Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8

2014 ◽  
Vol 60 (9) ◽  
pp. 585-591 ◽  
Author(s):  
Yan Long ◽  
Sheng Yang ◽  
Zhixiong Xie ◽  
Li Cheng
Keyword(s):  
Amino Acid ◽  
Candida Tropicalis ◽  
Amino Acid Identity ◽  
Phenol Hydroxylase ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Native Molecular Mass ◽  
Its Sequence Analysis

The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the Km and Vmax of recombinant phenol hydroxylase were 0.21 mmol·L–1 and 0.077 μmol·L–1·min−1, respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

1998 ◽  
Vol 64 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Ji-Quan Liu ◽  
Saeko Ito ◽  
Tohru Dairi ◽  
Nobuya Itoh ◽  
Michihiko Kataoka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Significant Loss ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequencing ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

scholarly journals Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

1998 ◽  
Vol 180 (17) ◽  
pp. 4387-4391 ◽  
Author(s):  
Sandra Achterholt ◽  
Horst Priefert ◽  
Alexander Steinbüchel
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ferulic Acid ◽  
Aldehyde Dehydrogenase ◽  
Genomic Library ◽  
Aromatic Aldehydes ◽  
Amino Acid Identity ◽  
Reading Frame ◽  
Relative Molecular Weight

ABSTRACT The coniferyl aldehyde dehydrogenase (CALDH) ofPseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD+-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α2 structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The Km values for coniferyl aldehyde and NAD+ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH2-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD+-dependent aldehyde dehydrogenases from different sources.

scholarly journals Characterization of a DivergentvanD-Type Resistance Element from the First Glycopeptide-Resistant Strain of Enterococcus faeciumIsolated in Brazil

2000 ◽  
Vol 44 (12) ◽  
pp. 3444-3446 ◽  
Author(s):  
Libera M. Dalla Costa ◽  
Peter E. Reynolds ◽  
Helena A. P. H. M. Souza ◽  
Dilair C. Souza ◽  
Marie-France I. Palepou ◽  
...  
Keyword(s):  
Amino Acid ◽  
Resistant Strain ◽  
Enterococcus Faecium ◽  
Amino Acid Identity ◽  
Open Reading Frame ◽  
Glycopeptide Resistance ◽  
Reading Frame ◽  
Acid Identity ◽  
Resistance Phenotype

ABSTRACT Enterococcus faecium 10/96A from Brazil was resistant to vancomycin (MIC, 256 μg/ml) but gave no amplification products with primers specific for known van genotypes. A 2,368-bp fragment of a van cluster contained one open reading frame encoding a peptide with 83% amino acid identity to VanHD, and a second encoding a d-alanine-d-lactate ligase with 83 to 85% identity to VanD. The divergent glycopeptide resistance phenotype was designated VanD4.

scholarly journals Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis

1996 ◽  
Vol 318 (1) ◽  
pp. 157-162 ◽  
Author(s):  
Brunella PERITO ◽  
Nerino ALLOCATI ◽  
Enrico CASALONE ◽  
Michele MASULLI ◽  
Beatrice DRAGANI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteus Mirabilis ◽  
Burkholderia Cepacia ◽  
Glutathione Transferase ◽  
Amino Acid Identity ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Promoter Sequences ◽  
E Coli ◽  
Cloned Gene

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421–425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli σ70 consensus promoter and to promoters of P. mirabiliscat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-β-d-thiogalactopyranoside and growth at 37 °C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.

Molecular cloning and characterization of chemokine-like factor 1 (CKLF1), a novel human cytokine with unique structure and potential chemotactic activity

2001 ◽  
Vol 357 (1) ◽  
pp. 127-135 ◽  
Author(s):  
Wenling HAN ◽  
Yaxin LOU ◽  
Junmin TANG ◽  
Yingmei ZHANG ◽  
Yingyu CHEN ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Inflammatory Responses ◽  
Interleukin 10 ◽  
Amino Acid Residues ◽  
Significant Similarity ◽  
Reading Frame ◽  
Small Proteins ◽  
Murine Skeletal Muscle

Cytokines are small proteins that have an essential role in the immune and inflammatory responses. The repertoire of cytokines is becoming diverse and expanding. Here we report the identification and characterization of a novel cytokine designated as chemokine-like factor 1 (CKLF1). The full-length cDNA of CKLF1 is 530bp long and a single open reading frame encoding 99 amino acid residues. CKLF1 bears no significant similarity to any other known cytokine in its amino acid sequence. Expression of CKLF1 can be partly inhibited by interleukin 10 in PHA-stimulated U937 cells. Recombinant CKLF1 is a potent chemoattractant for neutrophils, monocytes and lymphocytes; moreover, it can stimulate the proliferation of murine skeletal muscle cells. These results suggest that CKLF1 might have important roles in inflammation and in the regeneration of skeletal muscle.

scholarly journals Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme

2002 ◽  
Vol 366 (3) ◽  
pp. 817-824 ◽  
Author(s):  
Jianxia GUO ◽  
Ludwika ZIMNIAK ◽  
Piotr ZIMNIAK ◽  
John L. ORCHARD ◽  
Shivendra V. SINGH
Keyword(s):  
Small Intestine ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glutathione Transferase ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Amino Acid Residues ◽  
Reverse Transcription Pcr ◽  
Mouse Small Intestine

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1—mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67—78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.

scholarly journals groEL Encodes a Highly Antigenic Protein in Burkholderia pseudomallei

2001 ◽  
Vol 8 (4) ◽  
pp. 832-836 ◽  
Author(s):  
Patrick C. Y. Woo ◽  
Patricia K. L. Leung ◽  
Samson S. Y. Wong ◽  
Pak-Leung Ho ◽  
Kwok-Yung Yuen
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Burkholderia Cepacia ◽  
Burkholderia Pseudomallei ◽  
Amino Acid Identity ◽  
Local Alignment ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Acid Identity ◽  
Burkholderia Vietnamiensis

ABSTRACT No recombinant protein is available for serodiagnosis of melioidosis. In this study, we report the cloning of thegroEL gene, which encodes an immunogenic protein ofBurkholderia pseudomallei. Bidirectional DNA sequencing ofgroEL revealed that the gene contained a single open reading frame encoding 546 amino acid residues with a predicted molecular mass of 57.1 kDa. Basic Local Alignment Search Tool analysis showed that the putative protein encoded by groEL is homologous to the chaperonins encoded by the groEL genes of other bacteria. It has 98% amino acid identity with the GroEL ofBurkholderia cepacia, 98% amino acid identity with the GroEL of Burkholderia vietnamiensis, and 82% amino acid identity with the GroEL of Bordetella pertussis. Furthermore, it was observed that patients with melioidosis develop a strong antibody response against GroEL, suggesting that the recombinant protein and its monoclonal antibody may be useful for serodiagnosis in patients with melioidosis and that the protein may represent a good cell surface target for host humoral immunity. Further studies in these directions would be warranted.

Cloning, Expression, and Characterization of Mouse Tissue Factor Pathway Inhibitor (TFPI)

1998 ◽  
Vol 79 (02) ◽  
pp. 306-309 ◽  
Author(s):  
Dougald Monroe ◽  
Julie Oliver ◽  
Darla Liles ◽  
Harold Roberts ◽  
Jen-Yea Chang
Keyword(s):  
Amino Acid ◽  
Tissue Factor ◽  
Signal Peptide ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Mouse Tissue ◽  
Amino Acid Residues ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

scholarly journals Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhongying Wang ◽  
Qixuan Wang ◽  
Hao Wu ◽  
Zhiwu Huang
Keyword(s):  
Amino Acid ◽  
Inner Ear ◽  
Hair Cells ◽  
Rana Catesbeiana ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Rna Seq ◽  
American Bullfrog ◽  
Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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