Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

The Engineered Hexosaminidase TtOGA-D120N Is an Efficient O-/N-/S-Glycoligase That Also Catalyzes Formation and Release of Oxazoline Donors for Cascade Syntheses with Glycosynthases or Transglycosylases.

Engineering glycoside hydrolases is a major route to obtaining catalysts forming glycosidic bonds. Glycosynthases, thioglycoligases, and transglycosylases represent the main strategies, each having advantages and drawbacks. Here, we show that an engineered enzyme from the GH84 family, the acid-base mutant TtOGA-D120N, is an efficient O-, N-, and S-glycoligase, able to use Ssp3, Osp3, Nsp2, and Nsp nucleophiles. Moreover, TtOGA-D120N catalyzes the formation and release of N-acetyl-d-glucosamine 1,2-oxazoline, the intermediate of hexosaminidases displaying substrate-assisted catalysis. This release of an activated intermediate allows cascade synthesis by combination with transglycosylases or glycosynthases, here exemplified by synthesis of the human milk oligosaccharide lacto-N-triose II.

A healthy Bifidobacterium dentium caramel cocktail.

ß-d-fructofuranosyl glycosidases are enzymes that produce health-beneficial fructooligosaccharides from natural fructans. In a recent issue of JBC, Kashima et al. identified a novel α-d-fructofuranosyl-active enzyme, αFFase1, from the caries-associated bacterium Bifidobacterium dentium. αFFase1 reversibly forms a potential prebiotic also found in caramel, difructose dianhydride I, via intramolecular condensation of the substrate inulobiose. Kashima et al. elegantly combine NMR, X-ray crystallography, and molecular dynamics to describe an original mechanism for the reversible reactions catalyzed by αFFase1 that establishes the new glycoside hydrolase family GH172.

Three alginate lyases provide a new gut Bacteroides ovatus isolate with the ability to grow on alginate.

Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an endo-acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Whole-genome sequencing of this alginate-utilizing isolate suggested that it is a new Bacteroides ovatus strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The BoPL6 degraded polyG to oligosaccharides of DP 1-3, and BoPL17 released 4,5-unsaturated monouronate from polyM. BoPL38 degraded both alginates, polyM, polyG, and polyMG, in endo-mode; hence, it was assumed to deliver oligosaccharide substrates for BoPL6 and BoPL17, corresponding well with synergistic action on alginate. BoPL17 and BoPL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (α/α)6-barrel + anti-parallel ß-sheet and (α/α)7-barrel folds, distinctive for these PL families. BoPL17 had a more open active site than the two homologous structures. BoPL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium.IMPORTANCEHuman ingestion of sustainable biopolymers calls for insight into their utilization in our gut. Seaweed is one such resource with alginate, a major cell wall component, used as a food hydrocolloid and for encapsulation of pharmaceuticals and probiotics. Knowledge is sparse on the molecular basis for alginate utilization in the gut. We identified a new Bacteroides ovatus strain from human feces that grew on alginate and encoded three alginate lyases in a gene cluster. BoPL6 and BoPL17 show complementary specificity toward guluronate (G) and mannuronate (M) residues, releasing unsaturated oligosaccharides and monouronic acids. BoPL38 produces oligosaccharides degraded by BoPL6 and BoPL17 from both alginates, G-, M-, and MG-substrates. Enzymatic and structural characterization discloses the mode of action and synergistic degradation of alginate by these alginate lyases. Other bacteria were cross-feeding on alginate oligosaccharides produced by an endo-acting alginate lyase. Hence, there is an interdependent community in our guts that can utilize alginate.

Designing starch derivatives with desired structures and functional properties via rearrangements of glycosidic linkages by starch-active transglycosylases.

Modification of starch by transglycosylases from glycoside hydrolase families has attracted much attention recently; these enzymes can produce starch derivatives with novel properties, i.e. processability and functionality, employing highly efficient and safe methods. Starch-active transglycosylases cleave starches and transfer linear fragments to acceptors introducing α-1,4 and/or linear/branched α-1,6 glucosidic linkages, resulting in starch derivatives with excellent properties such as complexing and resistance to digestion characteristics, and also may be endowed with new properties such as thermo-reversible gel formation. This review summarizes the effects of variations in glycosidic linkage composition on structure and properties of modified starches. Starch-active transglycosylases are classified into 4 groups that form compounds: (1) in cyclic with α-1,4 glucosidic linkages, (2) with linear chains of α-1,4 glucosidic linkages, (3) with branched α-1,6 glucosidic linkages, and (4) with linear chains of α-1,6 glucosidic linkages. We discuss potential processability and functionality of starch derivatives with different linkage combinations and structures. The changes in properties caused by rearrangements of glycosidic linkages provide guidance for design of starch derivatives with desired structures and properties, which promotes the development of new starch products and starch processing for the food industry.

Tuning alginate ß-lactoglobulin complex coacervation by modulating pH and temperature.

The use of biomolecules in food matrices and encapsulation systems is, as in other areas, moving towards greener solutions and a center piece here is the complex coacervation between natural anionic polysaccharides and proteins. Both alginate and ß-lactoglobulin (ß-Lg) are used in different sectors and have been shown to coacervate at pH < 5.2. Albeit with increased interest, complex coacervation has almost exclusively been studied from a macromolecular perspective, and described as an interaction based on charge-charge attraction. Here, we show that through changes in pH and temperature, alginate ß-Lg complex coacervation can be tuned to purpose. By detailed biophysical and chemical characterization of coacervation and coacervate particles, insights into the molecular interaction and effect of external factors are obtained. We find that carboxylate resonance stabilization causes a release of protons at pH < pKa,alginate and an uptake of protons at pH > pKa,alginate upon coacervation. Proton release and uptake were quantified at pH 2.65 and 4.00 by isothermal titration calorimetry to be 4 and 2 protons per ß-Lg molecule, respectively. By increasing the temperature to 65 °C, we discovered a secondary ß-Lg concentration dependent coacervation step, where the formed particles change into large assemblies driven by entropy. These findings bring new insights to complex coacervation and its applicability in microencapsulation and drug delivery.

Increasing Protein Content of Rice Flour with Maintained Processability by Using Granular Starch Hydrolyzing Enzyme.

Rice flour (RF) has become a promising food material. In the present study, RF with higher protein content was prepared using a granular starch hydrolyzing enzyme (GSHE). Particle size, morphology, crystallinity, and molecular structures of RF and rice starch (RS) were characterized to establish a hydrolytic mechanism; thermal, pasting, and rheological properties were determined to evaluate processability using differential scanning calorimetry (DSC), rapid viscosity analysis (RVA), and rheometer, respectively. The GSHE treatment resulted in pinholes, pits, and surface erosion through sequential hydrolysis of crystalline and amorphous areas on the starch granule surface. The amylose content decreased with hydrolysis time, while the very short chains (DP < 6) increased rapidly at 3 h but decreased slightly later. After hydrolysis for 24 h, the protein content in RF increased from 8.52% to 13.17%. However, the processability of RF was properly maintained. Specifically, the data from DSC showed that the conclusion temperature and endothermic enthalpy of RS barely changed. The result of rapid RVA and rheological measurement indicated that RF paste viscosity and viscoelastic properties dropped rapidly after 1 h hydrolysis and thereafter recovered slightly. This study provided a new RF raw material useful for improving and developing RF-based foods.

Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBDGA-TuαGT and-SBDSt2-TuαGT showed Kd of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBDSt1-TuαGT had 3-5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of TuαGT fusions.

Interfacial Catalysis during Amylolytic Degradation of Starch Granules: Current Understanding and Kinetic Approaches.

Enzymatic hydrolysis of starch granules forms the fundamental basis of how nature degrades starch in plant cells, how starch is utilized as an energy resource in foods, and develops efficient, low-cost saccharification of starch, such as bioethanol and sweeteners. However, most investigations on starch hydrolysis have focused on its rates of degradation, either in its gelatinized or soluble state. These systems are inherently more well-defined, and kinetic parameters can be readily derived for different hydrolytic enzymes and starch molecular structures. Conversely, hydrolysis is notably slower for solid substrates, such as starch granules, and the kinetics are more complex. The main problems include that the surface of the substrate is multifaceted, its chemical and physical properties are ill-defined, and it also continuously changes as the hydrolysis proceeds. Hence, methods need to be developed for analyzing such heterogeneous catalytic systems. Most data on starch granule degradation are obtained on a long-term enzyme-action basis from which initial rates cannot be derived. In this review, we discuss these various aspects and future possibilities for developing experimental procedures to describe and understand interfacial enzyme hydrolysis of native starch granules more accurately.

Importance of Inactivation Methodology in Enzymatic Processing of Raw Potato Starch: NaOCl as Efficient α-Amylase Inactivation Agent.

Efficient inactivation of microbial α-amylases (EC 3.2.1.1) can be a challenge in starch systems as the presence of starch has been shown to enhance the stability of the enzymes. In this study, commonly used inactivation methods, including multistep washing and pH adjustment, were assessed for their efficiency in inactivating different α-amylases in presence of raw potato starch. Furthermore, an effective approach for irreversible α-amylase inactivation using sodium hypochlorite (NaOCl) is demonstrated. Regarding inactivation by extreme pH, the activity of five different α-amylases was either eliminated or significantly reduced at pH 1.5 and 12. However, treatment at extreme pH for 5 min, followed by incubation at pH 6.5, resulted in hydrolysis yields of 42-816% relative to controls that had not been subjected to extreme pH. "Inactivation" by multistep washing with water, ethanol, and acetone followed by gelatinization as preparation for analysis gave significant starch hydrolysis compared to samples inactivated with NaOCl before the wash. This indicates that the further starch degradation observed in samples subjected to washing only took place during the subsequent gelatinization. The current study demonstrates the importance of inactivation methodology in α-amylase-mediated raw starch depolymerization and provides a method for efficient α-amylase inactivation in starch systems.

Exploration of the Transglycosylation Activity of Barley Limit Dextrinase for Production of Novel Glycoconjugates.

A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (HvLD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different p-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. HvLD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of HvLD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, HvLD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of HvLD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization.

Functional diversity of three tandem C-terminal carbohydrate-binding modules of a ß-mannanase.

Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating noncatalytic carbohydrate-binding modules (CBMs) to compensate for low-affinity binding typical of protein-carbohydrate interactions. The bacterium Saccharophagus degradans produces an endo-ß-mannanase of glycoside hydrolase family 5 subfamily 8 with three phylogenetically distinct family 10 CBMs located C-terminally from the catalytic domain (SdGH5_8-CBM10x3). However, the functional roles and cooperativity of these CBM domains in polysaccharide binding are not clear. To learn more, we studied the full-length enzyme, three stepwise CBM family 10 (CBM10) truncations, and GFP fusions of the individual CBM10s and all three domains together by pull-down assays, affinity gel electrophoresis, and activity assays. Only the C-terminal CBM10-3 was found to bind strongly to microcrystalline cellulose (dissociation constant, Kd = 1.48 µM). CBM10-3 and CBM10-2 bound galactomannan with similar affinity (Kd = 0.2-0.4 mg/ml), but CBM10-1 had 20-fold lower affinity for this substrate. CBM10 truncations barely affected specific activity on carob galactomannan and konjac glucomannan. Full-length SdGH5_8-CBM10x3 was twofold more active on the highly galactose-decorated viscous guar gum galactomannan and crystalline ivory nut mannan at high enzyme concentrations, but the specific activity was fourfold to ninefold reduced at low enzyme and substrate concentrations compared with the enzyme lacking CBM10-2 and CBM10-3. Comparison of activity and binding data for the different enzyme forms indicates unproductive and productive polysaccharide binding to occur. We conclude that the C-terminal-most CBM10-3 secures firm binding, with contribution from CBM10-2, which with CBM10-1 also provides spatial flexibility.

Characterization of five marine family 29 glycoside hydrolases reveals an α-L-fucosidase targeting specifically Fuc(α1,4)GlcNAc.

$\text{L} $ -Fucose is the most widely distributed $\text{L} $-hexose in marine and terrestrial environments and presents a variety of functional roles. $\text{L} $-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-$\text{L} $-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α-$\text{L} $-fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2'- and 3-fucosyllactose, while Fp284 degraded 2'-fucosyllactose and Fuc(α1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s-1 and Km = 660 µM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.

Mechanistic Basis for Understanding the Dual Activities of the Bifunctional Azotobacter vinelandii Mannuronan C-5-Epimerase and Alginate Lyase AlgE7.

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5-epimerases determine the monomer composition by catalyzing the epimerization of ß-d-mannuronic acid (M) residues into α-l-guluronic acid (G) residues. The molecular weight is affected by alginate lyases, which catalyze a ß-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5-epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by nuclear magnetic resonance (NMR) spectroscopy. Our results show that calcium promotes lyase activity, whereas NaCl reduces the lyase activity of AlgE7. By using defined polymannuronan (polyM) and polyalternating alginate (polyMG) substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study, we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 functions as the catalytic acid. IMPORTANCE Postharvest valorization and upgrading of algal constituents are promising strategies in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens further applications of bacterial epimerases and lyases in the enzymatic tailoring of alginate polymers.

Enzymatic preparation and potential applications of agar oligosaccharides: a review.

Oligosaccharides derived from agar, that is, agarooligosaccharides and neoagarooligosaccharides, have demonstrated various kinds of bioactivities which have been utilized in a variety of fields. Enzymatic hydrolysis is a feasible approach that principally allows for obtaining specific agar oligosaccharides in a sustainable way at an industrial scale. This review summarizes recent technologies employed to improve the properties of agarase. Additionally, the relationship between the degree of polymerization, bioactivities, and potential applications of agar-derived oligosaccharides for pharmaceutical, food, cosmetic, and agricultural industries are discussed. Engineered agarase exhibited general improvement of enzymatic performance, which is mostly achieved by truncation. Rational and semi-rational design assisted by computational methods present the latest strategy for agarase improvement with greatest potential to satisfy future industrial needs. Agarase immobilized on magnetic Fe3O4 nanoparticles via covalent bond formation showed characteristics well suited for industry. Additionally, albeit with the relationship between the degree of polymerization and versatile bioactivities like anti-oxidants, anti-inflammatory, anti-microbial agents, prebiotics and in skin care of agar-derived oligosaccharides are discussed here, further researches are still needed to unravel the complicated relationship between bioactivity and structure of the different oligosaccharides.

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d/l-, α/ß-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.

Impact of Alginate Mannuronic-Guluronic Acid Contents and pH on Protein Binding Capacity and Complex Size.

Alginates, serving as hydrocolloids in the food and pharma industries, form particles at pH < 4.5 with positively charged proteins, such as ß-lactoglobulin (ß-Lg). Alginates are linear anionic polysaccharides composed of 1,4-linked ß-d-mannuronate (M) and α-l-guluronate (G) residues. The impact of M and G contents and pH is investigated to correlate with the formation and size of ß-Lg alginate complexes under relevant ionic strength. It is concluded, using three alginates of M/G ratios 0.6, 1.1, and 1.8 and similar molecular mass, that ß-Lg binding capacity is higher at pH 4.0 than at pH 2.65 and for high M content. By contrast, the largest particles are obtained at pH 2.65 and with high G content. At pH 4.0 and 2.65, the stoichiometry was 28-48 and 3-10 ß-Lg molecules bound per alginate, respectively, increasing with higher M content. The findings will contribute to the design of formation of the desired alginate-protein particles in the acidic pH range.

Exceptionally rich keratinolytic enzyme profile found in the rare actinomycetes Amycolatopsis keratiniphila D2T.

The non-spore forming Gram-positive actinomycetes Amycolatopsis keratiniphila subsp. keratiniphila D2T (DSM 44,409) has a high potential for keratin valorization as demonstrated by a novel biotechnological microbial conversion process consisting of a bacterial growth phase and a keratinolytic phase, respectively. Compared to the most gifted keratinolytic Bacillus species, a very large number of 621 putative proteases are encoded by the genome of Amycolatopsis keratiniphila subsp. keratiniphila D2T, as predicted by using Peptide Pattern Recognition (PPR) analysis. Proteome analysis by using LC-MS/MS on aliquots of the supernatant of A. keratiniphila subsp. keratiniphila D2T culture on slaughterhouse pig bristle meal, removed at 24, 48, 96 and 120 h of growth, identified 43 proteases. This was supplemented by proteome analysis of specific fractions after enrichment of the supernatant by anion exchange chromatography leading to identification of 50 proteases. Overall 57 different proteases were identified corresponding to 30% of the 186 proteins identified from the culture supernatant and distributed as 17 metalloproteases from 11 families, including an M36 protease, 38 serine proteases from 4 families, and 13 proteolytic enzymes from other families. Notably, M36 keratinolytic proteases are prominent in fungi, but seem not to have been discovered in bacteria previously. Two S01 family peptidases, named T- and C-like proteases, prominent in the culture supernatant, were purified and shown to possess a high azo-keratin/azo-casein hydrolytic activity ratio. The C-like protease revealed excellent thermostability, giving promise for successful applications in biorefinery processes. Notably, the bacterium seems not to secrete enzymes for cleavage of disulfides in the keratinous substrates. KEY POINTS: • A. keratiniphila subsp. keratiniphila D2T is predicted to encode 621 proteases. • This actinomycete efficiently converts bristle meal to a protein hydrolysate. • Proteome analysis identified 57 proteases in its secretome.

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach.

Wheat represents one of the most important cereals for mankind. However, since wheat proteins are also the causative agent of several adverse reactions, during the last decades, consumers have shown an increasing interest in the old wheat genotypes, which are generally perceived as more "natural" and healthier than the modern ones. Comparison of nutritional value for modern and old wheat genotypes is still controversial, and to evaluate the real impact of these foods on human health comparative experiments involving old and modern genotypes are desirable. The nutritional quality of grain is correlated with its proteomic composition that depends on the interplay between the genetic characteristics of the plant and external factors related to the environment. We report here the label-free shotgun quantitative comparison of the metabolic protein fractions of two old Sicilian landraces (Russello and Timilia) and the modern variety Simeto, from the 2010-2011 and 2011-2012 growing seasons. The overall results show that Timilia presents the major differences with respect to the other two genotypes investigated. These differences may be related to different defense mechanisms and some other peculiar properties of these genotypes. On the other hand, our results confirm previous results leading to the conclusion that with respect to a nutritional value evaluation, there is a substantial equivalence between old and modern wheat genotypes. Data are available via ProteomeXchange with identifier .

Substrate preference of an ABC importer corresponds to selective growth on ß-(1,6)-galactosides in Bifidobacterium animalis subsp. lactis.

Bifidobacteria are exposed to substantial amounts of dietary ß-galactosides. Distinctive preferences for growth on different ß-galactosides are observed within Bifidobacterium members, but the basis of these preferences remains unclear. We previously described the first ß-(1,6)/(1,3)-galactosidase from Bifidobacterium animalis subsp. lactis Bl-04. This enzyme is relatively promiscuous, exhibiting only 5-fold higher efficiency on the preferred ß-(1,6)-galactobiose than the ß-(1,4) isomer. Here, we characterize the solute-binding protein (Bal6GBP) that governs the specificity of the ABC transporter encoded by the same ß-galactoside utilization locus. We observed that although Bal6GBP recognizes both ß-(1,6)- and ß-(1,4)-galactobiose, Bal6GBP has a 1630-fold higher selectivity for the former, reflected in dramatic differences in growth, with several hours lag on less preferred ß-(1,4)- and ß-(1,3)-galactobiose. Experiments performed in the presence of varying proportions of ß-(1,4)/ß-(1,6)-galactobioses indicated that the preferred substrate was preferentially depleted from the culture supernatant. This established that the poor growth on the nonpreferred ß-(1,4) was due to inefficient uptake. We solved the structure of Bal6GBP in complex with ß-(1,6)-galactobiose at 1.39 Å resolution, revealing the structural basis of this strict selectivity. Moreover, we observed a close evolutionary relationship with the human milk disaccharide lacto-N-biose-binding protein from Bifidobacterium longum, indicating that the recognition of the nonreducing galactosyl is essentially conserved, whereas the adjacent position is diversified to fit different glycosidic linkages and monosaccharide residues. These findings indicate that oligosaccharide uptake has a pivotal role in governing selectivity for distinct growth substrates and have uncovered evolutionary trajectories that shape the diversification of sugar uptake proteins within Bifidobacterium.