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 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 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.

scholarly journals New Enzymatic Assay for Glycohemoglobin

2003 ◽  
Vol 49 (2) ◽  
pp. 269-274 ◽  
Author(s):  
Ikunosuke Sakurabayashi ◽  
Tatsurou Watano ◽  
Satoshi Yonehara ◽  
Kaori Ishimaru ◽  
Kaoru Hirai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Blood Cells ◽  
Hplc Method ◽  
Enzymatic Assay ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Blood Samples ◽  
Total Hemoglobin ◽  
Patients With Diabetes ◽  
Fructosyl Amino Acid Oxidase

Abstract Background: Previous methods to measure glycohemoglobin (GHb) have been time-consuming or imprecise; we therefore developed a new enzymatic assay for GHb. Methods: Blood cells were first hemolyzed, and hemoglobin was digested with protease to yield fructosyl amino acid. Fructosyl amino acid oxidase acts on the fructosyl amino acid and generates hydrogen peroxide, which reacts with chromogens in the presence of peroxidase. Total hemoglobin was measured spectrometrically in the same reaction tube. The results were reported as the ratio of the concentrations of GHb and hemoglobin. Results: The measured values were comparable to those determined with a HPLC method and with an immunoassay in blood samples from 2854 patients with diabetes. Regression analysis for the enzymatic assay (y) vs the HPLC method (x) produced the following: r = 0.979; slope, 0.994 [95% confidence interval (CI), 0.986–1.001]; y-intercept, 0.04% (95% CI, −0.09% to 0.01%); n = 2854. For the enzymatic assay (y) vs the immunoassay (x), the regression statistics were as follows: r = 0.982; slope, 1.002 (95% CI, 0.995–1.009); y-intercept, 0% (95% CI, −0.05% to 0.05%); n = 2854. Conclusions: The values measured by the new enzymatic assay are sufficiently correlated with those of the conventional HPLC method and immunoassay, but the proposed assay for GHb is rapid and has high precision.

scholarly journals Review of Fructosyl Amino Acid Oxidase Engineering Research: A Glimpse into the Future of Hemoglobin A1c Biosensing

2009 ◽  
Vol 3 (3) ◽  
pp. 585-592 ◽  
Author(s):  
Stefano Ferri ◽  
Seungsu Kim ◽  
Wakako Tsugawa ◽  
Koji Sode
Keyword(s):  
Amino Acid ◽  
Hemoglobin A1c ◽  
Point Of Care ◽  
Detection Methods ◽  
Future Research ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Engineering Research ◽  
Glycated Proteins ◽  
Fructosyl Amino Acid Oxidase

Glycated proteins, particularly glycated hemoglobin A1c, are important markers for assessing the effectiveness of diabetes treatment. Convenient and reproducible assay systems based on the enzyme fructosyl amino acid oxidase (FAOD) have become attractive alternatives to conventional detection methods. We review the available FAOD-based assays for measurement of glycated proteins as well as the recent advances and future direction of FAOD research. Future research is expected to lead to the next generation of convenient, simple, and economical sensors for glycated protein, ideally suited for point-of-care treatment and self-monitoring applications.

Sign in / Sign up

Export Citation Format

Share Document