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 A New Technic for the Production of an in Vivo Labeled Fibrinogen

Blood ◽  
1967 ◽  
Vol 29 (4) ◽  
pp. 517-525 ◽  
Author(s):  
HENRY GANS ◽  
JAMES MC LEOD ◽  
JAMES T. LOWMAN
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Entire Period ◽  
Turnover Rates ◽  
Prolonged Infusion ◽  
Slow Infusion

Abstract The fact that in vitro labeled proteins, as a rule, exhibit faster turnover rates than in vivo labeled materials led us to explore means of obtaining in vivo labeled fibrinogen of high specific activity. It was found that defibrination of the rat provides a stimulus for the liver to regenerate fibrinogen at an accelerated rate. Administration of seleno75 methionine shortly after thrombin-induced defibrination of the animal resulted in the incorporation of large quantities of the label. The rate of incorporation was further increased if the amino acid was administered as a slow infusion during the entire period of fibrinogen regeneration. In addition, prior nephrectomy of the animal would appear to result in a slight increase in specific activity of the fibrinogen preparation obtained. The results of these studies indicate that defibrination, nephrectomy, and the prolonged infusion of the labeled amino acid selenomethionine provided us with a technic for obtaining a biosynthetically labeled, γ-emitting, fibrinogen preparation of high specific activity.

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 Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

2001 ◽  
Vol 183 (15) ◽  
pp. 4468-4476 ◽  
Author(s):  
Franz Kaufmann ◽  
Derek R. Lovley
Keyword(s):  
Amino Acid ◽  
Electron Donor ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Geobacter Sulfurreducens ◽  
Oxidoreductase Activity ◽  
Isolation And Characterization ◽  
Apparent Homogeneity

ABSTRACT NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.

scholarly journals Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D β-Lactamase, OXA-436, Found in Isolates from Four Patients at Six Different Hospitals in Denmark

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Ørjan Samuelsen ◽  
Frank Hansen ◽  
Bettina Aasnæs ◽  
Henrik Hasman ◽  
Bjarte Aarmo Lund ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Type ◽  
Amino Acid Sequences ◽  
Citrobacter Freundii ◽  
Activity Profile ◽  
Content Type ◽  
Class D ◽  
Chromosomal Genes ◽  
Enterobacter Asburiae

ABSTRACT The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D β-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The bla OXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to bla OXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of bla OXA-436 illustrate its potential for further dissemination.

scholarly journals Arabinoxylan Oligosaccharide Hydrolysis by Family 43 and 51 Glycosidases from Lactobacillus brevis DSM 20054

2013 ◽  
Vol 79 (21) ◽  
pp. 6747-6754 ◽  
Author(s):  
Herbert Michlmayr ◽  
Johannes Hell ◽  
Cindy Lorenz ◽  
Stefan Böhmdorfer ◽  
Thomas Rosenau ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Functional Food ◽  
Specific Activity ◽  
High Specific Activity ◽  
Lactobacillus Brevis ◽  
Glycoside Hydrolases ◽  
Content Type ◽  
Food And Feed ◽  
Oligosaccharide Hydrolysis

ABSTRACTDue to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism ofBifidobacteriaceaehas been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome ofLactobacillus brevisDSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases fromLb. breviswere able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply thatLb. brevisis at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes inLactobacillalesgenomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentativeLactobacillusspp.,Pediococcusspp., members of theLeuconostoc/Weissellabranch, andEnterococcusspp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

scholarly journals Overproduction of l-Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

2001 ◽  
Vol 67 (7) ◽  
pp. 3064-3070 ◽  
Author(s):  
Hiroaki Motoyama ◽  
Hiroshi Yano ◽  
Yoko Terasaki ◽  
Hideharu Anazawa
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Test Tube ◽  
Amino Acid Sequences ◽  
Nutritional Requirement ◽  
Wild Type ◽  
Gene Encoding ◽  
Lysine Production ◽  
Obligate Methylotroph ◽  
In The Wild

ABSTRACT The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by l-lysine, was cloned from an l-threonine- andl-lysine-coproducing mutant of the obligate methylotrophMethylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapAmutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an l-threonine producing mutant, elevated the specific activity of DDPS 20-fold andl-lysine production 2- to 3-fold with concomitant reduction of l-threonine in test tube cultures. AL119 containing thedapA gene produced 8 g of l-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M r of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition byl-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector,l-lysine.

scholarly journals Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
L. Dabos ◽  
A. B. Jousset ◽  
R. A. Bonnin ◽  
N. Fortineau ◽  
A. Zavala ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequence Identity ◽  
Biochemical Characterization ◽  
Content Type ◽  
Sequence Identity

ABSTRACT OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most β-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of blaOXA-535 and blaOXA-436 genetic environments suggests that an ISCR1 may be responsible for blaOXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.

scholarly journals Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35

1999 ◽  
Vol 340 (3) ◽  
pp. 793-801 ◽  
Author(s):  
Andrew BINIEDA ◽  
Martin FUHRMANN ◽  
Bruno LEHNER ◽  
Claudine REY-BERTHOD ◽  
Séverine FRUTIGER-HUGHES ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequence ◽  
Specific Activity ◽  
Amino Acid Sequences ◽  
Active Enzyme ◽  
Gene Probe ◽  
Pimelic Acid ◽  
Pseudomonas Mendocina ◽  
Ph Optimum ◽  
E Coli

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of ≈ 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of ≈ 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, imelic cid-tilizing ) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

scholarly journals Relatedness of type IV pilin PilA amongst geographically diverse Moraxella bovoculi isolated from cattle with infectious bovine keratoconjunctivitis

2021 ◽  
Author(s):  
John A. Angelos ◽  
Kristin A. Clothier ◽  
Regina L. Agulto ◽  
Boguslav Mandzyuk ◽  
Morten Tryland
Keyword(s):  
Amino Acid ◽  
Type Species ◽  
Amino Acid Sequences ◽  
Similar Degree ◽  
Content Type ◽  
Infectious Bovine Keratoconjunctivitis ◽  
Link Type ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Moraxella Bovoculi

Introduction. Moraxella bovoculi is frequently isolated from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK; pinkeye). As with M. bovis, which has been causally linked to IBK, M. bovoculi expresses an RTX (repeats in the structural toxin) cytotoxin that is related to M. bovis cytotoxin. Pilin, another pathogenic factor in M. bovis , is required for corneal attachment. Seven antigenically distinct pilin serogroups have been described in M. bovis . Hypothesis/Gap Statement. Multiple different serogroups exist amongst type IV pilin encoded by M. bovis , however, it is not known whether M. bovoculi exhibits a similar degree of diversity in type IV pilin that it encodes. Aim. This study was done to characterize a structural pilin (PilA) encoded by M. bovoculi isolated from cases of IBK to determine if diversity exists amongst PilA sequences. Methodology. Ninety-four isolates of M. bovoculi collected between 2002 and 2017 from 23 counties throughout California and from five counties in four other Western states were evaluated. Results. DNA sequencing and determination of deduced amino acid sequences revealed ten (designated groups A through J) unique PilA sequences that were ~96.1–99.3 % identical. Pilin groups A and C matched previously reported putative PilA sequences from M. bovoculi isolated from IBK-affected cattle in the USA (Virginia, Nebraska, and Kansas) and Asia (Kazakhstan). The ten pilin sequences identified were only ~74–76 % identical to deduced amino acid sequences of putative pilin proteins identified from the previously reported whole-genome sequences of M. bovoculi derived from deep nasopharyngeal swabs of IBK-asymptomatic cattle. Conclusions. Compared to the diversity reported between structural pilin proteins amongst different serogroups of M. bovis , M. bovoculi PilA from geographically diverse isolates derived from IBK-affected cattle are more conserved.

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