Mycobiota profile of oral fungal infections in head and neck cancer patients receiving radiotherapy: A 6-year retrospective MALDI-TOF mass spectrometry study.

OBJECTIVES: Head and neck cancer (HNC) impairs patient immunity and increases susceptibility to oral fungal infections (OFIs). Effectively treating such infections requires accurate identification of the causative pathogens. This study aimed to characterize the mycobiota profile of OFIs in HNC patients undergoing radiation treatment (RT). MATERIALS AND METHODS: A 6-year retrospective analysis of oral mucosal samples from HNC patients with a history of RT and OFIs between 2014 and 2019 was conducted using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) profiling. Samples from the Clinical Microbiology Laboratory at Karolinska University Hospital were evaluated for mycobiota diversity and species co-occurrence patterns in the ongoing-RT and post-RT groups. RESULTS: A total of 190 oral fungi (88% Candida, 5% Pichia) were isolated from 162 HNC patients receiving RT. In the ongoing-RT group, the emergent non-albicans Candida (NAC) species; F. solani and C. jadinii, were detected for the first time. The dominant pathogens in both ongoing and post-RT groups were C. albicans, C. glabrata, P. kudriavzevii, C. parapsilosis, and C. tropicalis, as shown by Venn analysis. Network analysis revealed greater fungi diversity and multi-species co-occurrence in the ongoing-RT group. C. albicans commonly co-occurred with C. glabrata in both ongoing-RT (21%) and post-RT groups (30%). CONCLUSION: MALDI-TOF MS identified a wide range of oral fungal species in HNC patients receiving RT. While C. albicans remains the most prevalent OFIs pathogen, multi-species co-occurrence and novel NACs were noted. Understanding the ecological interactions among these causative pathogens could significantly advance the development of effective therapeutics for treating OFIs in HNC patients.

Improved catalytic properties of Candida antarctica lipase B immobilized on cetyl chloroformate-modified cellulose nanocrystals.

The catalytic activity of Candida antarctica lipase B (CALB) immobilized on modified cellulose nanocrystals (CNC) with different hydrophobicity was investigated using experimental and theoretical approaches. Firstly, the modified CNC were characterized by multi-spectroscopic methods, water contact angle, scanning electron microscopy and thermogravimetric analysis. Moderately hydrophobic CNC were found to be an optimal support for CALB immobilization. Secondly, model systems contained a CALB molecule and different numbers of modified CNC molecules (CALB@3CNC-C16, CALB@10CNC-C16 and CALB@15CNC-C16) were prepared for molecular dynamics (MD) simulation. Root-mean-square fluctuation values (0.61-2.61 Å) of lid region were relatively high in CALB@10CNC-C16, indicating that modified CNC with moderate hydrophobicity favored forming a lid-open conformation of CALB. Finally, the esterification of oleic acid catalyzed by the immobilized CALB showed higher conversion (54.68 %) than free CALB (12.98 %). Insights into modified CNC with tunable properties provided by this study may be a potential support for improving the catalytic performance of lipases.

Raman Spectroscopy of Oral Candida Species: Molecular-Scale Analyses, Chemometrics, and Barcode Identification.

Oral candidiasis, a common opportunistic infection of the oral cavity, is mainly caused by the following four Candida species (in decreasing incidence rate): Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei. This study offers in-depth Raman spectroscopy analyses of these species and proposes procedures for an accurate and rapid identification of oral yeast species. We first obtained average spectra for different Candida species and systematically analyzed them in order to decode structural differences among species at the molecular scale. Then, we searched for a statistical validation through a chemometric method based on principal component analysis (PCA). This method was found only partially capable to mechanistically distinguish among Candida species. We thus proposed a new Raman barcoding approach based on an algorithm that converts spectrally deconvoluted Raman sub-bands into barcodes. Barcode-assisted Raman analyses could enable on-site identification in nearly real-time, thus implementing preventive oral control, enabling prompt selection of the most effective drug, and increasing the probability to interrupt disease transmission.

Effects of Yeast Species and Processing on Intestinal Health and Transcriptomic Profiles of Atlantic Salmon (Salmo salar) Fed Soybean Meal-Based Diets in Seawater.

The objective of the current study was to examine the effects of yeasts on intestinal health and transcriptomic profiles from the distal intestine and spleen tissue of Atlantic salmon fed SBM-based diets in seawater. Cyberlindnera jadinii (CJ) and Wickerhamomyces anomalus (WA) yeasts were heat-inactivated with spray-drying (ICJ and IWA) or autolyzed at 50 °C for 16 h (ACJ and AWA), followed by spray-drying. Six diets were formulated, one based on fishmeal (FM), a challenging diet with 30% soybean meal (SBM) and four other diets containing 30% SBM and 10% of each of the four yeast fractions (i.e., ICJ, ACJ, IWA and AWA). The inclusion of CJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α labeled cells present in the lamina propria of fish fed the SBM diet. The CJ yeasts controlled the inflammatory responses of fish fed SBM through up-regulation of pathways related to wound healing and taurine metabolism. The WA yeasts dampened the inflammatory profile of fish fed SBM through down-regulation of pathways related to toll-like receptor signaling, C-lectin receptor, cytokine receptor and signal transduction. This study suggests that the yeast species, Cyberlindnera jadinii and Wickerhamomyces anomalus are novel high-quality protein sources with health-beneficial effects in terms of reducing inflammation associated with feeding plant-based diets to Atlantic salmon.

Development of a Highly Sensitive ß-Glucan Detection System Using Scanning Single-Molecule Counting Method.

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant ß-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-ß-D-glucan recognition protein (S-BGRP) and a (1→6)-ß-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-ß-D-glucan from fungi. S-BGRP and (1→6)-ß-glucanase mutant proteins reacted with ß-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived ß-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-ß-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of ß-glucan levels by the SSMC method using recombinant ß-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.

Chemoenzymatic Synthesis of the New 3-((2,3-Diacetoxypropanoyl)oxy)propane-1,2-diyl Diacetate Using Immobilized Lipase B from Candida antarctica and Pyridinium Chlorochromate as an Oxidizing Agent.

To exploit the hydrolytic activity and high selectivity of immobilized lipase B from Candida antarctica on octyl agarose (CALB-OC) in the hydrolysis of triacetin and also to produce new value-added compounds from glycerol, this work describes a chemoenzymatic methodology for the synthesis of the new dimeric glycerol ester 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate. According to this approach, triacetin was regioselectively hydrolyzed to 1,2-diacetin with CALB-OC. The diglyceride product was subsequently oxidized with pyridinium chlorochromate (PCC) and a dimeric ester was isolated as the only product. It was found that the medium acidity during the PCC treatment and a high 1,2-diacetin concentration favored the formation of the ester. The synthesized compounds were characterized using IR, MS, HR-MS, and NMR techniques. The obtained dimeric ester was evaluated at 100 ppm against seven bacterial strains and two Candida species to identify its antimicrobial activity. The compound has no inhibitory activity against the bacterial strains used but decreased C. albicans and C. parapsilosis growth by 49% and 68%, respectively. Hemolytic activity was evaluated, and the results obtained support the use of the dimeric ester to control C. albicans and C. parapsilosis growth in non-intravenous applications because the compound shows hemolytic activity.

A detailed lipidomic study of human pathogenic fungi Candida auris.

The present study is an attempt to determine the lipid composition of Candida auris and to highlight if the changes in lipids can be correlated to high drug resistance encountered in C. auris. For this, the comparative lipidomics landscape between drug-susceptible (CBS10913T) and a resistant hospital isolate (NCCPF_470033) of C. auris was determined by employing high throughput mass spectrometry. All major groups of phosphoglycerides (PGL), sphingolipids, sterols, diacylglycerols (DAG) and triacylglycerols (TAG), were quantitated along with their molecular lipid species. Our analyses highlighted several key changes where the NCCPF_470033 showed an increase in PGL content, specifically phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine; odd chain containing lipids and accumulation of 16:1-DAG and 16:0-DAG; depletion of 18:1-TAG and 18:0-TAG. The landscape of molecular species displayed a distinct imprint between isolates. For example, the levels of unsaturated PGLs, contributed by both odd and even-chain fatty acyls were higher in resistant NCCPF_470033 isolate, resulting in a higher unsaturation index. Notwithstanding, several commonalities of lipid compositional changes between resistant C. auris and other Candida spp., the study could also identify distinguishable changes in specific lipid species in C. auris. Together, the data highlights the modulation of membrane lipid homeostasis associated with drug-resistant phenotype of C. auris.

Effect of pH on xylitol production by Candida species from a prairie cordgrass hydrolysate.

Using hydrolysates of the North American prairie grass prairie cordgrass buffered at pH 4.5, 5.0, 5.5 or 6.0, xylitol production, xylitol yield, cell biomass production and productivity were investigated for three strains of yeast Candida. Of the three strains, the highest xylitol concentration of 20.19 g xylitol (g xylose consumed)-1 and yield of 0.89 g xylitol (g xylose consumed)-1 were produced by Candida mogi ATCC 18364 when grown for 120 h at 30° C on the pH 5.5-buffered hydrolysate-containing medium. The highest biomass level being 7.7 g cells (kg biomass)-1 was observed to be synthesized by Candida guilliermondii ATCC 201935 after 120 h of growth at 30° C on a pH 5.5-buffered hydrolysate-containing medium. The highest xylitol specific productivity of 0.73 g xylitol (g cells h)-1 was determined for C. guilliermondii ATCC 20216 after 120 h of growth at 30°C on a pH 5.0-buffered hydrolysate-containing medium. Xylitol production and yield by the three Candida strains was higher on prairie cordgrass than what was previously observed for the same strains after 120 h at 30° C when another North American prairie grass big bluestem served as the plant biomass hydrolysate indicating that prairie cordgrass may be a superior plant biomass substrate.

Comparison of simple and rapid cell wall disruption methods for improving lipid extraction from yeast cells.

The present study examined the effect of six disruption methods of the cell wall (acid hydrolysis, ultrasonication, osmotic shock, pasteurization, homogenization with zirconia balls, and freezing/defrosting) on the efficiency of lipid extraction from yeast cells and the composition of fatty acids. Acid hydrolysis and sonication led to a significant increase in lipid extraction from Cyberlindnera jadinii ATCC 9950 and Rhodotorula glutinis LOCKR13 yeast cells. The amount of lipids extracted in these conditions increased for C. jadinii from 12.46 (biomass not subjected to any pretreatment) to 20.37 and 19.53 g/100 gd.w. after the application of acid hydrolysis and sonication, respectively, and for R. glutinis strain from 13.95 to 21.20 and 17.22 g/100 gd.w., respectively, for the same methods. Initial sonication of biomass led to a significant reduction in the percentage of unsaturated fatty acids. The largest differences in fatty acid composition were found for the sample homogenized with zirconium balls. This process resulted in the degradation of both oleic acid and linolenic acid. The obtained results revealed that the method that significantly increases lipid extraction and does not change the composition of fatty acids is acid hydrolysis with hydrochloric acid. In addition, it is easy, cheap, does not require specialized equipment, and therefore can be implemented in any laboratory.

Cyberlindnera jadinii yeast as a protein source for broiler chickens: effects on growth performance and digestive function from hatching to 30 days of age.

Europe is heavily dependent on imported feed protein sources such as soybean meal (SBM); thus, investigating local sustainable alternatives is crucial to increase self-sufficiency. This study evaluated the effects of the inactivated yeast Cyberlindnera jadinii grown on local lignocellulosic sugars on the growth performance and digestive function of Ross 308 broiler chickens. A total of 1,000 male chicks were allocated to 20 pens. There were 5 replicate pens with 50 birds each, from 1 to 30 D after hatch. The birds were offered one conventional wheat-oat-SBM-based control diet and 3 diets with increasing levels of C. jadinii replacing 10, 20, and 30% of dietary crude protein (CP), whereas SBM levels were gradually decreased. The feed intake and weight gain of the birds decreased linearly, and feed conversion ratio increased linearly (P < 0.01) with increasing dietary levels of C. jadinii. Nevertheless, growth performance and feed intake were similar between the birds fed with control diets and diets containing 10% CP from C. jadinii in the starter and grower periods. The apparent ileal digestibility (AID) of dry matter, crude fat, organic matter, and carbohydrates was higher in control diets than in diets with 30% C. jadinii CP (P < 0.05) and decreased (P < 0.01) with incremental levels of dietary C. jadinii. Regardless, the AID of CP, starch, ash, and phosphorus was unaffected. Ileal villus height on day 10 was maintained in birds fed with diets containing 30% C. jadinii CP compared with the birds fed with control diets but was lower for birds fed with diets containing 10 and 20% C. jadinii protein (P < 0.05). To conclude, up to 10% C. jadinii CP can replace SBM CP in broiler chicken diets, maintaining growth performance and digestive function, whereas higher levels of C. jadinii may decrease bird performance. Altogether, this suggests the potential of C. jadinii as a local-based protein source in broiler chicken diets, contributing to a more sustainable feed.

Carboxylic Acid Transporters in Candida Pathogenesis.

Opportunistic pathogens such as Candida species can use carboxylic acids, like acetate and lactate, to survive and successfully thrive in different environmental niches. These nonfermentable substrates are frequently the major carbon sources present in certain human body sites, and their efficient uptake by regulated plasma membrane transporters plays a critical role in such nutrient-limited conditions. Here, we cover the physiology and regulation of these proteins and their potential role in Candida virulence. This review also presents an evolutionary analysis of orthologues of the Saccharomyces cerevisiae Jen1 lactate and Ady2 acetate transporters, including a phylogenetic analysis of 101 putative carboxylate transporters in twelve medically relevant Candida species. These proteins are assigned to distinct clades according to their amino acid sequence homology and represent the major carboxylic acid uptake systems in yeast. While Jen transporters belong to the sialate:H+ symporter (SHS) family, the Ady2 homologue members are assigned to the acetate uptake transporter (AceTr) family. Here, we reclassify the later members as ATO (acetate transporter ortholog). The new nomenclature will facilitate the study of these transporters, as well as the analysis of their relevance for Candida pathogenesis.

Raman micro-spectroscopy monitoring of cytochrome c redox state in Candida utilis during cell death under low-temperature plasma-induced oxidative stress.

Oxidative stress may result in different modes of cell death, such as necrosis, apoptosis and necroptosis. Currently, researchers are still striving to develop efficient tools/methods to distinguish the cell death modes in direct and label-free ways. In this study, we attempted to employ Raman micro-spectroscopy to observe the molecular changes in Candida utilis cells under oxidative stress induced by low-temperature plasma (LTP) and explore the spectroscopic biomarkers for the modes of cell death under oxidative stress. In this research, we confirmed that LTP could impose oxidative stress on the yeast cells, and recorded the changes of Raman signals of cytochrome c in the cells under LTP oxidative stress. Subsequently, we identified the biochemical and morphological characteristic features corresponding to different modes of cell death. Interestingly, we found that LTP under certain conditions could induce oxidative stress which caused the yeast cell death mainly by means of necroptosis, which was verified by Annexin V/PI, HMGB1 location assay and immunoprecipitation assay of the RIP1/RIP3 necrosome. Correspondingly, we also showed that the LTP induced necroptosis, associated with the increase of cytoplasmic Ca2+ and mitochondrial ROS, the decrease of mitochondrial membrane potential, the release of oxidized cytochrome c from the mitochondrion to the cytoplasm, and the destruction of mitochondria in yeast cells. This work has therefore demonstrated that monitoring the redox state of cytochrome c using Raman micro-spectroscopy is very useful for distinguishing the modes of cell death and particularly may unveil the unique necroptosis process of cells under extrinsic oxidative stress.

Influence of the Sample Preparation Method in Discriminating Candida spp. Using ATR-FTIR Spectroscopy.

Several studies have investigated the capacity of ATR-FTIR spectroscopy for fungal species discrimination. However, preparation methods vary among studies. This study aims to ascertain the effect of sample preparation on the discriminatory capacity of ATR-FTIR spectroscopy. Candida species were streaked to obtain colonies and spectra were collected from each preparation type, which included: (a) untreated colonies being directly transferred to the ATR crystal, (b) following washing and (c) following 24-h fixation in formalin. Spectra were pre-processed and principal component analysis (PCA) and K-means cluster analysis (KMC) were performed. Results showed that there was a clear discrimination between preparation types. Groups of spectra from untreated and washed isolates clustered separately due to intense protein, DNA and polysaccharide bands, whilst fixed spectra clustered separately due to intense polysaccharide bands. This signified that sample preparation had influenced the chemical composition of samples. Nevertheless, across preparation types, significant species discrimination was observed, and the polysaccharide (1200-900 cm-1) region was a common critical marker for species discrimination. However, different discriminatory marker bands were observed across preparation methods. Thus, sample preparation appears to influence the chemical composition of Candida samples; however, does not seem to significantly impact the species discrimination potential for ATR-FTIR spectroscopy.

Crystal structure and transient dimerization for the FKBP12 protein from the pathogenic fungus Candida auris.

International concern over the recent emergence of Candida auris infections reflects not only its comparative ease of transmission and substantial mortality but the increasing level of resistance observed to all three major classes of antifungal drugs. Diminution in virulence has been reported for a wide range of fungal pathogens when the FK506-binding protein FKBP12 binds to that immunosuppressant drug and the binary complex then inhibits the fungal calcineurin signaling pathway. Structure-based drug design efforts have described modifications of FK506 which modestly reduce virulence for a number of fungal pathogens while also lessening the side effect of suppressing the tissue immunity response in the patient. To aid in such studies, we report the crystal structure of Candida auris FKBP12. As physiological relevance has been proposed for transient homodimerization interactions of distantly related fungal FKBP12 proteins, we report the solution NMR characterization of the homodimerization interactions of the FKBP12 proteins from both Candida auris and Candida glabrata.

Evaluating glucose and mannose profiles in Candida species using quantum dots conjugated with Cramoll lectin as fluorescent nanoprobes.

Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 µm) was smaller when compared to C. albicans (4.0 µm) and C. parapsilosis sensu stricto (3.8 µm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.

Biomarkers of fungal infection: Expert opinion on the current situation.

The introduction of non-culture-based diagnostic techniques is revolutionizing the world of microbiological diagnosis and infection assessment. Fungi are no exception, and the introduction of biomarkers has opened up enormous expectations for better management of these entities. Biomarkers are diverse, their targets are also diverse and their evaluation has been done preferably in an individualized use and with deficient designs. Less is known about the value of the combined use of biomarkers and the impact of the negativity of two or more biomarkers on antifungal treatment decisions has been poorly studied. Given the paucity of prospective, randomized and definitive studies, we have convened experts from different fields, with an interest in invasive fungal infections, to answer some questions about the current relevant use of fungal biomarkers. This document summarizes the answers of these experts to the different questions.

Rapid identification of pathogenic bacteria using Raman spectroscopy and deep learning.

Raman optical spectroscopy promises label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step. However, achieving clinically relevant speeds and accuracies remains challenging due to weak Raman signal from bacterial cells and numerous bacterial species and phenotypes. Here we generate an extensive dataset of bacterial Raman spectra and apply deep learning approaches to accurately identify 30 common bacterial pathogens. Even on low signal-to-noise spectra, we achieve average isolate-level accuracies exceeding 82% and antibiotic treatment identification accuracies of 97.0±0.3%. We also show that this approach distinguishes between methicillin-resistant and -susceptible isolates of Staphylococcus aureus (MRSA and MSSA) with 89±0.1% accuracy. We validate our results on clinical isolates from 50 patients. Using just 10 bacterial spectra from each patient isolate, we achieve treatment identification accuracies of 99.7%. Our approach has potential for culture-free pathogen identification and antibiotic susceptibility testing, and could be readily extended for diagnostics on blood, urine, and sputum.

Multiscale design of a dairy beverage model composed of Candida utilis single cell protein supplemented with oleic acid.

One of the main challenges in the food industry is to design strategies for the successful incorporation of natural sources of bioactive compounds. Recently, yogurts and other fermented dairy beverages have been proposed as ideal carriers of such bioactive compounds such as fatty acids and antioxidants that could improve consumers' health. However, the incorporation of new ingredients causes functional and structural modifications that may affect the consumers' preferences. In this work, a dairy beverage model supplemented with oleic acid has been designed by partial substitution of milk by Candida utilis single-cell protein extract. The changes in the structural properties of this new beverage were evaluated by following the fermentation process, pH, aggregate size, microstructure, and changes in rheological properties. Furthermore, molecular dynamics simulations were carried out to analyze the interaction between its main components. Our data revealed that samples with a percentage of milk substitution of 30% showed a higher viscosity as compared with the other percentages and less viscosity than the control (no substitution). These samples were then selected for fortification by incorporating oleic acid microcapsules. A concentration of 1.5 g/100 g was shown to be the optimal quantity of microcapsules for oleic acid supplementation. Molecular dynamic simulations revealed glutathione as an important component of the micro-gel structure. The present study forms the basis for novel studies where Candida utilis single-cell protein and microencapsulated essential oils could be used to design innovative bioproducts.

Meso-Raman approach for rapid yeast cells identification.

An increasing effort is currently devoted to developing Raman spectroscopy for identification of microorganisms. Micro-Raman setups are typically used for this purpose with the limit that the intra-species and inter-species spectral variability are comparable, thus limiting the identification capability. To overcome this limit a meso-Raman approach is here implemented. Thin films of planktonic cells are analyzed throughout the collection of back-scattered light providing a Raman signal already averaged over tens of cells. The collecting of unpolarized (VU) and depolarized (HV) Raman signals increased the spectral information obtainable from the data, demonstrating the ability of the principal component analysis to differentiate the most common Candida species, namely C. glabrata, C. albicans, C. parapsilosis and C. tropicalis. The proposed method can contribute to bring Raman spectroscopy closer to its potential clinical use for fast identification of yeast cells.

Characterization of non-covalent immobilized Candida antartica lipase b over PS-b-P4VP as a model bio-reactive porous interface.

The design of interfaces that selectively react with molecules to transform them into compounds of industrial interest is an emerging area of research. An example of such reactions is the hydrolytic conversion of ester-based molecules to lipids and alcohols, which is of interest to the food, and pharmaceutical industries. In this study, a functional bio-interfaced layer was designed to hydrolyze 4-nitrophenyl acetate (pNPA) and Ricinus Communis (castor) oil rich in triglycerides using lipase b from Candida antarctica (CALB, EC 3.1.1.3). The attachment of CALB was performed via non-covalent immobilization over a polymer film of vertically aligned cylinders that resulted from the self-assembly of the di-block copolymer polystyrene-block-poly(4-vinyl pyridine) (PS-b-P4VP). This polymer-lipase model will serve as the groundwork for the design of further bioactive layers for separation applications requiring similar hydrolytic processes. Results from the fabricated functional bio-interfaced material include cylinders with featured pore size of 19 nm, d spacing of 34 nm, and ca. 40 nm of thickness. The polymer-enzyme layers were physically characterized using AFM, XPS, and FTIR. The immobilized enzyme was able to retain 91% of the initial enzymatic activity when using 4-nitrophenyl acetate (pNPA) and 78% when exposed to triglycerides from castor oil.