Iron bioleaching and polymers accumulation by an extreme acidophilic bacterium.

In many European regions, both local metallic and non-metallic raw materials are poorly exploited due to their low quality and the lack of technologies to increase their economic value. In this context, the development of low cost and eco-friendly approaches, such as bioleaching of metal impurities, is crucial. The acidophilic strain Acidiphilium sp. SJH reduces Fe(III) to Fe(II) by coupling the oxidation of an organic substrate to the reduction of Fe(III) and can therefore be applied in the bioleaching of iron impurities from non-metallic raw materials. In this work, the physiology of Acidiphilium sp. SJH and the reduction of iron impurities from quartz sand and its derivatives have been studied during growth on media supplemented with various carbon sources and under different oxygenation conditions, highlighting that cell physiology and iron reduction are tightly coupled. Although the organism is known to be aerobic, maximum bioleaching performance was obtained by cultures cultivated until the exponential phase of growth under oxygen limitation. Among carbon sources, glucose has been shown to support faster biomass growth, while galactose allowed highest bioleaching. Moreover, Acidiphilium sp. SJH cells can synthesise and accumulate Poly-ß-hydroxybutyrate (PHB) during the process, a polymer with relevant application in biotechnology. In summary, this work gives an insight into the physiology of Acidiphilium sp. SJH, able to use different carbon sources and to synthesise a technologically relevant polymer (PHB), while removing metals from sand without the need to introduce modifications in the process set up.

Cold-Active ß-Galactosidases: Insight into Cold Adaption Mechanisms and Biotechnological Exploitation.

ß-galactosidases (EC 3.2.1.23) catalyze the hydrolysis of ß-galactosidic bonds in oligosaccharides and, under certain conditions, transfer a sugar moiety from a glycosyl donor to an acceptor. Cold-active ß-galactosidases are identified in microorganisms endemic to permanently low-temperature environments. While mesophilic ß-galactosidases are broadly studied and employed for biotechnological purposes, the cold-active enzymes are still scarcely explored, although they may prove very useful in biotechnological processes at low temperature. This review covers several issues related to cold-active ß-galactosidases, including their classification, structure and molecular mechanisms of cold adaptation. Moreover, their applications are discussed, focusing on the production of lactose-free dairy products as well as on the valorization of cheese whey and the synthesis of glycosyl building blocks for the food, cosmetic and pharmaceutical industries.

Conversion of sugar beet residues into lipids by Lipomyces starkeyi for biodiesel production.

BACKGROUND: Lipids from oleaginous yeasts emerged as a sustainable alternative to vegetable oils and animal fat to produce biodiesel, the biodegradable and environmentally friendly counterpart of petro-diesel fuel. To develop economically viable microbial processes, the use of residual feedstocks as growth and production substrates is required. RESULTS: In this work we investigated sugar beet pulp (SBP) and molasses, the main residues of sugar beet processing, as sustainable substrates for the growth and lipid accumulation by the oleaginous yeast Lipomyces starkeyi. We observed that in hydrolysed SBP the yeast cultures reached a limited biomass, cellular lipid content, lipid production and yield (2.5 g/L, 19.2%, 0.5 g/L and 0.08 g/g, respectively). To increase the initial sugar availability, cells were grown in SBP blended with molasses. Under batch cultivation, the cellular lipid content was more than doubled (47.2%) in the presence of 6% molasses. Under pulsed-feeding cultivation, final biomass, cellular lipid content, lipid production and lipid yield were further improved, reaching respectively 20.5 g/L, 49.2%, 9.7 g/L and 0.178 g/g. Finally, we observed that SBP can be used instead of ammonium sulphate to fulfil yeasts nitrogen requirement in molasses-based media for microbial oil production. CONCLUSIONS: This study demonstrates for the first time that SBP and molasses can be blended to create a feedstock for the sustainable production of lipids by L. starkeyi. The data obtained pave the way to further improve lipid production by designing a fed-batch process in bioreactor.

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.

Saturn-Shaped Ice Burst Pattern and Fast Basal Binding of an Ice-Binding Protein from an Antarctic Bacterial Consortium.

Ice-binding proteins (IBPs) bind to ice crystals and control their growth, enabling host organisms to adapt to subzero temperatures. By binding to ice, IBPs can affect the shape and recrystallization of ice crystals. The shapes of ice crystals produced by IBPs vary and are partially due to which ice planes the IBPs are bound to. Previously, we have described a bacterial IBP found in the metagenome of the symbionts of Euplotes focardii ( EfcIBP). EfcIBP shows remarkable ice recrystallization inhibition activity. As recrystallization inhibition of IBPs and other materials are important to the cryopreservation of cells and tissues, we speculate that the EfcIBP can play a future role as an ice recrystallization inhibitor in cryopreservation applications. Here we show that EfcIBP results in a Saturn-shaped ice burst pattern, which may be due to the unique ice-plane affinity of the protein that we elucidated using the fluorescent-based ice-plane affinity analysis. EfcIBP binds to ice at a speed similar to that of other moderate IBPs (5 ± 2 mM-1 s-1); however, it is unique in that it binds to the basal and previously unobserved pyramidal near-basal planes, while other moderate IBPs typically bind to the prism and pyramidal planes and not basal or near-basal planes. These insights into EfcIBP allow a better understanding of the recrystallization inhibition for this unique protein.

Aggregation properties of a disordered protein are tunable by pH and depend on its net charge per residue.

Intrinsically disordered proteins (IDPs) possess a peculiar amino acid composition that makes them very soluble. Nevertheless, they can encounter aggregation in physiological and pathological contexts. In this work, we addressed the issue of how electrostatic charges can influence aggregation propensity by using the N-terminus moiety of the measles virus phosphoprotein, PNT, as a model IDP. Taking advantage of the high sequence designability of IDPs, we have produced an array of PNT variants sharing the same hydrophobicity, but differing in net charges per residue and isoelectric points (pI). The solubility and conformational properties of these proteins were analysed through biochemical and biophysical techniques in a wide range of pH values and compared with those of the green fluorescence protein (GFP), a globular protein with lower net charge per residue, but similar hydrophobicity. Tested proteins showed a solubility minimum close to their pI, as expected, but the pH-dependent decrease of solubility was not uniform and driven by the net charge per residue of each variant. A parallel behaviour was observed also in fusion proteins between PNT variants and GFP, which minimally contributes to the solubility of chimeras. Our data suggest that the overall solubility of a protein can be dictated by protein regions endowed with higher net charge per residue and, hence, prompter to respond to pH changes. This finding could be exploited for biotechnical purposes, such as the design of solubility/aggregation tags, and in studies aimed to clarify the pathological and physiological behaviour of IDPs.

Sulfated and sulfonated polymers are able to solubilize efficiently the protein aggregates of different nature.

The search for new ways to suppress unwanted protein aggregation represents an important problem in modern biochemistry, bioengineering, and even medicine. Recently we succeeded in preventing the aggregation using synthetic polyelectrolytes. The present work describes a new approach to solubilizing pre-formed protein aggregates with sulfated or sulfonated polymers (polysulfoanions). For the first time it was shown that polysulfoanions are capable of solubilizing amorphous and amyloid protein inclusion bodies as well as thermal aggregates. Treatment of prion protein inclusion bodies with sulfonated polymers was shown to cause significant decrease in amyloid structure content, whereas in case of thermal aggregates of glyceraldehyde-3-phosphate dehydrogenase the observed solubilization was accompanied by a partial recovery of enzymatic activity. The suggested approach could be relevant in the task of extracting recombinant proteins from inclusion bodies and also useful in the development of amyloid disease therapy.

Structural determinants of cold activity and glucose tolerance of a family 1 glycoside hydrolase (GH1) from Antarctic Marinomonas sp. ef1.

Cold-active enzymes support life at low temperatures due to their ability to maintain high activity in the cold and can be useful in several biotechnological applications. Although information on the mechanisms of enzyme cold adaptation is still too limited to devise general rules, it appears that very diverse structural and functional changes are exploited in different protein families and within the same family. In this context, we studied the cold adaptation mechanism and the functional properties of a member of the glycoside hydrolase family 1 (GH1) from the Antarctic bacterium Marinomonas sp. ef1. This enzyme exhibits all typical functional hallmarks of cold adaptation, including high catalytic activity at 5 °C, broad substrate specificity, low thermal stability, and higher lability of the active site compared to the overall structure. Analysis of the here-reported crystal structure (1.8 Å resolution) and molecular dynamics simulations suggest that cold activity and thermolability may be due to a flexible region around the active site (residues 298-331), whereas the dynamic behavior of loops flanking the active site (residues 47-61 and 407-413) may favor enzyme-substrate interactions at the optimal temperature of catalysis (Topt) by tethering together protein regions lining the active site. Stapling of the N-terminus onto the surface of the ß-barrel is suggested to partly counterbalance protein flexibility, thus providing a stabilizing effect. The tolerance of the enzyme to glucose and galactose is accounted for by the presence of a "gatekeeping" hydrophobic residue (Leu178), located at the entrance of the active site.

Evolutionary history and activity towards oligosaccharides and polysaccharides of GH3 glycosidases from an Antarctic marine bacterium.

Glycoside hydrolases (GHs) are pivotal in the hydrolysis of the glycosidic bonds of sugars, which are the main carbon and energy sources. The genome of Marinomonas sp. ef1, an Antarctic bacterium, contains three GHs belonging to family 3. These enzymes have distinct architectures and low sequence identity, suggesting that they originated from separate horizontal gene transfer events. M-GH3_A and M-GH3_B, were found to differ in cold adaptation and substrate specificity. M-GH3_A is a bona fide cold-active enzyme since it retains 20 % activity at 10 °C and exhibits poor long-term thermal stability. On the other hand, M-GH3_B shows mesophilic traits with very low activity at 10 °C (< 5 %) and higher long-term thermal stability. Substrate specificity assays highlight that M-GH3_A is a promiscuous ß-glucosidase mainly active on cellobiose and cellotetraose, whereas M-GH3_B is a ß-xylosidase active on xylan and arabinoxylan. Structural analysis suggests that such functional differences are due to their differently shaped active sites. The active site of M-GH3_A is wider but has a narrower entrance compared to that of M-GH3_B. Genome-based prediction of metabolic pathways suggests that Marinomonas sp. ef1 can use monosaccharides derived from the GH3-catalyzed hydrolysis of oligosaccharides either as a carbon source or for producing osmolytes.

A novel laccase from Trametes polyzona with high performance in the decolorization of textile dyes.

Laccases are multicopper oxidases able to oxidize several phenolic compounds and find application in numerous industrial applications. Among laccase producers, white-rot fungi represent a valuable source of multiple isoforms and isoenzymes of these multicopper oxidases. Here we describe the identification, biochemical characterization, and application of laccase 2 from Trametes polyzona (TP-Lac2), a basidiomycete fungus emerged among others that have been screened by plate assay. This enzyme has an optimal temperature of 50 °C and in acidic conditions it is able to oxidize both phenolic and non-phenolic compounds. The ability of TP-Lac2 to decolorize textile dyes was tested in the presence of natural and synthetic mediators at 30 °C and 50 °C. Our results indicate that TP-Lac2 most efficiently decolorizes (decolorization rate > 75%) malachite green oxalate, orange G, amido black10B and bromocresol purple in the presence of acetosyringone and 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonate)-ABTS. Overall, the laccase mediator system consisting of TP-Lac2 and the natural mediator acetosyringone has potential as an environmentally friendly alternative for wastewater treatment in the textile industry.

Mechanism of non-phenolic substrate oxidation by the fungal laccase Type 1 copper site from Trametes versicolor: the case of benzo[a]pyrene and anthracene.

Laccases (EC 1.10.3.2) are multicopper oxidases with the capability to oxidize diverse phenolic and non-phenolic substrates. While the molecular mechanism of their activity towards phenolic substrates is well-established, their reactivity towards non-phenolic substrates, such as polycyclic aromatic hydrocarbons (PAHs), remains unclear. To elucidate the oxidation mechanism of PAHs, particularly the activation mechanism of the sp2 aromatic C-H bond, we conducted a density functional theory investigation on the oxidation of two PAHs (anthracene and benzo[a]pyrene) using an extensive model of the T1 copper catalytic site of the fungal laccase from Trametes versicolor.

Sustainable production of a biotechnologically relevant ß-galactosidase in Escherichia coli cells using crude glycerol and cheese whey permeate.

Responsible use of natural resources and waste reduction are key concepts in bioeconomy. This study demonstrates that agro-food derived-biomasses from the Italian food industry, such as crude glycerol and cheese whey permeate (CWP), can be combined in a high-density fed-batch culture to produce a recombinant ß-galactosidase from Marinomonas sp. ef1 (M-ßGal). In a small-scale process (1.5 L) using 250 mL of crude glycerol and 300 mL of lactose-rich CWP, approximately 2000 kU of recombinant M-ßGal were successfully produced along with 30 g of galactose accumulated in the culture medium. The purified M-ßGal exhibited high hydrolysis efficiency in lactose-rich matrices, with hydrolysis yields of 82 % in skimmed milk at 4 °C and 94 % in CWP at 50 °C, highlighting its biotechnological potential. This approach demonstrates the effective use of crude glycerol and CWP in sustainable and cost-effective high-density Escherichia coli cultures, potentially applicable to recombinant production of various proteins.

Why and how protein aggregation has to be studied in vivo.

The understanding of protein aggregation is a central issue in different fields of protein science, from the heterologous protein production in biotechnology to amyloid aggregation in several neurodegenerative and systemic diseases. To this goal, it became more and more evident the crucial relevance of studying protein aggregation in the complex cellular environment, since it allows to take into account the cellular components affecting protein aggregation, such as chaperones, proteases, and molecular crowding. Here, we discuss the use of several biochemical and biophysical approaches that can be employed to monitor protein aggregation within intact cells, focusing in particular on bacteria that are widely employed as microbial cell factories.

Effects of methanol on a methanol-tolerant bacterial lipase.

Methanol is often employed in biocatalysis with the purpose of increasing substrates solubility or as the acyl acceptor in transesterification reactions, but inhibitory effects are observed in several cases. We have studied the influence of methanol on the catalytic activity and on the conformation of the lipase from Burkholderia glumae, which is reported to be highly methanol tolerant if compared with other lipases. We detected highest activity in the presence of 50-70 % methanol. Under these conditions, however, the enzyme stability is perturbed, leading to gradual protein unfolding and finally to aggregation. These results surmise that, for this lipase, methanol-induced deactivation does not depend on inhibition of catalytic activity but rather on negative effects on the conformational stability of the catalyst.

A cold-active esterase enhances mesophilic properties through Mn2+ binding.

A key aspect of adaptation to cold environments is the production of cold-active enzymes by psychrophilic organisms. These enzymes not only have high activity at low temperatures, but also exhibit remarkable structural flexibility and thermolability. In this context, the role of metal ions has been little explored, and the few available studies seem to suggest that metal binding counteracts structural flexibility. This article reports an investigation into the role of the binding of manganese ion (Mn2+ ) in the thermal adaptation of an esterase (M-Est) of the GDSx family, identified in the genome of the Antarctic bacterium Marinomonas sp. ef1. M-Est is specific for esters containing acetate groups and turned out to be a highly thermolabile cold-active enzyme, with a catalysis optimum temperature of 5 °C and a melting temperature of 31.7 °C. A combination of biochemical and computational analyses, including molecular dynamics simulations, revealed that M-Est binds Mn2+ ions via a single binding site located on the surface of the enzyme, close to the active site. Although the interaction between M-Est and Mn2+ induces only local conformational changes involving the active site, quite surprisingly they trigger an improvement in both thermal stability and catalytic efficiency under mild temperature conditions. These results, together with the conservation of the Mn2+ binding site among psychrophilic and psychrotolerant homologues, suggest that Mn2+ binding may be a useful, albeit atypical, strategy to mitigate the detrimental effects of temperature on true cold-active enzymes.

Cheese-whey permeate improves the fitness of Escherichia coli cells during recombinant protein production.

BACKGROUND: Escherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose. RESULTS: The effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness. CONCLUSIONS: CWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

Amplification of the CUP1 gene is associated with evolution of copper tolerance in Saccharomyces cerevisiae.

In living organisms, copper (Cu) contributes to essential functions but at high concentrations it may elicit toxic effects. Cu-tolerant yeast strains are of relevance for both biotechnological applications and studying physiological and molecular mechanisms involved in stress resistance. One way to obtain tolerant strains is to exploit experimental methods that rely on the principles of natural evolution (evolutionary engineering) and allow for the development of complex phenotypic traits. However, in most cases, the molecular and physiological basis of the phenotypic changes produced have not yet been unravelled. We investigated the determinants of Cu resistance in a Saccharomyces cerevisiae strain that was evolved to tolerate up to 2.5 g CuSO(4) l(-1) in the culture medium. We found that the content of intracellular Cu and the expression levels of several genes encoding proteins involved in Cu metabolism and oxidative stress response were similar in the Cu-tolerant (evolved) and the Cu-sensitive (non-evolved) strain. The major difference detected in the two strains was the copy number of the gene CUP1, which encodes a metallothionein. In evolved cells, a sevenfold amplification of CUP1 was observed, accounting for its strongly and steadily increased expression. Our results implicate CUP1 in protection of the evolved S. cerevisiae cells against Cu toxicity. In these cells, robustness towards Cu is stably inheritable and can be reproducibly selected by controlling environmental conditions. This finding corroborates the effectiveness of laboratory evolution of whole cells as a tool to develop microbial strains for biotechnological applications.

Laboratory evolution of copper tolerant yeast strains.

BACKGROUND: 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. RESULTS: We characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L-1 CuSO4 in 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 S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida 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. CONCLUSIONS: Following evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L-1 CuSO4 and 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.

Short-chain alcohols inactivate an immobilized industrial lipase through two different mechanisms.

Broadly used in biocatalysis as acyl acceptors or (co)-solvents, short-chain alcohols often cause irreversible loss of enzyme activity. Understanding the mechanisms of inactivation is a necessary step toward the optimization of biocatalytic reactions and the design of enzyme-based sustainable processes. The functional and structural responses of an immobilized enzyme, Novozym 435 (N-435), exposed to methanol, ethanol, and tert-butanol, are explored in this work. N-435 consists of Candida antarctica lipase B (CALB) adsorbed on polymethacrylate beads and finds application in a variety of processes involving the presence of short-chain alcohols. The nature of the N-435 material required the development of an ad hoc method of structural analysis, based on Fourier transform infrared microspectroscopy, which was complemented by catalytic activity assays and by morphological observation by transmission electron microscopy. The inactivation of N-435 was found to be highly dependent on alcohol concentration and occurs through two different mechanisms. Short-chain alcohols induce conformational changes leading to CALB aggregation, which is only partially prevented by immobilization. Moreover, alcohol modifies the texture of the solid support promoting the enzyme release. Overall, knowledge of the molecular mechanisms underlying N-435 inactivation induced by short-chain alcohols promises to overcome the limitations that usually occur during industrial processes.

Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain.

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.