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Lower Km (lower + km)
Selected AbstractsEngineered Pyranose 2-Oxidase: Efficiently Turning Sugars into Electrical EnergyELECTROANALYSIS, Issue 7-8 2010Oliver Spadiut Abstract Due to the recent interest in enzymatic biofuel cells (BFCs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale-devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi-rational protein design to improve the catalytic activity of the enzyme pyranose 2-oxidase (P2Ox) with its alternative soluble electron acceptors 1,4-benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BFCs. Using a screening assay based on 96-well plates, we identified the variant H450G, which showed lower KM and higher kcat values for both 1,4-benzoquinone and ferricenium ion compared to the wild-type enzyme, when either D -glucose or D -galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BFCs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either "wired" to an osmium redox polymer or using soluble 1,4-benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4-fold compared to systems employing the wild-type enzyme. [source] Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesisFEBS JOURNAL, Issue 22 2001Xing-Guo Wang In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards l -methionine, l -norleucine and l -norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used l -aspartate and l -leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher Km values for glutamate/2-oxoglutarate at pH 7.0, but a similar kcat/Km value and lower Km at pH 8.0, and a nearly 22-fold lower S0.5 (substrate concentration giving half-saturation under conditions where Michaelis,Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100,1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially l -norleucine and l -methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate kcat and Km separately, but for reductive amination the additional mutations have no significant effect on kcat at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in Km. At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine ,,2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants. [source] Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiaeBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2001Birgit Morawski Abstract Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower Km for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50°C. A single mutation (N175S) in the HRP active site was found to improve thermal stability. Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60°C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H2O2, SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 99,107, 2001. [source] Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicumFEBS JOURNAL, Issue 3 2005Gloria Herrmann The fermentation of ,-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation of l -alanine. By shifting the organism from the carbon and energy source ,-alanine to ,-alanine, the enzyme ,-alanyl-CoA:ammonia lyase is induced 300-fold (, 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on ,-alanine. The deduced ,-alanyl-CoA:ammonia lyase proteins are related to putative ,-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. ,-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent Km value for acryloyl-CoA was measured as 23 ± 4 µm, independent of the concentration of the second substrate ammonia; kcat/Km was calculated as 107 m,1·s,1. The apparent Km for ammonia was much higher, 70 ± 5 mm at 150 µm acryloyl-CoA with a much lower kcat/Km of 4 × 103 m,1·s,1. In the reverse reaction, a Km of 210 ± 30 µM was obtained for ,-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of ,-alanyl-CoA:ammonia lyase in ,-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between l -alanine/,-alanine or d -lactate/3-hydroxypropionate. [source] | ||||||||||