1,720,958 research outputs found
In vitro evolution of an l-amino acid deaminase active on l-1-naphthylalanine
No full textl-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a promising biocatalyst for enantioselective biocatalysis that can be exploited to produce optically pure d-amino acids or -keto acids. In this study, we improved the catalytic efficiency of PmaLAAD on l-1-naphthylalanine (l-1-Nal), a synthetic amino acid of biotechnological interest. Eight evolvable positions were identified by a molecular docking and evolutionary conservation analysis. These positions were subjected to site-saturation mutagenesis, producing smaller but smarter libraries of variants. The best variant (F318A/V412A/V438P PmaLAAD) possesses a approximate to 5-fold lower K-m (0.17 mM) and a approximate to 7-fold higher catalytic efficiency (9.2 s(-1) mM(-1)) on l-1-Nal than the wild-type enzyme. Molecular docking analysis suggests that the substitutions increase the active site volume, allowing better binding of the bulky l-1-Nal substrate. Bioconversion reactions showed that the F318A/V412A/V438P PmaLAAD variant outperforms the wild-type enzyme in the deracemization of d,l-1-Nal: the complete conversion of 0.6 mM of the l-enantiomer was achieved in about 15 min, which is approximate to 7.5-fold faster than the wild-type enzyme. In addition, the F318A/V412A/V438P PmaLAAD is efficiently employed, together with the M213G d-amino acid oxidase variant, to produce 1-naphtylpyruvate from racemic d,l-1-Nal in one pot
‐cutinase highlights the key role of residue 243
Get PDFEnzymatic degradation of poly(ethylene terephthalate) (PET) is becoming a reality because of the identification of novel PET-hydrolysing enzymes (PHEs) and the engineering of evolved enzyme variants. Here, improved variants of leaf-branch compost cutinase (LCC), a thermostable enzyme isolated by a metagenomic approach, were generated by a semi-rational protein engineering approach. Starting from a deleted LCC form lacking the secretion signal (Delta LCC), single and double variants possessing a higher activity on PET were isolated. The single-point F243T Delta LCC variant partially (similar to 67%) depolymerized amorphous PET film producing similar to 21.9 mm of products after 27 h of reaction at 72 degrees C. The S101N/F243T Delta LCC double variant reached a further increase in activity on PET. Notably, for both single and double variants the highest conversion yield was obtained at 55 degrees C. Kinetics studies and molecular dynamics simulations support that a slight decreased affinity for PET is responsible for the superior degradation performance of the S101N/F243T variant and that this stems from a slightly higher flexibility of the active site region close to position 243. Furthermore, our findings question the need for a high reaction temperature for PET degradation, at least for LCC: at >= 70 degrees C, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favoured. The evolved S101N/F243T Delta LCC variant is able to depolymerize fully 1.3 g of untreated postconsumer PET waste in <= 3 days at 55 degrees C (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process
An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes
Get PDFEnzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering com-bined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-deltaIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-deltaIsPET variant. By using 0.1 mg mL−1 of TS-deltaIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL−1 PET microparticles (~26% depoly-merization yield). Indeed, TS-deltaIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL−1. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation
Playing on both sides of the mirror: engineered amino acid oxidases for chiral biocatalysis
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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