scholarly journals Influence of CaCl2 and EDTA on reversible thermal inactivation of recombinant wild-type and mutant (E40H/E44H) Phlebia radiata manganese peroxidase 3 (rPr-MnP3)

2014 ◽  
Vol 8 (6) ◽  
pp. 118-126
Author(s):  
F. Ufot Usenobong ◽  
I. Akpanabiatu Monday
Keyword(s):  
Manganese Peroxidase ◽  
Thermal Inactivation ◽  
Wild Type ◽  
Phlebia Radiata

scholarly journals Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

2014 ◽  
Vol 80 (7) ◽  
pp. 2158-2165 ◽  
Author(s):  
Kun Wang ◽  
Huiying Luo ◽  
Jian Tian ◽  
Ossi Turunen ◽  
Huoqing Huang ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Thermal Inactivation ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Scanning Calorimetry ◽  
Dynamic Simulations ◽  
Multiple Sequence ◽  
Content Type ◽  
Melting Temperatures ◽  
Loop Region

ABSTRACTProtein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability ofStreptomycessp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed inPichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.

scholarly journals Arthrobacter d-xylose isomerase: protein-engineered subunit interfaces

1993 ◽  
Vol 291 (2) ◽  
pp. 575-583 ◽  
Author(s):  
L Varsani ◽  
T Cui ◽  
M Rangarajan ◽  
B S Hartley ◽  
J Goldberg ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Ion ◽  
Thermal Inactivation ◽  
Xylose Isomerase ◽  
Crystallographic Analysis ◽  
Salt Bridges ◽  
Wild Type ◽  
Subunit Dissociation ◽  
Subunit Interface ◽  
A Subunit

Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253→>Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.

scholarly journals Conversion of Milled Pine Wood by Manganese Peroxidase from Phlebia radiata

2001 ◽  
Vol 67 (10) ◽  
pp. 4588-4593 ◽  
Author(s):  
Martin Hofrichter ◽  
Taina Lundell ◽  
Annele Hatakka
Keyword(s):  
Molecular Mass ◽  
Manganese Peroxidase ◽  
Unsaturated Fatty Acids ◽  
Tween 80 ◽  
White Rot ◽  
Size Exclusion ◽  
Tween 20 ◽  
Pine Wood ◽  
Phlebia Radiata

ABSTRACT Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn2+, Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid redsidues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata(MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37°C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (β-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.

scholarly journals Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

1997 ◽  
Vol 321 (2) ◽  
pp. 413-417 ◽  
Author(s):  
Marc VANHOVE ◽  
Gilliane GUILLAUME ◽  
Philippe LEDENT ◽  
John H. RICHARDS ◽  
Roger H. PAIN ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Guanidine Hydrochloride ◽  
Tryptophan Fluorescence ◽  
Disulphide Bond ◽  
Mutant Protein ◽  
Wild Type ◽  
High Concentration ◽  
Proline Isomerization ◽  
Drastic Increase ◽  
Time Courses

Class A α-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 α-lactamase, this bond was removed by introducing a Cys-77 → Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N ↔ H ↔ U). Both the folded mutant protein (N) and, to a lesser extent, the thermodynamically stable intermediate, H, were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.

scholarly journals Role of Nγ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine

1999 ◽  
Vol 65 (9) ◽  
pp. 3774-3779 ◽  
Author(s):  
David Cánovas ◽  
Nuno Borges ◽  
Carmen Vargas ◽  
Antonio Ventosa ◽  
Joaquín J. Nieto ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Thermal Inactivation ◽  
Alternative Pathway ◽  
Rabbit Muscle ◽  
Wild Type ◽  
Muscle Lactate ◽  
H Nmr ◽  
Halomonas Elongata ◽  
Stabilizing Effect

ABSTRACT Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts ofNγ-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794–25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A 1H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated thatH. elongata can synthesize hydroxyectoine by two different pathways—directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.

Ultrastructure of Melanin Formation in Curvularia sp., Alternaria sp., and Drechslera Sorokiniana

1977 ◽  
Vol 35 ◽  
pp. 398-399
Author(s):  
M. H. Wheeler ◽  
W. J. Tolmsoff ◽  
A. A. Bell
Keyword(s):  
Potassium Phosphate ◽  
Thielaviopsis Basicola ◽  
Wild Type ◽  
Potassium Phosphate Buffer ◽  
Transmission Microscopy ◽  
Electron Transmission ◽  
Melanin Formation ◽  
Before And After ◽  
Alternaria Sp ◽  
Electron Transmission Microscopy

(+)-Scytalone [3,4-dihydro-3,6,8-trihydroxy-l-(2Hj-naphthalenone] and 1,8-di- hydroxynaphthalene (DHN) have been proposed as intermediates of melanin synthesis in the fungi Verticillium dahliae (1, 2, 3, 4) and Thielaviopsis basicola (4, 5). Scytalone is enzymatically dehydrated by V. dahliae to 1,3,8-trihydroxynaphthalene which is then reduced to (-)-vermelone [(-)-3,4- dihydro-3,8-dihydroxy-1(2H)-naphthalenone]. Vermelone is subsequently dehydrated to DHN which is enzymatically polymerized to melanin.Melanin formation in Curvularia sp., Alternaria sp., and Drechslera soro- kiniana was examined by light and electron-transmission microscopy. Wild-type isolates of each fungus were compared with albino mutants before and after treatment with 1 mM scytalone or 0.1 mM DHN in 50 mM potassium phosphate buffer, pH 7.0. Both chemicals were converted to dark pigments in the walls of hyphae and conidia of the albino mutants. The darkened cells were similar in appearance to corresponding cells of the wild types under the light microscope.

Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

1976 ◽  
Vol 34 ◽  
pp. 54-55
Author(s):  
Karen S. Howard ◽  
H. D. Braymer ◽  
M. D. Socolofsky ◽  
S. A. Milligan
Keyword(s):  
Cell Wall ◽  
Neurospora Crassa ◽  
Wild Type ◽  
Morphological Comparison ◽  
Small Areas ◽  
Solid Media ◽  
Thin Sectioning ◽  
X Cells ◽  
Mutant Cells ◽  
Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

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