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1.
J Basic Microbiol ; 51(2): 129-35, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21298676

ABSTRACT

L-amino acid deaminases catalyze the deamination of natural L-amino acids. Two types of L-amino acid deaminase have been identified in Proteus species. One exhibits high levels of activity toward a wide range of aliphatic and aromatic L-amino acids, typically L-phenylalanine, whereas the other acts on a relatively narrow range of basic L-amino acids, typically L-histidine. In this study, we cloned, expressed, and characterized a second amino acid deaminase, termed Pm1, from P. mirabilis KCTC 2566. Homology alignment of the deduced amino acid sequence of Pm1 demonstrated that the greatest similarity (96%) was with the L-amino acid deaminase (LAD) of P. vulgaris, and that homology with Pma was relatively low (72%). Also, similar to LAD, Pm1 was most active on L-histidine, indicating that Pm1 belongs to the second type of amino acid deaminase. In agreement with this conclusion, the V(max) and K(m) values of Pm1 were 119.7 (µg phenylpyruvic acid/mg/min) and 31.55 mM phenylalanine, respectively, values lower than those of Pma. The Pml deaminase will be very useful industrially in the preparation of commercially valuable materials including urocanic acid and α-oxoglutarate.


Subject(s)
Ammonia-Lyases/metabolism , Proteus mirabilis/enzymology , Amino Acid Sequence , Ammonia-Lyases/biosynthesis , Ammonia-Lyases/genetics , Ammonia-Lyases/isolation & purification , Base Sequence , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Molecular Sequence Data , Polymerase Chain Reaction , Proteus mirabilis/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Alignment , Substrate Specificity
2.
Appl Microbiol Biotechnol ; 89(3): 697-703, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20890600

ABSTRACT

Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD(+) and NADP(+) as a cofactor. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37 °C. The K(m) and V(max) values for propionaldehyde in the presence of NAD(+) were 1.18 mM and 0.35 U mg⁻¹, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild-type strain (from 0.18 g L⁻¹ to 0.72 g L⁻¹) under shake-flask culture conditions, and the highest titer (1.38 g L⁻¹ 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.


Subject(s)
Aldehydes/metabolism , Bacterial Proteins/metabolism , Enzymes/metabolism , Glycerol/metabolism , Lactic Acid/analogs & derivatives , Limosilactobacillus reuteri/enzymology , Bacterial Proteins/genetics , Cloning, Molecular , Coenzymes/metabolism , Enzymes/genetics , Gene Expression , Kinetics , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , Lactic Acid/metabolism , Limosilactobacillus reuteri/metabolism , NAD/metabolism , NADP/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity
3.
Protein Expr Purif ; 75(2): 211-7, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20716445

ABSTRACT

The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to > 98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60 nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine.


Subject(s)
Capsid Proteins , Chromatography , Nucleopolyhedroviruses/genetics , Nucleopolyhedroviruses/isolation & purification , Papillomavirus Vaccines , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Vaccines, Virus-Like Particle , Animals , Capsid Proteins/chemistry , Capsid Proteins/isolation & purification , Chromatography/methods , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Female , Humans , Korea , Microscopy, Electron , Nucleopolyhedroviruses/growth & development , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/isolation & purification , Papillomavirus Infections/genetics , Papillomavirus Infections/immunology , Papillomavirus Infections/virology , Papillomavirus Vaccines/chemistry , Papillomavirus Vaccines/genetics , Recombinant Proteins/genetics , Spodoptera , Ultrafiltration , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/virology , Vaccines, Virus-Like Particle/chemistry , Vaccines, Virus-Like Particle/isolation & purification , Virus Assembly
4.
Appl Microbiol Biotechnol ; 85(3): 659-66, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19626321

ABSTRACT

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.


Subject(s)
Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Glycerol/metabolism , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Propylene Glycols/metabolism , Alcohol Dehydrogenase , Biosynthetic Pathways/genetics , Escherichia coli/genetics , Fermentation , Gene Deletion , Gene Dosage , Gene Expression , Genetic Complementation Test , Isoenzymes/genetics , Isoenzymes/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid
5.
Appl Microbiol Biotechnol ; 84(3): 527-34, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19352645

ABSTRACT

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae involves the formation of various by-products, which are synthesized through the oxidative pathway. To eliminate the by-products synthesis, the oxidative branch of glycerol metabolism was inactivated by constructing two mutant strains. In one of the mutant strains, the structural genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were deleted from the chromosomal DNA, whereas in the second mutant strain dhaR, which is a putative transcription factor that activates, gene expression was deleted from the chromosomal DNA. In the resultant mutant strains lacking the dhaT gene encoding 1,3-PD oxidoreductase, which was simultaneously deleted while replacing the native promoter with the lacZ promoter, the by-product formation except for acetate was eliminated, but it still produced 1,3-PD at a lower level, which might be due to a putative oxidoreductase that catalyzes the production of 1,3-PD. The recombinant strains in which the reductive pathway was recovered produced slightly lower amount of 1,3-PD as compared to the parent strain, which might be due to the reduced activity of DhaB caused by the substitution of the promoter. However, the production yield was higher in the recombinant strain (0.57 mol mol(-1)) than the wild type Cu strain (0.47 mol mol(-1)).


Subject(s)
Gene Expression Regulation, Bacterial , Glycerol/metabolism , Klebsiella pneumoniae/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Propylene Glycols/metabolism , Sugar Alcohol Dehydrogenases/genetics , Biotechnology/methods , Gene Deletion , Genetic Engineering , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/growth & development , Mutation , Oxidation-Reduction , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Sugar Alcohol Dehydrogenases/metabolism
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