organic & Biomolecular Chemistry
Chao Zhao, Yijing Wu, Hai Yu, Ishita M Shah, Yanhong Li, Jie Zeng, Bin Liu, David A Mills, Xi Chen
A novel α1-2-fucosyltransferase from Thermosynechococcus elongatus BP-1 (Te2FT) with high fucosyltransferase activity and low donor hydrolysis activity was discovered and characterized. It was used in an efficient one-pot multienzyme (OPME) fucosylation system for the high-yield synthesis of human blood group H antigens containing β1-3-linked galactosides and an important human milk oligosaccharide (HMOS) lacto-N-fucopentaose I (LNFP I) on preparative and gram scales. LNFP I was shown to be selectively consumed by Bifidobacterium longum subsp...
March 11, 2016: Chemical Communications: Chem Comm
Lei Guo, Xi Chen, Li-Na Li, Wei Tang, Yi-Ting Pan, Jian-Qiang Kong
BACKGROUND: (2S)-Pinocembrin is a chiral flavanone with versatile pharmacological and biological activities. Its health-promoting effects have spurred on research effects on the microbial production of (2S)-pinocembrin. However, an often-overlooked salient feature in the analysis of microbial (2S)-pinocembrin is its chirality. RESULTS: Here, we presented a full characterization of absolute configuration of microbial (2S)-pinocembrin from engineered Escherichia coli...
2016: Microbial Cell Factories
Daniel Sojka, Jana Pytelková, Jan Perner, Martin Horn, Jitka Konvičková, Jana Schrenková, Michael Mareš, Petr Kopáček
Host blood proteins, represented mainly by hemoglobin and serum albumin, serve as the ultimate source of amino acids needed for de novo protein synthesis during tick development and reproduction. While uptake and processing of hemoglobin by tick gut cells have been studied in detail, molecular mechanisms of host serum albumin degradation remain unknown. In this work, we have used artificial membrane feeding of Ixodes ricinus females on a hemoglobin-free diet in order to characterize the proteolytic machinery involved in albuminolysis...
December 19, 2015: Ticks and Tick-borne Diseases
Sonia Del Prete, Viviana De Luca, Clemente Capasso, Claudiu T Supuran, Vincenzo Carginale
With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes...
January 15, 2016: Bioorganic & Medicinal Chemistry
Peter Both, Hanna Busch, Paul P Kelly, Francesco G Mutti, Nicholas J Turner, Sabine L Flitsch
Enantiomerically pure chiral amines are ubiquitous chemical building blocks in bioactive pharmaceutical products and their synthesis from simple starting materials is of great interest. One of the most attractive strategies is the stereoselective installation of a chiral amine through C-H amination, which is a challenging chemical transformation. Herein we report the application of a multienzyme cascade, generated in a single bacterial whole-cell system, which is able to catalyze stereoselective benzylic aminations with ee values of 97...
January 22, 2016: Angewandte Chemie
Christopher C Roberts, Chia-en A Chang
Colocalized multistep enzymatic reaction pathways within biological catabolic and metabolic processes occur with high yield and specificity. Spatial organization on membranes or surfaces may be associated with increased efficiency of intermediate substrate transfer. Using a new Brownian dynamics package, GeomBD, we explored the geometric features of a surface-anchored enzyme system by parallel coarse-grained Brownian dynamics simulations of substrate diffusion over microsecond (μs) to millisecond (ms) time scales...
January 13, 2015: Journal of Chemical Theory and Computation
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