Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins' sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25-35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation

NLOS Localization Exploiting Frequency-selective Metasurfaces

This paper introduces a new approach to localize user devices located in non-line-of-sight (NLOS) areas using a passive, non-reconfigurable, and frequency-selective metasurface called metaprism. By analyzing the spatial filtering of subcarriers in the orthogonal frequency division multiplexing (OFDM) signal transmitted by each user device, the base station can estimate the device's angle of view, distance, and subsequently its position. Two different criteria are proposed for designing the frequency response of the metaprism, depending on whether the users operate in the far-field or near-field region of the metaprism. Simulation results in the millimeter-wave band demonstrate that the system can achieve an accuracy of less than 2 degrees in angle estimation and in the order of decimeters in position estimation

Alkaline phosphatase activity in cheese as a tracer for cheese milk pasteurization

Abstract Alkaline phosphatase (ALP) activity is used throughout the world as a marker for the proper pasteurization of milk, to guarantee its hygienic safety. The Standard ISO 11816-2/IDF 155-2 describes the analysis of ALP in cheese. However, the method has been questioned in the past because there have sometimes been ambiguous results. The critical operations of the analytical procedure are more precisely defined and a zonal cheese sampling adopted. ALP inactivation is firstly evaluated in the relevant steps of controlled cheese makings of hard (Emmental), semi-hard (Raschera) and soft (Chaource) cheeses. Application of the improved procedure in over 700 samples of typical cheeses from France, Italy and Switzerland proved the applicability of the method. Based on this large study, a limit for ALP activity in cheese from pasteurized milk is proposed at 10 mU/g

Components of the E. coli envelope are affected by and can react to protein over-production in the cytoplasm.

BACKGROUND: Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway. In this context, we focused on the still poorly addressed relationship between protein production in the cytoplasm and the bacterial envelope, an active and reactive cell compartment that controls interactions with the environment and several major cellular processes. Results available to date show that the accumulation of foreign proteins in the cytoplasm induces changes in the membrane lipids and in the levels of mRNAs for some membrane proteins. However, a direct connection between membrane protein expression levels and soluble/aggregated protein accumulation in the cytoplasm has never been reported. RESULTS: By the use of a combined physiological and proteomic approach, we investigated the effects on the cell membrane of E. coli of the overexpression of two recombinant proteins, the B. cepacia lipase (BCL) and the green fluorescent protein (GFP). Both polypeptides are expressed in the cytoplasm at similar levels but GFP is fully soluble whereas inactive BCL accumulates in inclusion bodies.Growth and viability of the transformed cells were tested in the presence of different drugs. We found that chloramphenycol preferentially inhibited the strain over-producing GFP while SDS was more effective when BCL inclusion bodies accumulated in the cytoplasm. In contrast, both proteins induced a similar response in the membrane proteome, i.e. increased levels of LamB, OmpF, OmpA and TolC. Under all tested conditions, the lipopolysaccharide was not affected, suggesting that a specific rather than a generalized rearrangement of the envelope was induced. CONCLUSION: Taking together physiological and biochemical evidence, our work indicates that the E. coli envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Components of the E. coli envelope are affected by and can react to protein over-production in the cytoplasm

<p>Abstract</p> <p>Background</p> <p>Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway. In this context, we focused on the still poorly addressed relationship between protein production in the cytoplasm and the bacterial envelope, an active and reactive cell compartment that controls interactions with the environment and several major cellular processes. Results available to date show that the accumulation of foreign proteins in the cytoplasm induces changes in the membrane lipids and in the levels of mRNAs for some membrane proteins. However, a direct connection between membrane protein expression levels and soluble/aggregated protein accumulation in the cytoplasm has never been reported.</p> <p>Results</p> <p>By the use of a combined physiological and proteomic approach, we investigated the effects on the cell membrane of <it>E. coli </it>of the overexpression of two recombinant proteins, the <it>B. cepacia </it>lipase (BCL) and the green fluorescent protein (GFP). Both polypeptides are expressed in the cytoplasm at similar levels but GFP is fully soluble whereas inactive BCL accumulates in inclusion bodies.</p> <p>Growth and viability of the transformed cells were tested in the presence of different drugs. We found that chloramphenycol preferentially inhibited the strain over-producing GFP while SDS was more effective when BCL inclusion bodies accumulated in the cytoplasm. In contrast, both proteins induced a similar response in the membrane proteome, i.e. increased levels of LamB, OmpF, OmpA and TolC. Under all tested conditions, the lipopolysaccharide was not affected, suggesting that a specific rather than a generalized rearrangement of the envelope was induced.</p> <p>Conclusion</p> <p>Taking together physiological and biochemical evidence, our work indicates that the <it>E. coli </it>envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment.</p

The "cold revolution". Present and future applications of cold-active enzymes and ice-binding proteins.

Psychrophilic organisms adapted to cold environments produce molecules of relevance for biotechnological application, in particular enzymes active at low temperatures and ice-binding proteins that control the growth of ice crystals. The use of cold-active enzymes supports low temperature processes that preserve heat labile compounds and can result, in some circumstances, in energy saving. Among the several possible applications in biotransformations, this paper focuses on reactions of relevance for the food industry and in molecular biology, representative of different market segments. Ice-binding proteins reduce tissues damage provoked by ice crystals and are therefore of relevance for frozen foods and for the cryopreservation of organs and tissues in the biomedical sector

Laboratory evolution of copper tolerant yeast strains

<p>Abstract</p> <p>Background</p> <p>Yeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts.</p> <p>Results</p> <p>We characterized the effects elicited in natural strains of <it>Candida humilis </it>and <it>Saccharomyces cerevisiae </it>by the exposure to copper in the culture broth. We observed that, whereas the growth of <it>Saccharomyces </it>cells was inhibited already at low Cu concentration, <it>C. humilis </it>was naturally robust and tolerated up to 1 g · L^-1CuSO₄in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in <it>S. cerevisiae </it>the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant <it>Candida </it>cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts.</p> <p>Conclusions</p> <p>Following evolution, both <it>Candida </it>and <it>Saccharomyces </it>cells were able to proliferate up to 2.5 g · L^-1CuSO₄and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper-binding proteome. However, copper elicits different physiological and molecular reactions in yeasts with different backgrounds.</p

Kinetics of aggregation and structural properties of proteins in inclusion bodies studied by Fourier transform infrared spectroscopy

n/

FT-IR spectroscopy for the study of bacterial membrane stress induced by recombinant protein production

on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meetin