Solid-binding peptides for immobilisation of thermostable enzymes to hydrolyse biomass polysaccharides.

BACKGROUND: Solid-binding peptides (SBPs) bind strongly to a diverse range of solid materials without the need for any chemical reactions. They have been used mainly for the functionalisation of nanomaterials but little is known about their use for the immobilisation of thermostable enzymes and their feasibility in industrial-scale biocatalysis. RESULTS: A silica-binding SBP sequence was fused genetically to three thermostable hemicellulases. The resulting enzymes were active after fusion and exhibited identical pH and temperature optima but differing thermostabilities when compared to their corresponding unmodified enzymes. The silica-binding peptide mediated the efficient immobilisation of each enzyme onto zeolite, demonstrating the construction of single enzyme biocatalytic modules. Cross-linked enzyme aggregates (CLEAs) of enzyme preparations either with or without zeolite immobilisation displayed greater activity retention during enzyme recycling than those of free enzymes (without silica-binding peptide) or zeolite-bound enzymes without any crosslinking. CLEA preparations comprising all three enzymes simultaneously immobilised onto zeolite enabled the formation of multiple enzyme biocatalytic modules which were shown to degrade several hemicellulosic substrates. CONCLUSIONS: The current work introduced the construction of functional biocatalytic modules for the hydrolysis of simple and complex polysaccharides. This technology exploited a silica-binding SBP to mediate effectively the rapid and simple immobilisation of thermostable enzymes onto readily-available and inexpensive silica-based matrices. A conceptual application of biocatalytic modules consisting of single or multiple enzymes was validated by hydrolysing various hemicellulosic polysaccharides

Decomposition cross-correlation for analysis of collagen matrix deformation by single smooth muscle cells

Microvascular remodeling is known to depend on cellular interactions with matrix tissue. However, it is difficult to study the role of specific cells or matrix elements in an in vivo setting. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local collagen matrix remodeling by single smooth muscle cells. We combined a motorized microscopic setup and time-lapse video microscopy with a new cross-correlation based image analysis algorithm to enable automated recording of cell-induced matrix reorganization. This method rendered 60–90 single cell studies per experiment, for which collagen deformation over time could be automatically derived. Thus, the current setup offers a tool to systematically study different components active in matrix remodeling

Paxillin Mediates Sensing of Physical Cues and Regulates Directional Cell Motility by Controlling Lamellipodia Positioning

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5–10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax−/− and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax−/− cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax−/− and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices

Disinfection of Ocular Cells and Tissues by Atmospheric-Pressure Cold Plasma

Background: Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage. Methodology and Principal Findings: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2) was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis. Conclusions: A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses

A novel framework for the cell-free enzymatic production of glucaric acid.

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ± 0.9 mM (3.0 ± 0.2 g l-1) and a molar yield of 35.2 ± 2.3% using non-immobilised enzymes, and a titre of 8.1 ± 0.2 mM (1.70 ± 0.04 g l-1) and a molar yield of 20.2 ± 0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ± 0.02 g/h/l for free enzymes and 0.170 ± 0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway