scholarly journals Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

2020 ◽  
Vol 21 (2) ◽  
pp. 416
Author(s):  
Angel De La Cruz Pech-Canul ◽  
Javier Carrillo-Campos ◽  
María de Lourdes Ballinas-Casarrubias ◽  
Rosa Lidia Solis-Oviedo ◽  
Selena Karina Hernández-Rascón ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Protein Purification ◽  
Phanerochaete Chrysosporium ◽  
Manganese Peroxidase ◽  
Expression System ◽  
White Rot Fungi ◽  
Functional Expression ◽  
White Rot ◽  
E Coli ◽  
One Step

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Establishing molecular genetics for Phanerochaete chrysosporium

1987 ◽  
Vol 321 (1561) ◽  
pp. 475-483 ◽  
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Complex Function ◽  
Strain Improvement ◽  
White Rot Fungi ◽  
Point Of View ◽  
Site Directed Mutagenesis ◽  
White Rot ◽  
General Strategy ◽  
Lignin Biodegradation ◽  
Control Of Expression

Genetics provides an approach to the analysis of the complex function of lignin biodegradation, through the isolation of mutants and the creation of gene libraries for the identification of genes and their products. However, white-rot fungi (for example, Phanerochaete chrysosporium ) have not so far been analysed from this point of view, and there is the challenge of establishing such genetics. P. chrysosporium is convenient experimentally because relatively few genes are switched on at the onset of ligninolytic activity. We describe the isolation of clones carrying genes expressed specifically in the ligninolytic phase, the development of a general strategy for mapping such clones, and the elucidation of the mating system of this organism. Another objective is the development of methods for transforming DNA into P. chrysosporium . This would allow the use of site-directed mutagenesis to analyse the functioning of ligninases, and the control of expression of the corresponding genes. The use of genetic crosses for strain improvement and the identification of components of the system are also discussed.

Production of laccase and manganese peroxidase by white-rot fungi from sugarcane bagasse in solid bed: Use for dyes decolourisation

Sugar Tech ◽  
2008 ◽  
Vol 10 (3) ◽  
pp. 260-264 ◽  
Author(s):  
Gilda Guerra ◽  
Osmel Domínguez ◽  
Miguel Ramos-Leal ◽  
Ana M. Manzano ◽  
María I. Sánchez ◽  
...  
Keyword(s):  
Sugarcane Bagasse ◽  
Manganese Peroxidase ◽  
White Rot Fungi ◽  
White Rot

scholarly journals pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

2001 ◽  
Vol 67 (3) ◽  
pp. 1262-1267 ◽  
Author(s):  
Shuhei Fujimoto ◽  
Yasuyoshi Ike
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Enterococcus Faecalis ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Chromatography Method ◽  
Shuttle Vectors ◽  
E Coli ◽  
One Step

ABSTRACT Two novel Enterococcus faecalis-Escherichia colishuttle vectors that utilize the promoter and ribosome binding site ofbacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli andE. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coliand E. faecalis at levels of 103 and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

scholarly journals Identification and Expression of Ligninase Enzymes from Tropical Asia Wood Insect for Agro-Pulp Biodelignification: A Theoretical Framework

2015 ◽  
Vol 773-774 ◽  
pp. 1380-1383
Author(s):  
Nadiah Ishak ◽  
Angzzas Sari Mohd Kassim ◽  
Ashuvila Mohd Aripin ◽  
Dayang Norulfairuz Abang Zaidel ◽  
Muhd Hafeez Zainulabidin
Keyword(s):  
Expression System ◽  
White Rot Fungi ◽  
Theoretical Framework ◽  
Industrial Applications ◽  
White Rot ◽  
Tropical Asia ◽  
Genetic Manipulations ◽  
Lignin Modification ◽  
Pulp Processing ◽  
Insect Fungi

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

scholarly journals Potensi fungi pelapuk putih asal lingkungan tropik untuk bioremediasi herbisida The potential white-rot fungi native of tropical environment for herbicides bioremediation

2016 ◽  
Vol 75 (1) ◽  
Author(s):  
Laksmita Prima SANTI ◽  
Lisdar Idwan SUDIRMAN ◽  
Didiek Hadjar GOENADI
Keyword(s):  
Laboratory Experiment ◽  
Phanerochaete Chrysosporium ◽  
White Rot Fungi ◽  
Tropical Environment ◽  
White Rot ◽  
Malt Extract ◽  
Ceriporiopsis Subvermispora ◽  
Pleurocybella Porrigens

SummaryFungal treatment by using white-rot fungito reduce a wide variety of herbicide com-pounds is a specialized bioremediation pro-cess. A laboratory experiment was conductedto determine the ability of Phanerochaetechrysosporium, Ceriporiopsis subvermispora,and Pleurocybella porrigens and seven white-rot fungi isolated from a native of tropicalenvironment to grow on yeast malt extractglucose (YMG) agar containing highconcentration of (I) 2,4-dichlorophenoxy aceticacid, (R) glyphosate, and (G) paraquat. Thedata indicated that P. chrysosporium couldgrow on YMG media containing 5000 ppm of(I) 2,4-D, whereas BPBPI 02/04 isolate onYMG 250 ppm of (R) glyphosate or (G)paraquat. Relative values of growth inhibitionof these fungi are 81.1; 27.8; and 50.0%respectively. Biodegradation capability ofherbicides by candidate inoculants in soil-sandmedia was also determined in greenhouseexperiment by using peanut, sorghum, corn,and Borreria alata as bio-indicators. Peanutand B. alata were found to be the bestresponsive seedlings as bio-indicator on thepresence of (I) 2,4-D herbicide in soil-sandmedia.RingkasanTeknologi bioremediasi dengan fungipelapuk putih (FPP) digunakan untuk me-reduksi sejumlah senyawa herbisida. Kegiatanpenelitian yang dilakukan di laboratoriumbertujuan untuk mengetahui kemampuan tum-buh Phanerochaete chrysosporium, Ceripo-riopsis subvermispora, dan Pleurocybellaporrigens serta tujuh isolat FPP yang diperolehdari lingkungan tropik secara in vitro padamedium agar yeast malt extract glucose(YMG) yang mengandung (I) 2,4-dikloro-fenoksi asam asetat, (R) glifosat, dan (G)parakuat konsentrasi tinggi. Dari data yangdiperoleh, diketahui bahwa Ph. chrysosporiummemiliki kemampuan tumbuh dalam mediumpadat YMG yang mengandung 5000 ppm (I)2,4-D dan isolat BPBPI 02/04 pada 250 ppm(R) glifosat dan (G) parakuat dengan nilaihambatan pertumbuhan relatif terhadap kontrol(HPR) masing-masing 81,1; 27,8; dan 50,0%.Pengujian isolat terpilih terhadap kemampuanmendegradasi herbisida di dalam mediumtanah dan pasir juga dilakukan di rumah kacadengan menggunakan kacang tanah, sorgum,jagung, dan Boreria alata sebagai bioindikator.Kacang tanah dan B. alata memberikan responterbaik terhadap keberadaan herbisida (I) 2,4-Ddi dalam medium tanah dan pasir .

Sign in / Sign up

Export Citation Format

Share Document