Facile fabrication of antibacterial and antiviral perhydrolase-polydopamine composite coatings.

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and HO, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer.

Engineering the cofactor specificity of an alcohol dehydrogenase via single mutations or insertions distal to the 2'-phosphate group of NADP(H).

There have been many reports exploring the engineering of the cofactor specificity of aldo-keto reductases (AKRs), as this class of proteins is ubiquitous and exhibits many useful activities. A common approach is the mutagenesis of amino acids involved in interactions with the 2'-phosphate group of NADP(H) in the cofactor binding pocket. We recently performed a 'loop-grafting' approach to engineer the substrate specificity of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus and we found that a loop insertion after residue 211, which is on the back side of the cofactor binding pocket, could also alter cofactor specificity.

Extreme makeover: Engineering the activity of a thermostable alcohol dehydrogenase (AdhD) from Pyrococcus furiosus.

Alcohol dehydrogenase D (AdhD) is a monomeric thermostable alcohol dehydrogenase from the aldo-keto reductase (AKR) superfamily of proteins. We have been exploring various strategies of engineering the activity of AdhD so that it could be employed in future biotechnology applications. Driven by insights made in other AKRs, we have made mutations in the cofactor-binding pocket of the enzyme and broadened its cofactor specificity.

High-throughput and combinatorial gene expression on a chip for metabolism-induced toxicology screening.

Differential expression of various drug-metabolizing enzymes (DMEs) in the human liver may cause deviations of pharmacokinetic profiles, resulting in interindividual variability of drug toxicity and/or efficacy. Here, we present the 'Transfected Enzyme and Metabolism Chip' (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip.

Immobilization of Candida rugosa lipase on superparamagnetic Fe3O4 nanoparticles for biocatalysis in low-water media.

A simple immobilization method for Candida rugosa lipase on superparamagnetic Fe3O4 nanoparticles is described. The Fe3O4 nanoparticles were coated with PEI and Candida rugosa lipase was adsorbed on these particles via electrostatic interactions. The immobilization resulted in marginal simultaneous purification.

Enzyme-based listericidal nanocomposites.

Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces. We report three facile routes for the surface incorporation of the listeria bacteriophage endolysin Ply500: covalent attachment onto FDA approved silica nanoparticles (SNPs), incorporation of SNP-Ply500 conjugates into a thin poly(hydroxyethyl methacrylate) film; and affinity binding to edible crosslinked starch nanoparticles via construction of a maltose binding protein fusion.

Examining structure-activity correlations of some high activity enzyme preparations for low water media.

A first study of the comparison of structures of enzymes (by FT-IR and CD) in different high activity (in low water media) preparations is reported. Using chymotrypsin and subtilisin as models, we have studied various factors that distinguish enzyme precipitated and rinsed with propanol (EPRP), crosslinked enzyme aggregates (CLEA), protein coated microcrystals (PCMC) and crosslinked protein coated microcrystals (CLPCMC). The suspensions in organic media were assayed for catalytic activity, and structures were probed by FT-IR and CD measurements.

A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents.

Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and complete activity.

Nanomaterials as matrices for enzyme immobilization.

Nanomaterials constitute novel and interesting matrices for enzyme immobilization. While their high surface to volume ratio is an obvious advantage, their Brownian motion can impact the behavior of enzymes immobilized on these matrices. Carbon nanotubes, superparamagnetic nanoparticles, and mesoporous materials constitute some important classes of matrices.

Process optimization for biodiesel production.

Biodiesel is an alkyl ester of long chain fatty acids and is considered to leave smaller footprint on the environment. It is produced by transesterification of a fat/oil with a short chain primary alcohol like methanol or ethanol. The three routes to its preparation are: alkali catalyzed, acid catalyzed and lipase-catalyzed transesterification.

Optimising biocatalyst design for obtaining high transesterification activity by alpha-chymotrypsin in non-aqueous media.

Background: Enzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent.

Enhancement of lipase activity in non-aqueous media upon immobilization on multi-walled carbon nanotubes.

Background: Immobilization of biologically active proteins on nanosized surfaces is a key process in bionanofabrication. Carbon nanotubes with their high surface areas, as well as useful electronic, thermal and mechanical properties, constitute important building blocks in the fabrication of novel functional materials.

Results: Lipases from Candida rugosa (CRL) were found to be adsorbed on the multiwalled carbon nanotubes with very high retention of their biological activity (97%).

Microwave-assisted preparation of affinity medium.

Microwave assistance was used for preparing polyethylene glycol (PEG)-Cibacron blue 3GA and Sepharose CL-4B-Cibacron blue 3GA affinity materials. The former was used as the affinity macroligand in a PEG-dextran aqueous two-phase system for purification of alcohol dehydrogenase and EcoRI. The Sepharose CL-4B-Cibacron blue 3GA was used for affinity chromatography of the above two enzymes.