| 1. |
Zotzel J,
Pasternack R,
Pelzer C,
Ziegert D,
Mainusch M,
Fuchsbauer HL,
( 2003 ) Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step. PMID : 14519127 : DOI : 10.1046/j.1432-1033.2003.03809.x Abstract >>
Transglutaminase (TGase) from Streptomyces mobaraensis is secreted as a precursor protein which is completely activated by the endoprotease TAMEP, a member of the M4 protease family [Zotzel, J., Keller, P. & Fuchsbauer, H.-L. (2003) Eur. J. Biochem. 270, 3214-3222]. In contrast with the mature enzyme, TAMEP-activated TGase exhibits an additional N-terminal tetrapeptide (Phe-Arg-Ala-Pro) suggesting truncation, at least, by a second protease. We have now isolated from the culture broth of submerged colonies a tripeptidyl aminopeptidase (SM-TAP) that is able to remove the remaining tetrapeptide. The 53-kDa peptidase was purified by ion-exchange and phenyl-Sepharose chromatography and subsequently characterized. Its proteolytic activity was highest against chromophoric tripeptides at pH 7 in the presence of 2 mm CaCl2. EDTA and EGTA (10 mm) both diminished the proteolytic activity by half. Complete inhibition was only achieved with 1 mm phenylmethanesulfonyl fluoride, suggesting that SM-TAP is a serine protease. Alignment of the N-terminal sequence confirmed its close relation to the Streptomyces TAPs. That removal of Phe-Arg-Ala-Pro from TAMEP-activated TGase by SM-TAP occurs in a single step was confirmed by experiments using various TGase fragments and synthetic peptides. SM-TAP was also capable of generating the mature N-terminus by cleavage of RAP-TGase. However, AP-TGase remained unchanged. As SM-TAP activity against chromophoric amino acids such as Pro-pNA or Phe-pNA could not be detected, the tetrapeptide of TAMEP-activated TGase must be removed without formation of an intermediate.
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2. |
Hatano K,
Nishii T,
Kasai H,
( 2003 ) Taxonomic re-evaluation of whorl-forming Streptomyces (formerly Streptoverticillium) species by using phenotypes, DNA-DNA hybridization and sequences of gyrB, and proposal of Streptomyces luteireticuli (ex Katoh and Arai 1957) corrig., sp. nov., nom. rev. PMID : 13130042 : DOI : 10.1099/ijs.0.02238-0 DOI : 10.1099/ijs.0.02238-0 Abstract >>
The taxonomic status of 64 strains of whorl-forming Streptomyces (formerly Streptoverticillium) species was re-evaluated and strains were reclassified on the basis of their phenotypes, DNA-DNA hybridization data and partial sequences of gyrB, the structural gene of the B subunit of DNA gyrase. These strains, which consisted of 46 species and eight subspecies with validly published names and 13 species whose names have not been validly published [including 10 strains examined by the International Streptomyces Project (ISP)], were divided into two groups, namely typical and atypical whorl-forming Streptomyces species, based on their phenotypes and gyrB gene sequences. The typical whorl-forming species (59 strains) were divided into six major clusters of three or more species, seven minor clusters of two species and five single-member clusters, based on the threshold value of 97 % gyrB sequence similarity. Major clusters were typified by Streptomyces abikoensis, Streptomyces cinnamoneus, Streptomyces distallicus, Streptomyces griseocarneus, Streptomyces hiroshimensis and Streptomyces netropsis. Phenotypically, members of each cluster resembled each other closely except for the S. distallicus cluster, which was divided phenotypically into the S. distallicus and Streptomyces stramineus subclusters, and the S. netropsis cluster, which was divided into the S. netropsis and Streptomyces eurocidicus subclusters. Strains in each minor cluster closely resembled each other phenotypically. DNA-DNA relatedness between the representative species and others in each major cluster and/or subcluster, and between strains in the minor clusters, was >70 %, indicating that the major clusters and/or subclusters and the minor clusters each comprise a single species. It was concluded that 59 strains of typical whorl-forming Streptomyces species consisted of the following 18 species, including subjective synonym(s): S. abikoensis, Streptomyces ardus, Streptomyces blastmyceticus, S. cinnamoneus, S. eurocidicus, S. griseocarneus, S. hiroshimensis, Streptomyces lilacinus, 'Streptomyces luteoreticuli', Streptomyces luteosporeus, Streptomyces mashuensis, Streptomyces mobaraensis, Streptomyces morookaense, S. netropsis, Streptomyces orinoci, S. stramineus, Streptomyces thioluteus and Streptomyces viridiflavus.
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3. |
Zotzel J,
Keller P,
Fuchsbauer HL,
( 2003 ) Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease. PMID : 12869197 : DOI : 10.1046/j.1432-1033.2003.03703.x Abstract >>
Streptomyces mobaraensis secretes a Ca2+-independent transglutaminase (TGase) that is activated by removing an N-terminal peptide from a precursor protein during submerged culture in a complex medium [Pasternack, R., Dorsch, S., Otterbach, J. T., Robenek, I. R., Wolf, S. & Fuchsbauer, H.-L. (1998) Eur. J. Biochem. 257, 570-576]. However, an activating protease could not be identified, probably because of the presence of a 14-kDa protein (P14) belonging to the Streptomyces subtilisin inhibitor family. In contrast, if the microorganism was allowed to grow on a minimal medium, several soluble proteases were extracted, among them the TGase-activating protease (TAMEP). TAMEP was purified by sequential chromatography on DEAE- and Arg-Sepharose and used to determine the cleavage site of TGase. It was clearly shown that the peptide bond between Phe(-4) and Ser(-5) was hydrolyzed, indicating that at least one additional peptidase is necessary to complete TGase processing, even if TAMEP cleavage was sufficient to obtain total activity. Sequence analysis from the N-terminus of TAMEP revealed the close relationship to a zinc endo-protease from S. griseus. The S. griseus protease differs from other members of the M4 protease family, such as thermolysin, in that it may be inhibited by the Streptomyces subtilisin inhibitor. P14 likewise inhibits TAMEP in approximately equimolar concentrations, suggesting its important role in regulating TGase activity.
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4. |
Kikuchi Y,
Date M,
Yokoyama K,
Umezawa Y,
Matsui H,
( 2003 ) Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus. PMID : 12514016 : DOI : 10.1128/aem.69.1.358-366.2003 PMC : PMC152470 Abstract >>
The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C. glutamicum but had no enzymatic activity. However, when the plasmid carrying the S. mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that C. glutamicum has potential as a host for industrial-scale protein production.
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5. |
Kashiwagi T,
Yokoyama K,
Ishikawa K,
Ono K,
Ejima D,
Matsui H,
Suzuki E,
( 2002 ) Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. PMID : 12221081 : DOI : 10.1074/jbc.M203933200 Abstract >>
The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 A resolution. The protein folds into a plate-like shape, and has one deep cleft at the edge of the molecule. Its overall structure is completely different from that of the factor XIII-like transglutaminase, which possesses a cysteine protease-like catalytic triad. The catalytic residue, Cys(64), exists at the bottom of the cleft. Asp(255) resides at the position nearest to Cys(64) and is also adjacent to His(274). Interestingly, Cys(64), Asp(255), and His(274) superimpose well on the catalytic triad "Cys-His-Asp" of the factor XIII-like transglutaminase, in this order. The secondary structure frameworks around these residues are also similar to each other. These results imply that both transglutaminases are related by convergent evolution; however, the microbial transglutaminase has developed a novel catalytic mechanism specialized for the cross-linking reaction. The structure accounts well for the catalytic mechanism, in which Asp(255) is considered to be enzymatically essential, as well as for the causes of the higher reaction rate, the broader substrate specificity, and the lower deamidation activity of this enzyme.
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6. |
Sánchez C,
Du L,
Edwards DJ,
Toney MD,
Shen B,
( 2001 ) Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin. PMID : 11451672 : Abstract >>
Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.
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7. |
Zhang D,
Koreishi M,
Imanaka H,
Imamura K,
Nakanishi K,
( 2007 ) Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis. PMID : 17289203 : DOI : 10.1016/j.jbiotec.2006.12.017 Abstract >>
We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-l-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an alpha subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively.
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8. |
Fu RY,
Chen J,
Li Y,
( 2005 ) Heterologous leaky production of transglutaminase in Lactococcus lactis significantly enhances the growth performance of the host. PMID : 16332889 : DOI : 10.1128/AEM.71.12.8911-8919.2005 PMC : PMC1317339 Abstract >>
This study describes a novel strategy to improve the growth performance of Lactococcus lactis by heterologous production of food-grade transglutaminase. The mtg gene from Streptoverticillium mobaraense that encodes the transglutaminase mature protein was cloned into a nisin-inducible expression vector and transformed into L. lactis subsp. cremoris NZ9000. The leaky expression of the mtg gene from the nisA promoter resulted in ammonia formation and carbon flux redistribution at the pyruvate branch. As a consequence, medium acidification was lessened and energy utilization was improved. This led to significantly higher biomass production under aerobic conditions and particularly under non-pH-controlled conditions (up to a 12-fold increase). The results presented here provide a novel way to enhance the growth yield of L. lactis, which is an important step for the purposes of producing proteins of commercial interest using L. lactis as a host.
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9. |
Umezawa Y,
Yokoyama K,
Kikuchi Y,
Date M,
Ito K,
Yoshimoto T,
Matsui H,
( 2004 ) Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning. PMID : 15598885 : DOI : 10.1093/jb/mvh129 Abstract >>
The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.
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10. |
Wang H,
Liu N,
Xi L,
Rong X,
Ruan J,
Huang Y,
( 2011 ) Genetic screening strategy for rapid access to polyether ionophore producers and products in actinomycetes. PMID : 21421776 : DOI : 10.1128/AEM.02915-10 PMC : PMC3126441 Abstract >>
Polyether ionophores are a unique class of polyketides with broad-spectrum activity and outstanding potency for the control of drug-resistant bacteria and parasites, and they are produced exclusively by actinomycetes. A special epoxidase gene encoding a critical tailoring enzyme involved in the biosynthesis of these compounds has been found in all five of the complete gene clusters of polyether ionophores published so far. To detect potential producer strains of these antibiotics, a pair of degenerate primers was designed according to the conserved regions of the five known polyether epoxidases. A total of 44 putative polyether epoxidase gene-positive strains were obtained by the PCR-based screening of 1,068 actinomycetes isolated from eight different habitats and 236 reference strains encompassing eight major families of Actinomycetales. The isolates spanned a wide taxonomic diversity based on 16S rRNA gene analysis, and actinomycetes isolated from acidic soils seemed to be a promising source of polyether ionophores. Four genera were detected to contain putative polyether epoxidases, including Micromonospora, which has not previously been reported to produce polyether ionophores. The designed primers also detected putative epoxidase genes from diverse known producer strains that produce polyether ionophores unrelated to the five published gene clusters. Moreover, phylogenetic and chemical analyses showed a strong correlation between the sequence of polyether epoxidases and the structure of encoded polyethers. Thirteen positive isolates were proven to be polyether ionophore producers as expected, and two new analogues were found. These results demonstrate the feasibility of using this epoxidase gene screening strategy to aid the rapid identification of known products and the discovery of unknown polyethers in actinomycetes.
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11. |
Sarafeddinov A,
Arif A,
Peters A,
Fuchsbauer HL,
( 2011 ) A novel transglutaminase substrate from Streptomyces mobaraensis inhibiting papain-like cysteine proteases. PMID : 21715969 : Abstract >>
Transglutaminase from Streptomyces mobaraensis is an enzyme of unknown function that cross-links proteins to high molecular weight aggregates. Previously, we characterized two intrinsic transglutaminase substrates with inactivating activities against subtilisin and dispase. This report now describes a novel substrate that inhibits papain, bromelain, and trypsin. Papain was the most sensitive protease; thus, the protein was designated Streptomyces papain inhibitor (SPI). To avoid transglutaminase-mediated glutamine deamidation during culture, SPI was produced by Streptomyces mobaraensis at various growth temperatures. The best results were achieved by culturing for 30-50 h at 42 degrees C, which yielded high SPI concentrations and negligibly small amounts of mature transglutaminase. Transglutaminasespecific biotinylation displayed largely unmodified glutamine and lysine residues. In contrast, purified SPI from the 28 degrees C culture lost the potential to be cross-linked, but exhibited higher inhibitory activity as indicated by a significantly lower Ki (60 nM vs. 140 nM). Despite similarities in molecular mass (12 kDa) and high thermostability, SPI exhibits clear differences in comparison with all members of the wellknown family of Streptomyces subtilisin inhibitors. The neutral protein (pI of 7.3) shares sequence homology with a putative protein from Streptomyces lavendulae, whose conformation is most likely stabilized by two disulfide bridges. However, cysteine residues are not localized in the typical regions of subtilisin inhibitors. SPI and the formerly characterized dispase-inactivating substrate are unique proteins of distinct Streptomycetes such as Streptomyces mobaraensis. Along with the subtilisin inhibitory protein, they could play a crucial role in the defense of vulnerable protein layers that are solidified by transglutaminase.
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12. |
Sarafeddinov A,
Schmidt S,
Adolf F,
Mainusch M,
Bender A,
Fuchsbauer HL,
( 2009 ) A novel transglutaminase substrate from Streptomyces mobaraensis triggers autolysis of neutral metalloproteases. PMID : 19420706 : DOI : 10.1271/bbb.80769 Abstract >>
Transglutaminase (TGase) from Streptomyces mobaraensis is a Ca(2+) independent enzyme that cross-links proteins to high molecular weight aggregates. A dispase autolysis inducing protein (DAIP) was identified as an intrinsic TGase substrate exhibiting accessible glutamine and lysine residues. DAIP modification during culture by TGase resulted in deamidation of reactive glutamines, formation of glutamic/lysine residue pairs, and failure of cross-linking. The reactivity of modified DAIP can be restored to some extent by N-lauroylamido-3-N',N'-dimethylpropyl amine, thus exposing concertedly buried glutamines and lysines. The novel TGase substrate differs considerably from the well known Streptomyces subtilisin inhibitors in higher molecular mass (37 kDa), lower pI (7.1-7.2), moderate thermo-stability, and the mode of erasing dispase activity. Our experiments suggested that DAIP induces autolysis without removal of essential metals, such as Ca(2+) and Zn(2+). Among other endoproteases, only thermolysin was similarly affected, but at considerably higher DAIP concentrations, due to simultaneous degradation of DAIP.
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13. |
Koreishi M,
Kawasaki R,
Imanaka H,
Imamura K,
Takakura Y,
Nakanishi K,
( 2009 ) Efficient Nepsilon-lauroyl-L-lysine production by recombinant epsilon-lysine acylase from Streptomyces mobaraensis. PMID : 19433221 : DOI : 10.1016/j.jbiotec.2009.03.008 Abstract >>
epsilon-Lysine acylase from Streptomyces mobaraensis (Sm-ELA), which specifically catalyzes hydrolysis of the epsilon-amide bond in various Nepsilon-acyl-L-lysines, was cloned and sequenced. The Sm-ELA gene consists of a 1617-bp open reading frame that encodes a 538-amino acid protein with a molecular mass of 55,816Da. An NCBI protein-protein BLAST search revealed that the enzyme belongs to the YtcJ-like metal-dependent amidohydrolase family, which is further characterized as the metallo-dependent hydrolase superfamily. The Sm-ELA gene was ligated into a pUC702 vector for expression in Streptomyces lividans TK24. Expression of recombinant Sm-ELA in S. lividans was approximately 300-fold higher than that in wild-type S. mobaraensis. The recombinant Sm-ELAs from the cell-free extract and culture supernatant were purified to homogeneity. The specific activities of the purified Sm-ELAs were 2500-2800U/mg, which were similar to that obtained for the wild-type Sm-ELA. Using the cell-free extract of the recombinant S. lividans cells, Nepsilon-lauroyl-L-lysine was synthesized from 500mM L-lysine hydrochloride and 50, 100, or 250mM lauric acid in an aqueous buffer solution at 37 degrees C. The yields were close to 100% after 6 and 9h of reaction for 50 and 100mM lauric acid, respectively, and 90% after 24h for 250mM lauric acid.
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14. |
Gao P,
Huang Y,
( 2009 ) Detection, distribution, and organohalogen compound discovery implications of the reduced flavin adenine dinucleotide-dependent halogenase gene in major filamentous actinomycete taxonomic groups. PMID : 19447951 : DOI : 10.1128/AEM.02958-08 PMC : PMC2708417 Abstract >>
Halogenases have been shown to play a significant role in biosynthesis and introducing the bioactivity of many halogenated secondary metabolites. In this study, 54 reduced flavin adenine dinucleotide (FADH(2))-dependent halogenase gene-positive strains were identified after the PCR screening of a large collection of 228 reference strains encompassing all major families and genera of filamentous actinomycetes. The wide distribution of this gene was observed to extend to some rare lineages with higher occurrences and large sequence diversity. Subsequent phylogenetic analyses revealed that strains containing highly homologous halogenases tended to produce halometabolites with similar structures, and halogenase genes are likely to propagate by horizontal gene transfer as well as vertical inheritance within actinomycetes. Higher percentages of halogenase gene-positive strains than those of halogenase gene-negative ones contained polyketide synthase genes and/or nonribosomal peptide synthetase genes or displayed antimicrobial activities in the tests applied, indicating their genetic and physiological potentials for producing secondary metabolites. The robustness of this halogenase gene screening strategy for the discovery of particular biosynthetic gene clusters in rare actinomycetes besides streptomycetes was further supported by genome-walking analysis. The described distribution and phylogenetic implications of the FADH(2)-dependent halogenase gene present a guide for strain selection in the search for novel organohalogen compounds from actinomycetes.
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15. |
Koreishi M,
Nakatani Y,
Ooi M,
Imanaka H,
Imamura K,
Nakanishi K,
( 2009 ) Purification, characterization, molecular cloning, and expression of a new aminoacylase from Streptomyces mobaraensis that can hydrolyze N-(middle/long)-chain-fatty-acyl-L-amino acids as well as N-short-chain-acyl-L-amino acids. PMID : 19734688 : DOI : 10.1271/bbb.90081 Abstract >>
We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 degrees Celsius (at pH 7.5).
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16. |
Schneider P,
Bouhired S,
Hoffmeister D,
( 2008 ) Characterization of the atromentin biosynthesis genes and enzymes in the homobasidiomycete Tapinella panuoides. PMID : 18805498 : DOI : 10.1016/j.fgb.2008.08.009 Abstract >>
This report highlights the first biochemical characterization of a multi-domain biosynthetic enzyme for basidiomycete secondary metabolism: the tri-domain enzyme atromentin synthetase AtrA, from Tapinella panuoides, which adenylates and dimerizes 4-hydroxyphenylpyruvic acid into atromentin. Also, the l-tyrosine:2-oxoglutarate aminotransferase AtrD, which provides the substrate for this dimerization step, has been characterized. AtrA and AtrD expand the shikimic acid pathway from l-tyrosine to atromentin, the central terphenylquinone intermediate for a prominent and widely occurring class of basidiomycete pigments, among them various pharmaceutically relevant compounds. The genes atrA and atrD were cloned and found to be clustered within one genetic locus. Given the broad distribution of atromentin-derived compounds among homobasidiomycetes we expect our system represents a widely applicable model.
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17. |
Liu Q,
Ma X,
Cheng H,
Xu N,
Liu J,
Ma Y,
( 2017 ) Co-expression of L-glutamate oxidase and catalase in Escherichia coli to produce �\-ketoglutaric acid by whole-cell biocatalyst. PMID : 28251390 : DOI : 10.1007/s10529-017-2314-5 Abstract >>
To improve the production of �\-ketoglutaric acid (�\-KG) from L-glutamate by whole-cell biocatalysis. A novel and highly active L-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 �� 2.7 U mg-1 at pH 6.0, 35 oC. The apparent Km and Vmax values of SmlGOX were 9.3 �� 0.5 mM and 159 �� 3 U mg-1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H2O2, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of �\-KG reached 77.4 g l-1 with a conversion rate from L-glutamate of 98.5% after 12 h. An efficient method for the production of �\-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.
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18. |
( 1993 ) Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112. PMID : 8099353 : Abstract >>
The complete amino acid sequence of transglutaminase (EC 2.3.2.13) (TGase), which is produced by a microorganism, Streptoverticillium sp. strain s-8112, and catalyzes the acyl transfer reaction between gamma-carboxyamide groups of glutamine residues in proteins and various primary amines, has been established by a combination of fast atom bombardment mass spectrometry and standard Edman degradation of peptide fragments produced by treatment of the TGase with various proteolytic enzymes and purified by a reversed-phase high performance liquid chromatography. The TGase consists of 331 amino acid residues with a chemical molecular weight of 37,863, in agreement with the observed molecular weight (37,869.2 +/- 8.8) determined from its electrospray ionization mass spectrum. The sequence of the enzyme is very different from those of mammalian TGases represented by guinea pig liver enzyme. The enzyme contains a sole Cys residue, which is essential for its catalytic activity. Hydropathy analysis indicated that the secondary structure of the region around the active site Cys residue is similar to those of mammalian TGases. These results suggest that this microbial protein evolved by a different pathway from that of mammalian TGases and acquired acyl transfer activity during the evolutional process.
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19. |
( 1998 ) Bacterial pro-transglutaminase from Streptoverticillium mobaraense--purification, characterisation and sequence of the zymogen. PMID : 9839945 : DOI : 10.1046/j.1432-1327.1998.2570570.x Abstract >>
The zymogen of bacterial transglutaminase was found during cultivation of Streptoverticillium mobaraense (DSMZ strain) using rabbit antibodies raised against the active enzyme. Ion-exchange chromatography at pH 5.0 yielded a highly purified pro-enzyme. Structure information was obtained by means of Edman degradation and analysis of PCR amplified nucleotide fragments. The data revealed an excess of negatively charged amino acids in the pro-region resulting in a decreased isoelectric point of the zymogen. Additionally, the new sequence gave rise to some modifications to the previously published hypothetical structure of prepro-transglutaminase derived from genomic DNA [Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Takagi, H., Motoki, M. & Takeuchi, K. (1994) Biosci. Biotechnol. Biochem. 58, 82-87]. Inactive transglutaminase, which carries an activation peptide of 45 amino acids, has a calculated molecular mass of 42445 Da. Its pro-region provides for both suppression of activity and increased thermostability. Furthermore, it could be shown that the micro-organism produces a protease which cleaves pro-transglutaminase at the C-side of Pro45. Rapid transformation of the mature enzyme also occurs by addition of other proteases. During conversion, 43 and 41 amino acid peptides are released by bovine trypsin and dispase from Bacillus polymyxa, respectively. The detection of endogenous substrates in the murein layer makes discussion of the physiological role of bacterial transglutaminases necessary.
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