scholarly journals Efficient production of α-keto acids by immobilized E. coli -pETduet-1- Pmi LAAO in a jacketed packed-bed reactor

2019 ◽  
Vol 6 (4) ◽  
pp. 182035 ◽  
Author(s):  
Licheng Wu ◽  
Xiaolei Guo ◽  
Gaobing Wu ◽  
Pengfu Liu ◽  
Ziduo Liu
Keyword(s):  
Amino Acid ◽  
Continuous Production ◽  
Packed Bed ◽  
Alginate Beads ◽  
Packed Bed Reactor ◽  
Efficient Production ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
E Coli ◽  
Keto Acids

α-keto acids are compounds of primary interest for the fine chemical, pharmaceutical and agrochemical sectors. l -amino acid oxidases as an efficient tool are used for α-keto acids preparation in this study. Firstly, an l -amino acid oxidase ( Pmi LAAO) from Proteus mirabilis was discovered by data mining . Secondly, by gene expression vector screening, pETDuet-1- Pmi LAAO activity improved by 130%, as compared to the pET20b- Pmi LAAO. Pmi LAAO production was increased to 9.8 U ml −1 by optimized expression condition (OD 600 = 0.65, 0.45 mmol l −1 IPTG, 20 h of induction). Furthermore, The Pmi LAAO was stabile in the pH range of 4.0–9.0 and in the temperature range of 10–40°C; the optimal pH and temperature of recombinant Pmi LAAO were 6.5 and 37°C, respectively. Afterwards, in order to simplify product separation process, E. coli -pETduet-1- Pmi LAAO was immobilized in Ca-alginate beads. Continuous production of 2-oxo-3-phenylpropanoic acid was conducted in a packed-bed reactor via immobilized E. coli -pETduet-1- Pmi LAAO. Significantly, 29.66 g l −1 2-oxo-3-phenylpropanoic acid with a substrate conversion rate of 99.5% was achieved by correspondingly increasing the residence time (25 h). This method holds the potential to be used for efficiently producing pure α-keto acids.

Role of renal d-amino-acid oxidase in pharmacokinetics of d-leucine

2004 ◽  
Vol 287 (1) ◽  
pp. E160-E165 ◽  
Author(s):  
Hiroshi Hasegawa ◽  
Takehisa Matsukawa ◽  
Yoshihiko Shinohara ◽  
Ryuichi Konno ◽  
Takao Hashimoto
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Renal Mass ◽  
Acid Oxidase ◽  
Mass Reduction ◽  
Chiral Inversion ◽  
Amino Acid Oxidase ◽  
Isotope Tracer ◽  
Keto Acids

d-Amino acids are now recognized to be widely present in mammals. Renal d-amino-acid oxidase (DAO) is associated with conversion of d-amino acids to the corresponding α-keto acids, but its contribution in vivo is poorly understood because the α-keto acids and/or l-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of d-amino acids to their α-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of d-[2H7]leucine to mutant mice lacking DAO activity (ddY/DAO−) and normal mice (ddY/DAO+), elimination of d-[2H7]leucine and formation of α-[2H7]ketoisocaproic acid ([2H7]KIC) and l-[2H7]leucine in plasma were determined. The ddY/DAO− mice, in contrast to ddY/DAO+ mice, failed to convert d-[2H7]leucine to [2H7]KIC and l-[2H7]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of d-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of d-[2H7]leucine and formation of [2H7]KIC and l-[2H7]leucine. Renal mass reduction slowed down the elimination of d-[2H7]leucine. The fraction of conversion of d-[2H7]leucine to [2H7]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of d-[2H7]leucine observed in rats suggested that kidney was the principal organ responsible for converting d-leucine to KIC.

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

Inhibition ofd-amino acid oxidase by α-keto acids analogs of amino acids

1996 ◽  
Vol 18 (5) ◽  
pp. 379-382 ◽  
Author(s):  
JoséA. Moreno ◽  
Francisco J. Montes ◽  
Jacinto Catalán ◽  
Miguel A. Galán
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Keto Acids

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 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.

THE METABOLISM OF ASCARIS LUMBRICOIDES OVARIES: II. AMINO ACID METABOLISM

1955 ◽  
Vol 33 (1) ◽  
pp. 307-316 ◽  
Author(s):  
John K. Pollak ◽  
Donald Fairbairn
Keyword(s):  
Amino Acid ◽  
Reductive Amination ◽  
Acid Oxidase ◽  
Active Protein ◽  
Amino Acid Oxidase ◽  
Keto Acids ◽  
Glutamic Dehydrogenase ◽  
A Minor ◽  
Pyruvic Acids ◽  
Minor Reaction

Homogenates of ascaris ovaries contained transamination, deamination, and reductive amination systems. Alanine–glutamic and aspartic–glutamic transaminases were active, whereas weaker transaminations occurred between glycine or serine and α-ketoglutaric or pyruvic acids. Sixteen other amino acids did not participate in transamination with these keto acids. A minor reaction, however, occurred between alanine or glutamic acid and α-ketovaleric acid. No amino acid oxidase activity was detected, and deamination appeared to be limited to a weakly positive glutamic dehydrogenase, which could be coupled to the alanine–glutamic and aspartic–glutamic transaminases to form a transdeaminase system. In the presence of pyruvate and ammonium chloride reductive amination occurred and alanine and aspartic acid were synthesized. This reaction was accelerated by bicarbonate, although oxalacetate could not be substituted successfully for pyruvate in the amination system. The results of the investigation are in accord with the probability that an active protein synthesis occurs in ascaris ovaries.

scholarly journals TINJAUAN, D-ASAM AMINO OKSIDASE DARI MIKROBA: PRODUKSI DAN APLIKASI

2015 ◽  
Vol 2 (2) ◽  
pp. 88
Author(s):  
Ahmad Wibisana ◽  
Indria Puti Mustika
Keyword(s):  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Carbon Source ◽  
Flavin Adenine Dinucleotide ◽  
Acid Oxidase ◽  
Amino Acid Production ◽  
Oxidative Deamination ◽  
Amino Acid Oxidase ◽  
Keto Acids ◽  
Biotechnology Application

D-amino acid oxidase (DAAO) is a flavin adenine dinucleotide-containing enzyme that catalyzes the oxidative deamination of amino acid D-isomers with high stereospecificity, which results in α-keto acids, ammonia and hydrogen peroxide. Having high stereospecificity, DAAO is used in a variety of applications such as drug, biocatalyst, biosensor and preparation of transgenic plants. DAAO is widespread in nature, found in microorganisms to mammals. Microbial DAAO is considered more important than mammalian DAAO for biotechnology application. DAAO production in submerged fermentation is influenced by several factors, such as carbon source, nitrogen source, inducer, dissolve oxygen, temperature and pH. The influence of those factors on DAAO production by microbial origin, DAAO production by microbial recombinant, and its application in biotechnology are discussed in this review.Keywords: Enzyme, DAAO, D-amino acid, production, application ABSTRAKEnzim D-asam amino oksidase (DAAO) merupakan enzim yang mengandung Flavin Adenine Dinucleotide yang bekerja mengkatalisis reaksi oksidasi deaminasi D-asam amino dengan stereospesifisitas yang tinggi menghasilkan α-asam keto, amonia dan hidrogen peroksida. Karena mempunyai karakteristik sreteospesifisitas yang tinggi, enzim DAAO banyak digunakan untuk berbagai aplikasi seperti obat, biokatalis, biosensor dan penyiapan tanaman transgenik. Enzim ini dapat dihasilkan oleh organisme mulai dari bakteri hingga mamalia, namun untuk aplikasi dibidang bioteknologi, enzim DAAO yang berasal dari mikroorganisme dipandang lebih penting dari pada yang berasal dari mamalia. Produksi enzim dari DAAO dari mikroorganisme dalam kultur cair dipengaruhi oleh beberapa faktor seperti sumber karbon, nitrogen, senyawa penginduksi, oksigen terlarut, temperatur dan pH medium. Pengaruh dari faktor-faktor tersebut terhadap produksi enzim DAAO, produksi enzim DAAO menggunakan mikroba rekombinan serta aplikasinya dalam bidang bioteknologi dibahas dalam tinjauan.Kata Kunci: Enzim, DAAO, D-asam amino, produksi, aplikasi

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