Enzyme immobilization and purification are two steps that are usually required in the development of any industrial biocatalyst. In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies (versus the target enzyme or versus some domains), the development of specific domains with affinity for some specific supports or just to increase the affinity for standard ones (ionic exchangers, silicates) will be revised. We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailormade heterofunctional supports as a tool to immobilize-stabilize-purify some proteins will be discussed in deep, using low concentration of adsorbents groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be a the last part of the review.

Antimicrobial peptides (AMP) are a promising source of antibiotics with a broad spectrum activity against bacteria and low incidence of developing resistance. The mechanism by which an AMP executes its function depends on a set of computable physicochemical properties from the amino acid sequence. The Peptides package was designed to allow the quick and easy computation of ten structural characteristics own of the antimicrobial peptides, with the aim of generating data to increase the accuracy in classification and design of new amino acid sequences. Moreover, the options to read and plot XVG output files from GROMACS molecular dynamics package are included. Installation and functions Peptides includes thirteen functions and is available for download and installation from CRAN, the Comprehensive R Archive Network. To install it, just type: > install.packages("Peptides") > library(Peptides) The Peptides package requires R version 1.2.2 or higher. Development releases of the package are available on the GitHub repository http://github.com/dosorio/peptides. > aacomp(seq = "GLPRKILCAIAKKKGKCKGPLKLVCKC"

A new heterofunctional support, octyl-glyoxyl agarose, is proposed in this study.

Loss of the CDKN2A tumor suppressor is associated with melanoma metastasis, but the mechanisms connecting the phenomena are unknown. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we discovered that a lineage-restricted transcription factor, BRN2, is downstream of CDKN2A and directly regulated by E2F1. In a cohort of melanocytic tumors that capture distinct progression stages, we observed that CDKN2A loss coincides with both the onset of invasive behavior and increased BRN2 expression. Loss of the CDKN2A protein product p16 permitted metastatic dissemination of human melanoma lines in mice, a phenotype rescued by inhibition of BRN2. These results demonstrate a mechanism by which CDKN2A suppresses the initiation of melanoma invasion through inhibition of BRN2.

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed.

Summary Mycobacterium tuberculosis M ycobacterial membrane protein Large (MmpL) proteins are important in substrate transport across the inner membrane. Herein, we show that MmpL proteins are classified into two phylogenetic clusters, where MmpL Cluster II contains three soluble domains (D1, D2, and D3) and has two full-length members, MmpL3 and MmpL11. Significantly, MmpL3 is currently the most druggable M. tuberculosis target. We have solved the 2.4 Å MmpL11-D2 crystal structure revealing structural homology to periplasmic porter subdomains of RND (multidrug) transporters. The resulting predicted Cluster II MmpL membrane topology has D1 and D2 residing, and possibly interacting, within the periplasm. Crosslinking and biolayer interferometry experiments confirm that Cluster II D1 and D2 bind with weak affinities, and guided D1-D2 heterodimeric model assemblies. The predicted full-length MmpL3 and MmpL11 structural models reveal key substrate binding and transport residues, and may serve as templates to set the stage for in silico anti-tuberculosis drug development.

Both the ability of bacteria to form biofilms and communicate through quorum sensing allows them to develop different survival or virulence traits that lead to increased bacterial resistance against conventional antibiotic therapy. Here, seventeen essential oils (EOs) were investigated for the antimicrobial, antibiofilm, and anti-quorum sensing activities on Escherichia. coli O157:H7, Escherichia coli O33, and Staphylococcus epidermidis ATCC 12228. All essential oils were isolated from plant material by using hydrodistillation and analyzed by GC-MS. The antimicrobial activity was performed by using the microdilution technique. Subinhibitory concentrations of each EO were assayed for biofilm inhibition in both bacterial strains. Quantification of violacein in Chromobacterium violaceum CV026 was performed for the anti-quorum sensing activity. The cytotoxicity activity of the EOs was evaluated on Vero cell line by using MTT method. Thymol-carvacrol-chemotype (I and II) oils from Lippia origanoides and Thymus vulgaris oil exhibited the higher antimicrobial activity with MIC values of 0.37–0.75 mg/mL. In addition, these EOs strongly inhibited the biofilm formation and violacein (QS) production in a concentration-dependent manner, highlighting thymol-carvacrol-chemotype (II) oil as the best candidate for further studies in antibiotic design and development against bacterial resistance.

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