Surrogate Substrates Research Articles

High-Throughput Absorbance-Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases.

Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51 % improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

High‐Throughput Absorbance‐Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases

AbstractRecent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy‐intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet‐based microfluidic systems have emerged as a powerful high‐throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet‐based microfluidic systems, we present an absorbance‐activated droplet sorter that allows droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105‐member aldehyde dehydrogenase library towards D‐glyceraldehyde using a NADH mediated coupled assay that generates WST‐1 formazan as the colorimetric product. We successfully identify a variant with a 51 % improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Characterisation of Modular Polyketide Synthases Designed to Make Pentaene Analogues of Amphotericin B.

Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved antibiotics. Here, Streptomyces nodosus was genetically re-programmed to synthesise pentaene analogues of the heptaene amphotericin B. These pentaenes are of interest as surrogate substrates for enzymes catalysing unusual, late-stage biosynthetic modifications. The previous deletion of amphotericin polyketide synthase modules 5 and 6 generated S. nodosus M57, which produces an inactive pentaene. Here, the chain-terminating thioesterase was fused to module 16 to generate strain M57-16TE, in which cycles 5, 6, 17 and 18 are eliminated from the biosynthetic pathway. Another variant of M57 was obtained by replacing modules 15, 16 and 17 with a single 15-17 hybrid module. This gave strain M57-1517, in which cycles 5, 6, 15 and 16 are deleted. M57-16TE and M57-1517 gave reduced pentaene yields. Only M57-1517 delivered its predicted full-length pentaene macrolactone in low amounts. For both mutants, the major pentaenes were intermediates released from modules 10, 11 and 12. Longer pentaene chains were unstable. The novel pentaenes were not glycosylated and were not active against Candida albicans. However, random mutagenesis and screening may yet deliver new antifungal producers from the M57-16TE and M57-1517 strains.

BODIPY-Labeled Estrogens for Fluorescence Analysis of Environmental Microbial Degradation.

Biodegradation of estrogen hormone micropollutants is a well-established approach toward their remediation. Fluorescently labeled substrates are used extensively for rapid, near-real-time analysis of biological processes and are a potential tool for studying biodegradation processes faster and more efficiently than conventional approaches. However, it is important to understand how the fluorescently tagged surrogates compare with the natural substrate in terms of chemical analysis and the intended application. We derivatized three natural estrogens with BODIPY fluorophores by azide-alkyne cycloaddition click reaction and developed an analytical workflow based on simple liquid-liquid extraction and HPLC-PDA analysis. The developed methods allow for concurrent analysis of both fluorescent and natural estrogens with comparable recovery, accuracy, and precision. We then evaluated the use of BODIPY-labeled estrogens as surrogate substrates for studying biodegradation using a model bacterium for estrogen metabolism. The developed analytical methods were successfully employed to compare the biological transformation of 17β-estradiol (E2), with and without the BODIPY fluorescent tag. Through measuring the complete degradation of E2 and the transformation of BODIPY-estradiol to BODIPY-estrone in the presence of a co-substrate, we found that BODIPY-labeled estrogens are biologically viable surrogates for investigating biodegradation in environmental bacteria.

Comparative Investigations on Different β-Glucosidase Surrogate Substrates

β-glucosidases are hydrolyzing enzymes which can release many aroma-active compounds from their glycoside form. Several yeasts produce these enzymes and thus are applied during the wine production process. To be able to test specific organisms for the presence of β-glucosidases and to investigate this enzyme activity, four main surrogate substrates have been described. The properties and applicability of these compounds, named arbutin (hydroquinone-β-D-glucopyranoside), esculin (6-O-(-D-glucosyl)aesculetin), 4-nitrophenyl-β-D-glucopyranoside (pNPG) and 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG), are discussed after comparing their advantages and disadvantages. Although all four substrates were found suitable for photometric assays, 4-MUG has proven to be most appropriate due to high sensitivity, high robustness and simple processing. Furthermore, the investigation of β-glucosidase product accumulation is described, which could be used to give indications about β-glucosidase localization.

Molluscs community as a keystone group for assessing the impact of urban sprawl at intertidal ecosystems

Mollusc communities are getting endangered in the aftermath of urban sprawl because artificial structures do not surrogate natural substrates. In this study, we compared the diversity, community and trophic arrangements of molluscs among different models of artificial substrate and their adjacent natural rock, to detect relationships between some abiotic variables and the mollusc communities. Complexity, chemical composition and age were tested as potential drivers of the community. Diversity, community and trophic structure differed between natural and artificial substrates. Complexity at the scale of cm was detected as the most important factor driving the community structure. In addition, a chemical composition based on silica and/or scarce calcium carbonates seems to be relevant for molluscs, as well as for the secondary substrate where they inhabit. However, age did not seem to be a driving factor. Among the different artificial structures, macroscale complexity was detected as the main factor diverging a drastically poor community at seawall from other artificial structures. In this context, macro and microscale complexity, chemical composition and mineral type are variables to consider in future designs of artificial substrates.

Biosynthetic incorporation for visualizing bacterial glycans.

Cell-surface glycans are central to many biological processes, yet methods for their site-selective modification are limited. Strategies for interrogating the structure and function of proteins have been enabled by chemoselective reactions of sidechain functionality for covalent modification, capture, or imaging. However, unlike protein sidechains, glycan building blocks lack distinguishing reactivity. Moreover, glycans are not primary gene products, so encoding glycan variants through genetic manipulation is challenging. Reactive functional groups can be introduced into glycans through metabolic engineering, which involves the generation of modified nucleotide-sugar building blocks. Lipid-linked building blocks, which are also used in glycan biosynthesis, have the advantage that they can be delivered directly to glycosyltransferases to function as surrogate substrates. This process, termed "biosynthetic incorporation," takes advantage of the properties of bacterial glycosyltransferase: they are selective for the products they generate yet promiscuous in their donor preferences. We describe how this strategy can be implemented to label arabinofuranose-containing glycans on the surface of mycobacterial cells. We anticipate that this platform can be expanded to develop chemoselective labeling agents for other important bacterial monosaccharides.

Noninvasive, label-free, and quantitative monitoring of lipase kinetics using terahertz emission technology.

Enzymes catalyze chemical transformations of great importance in many fields, and analysis of the rate of these transformations is equally important. The latter are typically monitored using surrogate substrates that produce quantifiable optical signals, owing to limitations associated with "label-free" techniques that could be used to monitor the transformation of original substrate molecules. In this study, terahertz (THz) emission technology is used as a noninvasive and label-free technique to monitor the kinetics of lipase-induced hydrolysis of several substrate molecules (including the complex substrate whole cow's milk) and horseradish peroxidase-catalyzed oxidation of o-phenylenediamine in the presence of H2 O2 . This technique was found to be quantitative, and kinetic parameters are compared to those obtained by proton NMR spectroscopy or UV/Vis spectroscopy. This study sets the stage for investigating THz emission technology as a tool for research and development involving enzymes, and for monitoring industrial processes in the food, cosmetic, detergent, pharmaceutical, and biodiesel sectors.

Characterization of black crusts developed on historic stones with diverse mineralogy under different air quality environments

Black crusts (BCs) are one of the most critical alteration forms found on stones belonging to architectural heritage. Since they could be considered as passive samplers of atmospheric pollution, it would be plausible to establish relations between the air contamination and the BCs. With this aim, we have characterized BCs collected on historic buildings from two Spanish cities (Granada and Vigo) with different polluted atmospheres, as well as formed on stone substrates of varied mineralogy and texture. Likewise, in order to assess the impact of the atmospheric pollutants on the growth of BCs, quartz fiber filters were used as surrogate substrates and placed nearby the studied buildings to collect and analyze the aerosol particulate matter (PM). To this end, an array of complementary analytical techniques was used to evaluate the mineralogy, chemical composition, and texture of the BCs and to establish the correlation with the ions, OC (organic carbon), and EC (elemental carbon) detected in the PM on the quartz fiber filters. As result, BCs developed on carbonate substrates from Granada show more complex structure than those from Vigo, which are thinner because of frequent rain episodes. In both cities, NaCl, Pb-Cl, and Ca-Cl-rich particles, Ca-phosphate particles and clusters of Ba-sulfate-rich particles were detected. However, metal-rich rounded particles were more abundant in Granada’s BCs, including soot particles. BCs from Granada were richer in carbonaceous components (OC and EC) than the Vigo’s BCs. Although in the filters PM did not show EC—mainly due to traffic—, in the BCs from both locations OC and EC were detected. Therefore, this different composition was related to the mineralogy of the stones and the higher pollution of Granada in contrast to the industrial and sea-exposed city of Vigo.

Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog

Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (thN) and isothiazole[4,3-d]pyrimidine (tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, thGN, the isothiazolo guanine surrogate, tzGN, does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, tzXN. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.

High-Throughput, Fluorescence-Based Esterase Activity Assay for Assessing Polysorbate Degradation Risk during Biopharmaceutical Development.

Hydrolytic degradation of polysorbate during 2-8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established. A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies. We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates. The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.

P-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA

The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C8) acyl chains into structurally-opposed hydrophobic channels, denoted A and B, to carry out a non-decarboxylative Claisen condensation reaction and initiate the biosynthesis of membrane hydrocarbons and β-lactone natural products. OleA has now been identified in hundreds of diverse bacteria via bioinformatics and high-throughput screening using p-nitrophenyl alkanoate esters as surrogate substrates. In the present study, p-nitrophenyl esters were used to probe the reaction mechanism of OleA and shown to be incorporated into Claisen condensation products for the first time. p-Nitrophenyl alkanoate substrates alone were shown not to undergo Claisen condensation, but co-incubation of p-nitrophenyl esters and CoA thioesters produced mixed Claisen products. Mixed product reactions were shown to initiate via acyl group transfer from a p-nitrophenyl carrier to the enzyme active site cysteine, C143. Acyl chains esterified to p-nitrophenol were synthesized and shown to undergo Claisen condensation with an acyl-CoA substrate, showing potential to greatly expand the range of possible Claisen products. Using p-nitrophenyl 1-13C-decanoate, the Channel A bound thioester chain was shown to act as the Claisen nucleophile, representing the first direct evidence for the directionality of the Claisen reaction in any OleA enzyme. These results both provide new insights into OleA catalysis and open a path for making unnatural hydrocarbon and β-lactone natural products for biotechnological applications using cheap and easily synthesized p-nitrophenyl esters.

Designed variants of ACE2-Fc that decouple anti-SARS-CoV-2 activities from unwanted cardiovascular effects

Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for SARS-CoV-2, and recombinant ACE2 decoys are being evaluated as new antiviral therapies. We designed and tested an antibody-like ACE2-Fc fusion protein, which has the benefit of long pharmacological half-life and the potential to facilitate immune clearance of the virus. Out of a concern that the intrinsic catalytic activity of ACE2 may unintentionally alter the balance of its hormonal substrates and cause adverse cardiovascular effects in treatment, we performed a mutagenesis screening for inactivating the enzyme. Three mutants, R273A, H378A and E402A, completely lost their enzymatic activity for either surrogate or physiological substrates. All of them remained capable of binding SARS-CoV-2 and could suppress the transduction of a pseudotyped virus in cell culture. This study established new ACE2-Fc candidates as antiviral treatment for SARS-CoV-2 without potentially harmful side effects from ACE2's catalytic actions toward its vasoactive substrates.

A phase Ib study of the safety and pharmacology of nilotinib to prevent paclitaxel-induced peripheral neuropathy in patients with breast cancer.

TPS12128 Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating adverse effect of paclitaxel, but convincing evidence for a preventative technique founded on mechanistic rationale is lacking. We previously report that paclitaxel accumulation is mediated by the murine organic anion transporting polypeptide, OATP1B2 in mice (OATP1B1 and OATP1B3 in humans) (Leblanc, J Clin Invest, 2018). We found that paclitaxel induces acute and chronic neurotoxicity in mice in an OATP1B2-dependent manner, which is reversed by pre-treatment with FDA-approved tyrosine kinase inhibitor, nilotinib. Methods: This phase Ib dose finding study of nilotinib in combination with weekly paclitaxel is investigating the hypothesis that intermittent dosing of nilotinib 24 hours before paclitaxel infusion (excluding C1D1) and again 30 minutes before paclitaxel infusion will inhibit OATP1B1 and prevent CIPN. This hypothesis will be assessed using an adaptive Bayesian method for dose finding based on efficacy-toxicity trade-offs in patients with breast cancer stages I-III who qualify for paclitaxel therapy. Patients with previous ≥ grade 2 neuropathy on breast cancer therapies will be excluded. The primary objectives are to find the recommended phase II dose of nilotinib in combination with paclitaxel for early stage breast cancer, defined as the lowest intermittent dose of nilotinib that temporarily inhibits OATP1B1 function without affecting paclitaxel plasma pharmacokinetics (PK), and to determine the toxicity profile of nilotinib in combination with paclitaxel. OATP1B1 inhibition by nilotinib will be assessed via validated surrogate endogenous substrates, glycochenodeoxycholate sulfate (GCDGA-S) and chenodeoxycholate-24-glucuronide (CDCA-24G). Effective OATP1B1 inhibition will be ≥5-fold increase in AUC of GCDCA-S from pre- to post- treatment or detectable CDCA-24G levels post-treatment. The 4 nilotinib dose levels are 50 mg (dose level -1), 100 mg (dose level 1), 200 mg (dose level 2), 300 mg (dose level 3). IV paclitaxel dose of 80 mg/m2 on D1,8,15 for 12 total weekly doses will be used. Oral nilotinib will be administered 24 hours before paclitaxel infusion on C1D7,D14 and again 30 minutes before infusion on C1D8,D15. Secondary objectives are to determine paclitaxel’s effect on PK of nilotinib and vice versa. Quality of life via CIPN-20 survey, disease free survival, event free survival, overall survival are exploratory objectives. NCT04205903 enrollment opened February 2020 and is accepting patients. Clinical trial information: NCT04205903 .

Biochemical and molecular dynamics studies of archaeal polyisoprenyl pyrophosphate phosphatase from Saccharolobus solfataricus

The undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane pyrophosphatase. In bacteria, UppP catalyzes the dephosphorylation of undecaprenyl pyrophosphate (C55-pp) to undecaprenyl phosphate (C55-P) in the periplasmic space, which is an essential step for the isoprenyl lipid carrier to reenter the peptidoglycan synthesis cycle. Besides bacteria, the UppP homologs are widely distributed in archaea genome. However, all archaea lack peptidoglycan structure in their cell wall components, and the major archaeal lipid carriers are dolichol phosphate (Dol-p) and dolichol pyrophosphate (Dol-pp), so the functions of the UppP homolog in archaea remain unclear. Here, we purified a recombinant polyisoprenyl pyrophosphatase of a thermoacidophilic archaeon, Saccharolobus solfataricus (SsUppP), and characterized its enzymatic properties. Two isoprenyl pyrophosphate, farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (Ggpp), were used as the surrogate substrates, simulating the bacterial and archaeal lipid carriers. SsUppP dephosphorylated Fpp and Ggpp at 37 °C, but retained the phosphatase activity at high temperatures. The optimal condition for the enzymatic activity was found to be at pH 7 and 70 °C. The thermostability of SsUppP was also supported by molecular dynamics simulation studies. Our results indicated that the archaeal SsUppP can dephosphorylate isoprenyl pyrophosphates at the natural environment of high temperature, and the possibility to catalyze the dephosphorylation of archaeal lipid carriers.

A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.

Alternative Splicing of the Aflatoxin-Associated Baeyer⁻Villiger Monooxygenase from Aspergillus flavus: Characterisation of MoxY Isoforms.

Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer–Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability.

Substrate and inhibitor selectivity, and biological activity of an epoxide hydrolase from Trichoderma reesei.

Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural substrates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible development of novel drugs for human diseases.

Study and Characterization of Environmental Deposition on Marble and Surrogate Substrates at a Monumental Heritage Site

In this study, the results of the field exposure activity conducted between 2014 and 2017 on the façade of the Milano cathedral (Italy) are reported. The main research aim was to characterize environmental deposition in real exposure conditions and for this purpose, both stone substrates (Candoglia marble) and surrogate substrates (quartz fibre filters) were exposed on the cathedral façade in two sites at different heights. A complete chemical characterization has been performed on quartz filters and marble substrates, i.e., quantification of the deposited aerosol particulate matter (PM) and of the main ions. On quartz filters, the carbonaceous component of deposits was also investigated, as well as the color change induced by soiling, by means of colorimetric measurements. The combined approach exploiting marble and surrogate substrates seems to be a suitable monitoring strategy, although some aspects should be taken into account. In particular, differences in the deposits composition have been highlighted mainly depending on the type of substrate. The environmental data related to atmospheric pollution in Milan for the same period have also been considered but no direct correlations were found between some atmospheric precursors and their related ions in solid deposits.

Chemically and size-resolved particulate matter dry deposition on stone and surrogate surfaces inside and outside the low emission zone of Milan: application of a newly developed "Deposition Box".

The collection of atmospheric particles on not-filtering substrates via dry deposition, and the subsequent study of the particle-induced material decay, is trivial due to the high number of variables simultaneously acting on the investigated surface. This work reports seasonally resolved data of chemical composition and size distribution of particulate matter deposed on stone and surrogate surfaces obtained using a new method, especially developed at this purpose. A "Deposition Box" was designed allowing the particulate matter dry deposition to occur selectively removing, at the same time, variables that can mask the effect of airborne particles on material decay. A pitched roof avoided rainfall and wind variability; a standardised gentle air exchange rate ensured a continuous "sampling" of ambient air leaving unchanged the sampled particle size distribution and, at the same time, leaving quite calm condition inside the box, allowing the deposition to occur. Thus, the "Deposition Box" represents an affordable tool that can be used complementary to traditional exposure systems. With this system, several exposure campaigns, involving investigated stone materials (ISMs) (Carrara Marble, Botticino limestone, Noto calcarenite and Granite) and surrogate (Quartz, PTFE, and Aluminium) substrates, have been performed in two different sites placed in Milan (Italy) inside and outside the low emission zone. Deposition rates (30-90μgcm-2month-1) showed significant differences between sites and seasons, becoming less evident considering long-period exposures due to a positive feedback on the deposition induced by the deposited particles. Similarly, different stone substrates influenced the deposition rates too. The collected deposits have been observed with optical and scanning electron microscopes and analysed by ion chromatography. Ion deposition rates were similar in the two sites during winter, whereas it was greater outside the low emission zone during summer and considering the long-period exposure. The dimensional distribution of the collected deposits showed a significant presence of fine particles in agreement with deposition rate of the ionic fraction. The obtained results allowed to point out the role of the fine particles fraction and the importance of making seasonal studies.