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 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 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 Induced immune response of Escherichia coli BL21 expressing recombinant MSP1a and MSP1b proteins of Anaplasma marginale

2009 ◽  
Vol 52 (spe) ◽  
pp. 113-120 ◽  
Author(s):  
Katia Tamekuni ◽  
Marilda Carlos Vidotto ◽  
Samuel Rodrigues Felix ◽  
Michelle Igarashi ◽  
João Luis Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Response ◽  
Western Blot ◽  
Molecular Mass ◽  
Outer Membrane ◽  
Humoral Response ◽  
Anaplasma Marginale ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Molecular Masses

This work aims to evaluate the potential of immunization with E. coli BL21 expressing the recombinant rMSP1a and rMSP1b proteins of Anaplasma marginale. E. coli BL21 was transformed with recombinant plasmids pET102/msp1α and pET101/msp1β, and rMSP1a and rMSP1b were expressed after induction by IPTG. BALB/c mice were vaccinated with formolized BL21/rMSP1a and BL21/rMSP1b, and the production in mice sera of whole IgG was determined by ELISA. The mice immunized with BL21/rMSP1a showed a better humoral response for whole IgG when compared to the mice immunized with BL21/rMSP1b; these mice exhibited a small response after the second vaccination. Sera of mice immunized with BL21/rMSP1a reacted via western blot with BL21 and rMSP1a, with molecular masses varying from 70 to 105 kDa. Sera of mice immunized with BL21/rMSP1b reacted with BL21 and rMSP1b with a molecular mass of 100 kDa. These results demonstrate that BL21 containing rMSP1a and rMSP1b in the outer membrane were able to produce an immune response in mice, reinforcing its use in vaccine models against bovine anaplasmosis.

Coexpression of Vitreoscilla Hemoglobin Reduces the Toxic Effect of Expression of D-Amino Acid Oxidase in E. coli

2004 ◽  
Vol 20 (5) ◽  
pp. 1359-1365 ◽  
Author(s):  
L.-J. Chien ◽  
J.-M. Wu ◽  
I.-C. Kuan ◽  
C.-K. Lee
Keyword(s):  
Amino Acid ◽  
Toxic Effect ◽  
Vitreoscilla Hemoglobin ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
E Coli

scholarly journals Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli

1987 ◽  
Vol 247 (1) ◽  
pp. 195-199 ◽  
Author(s):  
J L Schrimsher ◽  
K Rose ◽  
M G Simona ◽  
P Wingfield
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Natural Material ◽  
Animal Cells ◽  
Colony Stimulating Factors ◽  
E Coli ◽  
Macrophage Colony ◽  
The One ◽  
Human And Mouse

Human and mouse granulocyte-macrophage-colony-stimulating factors (hGM-CSF and mGM-CSF, respectively), isolated from Escherichia coli cells expressing the corresponding human and mouse genes, have been characterized. The observed properties of the proteins have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural GM-CSFs. The purified E. coli-derived proteins were found to have the expected molecular masses, amino acid compositions and N- and C-terminal amino acid sequences. The finding of 70-90% unprocessed N-terminal methionine for both proteins is discussed. The four Cys residues were found to be involved in two intramolecular disulphide bonds, linking the first and third, and second and fourth Cys residues. This disulphide bond arrangement is probably the one existing in natural material, since, although not glycosylated, both E. coli-derived proteins showed biological activity (colony stimulating assay for hGM-CSF, and cell proliferation assay for mGM-CSF) comparable with that reported for the respective proteins purified from animal cells.

scholarly journals The Effect of Various Concentrations of Ethanol Extract of the Leaves of Paederia foetida L. on the Growth of Escherichia Coli Bacteria

2021 ◽  
Vol 11 (6) ◽  
pp. 61-67
Author(s):  
Hertina Silaban
Keyword(s):  
Escherichia Coli ◽  
Ethanol Extract ◽  
Inhibition Zone ◽  
Positive Control ◽  
Negative Control ◽  
Rare Plants ◽  
E Coli ◽  
Paederia Foetida ◽  
Escherichia Coli Bacteria ◽  
The One

Bacterial infection of Escherichia coli (E. coli) as the cause of gastrointestinal disorders in humans has increased their prevalence. Treatment using natural ingredients can be a choice of therapy because of the minimal side effects. One of the rare plants believed by the community as an antibacterial is stinking vin’e known as the ‘leaf fart’. The purpose of this research is for knowing the activity of the ethanol extract of Paederia foetida L can affect the growth of E.coli. The serial diffusion disc method is being used as the antibacterial activity test. The concentration  of this extract are 10%, 20%, 40%, 80%, 100% with positive control (ciprofloxacin) and negative control (aqua dest). The inhibition zone diameter characterized the effect of Extract on bacterial growth were 6.16 mm of the concentration 10%, 6.667 mm of the concentration 20%, 7.10 mm of the concentration 40 %, 7.78 mm of the concentration 80%, and 10.03 mm of the concentration 100%. As for the negative control has no effect. The study stated that the higher concentration of antibacterial agent used, the greater the inhibition zone formed. Based on the result of the analysis of the data by using the One-Way ANOVA Test showed a probability value (p) = 0.000 or value (p) < 0.05, that H0 is rejected and H1 is accepted. The conclusion is that the Extract of stinking vin’e has an antibacterial effect on the growth of E.coli. Keywords: Antibacterial, E.coli, Extract of  Sembukan leaf

A Nursery Outbreak of Multiple-Aminoglycoside-Resistant Escherichia coli

1984 ◽  
Vol 5 (11) ◽  
pp. 519-524 ◽  
Author(s):  
Robert R Gaynes ◽  
Diane Simpson ◽  
Sally A. Reeves ◽  
Robert C. Noble ◽  
Clyde Thornsberry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Neonatal Intensive Care ◽  
Control Measures ◽  
Effective Control ◽  
Blood Isolate ◽  
E Coli ◽  
Feeding Duration ◽  
Matched Case ◽  
Continuous Feeding ◽  
The One

AbstractIn the neonatal intensive care unit (NICU) of one hospital, 16 infants became colonized or infected with multiply-resistant Escherichia coli (MR-E.coli) over an 8-month period. Isolates were obtained from blood, urine, and sputum of three patients and from rectal surveillance cultures of 13 patients. The one patient with the blood isolate died. A matched case-control study identified continuous feeding (nine of 16 cases vs. one of 16 controls, p ≤ 0.001) and receipt of aminoglycosides (p ≤ 0.03) as risk factors. For case-babies not exposed to continuous feeding, duration of bolus feeding was significantly greater than for their controls (cases, 22 days; controls, 7 days; p ≤ 0.02). All 16 isolates were the same serotype and were resistant to amikacin, tobramycin, kanamycin, and gentamicin. The epidemiologic investigation suggested that MR-E. coli may have spread from person-to-person on the hands of personnel and that MR-E. coli persisted in the NICU for 8 months until effective control measures were instituted.

A survival study of Escherichia coli in a south African river using membrane diffusion chambers

1995 ◽  
Vol 31 (5-6) ◽  
pp. 185-188 ◽  
Author(s):  
C. M. E. de Wet ◽  
S. N. Venter ◽  
N. Rodda ◽  
R. Kfir ◽  
M. C. Steynberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
South African ◽  
The Other ◽  
Membrane Diffusion ◽  
Diffusion Chambers ◽  
E Coli ◽  
Significant Difference ◽  
Survival Pattern ◽  
The One ◽  
Different Parts

Studies to describe the survival of Escherichia coli were performed at two sites in a river. The one site was dominated by domestic discharge and the other by industrial inputs. E coli suspensions within membrane diffusion chambers were immersed in the river at the selected sites. An identical chamber was submerged in river water in the laboratory as a comparison. Two test runs were performed, one during winter (July) and one during summer (December). Samples to determine the survival of E coli was taken on a scheduled basis. Results obtained showed no significant difference between the survival pattern of E coli as determined during the summer and winter periods or in the different parts of the river. The same survival pattern was observed for the studies performed in the laboratory.

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