Broad Substrate Specificity Research Articles

Biotransformation of gallate esters by a pH-stable tannase of mangrove-derived yeast Debaryomyces hansenii.

Introduction: Tannase is a crucial enzyme that finds wide applications in the pharmaceutical industry, feed processing, and beverage manufacturing. Although extensive studies have been conducted on tannases from fungi and bacteria, reports on tannases exhibiting favorable pH stability are relatively limited. Methods: In this study, a tannin-degrading strain Debaryomyces hansenii was screened to induce tannase production, and the corresponding tannase coding gene TANF was successfully cloned and expressed in Yarrowia lipolytica. SDS-PAGE analysis revealed that the purified TanF tannase had a molecular weight of approximately 70kDa. Results and Discussion: The enzyme demonstrated optimal activity at 40°C and retained over 80% of its activity in the range of 35°C-60°C. Of particular interest, TanF exhibited remarkable enzyme activity at pH 5.0 and retained more than 70% of its relative activity across a wide pH range of 3.0-8.0. Furthermore, TanF exhibited broad substrate specificity for gallate esters. The final gallic acid production by TanF from tannic acid achieved 18.32g/L. Therefore, the excellent properties TanF has been demonstrated to be an efficient tool for the preparation of gallic acid.

Conjugated Dienoic Acid Peroxides as Substrates in Chaetopterus Bioluminescence System.

Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fe2+ ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.

Identification of a mycobacterial hydrazidase, an isoniazid-hydrolyzing enzyme

There exists decades-old evidence that some mycobacteria, including Mycobacterium avium and Mycobacterium smegmatis, produce hydrazidase, an enzyme that can hydrolyze the first-line antitubercular agent isoniazid. Despite its importance as a potential resistance factor, no studies have attempted to reveal its identity. In this study, we aimed to isolate and identify M. smegmatis hydrazidase, characterize it, and evaluate its impact on isoniazid resistance. We determined the optimal condition under which M. smegmatis produced the highest amount of hydrazidase, purified the enzyme by column chromatography, and identified it by peptide mass fingerprinting. It was revealed to be PzaA, an enzyme known as pyrazinamidase/nicotinamidase whose physiological role remains unknown. The kinetic constants suggested that this amidase with broad substrate specificity prefers amides to hydrazides as a substrate. Notably, of the five tested compounds, including amides, only isoniazid served as an efficient inducer of pzaA transcription, as revealed by quantitative reverse transcription PCR. Moreover, high expression of PzaA was confirmed to be beneficial for the survival and growth of M. smegmatis in the presence of isoniazid. Thus, our findings suggest a possible role for PzaA, and other hydrazidases yet to be identified, as an intrinsic isoniazid resistance factor of mycobacteria.

Heterologous expression, purification, and characterization of a recombinant Cordyceps militaris lipase from Candida rugosa-like family in Pichia pastoris

Multiple sequence alignments of three lipase isoforms from the filamentous fungus, Cordyceps militaris, have revealed that the deduced protein from their common sequence belongs to the Candida rugosa lipase-like group. To express the protein in its active form, recombinant lipase from C. militaris (rCML) was extra cellularly expressed in Pichia pastoris X-33 after removing its signal peptide. Purified rCML was a stable monomeric protein with a molecular mass of 90 kDa, and was highly N-mannosylated compared to the native protein (69 kDa). The catalytic efficiency (kcat/Km) of rCML was greater than the native protein (1244.35 ± 50.88 and 1067.17 ± 29.07 mM−1·min−1, respectively), yet they had similar optimal pH values and temperatures (40 °C and pH 7.0–7.5), and showed preferences for Tween esters and short-chain triacylglycerols. Despite its monomeric conformation, interfacial activation was not observed for rCML, unlike the classical lipases. From the structural model of rCML, the binding pocket of rCML was predicted as a funnel-like structure consisting of a hollow space and an intramolecular tunnel, which is typical of C. rugosa lipase-like lipases. However, a blockage shortened the tunnel to 12–15 Å, which endows strict short-chain selectivity towards triacylglycerols and a perfect match for tricaproin (C6:0). The limited depth of the tunnel may enable accommodation of triacylglycerols with medium-to-long-chain fatty acids, which differentiates rCML from other C. rugosa lipase-like lipases with broad substrate specificities.

METTL7A (TMT1A) and METTL7B (TMT1B) Are Responsible for Alkyl S-Thiol Methyl Transferase Activity in Liver.

S-methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine (SAM)-dependent membrane-associated enzyme referred to as thiol methyltransferase (TMT). This putative TMT appeared to have a broad substrate specificity and methylated the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine prodrugs, clopidogrel, and prasugrel. Despite TMT's role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol methyltransferase. METTL7B is an endoplasmic reticulum-associated protein with similar biochemical properties and substrate specificity to the putative TMT. Yet, the historic TMT inhibitor 2,3-dichloro-α-methylbenzylamine (DCMB) did not inhibit METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, is also a SAM-dependent thiol methyltransferase. METTL7A exhibits similar biochemical properties to METTL7B and putative TMT, including inhibition by DCMB (IC50 = 1.17 μM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which are now renamed thiol methyltransferase 1A (TMT1A) and thiol methyltransferase 1B (TMT1B), respectively, that are responsible for thiol methylation activity in human liver microsomes. SIGNIFICANCE STATEMENT: We identified methyltransferase-like protein 7A (thiol methyltransferase 1A) and methyltransferase-like protein 7B (thiol methyltransferase 1B) as the enzymes responsible for the microsomal alkyl thiol methyltransferase (TMT) activity. These are the first two enzymes directly associated with microsomal TMT activity. S-methylation of commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible for this activity will improve our understanding of the drug metabolism and pharmacokinetic (DMPK) properties of alkyl- or phenolic thiol-containing therapeutics.

Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

Multipurpose cellulases of Promicromonospora sp. VP111, with broad substrate specificity and tolerance properties.

Cellulolytic actinobacterium, Promicromonospora sp. VP111 concomitantly produced cellulases (CELs), xylanase and pectinase when grown on commercial cellulose and untreated agricultural lignocellulosic residues (wheat straw and sugarcane bagasse). Secreted CELs hydrolyzed (enhanced with Co2+ ion) multiple cellulosic substrates, including sodium carboxymethyl cellulose (Na-CMC), Whatman filter paper no. 1, microcrystalline cellulose (avicel), p-nitrophenyl-β-D-glucopyranoside (pNPG), laminarin, and cellulose powder. The CELs showed stabilities in the presence of various chemicals, including glucose (0.2 M), detergents (1%, w/v or v/v), denaturants (1%, w/v or v/v), and sodium chloride (NaCl, 30%, w/v). The CELs were fractionated using ammonium sulfate precipitation and dialysis. Activities (%) of fractionated CELs were retained at 60°C for endoglucanase/carboxymethyl cellulase (CMCase) (88.38), filter paper cellulase (FPase) (77.55), and β-glucosidase (90.52), which indicated of thermo-stability. Similarly, the activities (%) for CMCase (85.79), FPase (82.48), and β-glucosidase (85.92) at pH 8.5 indicated of alkaline-stability. Kinetic factors, Km and Vmax for endoglucanase component of fractionated CELs were 0.014 g/l and 158.23 µM glucose/min/mL, respectively. Fractionated CELs yielded activation energies (kJ/mol) of 17.933, 6.294, and 4.207 for CMCase, FPase, and β-glucosidase activities, respectively in linear thermostable Arrhenius plots. Thus, this study reports on the multipurpose CELs from an untreated agricultural residue utilizing Promicromonospora in relation to broad substrate specificity, halo-tolerance, alkaline-tolerance, detergent-tolerance, thermo-tolerance, organic solvent-tolerance, and end product-tolerance.

Carbohydrate esterases involved in deacetylation of food components by the human gut microbiota.

Non-carbohydrate modifications such as acetylations are widespread in food stuffs as well as they play important roles in diverse biological processes. These modifications meet the gut environment and are removed from their carbohydrate substrates by the resident microbiota. Among the most abundant modifications are O-acetylations, contributing to polysaccharides physico-chemical properties such as viscosity and gelling ability, as well as reducing accessibility for glycosyl hydrolases, and thus hindering polysaccharide degradation. Of particular note, O-acetylations increase the overall complexity of a polymer, thus requiring a more advanced degrading machinery for microbes to utilize it. This minireview describes acetylesterases from the gut microbiota that deacetylate various food polysaccharides, either as natural components of food, ingredients, stabilizers of microbial origin, or as part of microbes for food and beverage preparations. These enzymes include members belonging to at least 8 families in the CAZy database, as well as a large number of biochemically characterized esterases that have not been classified yet. Despite different structural folds, most of these acetylesterases have a common acid-base mechanism and belong to the SGNH hydrolase superfamily. We highlight examples of acetylesterases that are highly specific to one substrate and to the position of the acetyl group on the glycosyl residue of the carbohydrate, while other members that have more broad substrate specificity. Current research aimed at unveiling the functions and regioselectivity of acetylesterases will help providing fundamental mechanistic understanding on how dietary components are utilized in the human gut and will aid developing applications of these enzymes to manufacture novel industrial products.

Crystal Structure and Functional Characterization of an S-Formylglutathione Hydrolase (BuSFGH) from Burkholderiaceae sp.

S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH (BuSFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of BuSFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that BuSFGH formed a stable dimer in solution. The enzyme activity assay indicated that BuSFGH has a high preference for short-chain p-nitrophenyl esters, such as p-nitrophenyl acetate. The activity of BuSFGH toward p-nitrophenyl acetate was five times higher than that of p-nitrophenyl butylate. Molecular modeling studies on the p-nitrophenyl acetate-bound BuSFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of BuSFGH. They can also provide useful insights for designing modified BuSFGHs with different substrate specificities.

Purification and characterization of lipase enzyme from endophytic Bacillus pumilus WSS5 for application in detergent industry

Lipolytic enzymes from endophytic microbes have been the focus of intense and growing research in view of the applicability of such enzymes in various industrial applications and endophytic microorganisms may emerge as a potential source of lipases with distinct characteristics. The current work involved purification and characterization of the lipase from an endophytic-bacteria isolated from the stem of Withania somnifera L. Dunal. Based on the analysis of morphology and 16S rDNA sequence, the endophytic bacteria was identified as Bacillus pumilus and designated as B. pumilus WSS5. Purified lipase from the B. pumilus WSS5 was found to be of 27KDa as revealed by SDS-PAGE with optimum activity at 37 °C and pH 8. The stability of the enzyme in presence of various metal ions, surfactants, inhibitors, and organic solvents suggested that this enzyme could be exploited in leather and detergent industries. In addition, the stain removal potential of the crude and purified lipase was determined on cotton fabric pieces stained with vegetable oil. Lipase added to water along with detergent significantly enhanced the stain removal efficiency of a commercially available detergent. The current findings suggest the potential of B. pumilusWSS5 lipase owing to promising properties such as the alkali-thermostability, organic solvent-tolerance, and broad substrate specificity that makes it a strong candidate for application in detergent formulations, biotransformation, industries, and medicine. To our knowledge, this is the first report on the purification and characterization of lipase from bacterial endophyte isolated from the stem of Withania somnifera L. Dunal.

Biochemical and structural basis of polyamine, lysine and ornithine acetylation catalyzed by spermine/spermidine N-acetyl transferase in moss and maize.

Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1 -acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N-AcetylTransferase Activity). Combining kinetics, HPLC-MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl-CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6 -acetyl-Lys, whereas N5 -acetyl-Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.

A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger

A putative virulence exoprotease designated as UcB5 was successfully purified from the bacterium Salmonella typhimurium to the electrophoretic homogeneity with 13.2-fold and 17.1% recovery by hydrophobic, ion-exchange, and gel permeation chromatography using Phenyl-Sepharose 6FF, DEAE-Sepharose CL-6B, and Sephadex G-75, respectively. By applying SDS-PAGE, the molecular weight was confirmed at 35 kDa. The optimal temperature, pH, and isoelectric point were 35 °C, 8.0, 5.6 ± 0.2, respectively. UcB5 was found to have a broad substrate specificity against almost all the tested chromogenic substrates with maximal affinity against N-Succ-Ala-Ala-Pro-Phe-pNA achieving Km of 0.16 mM, Kcat/Km of 3.01 × 105 S−1 M−1, and amidolytic activity of 28.9 µmol min−1 L−1. It was drastically inhibited by TLCK, PMSF, SBTI, and aprotinin while, DTT, β-mercaptoethanol, 2,2′-bipyridine, o-phenanthroline, EDTA, and EGTA had no effect, which suggested a serine protease-type. Also, it has shown a broad substrate specificity against a broad range of natural proteins including serum proteins. A cytotoxicity and electron microscopy study revealed that UcB5 could cause subcellular proteolysis that finally led to liver necrosis. For this, future research should focus on using a combination of external antiproteases and antimicrobial agents for the treatment of microbial diseases instead of using drugs alone.

Polymer-Degrading Enzymes of Pseudomonas chloroaphis PA23 Display Broad Substrate Preferences.

Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).

Electrochemically Enabled Intramolecular Amino- and Oxysulfonylation of Alkenes with Sodium Sulfinates to Access Sulfonylated Saturated Heterocycles.

A practical and efficient electrochemical intramolecular amino- or oxysulfonylation of internal alkenes equipped with pendant nitrogen or oxygen-centered nucleophiles with sodium sulfinate was developed. Under undivided electrolytic cell conditions, a variety of sulfonylated N-heterocycles and O-heterocycles, such as tetrahydrofurans, tetrahydropyrans, oxepanes, tetrahydropyrroles, piperidines, δ-valerolactones, etc., were efficiently prepared from easily accessible unsaturated alcohols, carboxylic acids, and N-tosyl amines without the need for additional metal or exogenous oxidant. The robust electrochemical transformation features excellent redox economy, high diastereoselectivity, and broad substrate specificity, which provide a general and practical access to sulfone-containing heterocycles and would facilitate the related synthetic and biological studies based on this electrosynthesis.

Exploring Class I polyhydroxyalkanoate synthases with broad substrate specificity for polymerization of structurally diverse monomer units.

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are key enzymes in PHA polymerization. PhaCs with broad substrate specificity are attractive for synthesizing structurally diverse PHAs. In the PHA family, 3-hydroxybutyrate (3HB)-based copolymers are industrially produced using Class I PhaCs and can be used as practical biodegradable thermoplastics. However, Class I PhaCs with broad substrate specificities are scarce, prompting our search for novel PhaCs. In this study, four new PhaCs from the bacteria Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, and Vibrio metschnikovii were selected via a homology search against the GenBank database, using the amino acid sequence of Aeromonas caviae PHA synthase (PhaCAc), a Class I enzyme with a wide range of substrate specificities, as a template. The four PhaCs were characterized in terms of their polymerization ability and substrate specificity, using Escherichia coli as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in E. coli with a high molecular weight, surpassing PhaCAc. The substrate specificity of PhaCs was evaluated by synthesizing 3HB-based copolymers with 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate, 3-hydroxy-2-methylbutyrate, and 3-hydroxypivalate monomers. Interestingly, PhaC from P. shigelloides (PhaCPs) exhibited relatively broad substrate specificity. PhaCPs was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.

Root-Expressed Rice PAP3b Enhances Secreted APase Activity and Helps Utilize Organic Phosphate.

Phosphate (Pi) deficiency leads to the induction of purple acid phosphatases (PAPs) in plants, which dephosphorylate organic phosphorus (P) complexes in the rhizosphere and intracellular compartments to release Pi. In this study, we demonstrate that OsPAP3b belongs to group III low-molecular weight PAP and is low Pi-responsive, preferentially in roots. The expression of OsPAP3b is negatively regulated with Pi resupply. Interestingly, OsPAP3b was found to be dual localized to the nucleus and secretome. Furthermore, OsPAP3b is transcriptionally regulated by OsPHR2 as substantiated by DNA-protein binding assay. Through in vitro biochemical assays, we further demonstrate that OsPAP3b is a functional acid phosphatase (APase) with broad substrate specificity. The overexpression (OE) of OsPAP3b in rice led to increased secreted APase activity and improved mineralization of organic P sources, which resulted in better growth of transgenics compared to the wild type when grown on organic P as an exogenous P substrate. Under Pi deprivation, OsPAP3b knock-down and knock-out lines showed no significant changes in total P content and dry biomass. However, the expression of other phosphate starvation-induced genes and the levels of metabolites were found to be altered in the OE and knock-down lines. In addition, in vitro pull-down assay revealed multiple putative interacting proteins of OsPAP3b. Our data collectively suggest that OsPAP3b can aid in organic P utilization in rice. The APase isoform behavior and nuclear localization indicate its additional role, possibly in stress signaling. Considering its important roles, OsPAP3b could be a potential target for improving low Pi adaptation in rice.

Synthesis of Pyrazolo[1,5-a]pyridinyl, Pyrazolo[1,5-a]quinolinyl, and Pyrazolo[5,1-a]isoquinolinyl Sulfonyl Fluorides via a [3 + 2] Annulation

A [3 + 2] cycloaddition reaction of N-aminopyridines, N-aminoquinolines, and N-aminoisoquinolines with 1-bromoethene-1-sulfonyl fluoride (BESF) was performed to obtain optimum yields of various useful pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]quinolinyl, and pyrazolo[5,1-a]isoquinolinyl sulfonyl fluorides (43-90% yield). The transformation process showed broad substrate specificity, mild reaction conditions, and operational simplicity. Therefore, the reaction has great applicable value in the field of medicinal chemistry and other disciplines.

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, it contains anti-nutritional compounds including phenolics (large and small molecular weight), which must be addressed to increase uptake by the industry. Microbial fermentation is currently used as a strategy to decrease larger molecular weight poly-phenolics, but results in the undesirable accumulation of small mono-phenolics. Here, we investigate cell-free biocatalytic reduction of phenolic content in faba bean (Vicia faba L.) meal. A representative phenolic ring-breaking catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Initially, large-scale fermentative recombinant production and purification of BLC23O was carried out, with functionality validated by in vitro kinetic analysis. When applied to faba bean meal, BLC23O yielded greatest reductions in phenolic content in a coarse air classified fraction (high carbohydrate), compared to either a fine fraction (high protein) or the original unfractionated meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (e.g. tannins, or complexes with proteins and carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Consistent with this, when applied to a selection of commercially available purified phenolic compounds, known to occur in faba bean, BLC23O was found to have high activity against monophenolic acids and little if any detectable activity against larger molecular weight compounds. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry.Graphical

Structural and Biochemical Analyses of the Butanol Dehydrogenase from Fusobacterium nucleatum.

Butanol dehydrogenase (BDH) plays a significant role in the biosynthesis of butanol in bacteria by catalyzing butanal conversion to butanol at the expense of the NAD(P)H cofactor. BDH is an attractive enzyme for industrial application in butanol production; however, its molecular function remains largely uncharacterized. In this study, we found that Fusobacterium nucleatum YqdH (FnYqdH) converts aldehyde into alcohol by utilizing NAD(P)H, with broad substrate specificity toward aldehydes but not alcohols. An in vitro metal ion substitution experiment showed that FnYqdH has higher enzyme activity in the presence of Co2+. Crystal structures of FnYqdH, in its apo and complexed forms (with NAD and Co2+), were determined at 1.98 and 2.72 Å resolution, respectively. The crystal structure of apo- and cofactor-binding states of FnYqdH showed an open conformation between the nucleotide binding and catalytic domain. Key residues involved in the catalytic and cofactor-binding sites of FnYqdH were identified by mutagenesis and microscale thermophoresis assays. The structural conformation and preferred optimal metal ion of FnYqdH differed from that of TmBDH (homolog protein of FnYqdH). Overall, we proposed an alternative model for putative proton relay in FnYqdH, thereby providing better insight into the molecular function of BDH.