scholarly journals Immobilization Screening and Characterization of an Alcohol Dehydrogenase and its Application to the Multi-Enzymatic Selective Oxidation of 1,-Omega-Diols

2021 ◽  
Vol 1 ◽  
Author(s):  
Javier Santiago-Arcos ◽  
Susana Velasco-Lozano ◽  
Eleftheria Diamanti ◽  
Aitziber L. Cortajarena ◽  
Fernando López-Gallego
Keyword(s):  
Alcohol Dehydrogenase ◽  
Nadh Oxidase ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Enzyme System ◽  
Life Time ◽  
Immobilized Enzymes ◽  
Heterogeneous Biocatalysts ◽  
Heterogeneous Biocatalyst

Alcohol dehydrogenase from Bacillus (Geobacillus) stearothermophilus (BsADH) is a NADH-dependent enzyme catalyzing the oxidation of alcohols, however its thermal and operational stabilities are too low for its long-term use under non-physiological conditions. Enzyme immobilizations emerges as an attractive tool to enhance the stability of this enzyme. In this work, we have screened a battery of porous carriers and immobilization chemistries to enhance the robustness of a His-tagged variant of BsADH. The selected carriers recovered close to 50% of the immobilized activity and increased enzyme stability from 3 to 9 times compared to the free enzyme. We found a trade-off between the half-life time and the specific activity as a function of the relative anisotropy values of the immobilized enzymes, suggesting that both properties are oppositely related to the enzyme mobility (rotational tumbling). The most thermally stable heterogeneous biocatalysts were coupled with a NADH oxidase/catalase pair co-immobilized on porous agarose beads to perform the batch oxidation of five different 1,ω-diols with in situ recycling of NAD+. Only when His-tagged BsADH was immobilized on porous glass functionalized with Fe3+, the heterogeneous biocatalyst oxidized 1, 5-pentanediol with a conversion higher than 50% after five batch cycles. This immobilized multi-enzyme system presented promising enzymatic productivities towards the oxidation of three different diols. Hence, this strategical study accompanied by a functional and structural characterization of the resulting immobilized enzymes, allowed us selecting an optimal heterogeneous biocatalyst and their integration into a fully heterogeneous multi-enzyme system.

Simultaneous optimization of activity and stability of xylose reductase from D. nepalensis NCYC 3413 using statistical experimental design

2020 ◽  
Vol 27 ◽  
Author(s):  
Shwethashree Malla ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Reductase Activity ◽  
Xylose Reductase ◽  
Life Time ◽  
Economic Feasibility ◽  
Simultaneous Optimization ◽  
Physical Parameters ◽  
Statistical Experimental Design

Background: Physical parameters like pH and temperature play a major role in the design of an industrial enzymatic process. Enzyme stability and activity are greatly influenced by these parameters; hence optimization and control of these parameters becomes a key point in determining the economic feasibility of the process. Objective: This study was taken up with the objective to optimize physical parameters for maximum stability and activity of xylose reductase from D. nepalensis NCYC 3413 through separate and simultaneous optimization studies and comparison thereof. Method: Effects of pH and temperature on the activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 were investigated by enzyme assays and independent variables were optimised using surface response methodology. Enzyme activity and stability were optimised separately and concurrently to decipher the appropriate conditions. Results: Optimized conditions of pH and temperature for xylose reductase activity were determined to be 7.1 and 27 ℃ respectively, with predicted responses of specific activity (72.3 U/mg) and half-life time (566 min). The experimental values (specific activity 50.2 U/mg, half-life time 818 min) were on par with predicted values indicating the significance of the model. Conclusion: Simultaneous optimization of xylose reductase activity and stability using statistical methods is effective as compared to optimisation of the parameters separately.

scholarly journals Spectroscopic Analysis of Heterogeneous Biocatalysts for Biodiesel Production from Expired Sunflower Cooking Oil

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Enoch Wembabazi ◽  
Patrick Joram Mugisha ◽  
Asumani Ratibu ◽  
Deborah Wendiro ◽  
Joseph Kyambadde ◽  
...  
Keyword(s):  
Calcium Oxide ◽  
Sunflower Oil ◽  
Active Form ◽  
Single Step ◽  
Biodiesel Production ◽  
Saw Dust ◽  
Heterogeneous Biocatalysts ◽  
Egg Shells ◽  
Heterogeneous Biocatalyst

The study characterized heterogeneous biocatalyst synthesized from sucrose, saw dust, and chicken egg shells using Fourier Transform Infrared (FTIR) spectroscopy coupled with Attenuated Total Reflectance (ATR) technique. Acidic sulphonate (–SO3H) groups were more visible in the spectrum generated for carbonized and sulphonated sucrose than in carbonized and sulphonated saw dust. This was highlighted further by the significantly higher conversion percentage achieved for sulphonated sucrose (62.5%) than sulphonated saw dust (46.6%) during esterification of expired sunflower oil (p=0.05). The spectra for calcinated egg shells also showed that the most active form of calcium oxide was produced at calcination temperature of 1000°C. This was confirmed in the single-step transesterification reaction in which calcium oxide generated at 1000°C yielded the highest biodiesel (87.8%) from expired sunflower oil. The study further demonstrated the versatility of the FTIR technique in qualitative analysis of biodiesel and regular diesel by confirming the presence of specific characteristic peaks of diagnostic importance. These findings therefore highlight the potential of FTIR-ATR as an inexpensive, fast, and accurate diagnostic means for easy identification and characterization of different materials and products.

scholarly journals Characterization of an Exceedingly Active NADH Oxidase from the Anaerobic Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 189 (8) ◽  
pp. 3312-3317 ◽  
Author(s):  
Xianqin Yang ◽  
Kesen Ma
Keyword(s):  
Nadh Oxidase ◽  
Specific Activity ◽  
Ph Value ◽  
Thermotoga Maritima ◽  
Cell Extract ◽  
Hyperthermophilic Bacterium ◽  
Its Gene ◽  
Molecular Masses ◽  
New Type

ABSTRACT An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80°C. The Vmax was 230 ± 14 μmol/min/mg (k cat/Km = 548,000 min−1 mM−1), and the Km values for NADH and oxygen were 42 ± 3 and 43 ± 4 μM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima.

scholarly journals Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Christopher L. Case ◽  
Jason R. Rodriguez ◽  
Biswarup Mukhopadhyay
Keyword(s):  
Coenzyme A ◽  
Nadh Oxidase ◽  
Specific Activity ◽  
Sulfenic Acid ◽  
Methanocaldococcus Jannaschii ◽  
Nitrobenzoic Acid ◽  
Disulfide Reductase ◽  
Group 3 ◽  
Nadh Oxidase Activity

Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit)−1, showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit)−1 and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were ≥10 and ≥95 °C, respectively. At pH 7 and 83 °C, apparent K m values for NADH and O2 were 3 μM and 1.9 mM, respectively, and the specific activity at 1.4 mM O2 was 60 μmol min−1 mg−1; 62 % of NADH-derived reducing equivalents were recovered as H2O2 and the rest probably generated H2O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,5′-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high K m, O2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

Activity of NAD-Dependent Dehydrogenase Enzyme of Lactic Streptococci on Acetaldehyde and n-Hexanal1

1979 ◽  
Vol 42 (10) ◽  
pp. 778-779 ◽  
Author(s):  
R. H. SCHMIDT ◽  
L. K. FARRON ◽  
T. R. BATEH ◽  
P. E. ARAUJO
Keyword(s):  
Alcohol Dehydrogenase ◽  
Centrifugal Force ◽  
Aldehyde Dehydrogenase ◽  
Specific Activity ◽  
Enzyme System ◽  
Substrate Inhibition ◽  
Cell Extract ◽  
Cell Extracts ◽  
Dehydrogenase Enzyme

Alcohol dehydrogenase (alcohol:NAD oxidoreductase, E.C. 1.1.1.1) activity was observed on acetaldehyde and n-hexanal in homogenized cell extract of Steptococcus lactis C2. Substrate inhibition was apparent at levels of n-hexanal above 4.0 mM. Increased centrifugal force from 12.000 × g for 20 min to 350,000 × g for 1 h resulted in increased specific activity in the cell-extract supernatant fluids. Aldehyde dehydrogenase (aldehyde: NAD oxidoreductose; E.C. 1.2.1.3) was not detected in any of the cell extracts. A possible involvement of the enzyme system with flavor modification in lactic-fermented oilseed milk is suggested.

scholarly journals Pereparation and characterization of two types of covalently immobilized amyloglucosidase

2005 ◽  
Vol 70 (5) ◽  
pp. 713-719 ◽  
Author(s):  
Nenad Milosavic ◽  
Radivoje Prodanovic ◽  
Slobodan Jovanovic ◽  
Irena Novakovic ◽  
Zoran Vujcic
Keyword(s):  
Specific Activity ◽  
Periodate Oxidation ◽  
Immobilized Enzymes ◽  
Soluble Starch ◽  
Soluble Enzyme ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Higher Temperature ◽  
Hydrolysis Of

Amyloglucosidase from A. niger was covalently immobilized onto poly( GMA-co-EGDMA) by the glutaraldehyde and periodate method. The immobilization of amyloglucosidase after periodate oxidation gave a preparate with the highest specific activity reported so far on similar polymers. The obtained immobilized preparates show the same pH optimum, but a higher temperature optimum compared with the soluble enzyme. The kinetic parameters for the hydrolysis of soluble starch by free and both immobilized enzymes were determined. .

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

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