Single nucleotide variants represent a prevalent form of genetic variation. Mutations in the coding regions are frequently associated with the development of various genetic diseases. Computational tools for the prediction of the effects of mutations on protein function are very important for analysis of single nucleotide variants and their prioritization for experimental characterization. Many computational tools are already widely employed for this purpose. Unfortunately, their comparison and further improvement is hindered by large overlaps between the training datasets and benchmark datasets, which lead to biased and overly optimistic reported performances. In this study, we have constructed three independent datasets by removing all duplicities, inconsistencies and mutations previously used in the training of evaluated tools. The benchmark dataset containing over 43,000 mutations was employed for the unbiased evaluation of eight established prediction tools: MAPP, nsSNPAnalyzer, PANTHER, PhD-SNP, PolyPhen-1, PolyPhen-2, SIFT and SNAP. The six best performing tools were combined into a consensus classifier PredictSNP, resulting into significantly improved prediction performance, and at the same time returned results for all mutations, confirming that consensus prediction represents an accurate and robust alternative to the predictions delivered by individual tools. A user-friendly web interface enables easy access to all eight prediction tools, the consensus classifier PredictSNP and annotations from the Protein Mutant Database and the UniProt database. The web server and the datasets are freely available to the academic community at http://loschmidt.chemi.muni.cz/predictsnp.

/pdf/predictsnp-robust-and-accurate-consensus-classifier-for-cpuwcg3sim.pdf

PredictSNP: robust and accurate consensus classifier for prediction of disease-related mutations.

Enzymes as catalysts in synthetic organic chemistry gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technology changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic organic chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixtures in vitro and of metabolic engineering of pathways in cells as factories for the production o...

Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future

Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability

Asymmetric catalysis plays a key role in modern synthetic organic chemistry, with synthetic catalysts and enzymes being the two available options. During the latter part of the last century the use of enzymes in organic chemistry and biotechnology experienced a period of rapid growth. However, these biocatalysts have traditionally suffered from several limitations, including in many cases limited substrate scope, poor enantioselectivity, insufficient stability, and sometimes product inhibition. During the last 15 years, the genetic technique of directed evolution has been developed to such an extent that all of these long-standing problems can be addressed and solved. It is based on repeated cycles of gene mutagenesis, expression, and screening (or selection). This Review focuses on the directed evolution of enantioselective enzymes, which constitutes a fundamentally new approach to asymmetric catalysis. Emphasis is placed on the development of methods to make laboratory evolution faster and more efficient, thus providing chemists and biotechnologists with a rich and non-ending source of robust and selective catalysts for a variety of useful applications.

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

/pdf/consensus-guided-mutagenesis-of-renilla-luciferase-yields-2ntut62ff2.pdf

Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.

Variants of Bacillus sp. TS-23 strain alpha-amylases exhibit improved enzymatic performance, including increased themostability, reduced calcium dependence, increased washing / cleaning performance, and baking ability. Compositions comprising these variants are useful in methods of starch processing, starch liquefaction, fermatation, starch saccharification, cleaning, laundrying, textile desizing, baking, and biofilm removal.

Variants of Bacillus sp. TS-23 alpha-amylase with altered properties

The present invention provides methods and compositions for the production of stabilized proteins. In particular, the present invention provides methods and compositions for the generation of combinatorial libraries of consensus mutations and screening for improved protein variants.

Generation of stabilized proteins by combinatorial consensus mutagenesis

Described are variants of a parent alpha-amylase that exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: specific activity, substrate specificity, substrate binding, substrate cleavage, thermal stability, pH-dependent activity, pH-dependent stability, oxidative stability, calcium dependency, pI, and wash performance. The variants are suitable for starch conversion, ethanol production, laundry washing, dish washing, hard surface cleaning, textile desizing, and/or sweetener production.

Geobacillus stearothermophilus alpha-amylase (AMYS) variants with improved properties

Disclosed alpha-amylase/variant alpha-amylase enzymes, nucleic acids encoding the same, and compositions and methods related to the production and use thereof. The alpha-amylase/variant alpha-amylases are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry or dishwashing, for textile processing (e.g., desizing), in animal feed for improving digestibility, and for baking and brewing.

Alpha-amylase variants

An α-amylase from Bacillus subtilis (AmyE) produces significant amounts of glucose from various carbohydrate substrates, including vegetable starch, maltoheptaose, and maltotriose. Among other things, this advantageous property allows AmyE or variants thereof to be used in a saccharification reaction having a reduced or eliminated requirement for glucoamylase. The reduction or elimination of the glucoamylase requirement significantly improves the efficiency of the production of ethanol or high fructose corn syrup, for example.

Production of glucose from starch using alpha-amylases from Bacillus subtilis

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