scholarly journals Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

2013 ◽  
Vol 79 (9) ◽  
pp. 3049-3058 ◽  
Author(s):  
Haiquan Yang ◽  
Xinyao Lu ◽  
Long Liu ◽  
Jianghua Li ◽  
Hyun-dong Shin ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Specific Activity ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Content Type ◽  
Resistance To Oxidation ◽  
Catalytic Constant ◽  
N Terminus ◽  
Ph Range

ABSTRACTIn this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) fromAlkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (kcat) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK:kcat/Kmincreased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H2O2, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.

scholarly journals ENHANCED ENZYMATIC ACTIVITY OF STREPTOMYCES GRISEOPLANUS L-ASPARGINASE VIA ITS INCORPORATION IN AN OIL-BASED NANOCARRIER

2020 ◽  
pp. 203-210
Author(s):  
ABEER A. EL-HADI ◽  
HANAN MOSTAFA AHMED ◽  
RANIA A. ZAKI ◽  
AMIRA MOHAMED MOHSEN
Keyword(s):  
Thermal Stability ◽  
Oleic Acid ◽  
Enzymatic Activity ◽  
Specific Activity ◽  
Residual Activity ◽  
Tween 80 ◽  
Biochemical Properties ◽  
Lymphocytic Leukemia ◽  
Tween 20 ◽  
Ph Range

Objective: L-asparaginase (L-asp) is a vital enzyme used as a therapeutic agent in combination with other drugs in the treatment of acute lymphoma, melanosarcoma and lymphocytic leukemia. Immobilization of enzymes through loading on nanoemulsion (NE) results in some advantages such as enhancing their stability and increasing their resistance to proteases. Aim of the present study is to formulate L-asp loaded nanoemulsion to enhance its efficiency and thermal stability. Methods: Nanoemulsion loaded with L-asp crude extract (specific activity 13.23U/mg protein) was prepared employing oleic acid as oil, tween 20/tween 80 as surfactants and propylene glycol (PG) as co-surfactant. L-asp loaded NE underwent several thermodynamic stability studies and the optimized formulae were further examined for their biochemical properties and thermal stability. Results The developed formulations were spherical in shape and their sizes were in the nanometric dimensions with negatively charged zeta potential values. Upon comparing the enzyme activity of L-asp loaded NE employing tween 20 (F1) or tween80 (F4) at different concentrations, the results revealed that F4 NE showed higher enzymatic activity [323 U/ml] compared to F1 NE [197 U/ml] at the same concentration. The nanosized immobilized L-asp was more stable in the pH range from 8 to 8.5 as compared to free L-asp. The immobilized enzyme preserved about 59.11% of its residual activity at 50 °C; while free L-asp preserved about 33.84%. Conclusion: In the view of these results, NE composed of oleic acid, tween 80 and PG represents a promising dosage form for enhancing the activity and stability of Streptomyces griseoplanus L-asp.

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 Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

Influence of WS2/SnS2 on the tribological performance of copper-free brake pads

2019 ◽  
Vol 71 (3) ◽  
pp. 398-405 ◽  
Author(s):  
Justin Antonyraj I. ◽  
Vijay R. ◽  
Lenin Singaravelu D.
Keyword(s):  
Thermal Stability ◽  
Profile Analysis ◽  
Film Formation ◽  
Three Dimensional ◽  
Solid Lubricants ◽  
Tribological Performance ◽  
Mechanical And Thermal Properties ◽  
Content Type ◽  
Brake Pads ◽  
Lubrication Film

Purpose The purpose of this study is to investigate the influence of solid lubricants (tungsten disulfide [WS2]/ Tin disulfide [SnS2]) on the tribological performance of brake pads. Design/methodology/approach In this study, the brake pads were developed by varying the solid lubricants (WS2/SnS2) without varying the other ingredients. The brake pads were developed as per the industrial procedure. Thermal stability was found for varying ingredients and developed pads. The physical, mechanical and thermal properties of the developed brake pads were analyzed as per the industrial standards. The tribological properties were analyzed using the Chase test. The worn surface analysis was done using scanning electron microscopy, elemental mapping and three-dimensional profile analysis. Findings The experimental results indicate that the WS2-based brake pads possess good physical, chemical and mechanical properties with stable friction and less wear rate due to its good lubrication film formation and thermal stability natures of WS2. Originality/value This paper explains the effect of solid lubricants in brake pads for enhancing the tribological performance by the shearing of crystal structure, thermal stability and tribo film properties of the lubricants.

The role of shell/core saturation level on the accuracy and mechanical characteristics of porous calcium phosphate models produced by 3Dprinting

2015 ◽  
Vol 21 (1) ◽  
pp. 43-55 ◽  
Author(s):  
Miguel Castilho ◽  
Barbara Gouveia ◽  
Inês Pires ◽  
Jorge Rodrigues ◽  
Manuel Pereira
Keyword(s):  
Calcium Phosphate ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Sem Analysis ◽  
Strength Distribution ◽  
Saturation Level ◽  
Content Type ◽  
Core Saturation ◽  
Size And Morphology ◽  
Binder Saturation

Purpose – This paper aims to study the influence of the binder saturation level on the accuracy and on the mechanical properties of three-dimensional (3D)-printed scaffolds for bone tissue engineering. Design/methodology/approach – To study the influence of the liquid binder volume on the models accuracy, two quality test plates with different macropore sizes were designed and produced. For the mechanical and physical characterisation, cylindrical specimens were used. The models were printed using a calcium phosphate powder, which was characterised in terms of composition, particle size and morphology, by X-ray diffraction (XRD), laser diffraction and Scanning electron microscopy (SEM) analysis. The sample’s physical characterisation was made using the Archimedes method (porosity), SEM, micro-computer tomography (CT) and digital scan techniques, while the mechanical characterisation was performed by means of uniaxial compressive tests. Strength distribution was analysed using a statistical Weibull approach, and the dependence of the compressive strength on the porosity was discussed. Findings – The saturation level is determinant for the structural characteristics, accuracy and strength the models produced by three-dimensional printing (3DP). Samples printed with the highest saturation showed higher compressive strengths (24 MPa), which are over the human trabecular bone. The models printed with lower saturations presented the highest accuracy and pore interconnectivity. Originality/value – This study allowed to acquire important knowledge concerning the effects of shell/core saturation on the overall performance of the 3DP. With this information it is possible to devise scaffolds with the required properties for bone scaffold engineering.

scholarly journals A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases

2014 ◽  
Vol 80 (11) ◽  
pp. 3426-3432 ◽  
Author(s):  
Zhongyuan Li ◽  
Xianli Xue ◽  
Heng Zhao ◽  
Peilong Yang ◽  
Huiying Luo ◽  
...  
Keyword(s):  
Specific Activity ◽  
Glycosyl Hydrolase ◽  
Beech Wood ◽  
Catalytic Efficiency ◽  
Cd Spectroscopy ◽  
Titration Calorimetry ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Sheep Rumen ◽  
Hydrolase Family

ABSTRACTEfficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed inEscherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

Archiving experience: an exploration of the challenges of preserving virtual reality

2020 ◽  
Vol 30 (2) ◽  
pp. 253-274
Author(s):  
Zack Lischer-Katz
Keyword(s):  
Virtual Reality ◽  
Multiple Scales ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Central Element ◽  
Theory And Practice ◽  
Archival Theory ◽  
Content Type ◽  
Archival Practice ◽  
The Impact

Purpose This paper aims to explore the opportunities and challenges that immersive virtual reality (VR) technologies pose for archival theory and practice. Design/methodology/approach This conceptual paper reviews research on VR adoption in information institutions and the preservation challenges of VR to identify ways in which VR has the potential to disrupt existing archival theory and practice. Findings Existing archival approaches are found to be disrupted by the multi-layered structural characteristics of VR, the part–whole relationships between the technological elements of VR environments and the three-dimensional content they contain and the immersive, experiential nature of VR experiences. This paper argues that drawing on perspectives from phenomenology and digital materiality is helpful for addressing the preservation challenges of VR. Research limitations/implications The findings extend conceptualizations of preservation by identifying gaps in existing preservation approaches to VR and stressing the importance of “experience” as a central element of archival practice and by emphasizing the embodied dimensions of interpreting archival records and the multiple scales of materiality that archival researchers and practitioners should consider to preserve VR. Practical implications These findings provide guidance for digital curators and preservationists by outlining the current thinking on VR preservation and the impact of VR on digital preservation strategies. Originality/value This paper gives new insight into VR as an emerging area of concern to digital curation and preservation and expands archival thinking with new conceptualizations that disrupt existing paradigms.

scholarly journals Biochemical Characterization of the FEZ-1 Metallo-β-Lactamase of Legionella gormanii ATCC 33297T Produced in Escherichia coli

2001 ◽  
Vol 45 (4) ◽  
pp. 1254-1262 ◽  
Author(s):  
Paola Sandra Mercuri ◽  
Fabrice Bouillenne ◽  
Letizia Boschi ◽  
Josette Lamotte-Brasseur ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Expression System ◽  
Three Dimensional ◽  
Zinc Content ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Gene Coding ◽  
Spectrum Activity ◽  
Ph Range

ABSTRACT The bla FEZ-1 gene coding for the metallo-β-lactamase of Legionella (Fluoribacter) gormaniiATCC 33297T was overexpressed via a T7 expression system inEscherichia coli BL21(DE3)(pLysS). The product was purified to homogeneity in two steps with a yield of 53%. The FEZ-1 metallo-β-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime. Monobactams were not hydrolyzed. The β-lactamase was inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites. Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme. A model of the FEZ-1 three-dimensional structure was built.

scholarly journals Synthesis and Characterization of Chimeric Proteins Based on Cellulase and Xylanase from an Insect Gut Bacterium

2011 ◽  
Vol 77 (14) ◽  
pp. 4859-4866 ◽  
Author(s):  
Nidhi Adlakha ◽  
Raman Rajagopal ◽  
Saravanan Kumar ◽  
Vanga Siva Reddy ◽  
Syed Shams Yazdani
Keyword(s):  
Plant Biomass ◽  
Specific Activity ◽  
Bacterial Flora ◽  
Three Dimensional ◽  
Cd Spectroscopy ◽  
Gene Encoding ◽  
Content Type ◽  
Hydrolyzing Enzymes ◽  
Chimeric Model ◽  
Insect Gut

ABSTRACTInsects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated aPaenibacillusstrain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species ofPaenibacillus. Endo5A and Xyl11D were overexpressed inEscherichia coli, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.

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