scholarly journals EXPRESSION OF GLUTARYL7-AMINOCEPHALOSPORANIC ACID ACYLASE IN ESCHERICHIA COLI BL21(DE3) AND IMMOBILIZATION OF RECOMBINANT ENZYME ON NANOPOROUS MATERIALS

2018 ◽  
Vol 54 (4A) ◽  
pp. 123
Author(s):  
Vu Thi Hanh
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Expression Vector ◽  
Nanoporous Materials ◽  
Acid Oxidase ◽  
Nano Materials ◽  
Amino Acid Oxidase ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Gla Gene

The synthesis of 7-ACA from cephalosporin C (CPC) by a two-step bioconversion using D-amino acid oxidase (DAAO) and glutaryl 7-ACA acylase (GLA) has been effectively and largely applied in pharmaceutical industry. In this study, the gene gla coding for 720-amino acid GLA from plasmid pUC57::gla was analyzed and successfully inserted into vector pET22b(+) to form expression vector pET22b(+)::gla. The newly constructed expression vector pET22b(+)::gla was cloned and then transformed into Escherichia coli BL21(DE3) to generate recombinant strain E. coli BL21(DE3)[pET22b(+)::gla]. The suitable conditions for expression of gla gene were in LB medium at 30 oC and induced by 0.4 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hours. Under the chosen culturing parameters, expression of gla gene by E. coli BL21(DE3)/[pET22b(+)::gla] resulted in a recombinant GLA (rGLA) with molecular weight of 83 kDa and catalytic activity of 2.7 U/mg of total protein. Experimental research on immobilization of rGLA onto ten nanoporous materials were showed that, SBA-15 was the best one for immobilization of rGLA, reaching activity of immobilized enzyme of 22.2 U/g matrix. Furthermore, optimal conditions of procedure for immobilizing rGLA on nanomaterials (SBA-15) were determined as follows: temperature is 25 °C, pH7.0 and immobilization time –60 minutes. Therefore the results reported in this study revealed the successfully heterologous expression of GLA in recombinant E. coli and potential immobilization of enzyme on inorganic nano-materials.

PRODUKSI REKOMBINAN SEFALOSPORIN ASILASE SEBAGAI BIOKATALIS UNTUK PRODUKSI ASAM 7-AMINOSEFALOSPORANAT

2017 ◽  
Vol 4 (1) ◽  
pp. 28 ◽  
Author(s):  
Bima Wedana Isdiyono ◽  
Dudi Hardianto ◽  
Fransiskus Xaverius Ivan
Keyword(s):  
Escherichia Coli ◽  
Induction Time ◽  
Acid Oxidase ◽  
Enzymatic Method ◽  
Amino Acid Oxidase ◽  
Two Stage ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Cephalosporin Acylase ◽  
The One

Production of Cephalosporin Acylase Recombinant as Biocatalyst for 7-Aminocephalosporanic Acid Production7-aminocephalosporanic acid (7-ACA) is a precursor for the production of semisynthetic cephalosporin derivatives. The enzymatic 7-ACA production can use two-stage and one-step enzymatic methods. Two-stage enzymatic method uses D-amino acid oxidase (DAAO) enzyme to produce glutaryl-7-aminocephalosporanic acid (GL-7-ACA) in the first stage and glutaryl-7-aminocephalosporanic acid acylase to produce 7-ACA in the second stage. The one-stage enzymatic method using cephalosporin acylase (CPC acylase) converts the CPC to 7-ACA directly. The aim of this research was to produce recombinant CPC acylase in Escherichia coli BL21(DE3). Transformantion culture E. coli BL21(DE3) was induced with concentrations of IPTG 0; 0.25; 0.5; 0.75; 1; 2 mM for 5 hours. The induction time of IPTG was determined at 0, 1, 2, 3, 4, and 5 hours. The results showed that CPC acylase produced by E. coli BL21(DE3) with optimum condition of CPC acylase production was 0.5 mM IPTG and optimal induction time of IPTG was 5 hours.Keywords: Cephalosporin, cephalosporin acylase, 7-ACA, protein expression, Escherichia coli BL21(DE3) ABSTRAKAsam 7-aminosefalosporanat (7-ACA) merupakan prekursor untuk produksi turunan sefalosporin semisintetik. Produksi 7-ACA secara enzimatik dapat menggunakan metode dua tahap dan satu tahap enzimatik. Metode enzimatik secara dua tahap menggunakan enzim asam D-amino oksidase (DAAO) untuk menghasilkan asam glutaril-7-aminosefalosporinat (GL-7-ACA) pada tahap pertama dan menggunakan asam glutaril-7-aminosefalosporinat asilase untuk menghasilkan 7-ACA pada tahap kedua. Metode enzimatik satu tahap dengan sefalosporin asilase (CPC asilase) mengubah CPC menjadi 7-ACA secara langsung. Tujuan penelitian adalah memproduksi rekombinan CPC asilase di dalam sel Escherichia coli BL21(DE3). Kultur Transforman E. coli BL21(DE3) diinduksi dengan konsentrasi IPTG 0; 0,25; 0,5; 0,75; 1; 2 mM selama 5 jam. Waktu induksi IPTG ditentukan pada 0, 1, 2, 3, 4 dan 5 jam. Hasil penelitian menunjukan bahwa CPC asilase diproduksi oleh E. coli BL21(DE3) dengan kondisi optimal produksi CPC asilase adalah konsentrasi IPTG 0,5 mM dan waktu induksi IPTG optimal adalah 5 jam.

scholarly journals Cloning, expression and purification of fructosyl amino acid oxidase (FAOX) in Escherichia Coli

2021 ◽  
Vol 11 (1) ◽  
pp. 21-29
Author(s):  
Ho Ta Giap ◽  
Phan Ngoc Han ◽  
Tran Le Duy Phuong ◽  
Phung Thi Thu Phung ◽  
Vu Van Van
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Exchange Chromatography ◽  
Cloning And Expression ◽  
Future Application ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Colony Pcr ◽  
E Coli ◽  
Fructosyl Amino Acid Oxidase

Introduction: The level of serum HbA1c is an indicator of the average blood sugar level in the last three months. HbA1c can be quantified using assays involving the enzyme fructosyl amino acid oxidase (FAOX). This study aims to produce GST-tagged FAOX-TE (GST/FAOX-TE), a thermal stable and specific variant of FAOX, for future application studies. Materials and methods: The E. coli strains DH5α and BL21 (DE3) were used as cloning and expression hosts, respectively. The FAOX-TE sequence was synthesized at IDT (US) and clonned into pGEX-4T3 vector, which was confirmed by Colony PCR. The expression was induced at 16°C, 0.5 mM IPTG in LB media containing 50 µg/ml ampicilin. The protein expression profile was analyzed by SDS-PAGE. The cell pellet was sonicated and purified by Glutathione Sepharose 4 Fast Flow (Cytiva, US). The catalytic activity of GST/FAOX-TE with fructosyl valine was determined using high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD). Results: The fusion protein was successfully expressed in Escherichia coli using the plasmid pGEX-4T3 and purified to high purity 93%. Recombinant GST/FAOX-TE was shown to be active on fructosyl valine. Conclusions: Active GST/FAOX-TE was successfully expressed in E. coli BL21 (DE3) and purified, which will be used for future development of biosensors for fructosyl valine quantification.

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals Porcine D-amino acid oxidase: Production of the biologically active enzyme in Escherichia coli

1989 ◽  
Vol 161 (2) ◽  
pp. 865-872 ◽  
Author(s):  
E. Ciccarelli ◽  
M. Massaer ◽  
J.-P. Guillaume ◽  
A. Herzog ◽  
R. Loriau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Enzyme ◽  
Biologically Active ◽  
Acid Oxidase ◽  
Amino Acid Oxidase

scholarly journals Identification, Cloning, and Expression of L-Amino Acid Oxidase from MarinePseudoalteromonassp. B3

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Zhiliang Yu ◽  
Ning Zhou ◽  
Hua Qiao ◽  
Juanping Qiu
Keyword(s):  
Amino Acid ◽  
Cloning And Expression ◽  
Expression Level ◽  
Sequence Information ◽  
Acid Oxidase ◽  
Amino Acid Residues ◽  
Amino Acid Oxidase ◽  
Calculated Molecular Mass ◽  
E Coli ◽  
Sea Area

L-amino acid oxidase (LAAO) is attracting more attentions due to its broad and important biological functions. Recently, an LAAO-producing marine microorganism (strain B3) was isolated from the intertidal zone of Dinghai sea area, China. Physiological, biochemical, and molecular identifications together with phylogenetic analysis congruously suggested that it belonged to the genusPseudoalteromonas. Therefore, it was designated asPseudoalteromonassp. B3. Its capability of LAAO production was crossly confirmed by measuring the products of H2O2, a-keto acids, andNH4+in oxidization reaction. Two rounds of PCR were performed to gain the entire B3-LAAO gene sequence of 1608 bps in length encoding for 535 amino acid residues. This deduced amino acid sequence showed 60 kDa of the calculated molecular mass, supporting the SDS-PAGE result. Like most of flavoproteins, B3-LAAO also contained two conserved typical motifs, GG-motif andβαβ-dinucleotide-binding domain motif. On the other hand, its unique substrate spectra and sequence information suggested that B3-LAAO was a novel LAAO. Our results revealed that it could be functionally expressed inE. coliBL21(DE3) using vectors, pET28b(+) and pET20b(+). However, compared with the native LAAO, the expression level of the recombinant one was relatively low, most probably due to the formation of inclusion bodies. Several solutions are currently being conducted in our lab to increase its expression level.

scholarly journals Trigonopsis variabilis d-Amino Acid Oxidase: Control of Protein Quality and Opportunities for Biocatalysis through Production in Escherichia coli

2006 ◽  
Vol 73 (1) ◽  
pp. 331-333 ◽  
Author(s):  
Iskandar Dib ◽  
Damir Stanzer ◽  
Bernd Nidetzky
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Protein Quality ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Original Activity ◽  
Toxic Activity ◽  
Enzyme Preparations ◽  
Trigonopsis Variabilis ◽  
Natural Enzyme

ABSTRACT Trigonopsis variabilis d-amino acid oxidase accounts for 35% of Escherichia coli protein when added d-methionine suppresses the toxic activity of the recombinant product. Permeabilized E. coli cells are reusable and stabilized enzyme preparations. The purified oxidase lacks the microheterogeneity of the natural enzyme. Oriented immobilization of a chimeric oxidase maintains 80% of the original activity in microparticle-bound enzymes.

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