scholarly journals Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic ArchaeonPyrococcus furiosusby Covalent Immobilization

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Roberta V. Branco ◽  
Melissa L. E. Gutarra ◽  
Jose M. Guisan ◽  
Denise M. G. Freire ◽  
Rodrigo V. Almeida ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Enzyme Stability ◽  
Enantiomeric Excess ◽  
Residual Activity ◽  
Covalent Bond ◽  
Covalent Immobilization ◽  
Covalent Attachment ◽  
Optimal Temperature ◽  
Thermostable Lipase ◽  
Multipoint Covalent Attachment

A recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic ArchaeonPyrococcus furiosus(PFUL) was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL) and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment) (glyoxyl-DTT PFUL) and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment) (glyoxyl PFUL). The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity), whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity). Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. TheEvalue also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivityE=22and enantiomeric excess (ee (%) = 91).

scholarly journals Preparation and characterization of stable Core/Shell Fe3O4@Au ‎decorated with amine group for immobilization of lipase by covalent ‎attachment

2021 ◽  
Author(s):  
Marziyeh Aghamolaei ◽  
Amir Landarani-Isfahani ◽  
Mehrnaz Bahadori ◽  
Zahra Zamani Nori ◽  
Saghar Rezaei ◽  
...  
Keyword(s):  
Self Assembly ◽  
Immobilized Lipase ◽  
Residual Activity ◽  
Covalent Bond ◽  
Temperature Stability ◽  
Covalent Immobilization ◽  
Covalent Attachment ◽  
Core Shell ◽  
Amine Group ◽  
Wide Range

The self-assembly approach was used for amine decoration of core/shell Fe3O4@Au with 4-aminothiphenol. This structure was used for covalent immobilization of lipase using a Ugi 4-component reaction. The amine group on the structure and carboxylic group from lipase can react in the Ugi reaction and a firm and stable covalent bond creates between enzyme and support. The synthesized structure was fully characterized and its activity was explored in different situations. The results displayed the pH and temperature stability of immobilized lipase compared to free lipase in a wide range of pH and temperature. Also after 60 days, it showed excellent activity while residual activity for the free enzyme was only 10%. The synthesized structure was conveniently separated using an external magnetic field and reused 6 times without losing the activity of the immobilized enzyme.

Covalent Attachment of a Single Dextran Polymer Between the Tip of an Atomic Force Microscope and a Gold Surface

1999 ◽  
Vol 576 ◽  
Author(s):  
M. Grandbois ◽  
R. Décor ◽  
M. Rief ◽  
A. Wagner ◽  
C. Mioskowski ◽  
...  
Keyword(s):  
Atomic Force Microscope ◽  
Covalent Bond ◽  
Gold Surface ◽  
Covalent Immobilization ◽  
Covalent Attachment ◽  
Amino Groups ◽  
Fly Fishing ◽  
Atomic Force ◽  
Carbodiimide Hydrochloride ◽  
Measuring Force

ABSTRACTA method for covalent immobilization of a single dextran polymer between a gold surface and the tip of an atomic force microscope (AFM) is presented. Carboxymethylated dextran immobilized on gold by epoxythiol chemistry was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) in order to make the dextran polymer reactive for the amino groups present on the previously aminosilanized AFM tip. By measuring force vs extension curves we have shown that it is possible to catch such an activated dextran polymer with an AFM tip through the formation of a covalent bond. Dextran polymers were attached even without any detectable indentation of the tip in the dextran-coated gold surface. In this so-called fly-fishing mode, attachment of multiple dextran polymers, which typically occurs when the tip is indented into the surface, are efficiently avoided.

scholarly journals Immobilization and Characterization of a Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus on Supports with Different Degrees of Hydrophobicity

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Roberta Vieira Branco ◽  
Melissa Limoeiro Estrada Gutarra ◽  
Denise Maria Guimarães Freire ◽  
Rodrigo Volcan Almeida
Keyword(s):  
Storage Stability ◽  
Immobilized Enzyme ◽  
Pyrococcus Furiosus ◽  
Residual Activity ◽  
Maximum Activity ◽  
Thermostable Lipase ◽  
Hyperthermophilic Archaeon ◽  
Biotechnological Processes

We studied the immobilization of a recombinant thermostable lipase (Pf2001) from the hyperthermophilic archaeon Pyrococcus furiosus on supports with different degrees of hydrophobicity: butyl Sepabeads and octadecyl Sepabeads. The enzyme was strongly adsorbed in both supports. When it was adsorbed on these supports, the enzyme showed 140 and 237% hyperactivation, respectively. The assessment of storage stability showed that the octadecyl Sepabeads immobilized enzyme showed 100% of residual activity after 30 days of storage. However, the greatest stability at was obtained in butyl Sepabeads immobilized enzyme, which retained 77% activity after 1 hour incubation. The maximum activity of the immobilized preparations was obtained with the pH between 6 and 7, at . Thus, this study achieved a new extremophilic biocatalyst with greater stability, for use in several biotechnological processes.

scholarly journals Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Sylvia Maria Campbell Alquéres ◽  
Roberta Vieira Branco ◽  
Denise Maria Guimarães Freire ◽  
Tito Lívio Moitinho Alves ◽  
Orlando Bonifácio Martins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Optimum Temperature ◽  
Optimal Temperature ◽  
Thermostable Lipase ◽  
Fusion Tag ◽  
Triton X

In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX−PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX−PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100.

Advances in the design of new epoxy supports for enzyme immobilization–stabilization

2007 ◽  
Vol 35 (6) ◽  
pp. 1593-1601 ◽  
Author(s):  
C. Mateo ◽  
V. Grazú ◽  
B.C.C. Pessela ◽  
T. Montes ◽  
J.M. Palomo ◽  
...  
Keyword(s):  
Physical Adsorption ◽  
Immobilized Enzymes ◽  
Covalent Immobilization ◽  
Covalent Attachment ◽  
Industrial Enzymes ◽  
Protein Surface ◽  
Strong Stabilization ◽  
Support Surfaces ◽  
Multipoint Covalent Attachment ◽  
Epoxy Groups

Multipoint covalent immobilization of enzymes (through very short spacer arms) on support surfaces promotes a very interesting ‘rigidification’ of protein molecules. In this case, the relative positions of each residue of the enzyme involved in the immobilization process have to be preserved unchanged during any conformational change induced on the immobilized enzyme by any distorting agent (heat, organic solvents etc.). In this way, multipoint covalent immobilization should induce a very strong stabilization of immobilized enzymes. Epoxy-activated supports are able to chemically react with all nucleophile groups placed on the protein surface: lysine, histidine, cysteine, tyrosine etc. Besides, epoxy groups are very stable. This allows the performance of very long enzyme–support reactions, enabling us to get very intense multipoint covalent attachment. In this way, these epoxy supports seem to be very suitable to stabilize industrial enzymes by multipoint covalent attachment. However, epoxy groups exhibit a low intermolecular reactivity towards nucleophiles and hence the enzymes are not able to directly react with the epoxy supports. Thus a rapid physical adsorption of enzymes on the supports becomes a first step, followed by an additional rapid ‘intramolecular’ reaction between the already adsorbed enzyme and the activated support. In this situation, a suitable first orientation of the enzyme on the support (e.g. through regions that are very rich in nucleophiles) is obviously necessary to get a very intense additional multipoint covalent immobilization. The preparation of different ‘generations’ of epoxy supports and the design of different protocols to fully control the first interaction between enzymes and epoxy supports will be reviewed in this paper. Finally, the possibilities of a directed immobilization of mutated enzymes (change of an amino acid by cysteine on specific points of the protein surface) on tailor-made disulfide-epoxy supports will be discussed as an almost-ideal procedure to achieve very intense and very efficient rigidification of a desired region of industrial enzymes.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Epoxy Sepabeads: A Novel Epoxy Support for Stabilization of Industrial Enzymes via Very Intense Multipoint Covalent Attachment

2002 ◽  
Vol 18 (3) ◽  
pp. 629-634 ◽  
Author(s):  
C. Mateo ◽  
O. Abian ◽  
G. Fernandez-Lorente ◽  
J. Pedroche ◽  
R. Fernandez-Lafuente ◽  
...  
Keyword(s):  
Covalent Attachment ◽  
Industrial Enzymes ◽  
Multipoint Covalent Attachment
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