scholarly journals Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC

2005 ◽  
Vol 393 (1) ◽  
pp. 219-226 ◽  
Author(s):  
Eitaro Matsumura ◽  
Masashi Sakai ◽  
Katsuaki Hayashi ◽  
Shuichiro Murakami ◽  
Shinji Takenaka ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Gene Cluster ◽  
Specific Activity ◽  
Regulatory Protein ◽  
Constitutive Expression ◽  
Amino Acid Sequences ◽  
Upstream Region ◽  
Rhodococcus Species ◽  
Cat Gene ◽  
Rhodococcus Sp

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.

scholarly journals Isolation of a mouse theta glutathione S-transferase active with methylene chloride

1996 ◽  
Vol 314 (2) ◽  
pp. 445-448 ◽  
Author(s):  
Guy W. MAINWARING ◽  
John NASH ◽  
Matthew DAVIDSON ◽  
Trevor GREEN
Keyword(s):  
Molecular Mass ◽  
Mouse Liver ◽  
Methylene Chloride ◽  
Specific Activity ◽  
Active Form ◽  
Amino Acid Sequences ◽  
Terminal Sequence ◽  
Limited Evidence ◽  
Glutathione S Transferase ◽  
Similar Activity

A glutathione S-transferase metabolizing methylene chloride has been isolated from mouse liver using a variety of chromatographic methods. N-terminal and internal amino acid sequences show that the enzyme, designated GST T1-1*, is closely related to the rat Theta-class GST 5-5. The mouse enzyme, molecular mass 25000 Da, has been isolated to homogeneity in active form with an approximate yield of 2% of the cytosolic activity towards methylene chloride. GST T1-1* has a specific activity of about 5.5 μmol/min per mg of protein whereas the rat GST 5-5 is reported to have a specific activity of about 11 μmol/min per mg of protein [Meyer, Coles, Pemble, Gilmore, Fraser and Ketterer (1991) Biochem. J. 274, 409–414], demonstrating that both the rat and mouse enzymes have similar activity with this substrate. Limited evidence was obtained for a second enzyme, with a similar molecular mass (25400 Da), which had an N-terminal sequence identical to that of rat GST 12-12. This protein, which was sequenced from a band on a gel, was extremely labile and could not be isolated to homogeneity. The partially purified enzyme was not active with methylene chloride.

scholarly journals Identification and Utility of FdmR1 as a Streptomyces Antibiotic Regulatory Protein Activator for Fredericamycin Production in Streptomyces griseus ATCC 49344 and Heterologous Hosts

2008 ◽  
Vol 190 (16) ◽  
pp. 5587-5596 ◽  
Author(s):  
Yihua Chen ◽  
Evelyn Wendt-Pienkowski ◽  
Ben Shen
Keyword(s):  
Gene Cluster ◽  
Regulatory Protein ◽  
Constitutive Expression ◽  
Streptomyces Griseus ◽  
Biosynthetic Gene Cluster ◽  
Positive Control ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Protein Activator ◽  
Biosynthetic Machinery

ABSTRACT The fredericamycin (FDM) A biosynthetic gene cluster, cloned previously from Streptomyces griseus ATCC 49344, contains three putative regulatory genes, fdmR, fdmR1, and fdmR2. Their deduced gene products show high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family (FdmR1) or to MarR-like regulators (FdmR and FdmR2). Here we provide experimental data supporting FdmR1 as a SARP-type activator. Inactivation of fdmR1 abolished FDM biosynthesis, and FDM production could be restored to the fdmR1::aac(3)IV mutant by expressing fdmR1 in trans. Reverse transcription-PCR transcriptional analyses revealed that up to 26 of the 28 genes within the fdm gene cluster, with the exception of fdmR and fdmT2, were under the positive control of FdmR1, directly or indirectly. Overexpression of fdmR1 in S. griseus improved the FDM titer 5.6-fold (to about 1.36 g/liter) relative to that of wild-type S. griseus. Cloning of the complete fdm cluster into an integrative plasmid and subsequent expression in heterologous hosts revealed that considerable amounts of FDMs could be produced in Streptomyces albus but not in Streptomyces lividans. However, the S. lividans host could be engineered to produce FDMs via constitutive expression of fdmR1; FDM production in S. lividans could be enhanced further by overexpressing fdmC, encoding a putative ketoreductase, concomitantly with fdmR1. Taken together, these studies demonstrate the viability of engineering FDM biosynthesis and improving FDM titers in both the native producer S. griseus and heterologous hosts, such as S. albus and S. lividans. The approach taken capitalizes on FdmR1, a key activator of the FDM biosynthetic machinery.

scholarly journals An α-l-Arabinofuranosidase fromTrichoderma reesei Containing a Noncatalytic Xylan-Binding Domain

1999 ◽  
Vol 65 (9) ◽  
pp. 3964-3968 ◽  
Author(s):  
Masahiro Nogawa ◽  
Kenji Yatsui ◽  
Akiko Tomioka ◽  
Hirofumi Okada ◽  
Yasushi Morikawa
Keyword(s):  
Molecular Mass ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Crystalline Cellulose ◽  
Terminal Amino Acid ◽  
Catalytic Constant ◽  
Terminal Amino ◽  
Oat Spelt

ABSTRACT l-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced α-l-arabinofuranosidase (α-AF) activity. An α-AF induced by l-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported α-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported α-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters forp-nitrophenyl-α-l-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported α-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.

scholarly journals Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

1998 ◽  
Vol 64 (9) ◽  
pp. 3282-3289 ◽  
Author(s):  
Kazuaki Igarashi ◽  
Yuji Hatada ◽  
Hiroshi Hagihara ◽  
Katsuhisa Saeki ◽  
Mikio Takaiwa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Amino Acid Sequences ◽  
Ph Optimum ◽  
Calculated Molecular Mass

ABSTRACT A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein−1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)8 barrel motif in domain A.

scholarly journals Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 453
Author(s):  
Sebastian Estrada-Gómez ◽  
Leidy Johana Vargas-Muñoz ◽  
Cesar Segura Latorre ◽  
Monica Maria Saldarriaga-Cordoba ◽  
Claudia Marcela Arenas-Gómez
Keyword(s):  
Enzymatic Activity ◽  
Molecular Mass ◽  
Evolutionary Relationship ◽  
Venom Gland ◽  
Duplication Event ◽  
High Molecular Mass ◽  
Amino Acid Sequences ◽  
Secretory Proteins ◽  
Phospholipases A2 ◽  
Kunitz Type

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Ecdysterone regulatory elements function as both transcriptional activators and repressors

1991 ◽  
Vol 11 (4) ◽  
pp. 1846-1853
Author(s):  
L Dobens ◽  
K Rudolph ◽  
E M Berger
Keyword(s):  
Regulatory Element ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Transcriptional Activators ◽  
Reporter Plasmid ◽  
Bacterial Gene ◽  
Simplex Virus ◽  
Very High ◽  
Cat Gene ◽  
Cat Expression

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.

scholarly journals [Phosphotyrosine]protein phosphatase in rat brain. A major [phosphotyrosine]protein phosphatase is a 23 kDa protein distinct from acid phosphatase

1986 ◽  
Vol 239 (1) ◽  
pp. 155-162 ◽  
Author(s):  
M Okada ◽  
K Owada ◽  
H Nakagawa
Keyword(s):  
Acid Phosphatase ◽  
Molecular Mass ◽  
Rat Brain ◽  
Column Chromatography ◽  
Protein Phosphatase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Sodium Vanadate ◽  
Nitrophenyl Phosphate ◽  
Phosphotyrosine Protein Phosphatase

A [phosphotyrosine]protein phosphatase (PTPPase) was purified almost to homogeneity from rat brain, with [32P]p130gag-fps, an oncogene product of Fujinami sarcoma virus, as substrate. The characteristics of the purified preparation of PTPPase were as follows: the enzyme was a monomer with a molecular mass of 23 kDa; its optimum pH was 5.0-5.5; its activity was not dependent on bivalent cations; its activity was strongly inhibited by sodium vanadate, but was not inhibited by ZnCl2, L(+)-tartrate or NaF; it catalysed the dephosphorylation of [32P]p130gag-fps, [[32P]Tyr]casein, p-nitrophenyl phosphate and L-phosphotyrosine, but did not hydrolyse [[32P]Ser]tubulin, L-phosphoserine, DL-phosphothreonine, 5′-AMP, 2′-AMP or beta-glycerophosphate significantly. During the purification, most of the PTPPase activity was recovered in distinct fractions from those of conventional low-molecular-mass acid phosphatase (APase), which was reported to be a major PTPPase [Chernoff & Li (1985) Arch. Biochem. Biophys. 240, 135-145], from DE-52 DEAE-cellulose column chromatography, and those two enzymes could be completely separated by Sephadex G-75 column chromatography. APase also showed PTPPase activity with [32P]p130gag-fps, but the specific activity was lower than that of PTPPase with molecular mass of 23 kDa, and it was not sensitive to sodium vanadate. These findings suggested that PTPPase (23 kDa) was the major and specific PTPPase in the cell.

scholarly journals Genetic Characterization and Immunogenicity of Coli Surface Antigen 4 from Enterotoxigenic Escherichia coli when It Is Expressed in a Shigella Live-Vector Strain

2003 ◽  
Vol 71 (3) ◽  
pp. 1352-1360 ◽  
Author(s):  
Zeev Altboum ◽  
Myron M. Levine ◽  
James E. Galen ◽  
Eileen M. Barry
Keyword(s):  
Escherichia Coli ◽  
Shigella Flexneri ◽  
Regulatory Protein ◽  
Electron Microscopic Examination ◽  
Amino Acid Sequences ◽  
Enterotoxigenic Escherichia Coli ◽  
Electron Microscopic ◽  
Bacterial Surface ◽  
Fimbrial Subunit ◽  
Mucosal Antibody

ABSTRACT The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D′, were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD′). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5α and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.

scholarly journals Complete Genome Sequence of the Polychlorinated Biphenyl Degrader Rhodococcus sp. WB1

2016 ◽  
Vol 4 (5) ◽  
Author(s):  
Yuxin Xu ◽  
Man Yu ◽  
Alin Shen
Keyword(s):  
Gene Cluster ◽  
Genome Sequence ◽  
Contaminated Soil ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Polychlorinated Biphenyl ◽  
Xenobiotic Metabolism ◽  
Rhodococcus Sp

Rhodococcus sp. WB1 is a polychlorinated biphenyl degrader which was isolated from contaminated soil in Zhejiang, China. Here, we present the complete genome sequence. The analysis of this genome indicated that a biphenyl-degrading gene cluster and several xenobiotic metabolism pathways are harbored.

Purification and Partial Amino Acid Sequence of Pentocin 31-1, an Anti-Listeria Bacteriocin Produced by Lactobacillus pentosus 31-1

2009 ◽  
Vol 72 (12) ◽  
pp. 2524-2529 ◽  
Author(s):  
JINLAN ZHANG ◽  
GUORONG LIU ◽  
NAN SHANG ◽  
WANPENG CHENG ◽  
SHANGWU CHEN ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mass Spectrometry Analysis ◽  
Gel Chromatography ◽  
Original Activity ◽  
Lactobacillus Pentosus ◽  
Spectrometry Analysis

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class IIa of bacteriocins.

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