scholarly journals Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

2012 ◽  
Vol 78 (12) ◽  
pp. 4330-4337 ◽  
Author(s):  
Michel Oelschlägel ◽  
Janosch A. D. Gröning ◽  
Dirk Tischler ◽  
Stefan R. Kaschabek ◽  
Michael Schlömann
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Chemical Reactivity ◽  
Styrene Oxide ◽  
Active Form ◽  
Temperature Tolerance ◽  
Product Inhibition ◽  
Rhodococcus Opacus ◽  
Content Type ◽  
Rhodococcus Opacus 1Cp

ABSTRACTStyrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacteriumRhodococcus opacus1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process.

scholarly journals DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

2016 ◽  
Vol 82 (16) ◽  
pp. 4975-4981 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Hans Gerstmans ◽  
Simon Thorpe ◽  
Stéphane Mesnage ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thermal Resistance ◽  
High Performance ◽  
Antimicrobial Agents ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Gram Negative ◽  
Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

scholarly journals Biochemical Characterization of StyAB from Pseudomonas sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase

2004 ◽  
Vol 186 (16) ◽  
pp. 5292-5302 ◽  
Author(s):  
Katja Otto ◽  
Karin Hofstetter ◽  
Martina Röthlisberger ◽  
Bernard Witholt ◽  
Andreas Schmid
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Styrene Oxide ◽  
Sole Source ◽  
Nadh Oxidation ◽  
Flavin Mononucleotide ◽  
Wild Type ◽  
Oxidation Activity ◽  
Epoxidation Reaction ◽  
Sequential Binding

ABSTRACT Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of C═C double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg−1 (k cat = 1.6 s−1) and Km values for styrene of 0.45 ± 0.05 mM (wild type) and 0.38 ± 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg−1 (k cat [NADH] = 60 s−1). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

scholarly journals Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

2009 ◽  
Vol 191 (15) ◽  
pp. 4996-5009 ◽  
Author(s):  
Dirk Tischler ◽  
Dirk Eulberg ◽  
Silvia Lakner ◽  
Stefan R. Kaschabek ◽  
Willem J. H. van Berkel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Styrene Oxide ◽  
Functional Expression ◽  
Rhodococcus Opacus ◽  
Reading Frame ◽  
Arthrobacter Aurescens ◽  
C Terminus ◽  
Two Component ◽  
Rhodococcus Opacus 1Cp

ABSTRACT Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

scholarly journals Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

2013 ◽  
Vol 79 (17) ◽  
pp. 5137-5145 ◽  
Author(s):  
Caspar Schäfer ◽  
Bärbel Friedrich ◽  
Oliver Lenz
Keyword(s):  
Biochemical Characterization ◽  
Ralstonia Eutropha ◽  
Specific Activity ◽  
Atmospheric Oxygen ◽  
Oxidation Activity ◽  
Tetrazolium Chloride ◽  
Content Type ◽  
Activity Measurements ◽  
Group 5

ABSTRACTRecently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacteriumRalstonia eutrophaH16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by thein vivoH2uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed lowKmof group 5 hydrogenases and makes atmospheric H2uptake byR. eutrophamost unlikely. Amperometric activity measurements revealed that the AH maintains full H2oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.

scholarly journals Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production

2015 ◽  
Vol 81 (20) ◽  
pp. 7319-7329 ◽  
Author(s):  
K. M. Trollope ◽  
J. F. Görgens ◽  
H. Volschenk
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Direct Interaction ◽  
Product Inhibition ◽  
Single Amino Acid ◽  
Content Type ◽  
Aspergillus Japonicus ◽  
Enzyme Thermostability ◽  
Loop Regions ◽  
Computational Analyses

ABSTRACTTheAspergillus japonicusβ-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition.

Rhodococcus defluvii sp. nov., isolated from wastewater of a bioreactor and formal proposal to reclassify [Corynebacterium hoagii] and Rhodococcus equi as Rhodococcus hoagii comb. nov.

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 755-761 ◽  
Author(s):  
P. Kämpfer ◽  
W. Dott ◽  
K. Martin ◽  
S. P. Glaeser
Keyword(s):  
Type Species ◽  
Phylogenetic Trees ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Rhodococcus Equi ◽  
Rhodococcus Opacus ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Type Strains

A Gram-stain-positive, non-endospore-forming rod-shaped bacterium, strain Ca11T, was isolated from a bioreactor with extensive phosphorus removal and was studied in detail for its taxonomic allocation. 16S rRNA gene sequence analysis revealed closest sequence similarity of the strain to type strains of [ Corynebacterium hoagii ] and Rhodococcus equi (98.9 %), Rhodococcus koreensis and Rhodococcus wratislaviensis (both 98.4 %), Rhodococcus opacus and Rhodococcus canchipurensis (both 98.0 %) followed by Rhodococcus kunmingensis and Rhodococcus imtechensis (97.7 %). Phylogenetic trees showed a distinct clustering of strain Ca11T with the type strains of [ C. hoagii ], R. equi , and R. kunmingensis separate to all other species of the genus Rhodococcus . The quinone system of strain Ca11T was composed of dihydrogenated menaquinones with 8 (major amount) as well as 7 and 6 isoprenoid units [MK-8(H2), MK-7(H2), MK-6(H2)]. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, one unknown phospholipid and an unidentified glycolipid. The fatty acid profile was similar to that reported for R. equi and contained major amounts of C16 : 0, C18 : 1ω9c and 10-methyl C18 : 0, supporting the allocation of the strain to the genus Rhodococcus . Physiological and biochemical characterization and DNA–DNA hybridization with type strains of the most closely related species allowed clear phenotypic and genotypic differentiation of the isolate. On the basis of these results, strain Ca11T ( = DSM 45893T = LMG 27563T) represents a novel species of the genus Rhodococcus , with the proposed name Rhodococcus defluvii sp. nov. In addition, a polyphasic taxonomic analysis of [ Corynebacterium hoagii ] DSM 20295T and Rhodococcus equi DSM 20307T indicated that the two strains belong to the same species, for which the name Rhodococcus hoagii comb. nov. takes priority, according to the Rules of the Bacteriological Code.

scholarly journals Two Homologous Enzymes of the GalU Family in Rhodococcus opacus 1CP—RoGalU1 and RoGalU2

2019 ◽  
Vol 20 (22) ◽  
pp. 5809 ◽  
Author(s):  
Antje Kumpf ◽  
Anett Partzsch ◽  
André Pollender ◽  
Isabel Bento ◽  
Dirk Tischler
Keyword(s):  
Natural Products ◽  
Elevated Temperatures ◽  
Active Form ◽  
Residual Activity ◽  
Model Organism ◽  
Building Blocks ◽  
Rhodococcus Opacus ◽  
Sugar Moiety ◽  
Activity Factor ◽  
Rhodococcus Opacus 1Cp

Uridine-5’-diphosphate (UDP)-glucose is reported as one of the most versatile building blocks within the metabolism of pro- and eukaryotes. The activated sugar moiety is formed by the enzyme UDP-glucose pyrophosphorylase (GalU). Two homologous enzymes (designated as RoGalU1 and RoGalU2) are encoded by most Rhodococcus strains, known for their capability to degrade numerous compounds, but also to synthesize natural products such as trehalose comprising biosurfactants. To evaluate their functionality respective genes of a trehalose biosurfactant producing model organism—Rhodococcus opacus 1CP—were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of RoGalU2, the Vmax was determined to be 177 U mg−1 (uridine-5’-triphosphate (UTP)) and Km to be 0.51 mM (UTP), respectively. Like other GalUs this enzyme seems to be rather specific for the substrates UTP and glucose 1-phosphate, as it accepts only dTTP and galactose 1-phoshate in addition, but both with solely 2% residual activity. In comparison to other bacterial GalU enzymes the RoGalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, RoGalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.

scholarly journals Biochemical Characterization of a Novel l-Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

2013 ◽  
Vol 80 (5) ◽  
pp. 1561-1569 ◽  
Author(s):  
Linhua Huang ◽  
Yu Liu ◽  
Yan Sun ◽  
Qiaojuan Yan ◽  
Zhengqiang Jiang
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
High Specific Activity ◽  
Rhizomucor Miehei ◽  
Drug Dosage ◽  
Amino Acid Sequences ◽  
Content Type ◽  
Leukemia Treatment ◽  
Glutaminase Activity

ABSTRACTA novel fungal gene encoding theRhizomucor mieheil-asparaginase (RmAsnase) was cloned and expressed inEscherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reportedl-asparaginases. The purifiedl-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, thisl-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.

scholarly journals Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Peixian Bai ◽  
Liyuan Wang ◽  
Kang Wei ◽  
Li Ruan ◽  
Liyun Wu ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Camellia Sinensis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Sequences ◽  
Biochemical Properties ◽  
High Similarity ◽  
New Gene ◽  
Tea Plants

Abstract Background Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. Results The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0–8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5′-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). Conclusions Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

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