Overproduction and characterization of xylanase B fromAspergillus niger

2005 ◽  
Vol 51 (2) ◽  
pp. 177-183 ◽  
Author(s):  
Anthony Levasseur ◽  
Marcel Asther ◽  
Eric Record
Keyword(s):  
Expression System ◽  
Gene Promoter ◽  
Recombinant Enzyme ◽  
Glycoside Hydrolase Family ◽  
Metal Affinity ◽  
Homologous Expression ◽  
Dehydrogenase Gene ◽  
Beet Pulp ◽  
One Step ◽  
Ph Range

The xynB gene, which encodes endo-β-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg·L–1. The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 °C with stability in the pH range 4.0-7.0 and temperature up to 50 °C. Using soluble oat spelts xylan, the determined Kmand Vmaxvalues were 7.1 mg·mL–1and 3881 U·mg–1, respectively.Key words: homologous expression, glycoside hydrolase family 11, cell-wall degradation.

scholarly journals A Novel Dimeric Exoglucanase (GH5_38): Biochemical and Structural Characterisation towards its Application in Alkyl Cellobioside Synthesis

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 746 ◽  
Author(s):  
Mpho S. Mafa ◽  
Heinrich W. Dirr ◽  
Samkelo Malgas ◽  
Rui W. M. Krause ◽  
Konanani Rashamuse ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Kinetic Studies ◽  
Glycoside Hydrolase Family ◽  
Primary Alcohols ◽  
Structural Characterisation ◽  
Alkyl Glycosides ◽  
Glycoside Synthesis ◽  
One Step ◽  
Ph Range ◽  
The One

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s−1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s−1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography–mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.

scholarly journals Characterization of Fusarium oxysporum β-1,6-Galactanase, an Enzyme That Hydrolyzes Larch Wood Arabinogalactan

2007 ◽  
Vol 73 (9) ◽  
pp. 3109-3112 ◽  
Author(s):  
Tatsuji Sakamoto ◽  
Yuya Taniguchi ◽  
Shiho Suzuki ◽  
Hideshi Ihara ◽  
Haruhiko Kawasaki
Keyword(s):  
Fusarium Oxysporum ◽  
Glycoside Hydrolase ◽  
Recombinant Enzyme ◽  
Glycoside Hydrolase Family ◽  
Type Ii ◽  
High Similarity ◽  
Degrading Enzyme ◽  
Larch Wood ◽  
Hydrolase Family

ABSTRACT A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a β-1,6-galactanase that preferentially debranches β-1,6-galactobiose from the substrate.

scholarly journals TEMPO-Functionalized Nanoporous Au Nanocomposite for the Electrochemical Detection of H2O2

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Dongxiao Wen ◽  
Qianrui Liu ◽  
Ying Cui ◽  
Huaixia Yang ◽  
Jinming Kong
Keyword(s):  
Electrochemical Detection ◽  
Nanoporous Gold ◽  
Carboxyl Groups ◽  
Time Interval ◽  
Optimal Conditions ◽  
Sem Images ◽  
One Step ◽  
Ph Range ◽  
Synthesis Approach ◽  
Higher Sensitivity

A novel nanocomposite of nanoporous gold nanoparticles (np-AuNPs) functionalized with 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO) was prepared; assembled carboxyl groups on gold nanoporous nanoparticles surface were combined with TEMPO by the “bridge” of carboxylate-zirconium-carboxylate chemistry. SEM images and UV-Vis spectroscopies of np-AuNPs indicated that a safe, sustainable, and simplified one-step dealloying synthesis approach is successful. The TEMPO-np-AuNPs exhibited a good performance for the electrochemical detection of H2O2 due to its higher number of electrochemical activity sites and surface area of 7.49 m2g-1 for load bigger amount of TEMPO radicals. The TEMPO-functionalized np-AuNPs have a broad pH range and shorter response time for H2O2 catalysis verified by the response of amperometric signal under different pH and time interval. A wide linear range with a detection limit of 7.8 × 10-7 M and a higher sensitivity of 110.403 μA mM-1cm-2 were obtained for detecting H2O2 at optimal conditions.

A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs

1994 ◽  
Vol 14 (7) ◽  
pp. 4360-4372
Author(s):  
M E Carter ◽  
T Gulick ◽  
D D Moore ◽  
D P Kelly
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Receptor ◽  
Receptor Binding ◽  
Gene Promoter ◽  
Response Element ◽  
Binding Motifs ◽  
Receptor Response ◽  
Dehydrogenase Gene ◽  
Chain Acyl ◽  
Acyl Coenzyme A

We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism.

scholarly journals Degradation of methylene blue with magnetic Co-doped Fe3O4@FeOOH nanocomposites as heterogeneous catalysts of peroxymonosulfate

RSC Advances ◽  
2019 ◽  
Vol 9 (31) ◽  
pp. 17664-17673 ◽  
Author(s):  
Kai Wang ◽  
Yi Yang ◽  
Tian C. Zhang ◽  
Ying Liang ◽  
Qingguo Wang
Keyword(s):  
Methylene Blue ◽  
Hydrothermal Synthesis ◽  
Heterogeneous Catalysts ◽  
Synthesis Process ◽  
Wide Ph Range ◽  
One Step ◽  
Ph Range ◽  
Co Doped

Magnetic Co-doped Fe3O4@FeOOH nanocomposites were prepared in one step using the hydrothermal synthesis process for catalyzing peroxymonosulfate (PMS) to degrade refractory methylene blue (MB) at a wide pH range (3.0–10.0).

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+.

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system

2004 ◽  
Vol 26 (18) ◽  
pp. 1453-1458 ◽  
Author(s):  
Yoshihiro Muneta ◽  
Hidekazu Nagaya ◽  
Yu Minagawa ◽  
Chiaki Enomoto ◽  
Sayaka Matsumoto ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Baculovirus Expression ◽  
One Step ◽  
Interleukin 21

scholarly journals A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.

1994 ◽  
Vol 14 (7) ◽  
pp. 4360-4372 ◽  
Author(s):  
M E Carter ◽  
T Gulick ◽  
D D Moore ◽  
D P Kelly
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Receptor ◽  
Receptor Binding ◽  
Gene Promoter ◽  
Response Element ◽  
Binding Motifs ◽  
Receptor Response ◽  
Dehydrogenase Gene ◽  
Chain Acyl ◽  
Acyl Coenzyme A

We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism.

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