Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 108 samples to be screened in one day. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-cell and single-molecule assays.

/pdf/droplet-microfluidics-for-high-throughput-biological-assays-14pgtmcykq.pdf

Droplet microfluidics for high-throughput biological assays

Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance.

Chemical modification and immobilization of enzymes have been usually considered unrelated tools to improve biocatalyst features. However, there are many examples where a chemically modified enzyme is finally used in an immobilized form, and that exemplifies how both tools may be complementary resulting in a synergism in the final results. In this review we present some of the strategies that may give that result. For example, the chemical modification of soluble enzymes may be used to improve their immobilization (reinforcing adsorption or improving multipoint covalent attachment), or just to improve enzyme stability and facilitate the selection of the immobilization conditions. Chemical modification of previously immobilized enzymes benefits from solid-phase chemistry due to the nature of enzymes (e.g., prevention of inactivation, aggregation, etc.). The use of different targets for chemical modifications with small molecules or multifunctional polymers are also discussed: intramolecular or intersubunit cross-linking, one-point modification, generation of artificial microenvironments, etc.

Coupling Chemical Modification and Immobilization to Improve the Catalytic Performance of Enzymes

Our previous contribution to increasing the efficiency of directed evolution is iterative saturation mutagenesis (ISM) as a systematic means of generating focused libraries for the control of substrate acceptance, enantioselectivity, or thermostability of enzymes. We have now introduced a crucial element to knowledge-guided targeted mutagenesis in general that helps to solve the numbers problem in directed evolution. We show that the choice of the amino acid (aa) alphabet, as specified by the utilized codon degeneracy, provides the experimenter with a powerful tool in designing "smarter" randomized libraries that require considerably less screening effort. A systematic comparison of two different codon degeneracies was made by examining the relative quality of the identically sized enzyme libraries in relation to the degree of oversampling required in the screening process. The specific example in our case study concerns the conventional NNK codon degeneracy (32 codons/20 aa) versus NDT (12 codons/12 aa). The model reaction is the hydrolytic kinetic resolution of a chiral trans-disubstituted epoxide, catalyzed by the epoxide hydrolase from Aspergillus niger. The NDT library proves to be of much higher quality, as measured by the dramatically higher frequency of positive variants and by the magnitude of catalyst improvement (enhanced rate and enantioselectivity). We provide a statistical analysis that constitutes a useful guide for the optimal design and generation of "smarter" focused libraries. This type of approach accelerates the process of laboratory evolution considerably and can be expected to be broadly applicable when engineering functional proteins in general.

Addressing the numbers problem in directed evolution

Historically, biocatalytic ketone reductions involved the use of Baker's yeast. Within the last five years, a significant and growing number of isolated ketoreductases have become available that have rendered yeast-based reductions obsolete. The broad substrate range and exquisite selectivities of these enzymes repeatedly outperform other ketone reduction chemistries, making biocatalysis the general method of choice for ketone reductions. Presented here is a summary of our understanding of the capabilities and limitations of these enzymes.

Advances in the Enzymatic Reduction of Ketones

Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.

/pdf/directed-evolution-an-approach-to-engineer-enzymes-4qjynfc6jc.pdf

Directed evolution: an approach to engineer enzymes

Abstract
The present study is an attempt to study the sex differences in minutiae frequencies of thumbprint with respect to dermatoglyphic patterns. A sample of 100 subjects (50 males and 50 females) were gathered from Shimla, Himachal Pradesh (North India). In regards to pattern type, maximum number of minutiae was present in loop pat-tern followed by whorls and least minutiae were in the arches in the right hand of both the sexes and left hand of females only, whereas in left hand of the males highest frequency of minutiae were noticed in whorls followed by loops and lowest in the arches, thereby indicating the lessened bimanual symmetry in males. It can be inferred from the present study that the simple pattern (arch) has less discontinuance in the regular flow of the ridges, while the complex patterns (loops and whorls) exhibited more interruptions in the dermal ridges.

Sex differences in minutiae frequencies of thumbprint with respect to dermatoglyphic patterns.

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Directed evolution: an approach to engineer enzymes

Sex differences in minutiae frequencies of thumbprint with respect to dermatoglyphic patterns.