A comprehensive trial on PFAS remediation: hemp phytoextraction and PFAS degradation in harvested plants.

Per- and polyfluoroalkyl substances (PFAS) are a class of recalcitrant, highly toxic contaminants, with limited remediation options. Phytoremediation - removal of contaminants using plants - is an inexpensive, community-friendly strategy for reducing PFAS concentrations and exposures. This project is a collaboration between the Mi'kmaq Nation, Upland Grassroots, and researchers at several institutions who conducted phytoremediation field trials using hemp to remove PFAS from soil at the former Loring Air Force base, which has now been returned to the Mi'kmaq Nation. PFAS were analyzed in paired hemp and soil samples using targeted and non-targeted analytical approaches. Additionally, we used hydrothermal liquefaction (HTL) to degrade PFAS in the harvested hemp tissue. We identified 28 PFAS in soil and found hemp uptake of 10 of these PFAS. Consistent with previous studies, hemp exhibited greater bioconcentration for carboxylic acids compared to sulfonic acids, and for shorter-chain compounds compared to longer-chain. In total, approximately 1.4 mg of PFAS was removed from the soil via uptake into hemp stems and leaves, with an approximate maximum of 2% PFAS removed from soil in the most successful area. Degradation of PFAS by HTL was nearly 100% for carboxylic acids, but a portion of sulfonic acids remained. HTL also decreased precursor PFAS and extractable organic fluorine. In conclusion, while hemp phytoremediation does not currently offer a comprehensive solution for PFAS-contaminated soil, this project has effectively reduced PFAS levels at the Loring site and underscores the importance of involving community members in research aimed at remediating their lands.

A genetically-encoded biosensor for direct detection of perfluorooctanoic acid.

Determination of per- and polyfluoroalkyl substances (PFAS) in drinking water at the low levels set by regulatory officials has been a major focus for sensor developing researchers. However, it is becoming more apparent that detection of these contaminants in soils, foods and consumer products is relevant and necessary at part per billion and even part per million levels. Here, a fluorescent biosensor for the rapid detection of PFOA was engineered based on human liver fatty acid binding protein (hLFABP). By conjugating circularly permuted green fluorescent protein (cp.GFP) to a split hLFABP construct, the biosensor was able to detect perfluorooctanoic acid PFOA in PBS as well as environmental water samples with LODs of 236 and 330 ppb respectively. Furthermore, E. coli cells cytosolically expressing the protein-based sensor were demonstrated to quickly detect PFOA, demonstrating feasibility of whole-cell sensing. Overall, this work demonstrates a platform technology utilizing a circularly permuted GFP and split hLFABP conjugate as a label-free optical biosensor for PFOA.

Structural Analyses of Substrate-pH Activity Pairing Observed across Diverse Polysaccharide Lyases.

Anionic polysaccharides found in nature are functionally and structurally diverse, and so are the polysaccharide lyases (PLs) that catalyze their degradation. Atomic superposition of various PL folds according to their cleavable substrate structure confirms the occurrence of structural convergence at PL active sites. This suggests that various PL folds have emerged to cleave a particular class of anionic polysaccharide during the course of evolution. Whereas the structural and mechanistic similarity of PL active site has been highlighted in earlier studies, a detailed understanding regarding functional properties of this catalytic convergence remains an open question, especially the role of extrinsic factors such as pH in the context of substrate binding and catalysis. Our earlier structural and functional work on pH directed multisubstrate specificity of Smlt1473 inspired us to regroup PLs according to substrate type to analyze the pH dependence of their catalytic activity. Interestingly, we find that particular groups of substrates are cleaved in a particular pH range (acidic/neutral/basic) irrespective of PL fold, boosting the idea of functional convergence as well. On the basis of this observation, we set out to define structurally and computationally the key constituents of an active site among PL families. This study delineates the structural determinants of conserved "substrate-pH activity pairing" within and between PL families.

Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.

Engineering human liver fatty acid binding protein for detection of poly- and perfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are a large group of synthetic fluorinated chemicals with surface active and water-repellent properties. The combination of wide-spread use in numerous consumer and industrial products and extended biological half-lives arising from strong carbon-fluorine bonds has led to significant accumulation of PFAS in humans. As most human interaction with PFAS comes from ingestion, it is important to be able to detect PFAS in drinking water as well as in agricultural water. Here we present an approach to designing a fluorescence-based biosensor for the rapid detection of PFAS based on human liver fatty acid binding protein (hLFABP). Introduction of solvatochromic fluorophores within the ligand binding pocket (L50) allowed for intrinsic detection of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and perfluorohexanesulfonic acid (PFHxS) via blue-shifts in fluorescence emission spectra. Initially, a single tryptophan mutation (L50W) was found to be able to detect PFOA with a limit of detection (LOD) of 2.8 ppm. We improved the sensitivity of the biosensor by exchanging tryptophan for the thiol reactive fluorophore, acrylodan. The acrylodan conjugated C69S/F50C hLFABP variant is capable of detecting PFOA, PFOS, and PFHxS in PBS with LODs of 112 ppb, 345 ppb, and 1.09 ppm, respectively. The protein-based sensor is also capable of detecting these contaminants at similar ranges in spiked environmental water samples, including samples containing an interfering anionic surfactant sodium dodecyl sulfate. Overall, this study demonstrates engineered hLFABP is a useful platform for detection of PFAS in environmental water samples and highlights its ease of use and versatility in field applications.

Structural insights into the mechanism of pH-selective substrate specificity of the polysaccharide lyase Smlt1473.

Polysaccharide lyases (PLs) are a broad class of microbial enzymes that degrade anionic polysaccharides. Equally broad diversity in their polysaccharide substrates has attracted interest in biotechnological applications such as biomass conversion to value-added chemicals and microbial biofilm removal. Unlike other PLs, Smlt1473 present in the clinically relevant Stenotrophomonas maltophilia strain K279a demonstrates a wide range of pH-dependent substrate specificities toward multiple, diverse polysaccharides: hyaluronic acid (pH 5.0), poly-ß-D-glucuronic (celluronic) acid (pH 7.0), poly-ß-D-mannuronic acid, and poly-α-L-guluronate (pH 9.0). To decode the pH-driven multiple substrate specificities and selectivity in this single enzyme, we present the X-ray structures of Smlt1473 determined at multiple pH values in apo and mannuronate-bound states as well as the tetra-hyaluronate-docked structure. Our results indicate that structural flexibility in the binding site and N-terminal loop coupled with specific substrate stereochemistry facilitates distinct modes of entry for substrates having diverse charge densities and chemical structures. Our structural analyses of wild-type apo structures solved at different pH values (5.0-9.0) and pH-trapped (5.0 and 7.0) catalytically relevant wild-type mannuronate complexes (1) indicate that pH modulates the catalytic microenvironment for guiding structurally and chemically diverse polysaccharide substrates, (2) further establish that molecular-level fluctuation in the enzyme catalytic tunnel is preconfigured, and (3) suggest that pH modulates fluctuations resulting in optimal substrate binding and cleavage. Furthermore, our results provide key insight into how strategies to reengineer both flexible loop and regions distal to the active site could be developed to target new and diverse substrates in a wide range of applications.

Disrupting Irreversible Bacterial Adhesion and Biofilm Formation with an Engineered Enzyme.

Biofilm formation is often attributed to postharvest bacterial persistence on fresh produce and food handling surfaces. In this study, a predicted glycosyl hydrolase enzyme was expressed, purified, and validated for the removal of microbial biofilms from biotic and abiotic surfaces under conditions used for chemical cleaning agents. Crystal violet biofilm staining assays revealed that 0.1 mg/ml of enzyme inhibited up to 41% of biofilm formation by Escherichia coli O157:H7, E. coli 25922, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Furthermore, the enzyme was effective at removing mature biofilms, providing a 35% improvement over rinsing with a saline solution alone. Additionally, a parallel-plate flow cell was used to directly observe and quantify the impact of enzyme rinses on E. coli O157:H7 cells adhering to spinach leaf surfaces. The presence of 1 mg/liter enzyme resulted in nearly 6-times-higher detachment rate coefficients than a deionized (DI) water rinse, while the total cells removed from the surface increased from 10% to 25% over the 30-min rinse time, reversing the initial phases of biofilm formation. Enzyme treatment of all 4 cell types resulted in significantly reduced cell surface hydrophobicity and collapse of negatively stained E. coli 25922 cells imaged by electron microscopy, suggesting potential polysaccharide surface modification of enzyme-treated bacteria. Collectively, these results point to the broad substrate specificity and robustness of the enzyme for different types of biofilm stages, solution conditions, and pathogen biofilm types and may be useful as a method for the removal or inhibition of bacterial biofilm formation. IMPORTANCE In this study, the ability of an engineered enzyme to reduce bacterial adhesion and biofilm formation of several foodborne pathogens was demonstrated, representing a promising option for enhancing or replacing chlorine and other chemical sanitizers in food processing applications. Specifically, significant reductions of biofilms of the pathogens Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes are observed, as are reductions in initial adhesion. Enzymes have the added benefits of being green, sustainable alternatives to chemical sanitizers, as well as having a minimal impact on food properties, in contrast to many alternative antimicrobial options such as bleach that aim to minimize food safety risks.

Disrupting the transmembrane domain-mediated oligomerization of protein tyrosine phosphatase receptor J inhibits EGFR-driven cancer cell phenotypes.

Receptor protein tyrosine phosphatases (RPTPs) play critical regulatory roles in mammalian signal transduction. However, the structural basis for the regulation of their catalytic activity is not fully understood, and RPTPs are generally not therapeutically targetable. This knowledge gap is partially due to the lack of known natural ligands or selective agonists of RPTPs. Contrary to what is known from structure-function studies of receptor tyrosine kinases (RTKs), RPTP activities have been reported to be suppressed by dimerization, which may prevent RPTPs from accessing their RTK substrates. We report here that homodimerization of protein tyrosine phosphatase receptor J (PTPRJ, also known as DEP-1) is regulated by specific transmembrane (TM) residues. We found that disrupting these interactions destabilizes homodimerization of full-length PTPRJ in cells, reduces the phosphorylation of the known PTPRJ substrate epidermal growth factor receptor (EGFR) and of other downstream signaling effectors, antagonizes EGFR-driven cell phenotypes, and promotes substrate access. We demonstrate these observations in human cancer cells using mutational studies and identified a peptide that binds to the PTPRJ TM domain and represents the first example of an allosteric agonist of RPTPs. The results of our study provide fundamental structural and functional insights into how PTPRJ activity is tuned by TM interactions in cells. Our findings also open up opportunities for developing peptide-based agents that could be used as tools to probe RPTPs' signaling mechanisms or to manage cancers driven by RTK signaling.

Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris.

Hydrophobins are small highly surface-active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression host Pichia pastoris. One-, 2-, and 3-copy-HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression of PDI1 or ERO1 caused insignificant changes in HFBI secretion in 1- and 2-copy strains, but a statistically significant HFBI secretion increase in 3-copy strain. KAR2 overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3-copy-HFBI secreting 22±1.6 fold more than the 1-copy-HFBI/no chaperone strain. The highest increase was seen in 3-copy-HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1-copy-HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small-scale format used in this study.

Hydrophobins: multifunctional biosurfactants for interface engineering.

Hydrophobins are highly surface-active proteins that have versatile potential as agents for interface engineering. Due to the large and growing number of unique hydrophobin sequences identified, there is growing potential to engineer variants for particular applications using protein engineering and other approaches. Recent applications and advancements in hydrophobin technologies and production strategies are reviewed. The application space of hydrophobins is large and growing, including hydrophobic drug solubilization and delivery, protein purification tags, tools for protein and cell immobilization, antimicrobial coatings, biosensors, biomineralization templates and emulsifying agents. While there is significant promise for their use in a wide range of applications, developing new production strategies is a key need to improve on low recombinant yields to enable their use in broader applications; further optimization of expression systems and yields remains a challenge in order to use designed hydrophobin in commercial applications.

A Structural and Functional Role for Disulfide Bonds in a Class II Hydrophobin.

Hydrophobins are multifunctional, highly surface active proteins produced in filamentous fungi and can be identified by eight conserved cysteine residues, which form four disulfide bridges. These proteins can be subdivided into two classes based on their hydropathy profiles, solubility, and structures formed upon interfacial assembly. Here, we probe the structural and functional roles of disulfide bonds for a class II hydrophobin in different interfacial contexts by reducing its disulfides with 1,4-dithiothreitol and blocking the free thiols with iodoacetamide and then examining the protein secondary structure, emulsification capability, hydrophobic surface wetting, and solution self-assembly. Changes in circular dichroism spectra upon reduction and blocking of disulfides are consistent with an increase in the level of random coil secondary structure. Emulsification of octane in water using reduced and unreduced forms of class II hydrophobin showed a substantial loss of emulsification ability without disulfides and stable emulsion formation for hydrophobin with disulfides. Additionally, water contact angle measurements performed on polytetrafluoroethylene treated with solutions of reduced and unreduced hydrophobin showed efficient wetting of the hydrophobic surface for unreduced samples only. Lastly, Förster resonance energy transfer (FRET) was used to assess the role of disulfides in self-assembly in solution, and near complete loss of the FRET signal is consistent with a model in which solution self-assembly does not occur after reduction and blocking of the disulfides. From this, we conclude that, in contrast to class I hydrophobins, the disulfides of this class II hydrophobin are required for protein structural stability, surface activity at both liquid-liquid and solid-liquid interfaces, and solution self-assembly.

Enzymatic biomineralization of biocompatible CuInS2, (CuInZn)S2 and CuInS2/ZnS core/shell nanocrystals for bioimaging.

This work demonstrates a bioenabled fully aqueous phase and room temperature route to the synthesis of CuInS2/ZnS core/shell quantum confined nanocrystals conjugated to IgG antibodies and used for fluorescent tagging of THP-1 leukemia cells. This elegant, straightforward and green approach avoids the use of solvents, high temperatures and the necessity to phase transfer the nanocrystals prior to application. Non-toxic CuInS2, (CuInZn)S2, and CuInS2/ZnS core/shell quantum confined nanocrystals are synthesized via a biomineralization process based on a single recombinant cystathionine γ-lyase (CSE) enzyme. First, soluble In-S complexes are formed from indium acetate and H2S generated by CSE, which are then stabilized by l-cysteine in solution. The subsequent addition of copper, or both copper and zinc, precursors then results in the immediate formation of CuInS2 or (CuInZn)S2 quantum dots. Shell growth is realized through subsequent introduction of Zn acetate to the preformed core nanocrystals. The size and optical properties of the nanocrystals are tuned by adjusting the indium precursor concentration and initial incubation period. CuInS2/ZnS core/shell particles are conjugated to IgG antibodies using EDC/NHS cross-linkers and then applied in the bioimaging of THP-1 cells. Cytotoxicity tests confirm that CuInS2/ZnS core/shell quantum dots do not cause cell death during bioimaging. Thus, this biomineralization enabled approach provides a facile, low temperature route for the fully aqueous synthesis of non-toxic CuInS2/ZnS quantum dots, which are ideal for use in bioimaging applications.

Single Enzyme Direct Biomineralization of CdSe and CdSe-CdS Core-Shell Quantum Dots.

Biomineralization is the process by which biological systems synthesize inorganic materials. Herein, we demonstrate an engineered cystathionine γ-lyase enzyme, smCSE that is active for the direct aqueous phase biomineralization of CdSe and CdSe-CdS core-shell nanocrystals. The nanocrystals are formed in an otherwise unreactive buffered solution of Cd acetate and selenocystine through enzymatic turnover of the selenocystine to form a reactive precursor, likely H2Se. The particle size of the CdSe core nanocrystals can be tuned by varying the incubation time to generated particle sizes between 2.74 ± 0.63 nm and 4.78 ± 1.16 nm formed after 20 min and 24 h of incubation, respectively. Subsequent purification and introduction of l-cysteine as a sulfur source facilitates the biomineralization of a CdS shell onto the CdSe cores. The quantum yield of the resulting CdSe-CdS core-shell particles is up to 12% in the aqueous phase; comparable to that reported for more traditional chemical synthesis routes for core-shell particles of similar size with similar shell coverage. This single-enzyme route to functional nanocrystals synthesis reveals the powerful potential of biomineralization processes.

Direct Single-Enzyme Biomineralization of Catalytically Active Ceria and Ceria-Zirconia Nanocrystals.

Biomineralization is an intriguing approach to the synthesis of functional inorganic materials for energy applications whereby biological systems are engineered to mineralize inorganic materials and control their structure over multiple length scales under mild reaction conditions. Herein we demonstrate a single-enzyme-mediated biomineralization route to synthesize crystalline, catalytically active, quantum-confined ceria (CeO2-x) and ceria-zirconia (Ce1-yZryO2-x) nanocrystals for application as environmental catalysts. In contrast to typical anthropogenic synthesis routes, the crystalline oxide nanoparticles are formed at room temperature from an otherwise inert aqueous solution without the addition of a precipitant or additional reactant. An engineered form of silicatein, rCeSi, as a single enzyme not only catalyzes the direct biomineralization of the nanocrystalline oxides but also serves as a templating agent to control their morphological structure. The biomineralized nanocrystals of less than 3 nm in diameter are catalytically active toward carbon monoxide oxidation following an oxidative annealing step to remove carbonaceous residue. The introduction of zirconia into the nanocrystals leads to an increase in Ce(III) concentration, associated catalytic activity, and the thermal stability of the nanocrystals.

A Secreted Ankyrin-Repeat Protein from Clinical Stenotrophomonas maltophilia Isolates Disrupts Actin Cytoskeletal Structure.

Stenotrophomonas maltophilia is an emerging, multidrug-resistant pathogen of increasing importance for the immunocompromised, including cystic fibrosis patients. Despite its significance as an emerging pathogen, relatively little is known regarding the specific factors and mechanisms that contribute to its pathogenicity. We identify and characterize a putative ankyrin-repeat protein (Smlt3054) unique to clinical S. maltophilia isolates that binds F-actin in vitro and co-localizes with actin in transfected HEK293a cells. Smlt3054 is endogenously expressed and secreted from clinical S. maltophilia isolates, but not an environmental isolate (R551-3). The in vitro binding of Smlt3054 to F-actin resulted in a thickening of the filaments as observed by TEM. Ectopic expression of Smlt3054-GFP exhibits strong co-localization with F-actin, with distinct, retrograde F-actin waves specifically associated with Smlt3054 in individual cells as well as formation of dense, internal inclusions at the expense of retrograde F-actin waves. Collectively, our results point to an interaction between Smlt3054 and F-actin. Furthermore, as a potentially secreted protein unique to clinical S. maltophilia isolates, Smlt3054 may serve as a starting point for understanding the mechanisms by which S. maltophilia has become an emergent pathogen.

Interplay of Specific Trans- and Juxtamembrane Interfaces in Plexin A3 Dimerization and Signal Transduction.

Plexins are transmembrane proteins that serve as guidance receptors during angiogenesis, lymphangiogenesis, neuronal development, and zebrafish fin regeneration, with a putative role in cancer metastasis. Receptor dimerization or clustering, induced by extracellular ligand binding but modulated in part by the plexin transmembrane (TM) and juxtamembrane (JM) domains, is thought to drive plexin activity. Previous studies indicate that isolated plexin TM domains interact through a conserved, small-x3-small packing motif, and the cytosolic JM region interacts through a hydrophobic heptad repeat; however, the roles and interplay of these regions in plexin signal transduction remain unclear. Using an integrated experimental and simulation approach, we find disruption of the small-x3-small motifs in the Danio rerio Plexin A3 TM domain enhances dimerization of the TM-JM domain by enhancing JM-mediated dimerization. Furthermore, mutations of the cytosolic JM heptad repeat that disrupt dimerization do so even in the presence of TM domain mutations. However, mutations to the small-x3-small TM interfaces also disrupt Plexin A3 signaling in a zebrafish axonal guidance assay, indicating the importance of this TM interface in signal transduction. Collectively, our experimental and simulation results demonstrate that multiple TM and JM interfaces exist in the Plexin A3 homodimer, and these interfaces independently regulate dimerization that is important in Plexin A3 signal transduction.

A specific, transmembrane interface regulates fibroblast activation protein (FAP) homodimerization, trafficking and exopeptidase activity.

Fibroblast activation protein (FAP) is a cell-surface serine protease which promotes invasiveness of certain epithelial cancers and is therefore a potential target for cancer drug development and delivery. Unlike dipeptidyl peptidase IV (DPPIV), FAP exhibits prolyl endopeptidase activity and is active as a homodimer with specificity for type I collagen. The mechanism that regulates FAP homodimerization and its relation to prolyl endopeptidase activity is not completely understood. Here, we investigate key residues in the FAP TM domain that may be significant for FAP homodimerization. Mutations to predicted TM interfacial residues (G10L, S14L, and A18L) comprising a small-X3-small motif reduced FAP TM-CYTO dimerization relative to wild type as measured using the AraTM assay, whereas predicted off-interface residues showed no significant change from wild type. The results implied that the predicted small-X3-small dimer interface affect stabilization of FAP TM-CYTO homodimerization. Compared with FAPwild-type, the interfacial TM residue G10L significantly decreased FAP endopeptidase activity more than 25%, and also reduced cell-surface versus intracellular expression relative to other interfacial residues S14L and A18L. Thus, our results suggest FAP dimerization is important for both trafficking and protease activity, and is dependent on a specific TM interface.

Single-enzyme biomineralization of cadmium sulfide nanocrystals with controlled optical properties.

Nature has evolved several unique biomineralization strategies to direct the synthesis and growth of inorganic materials. These natural systems are complex, involving the interaction of multiple biomolecules to catalyze biomineralization and template growth. Herein we describe the first report to our knowledge of a single enzyme capable of both catalyzing mineralization in otherwise unreactive solution and of templating nanocrystal growth. A recombinant putative cystathionine γ-lyase (smCSE) mineralizes CdS from an aqueous cadmium acetate solution via reactive H2S generation from l-cysteine and controls nanocrystal growth within the quantum confined size range. The role of enzymatic nanocrystal templating is demonstrated by substituting reactive Na2S as the sulfur source. Whereas bulk CdS is formed in the absence of the enzyme or other capping agents, nanocrystal formation is observed when smCSE is present to control the growth. This dual-function, single-enzyme, aerobic, and aqueous route to functional material synthesis demonstrates the powerful potential of engineered functional material biomineralization.

Engineering broad-spectrum digestion of polyuronides from an exolytic polysaccharide lyase.

BACKGROUND: Macroalgae represents a promising source of fermentable carbohydrates for use in the production of energy efficient biofuel. The primary carbohydrate in brown algae is the uronic acid-containing alginate, whereas green algae contains a significant amount of glucuronan. A necessary step in the conversion of these polyuronides to bioethanol is saccharification, which can be achieved by enzymatic or chemical degradation. RESULTS: Polysaccharide lyases are a class of enzymes which cleave uronic acid-containing glycans via a ß-elimination mechanism, acting both endo- and exolytically on their substrates. In the present work, we characterize a putative alginate lyase from Stenotrophomonas maltophilia K279a (Smlt2602) and describe a H208F mutant that, in addition to cleaving alginate-based substrates, displays significant, exolytic glucuronan activity. CONCLUSIONS: To our knowledge this is the first polysaccharide lyase to act exolytically on glucuronan and is an attractive candidate for the broad-spectrum digestion of polyuronides into fermentable monomers.