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Lignin is the second most abundant polymer in nature, which is also widely generated during biomass fractionation in lignocellulose biorefineries. At present, most of technical lignin is simply burnt for energy supply although it represents the richest natural source of aromatics, and thus it is a promising feedstock for generation of value-added compounds. Lignin is heterogeneous in composition and recalcitrant to degradation, with this substantially hampering its use. Notably, microbes have evolved particular enzymes and specialized metabolic pathways to degrade this polymer and metabolize its various aromatic components. In recent years, novel pathways have been designed allowing to establish engineered microbial cell factories able to efficiently funnel the lignin degradation products into few metabolic intermediates, representing suitable starting points for the synthesis of a variety of valuable molecules. This review focuses on recent success cases (at the laboratory/pilot scale) based on systems metabolic engineering studies aimed at generating value-added and specialty chemicals, with much emphasis on the production of cis,cis-muconic acid, a building block of recognized industrial value for the synthesis of plastic materials. The upgrade of this global waste stream promises a sustainable product portfolio, which will become an industrial reality when economic issues related to process scale up will be tackled.

Biomolecules are frequently chiral compounds, existing in enantiomeric forms. Amino acids represent a meaningful example of chiral biological molecules. Both L- and D-amino acids play key roles in the biochemical structure and metabolic processes of living organisms, from bacteria to mammals. In this review, we explore the enantiospecific interaction between proteins and chiral amino acids, introducing theoretical models and describing the molecular basis of the ability of some of the most important enzymes involved in the metabolism of amino acids (i.e., amino acid oxidases, dehydrogenases, and aminotransferases) to discriminate the opposite enantiomers. Our analysis showcases the power of natural evolution in shaping biological processes. Accordingly, the importance of amino acids spurred nature to evolve strictly enantioselective enzymes both through divergent evolution, starting from a common ancestral protein, or through convergent evolution, starting from different scaffolds: intriguingly, the active sites of these enzymes are frequently related by a mirror symmetry.

The oxidation therapy, based on the controlled production of Reactive Oxygen Species directly into the tumor site, was introduced as alternative antitumor approach. For this purpose, d-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, an enzyme able to efficiently catalyze the production of hydrogen peroxide from d-amino acids, was adsorbed onto multi-walled carbon nanotubes (MWCNTs), previously functionalized with polylactic-co-glycolic acid (PLGA) or polyethylene glycol (PEG) at different degrees to reduce their toxicity, to be targeted directly into the tumor. In vitro activity and cytotoxicity assays demonstrated that DAAO-functionalized nanotubes (f-MWCNTs) produced H2O2 and induced toxic effects to selected tumor cell lines. After incubation in human plasma, the protein corona was investigated by SDS-PAGE and mass spectrometry analysis. The enzyme nanocarriers generally seemed to favor their biocompatibility, promoting the interaction with dysopsonins. Despite this, PLGA or high degree of PEGylation promoted the adsorption of immunoglobulins with a possible activation of immune response and this effect was probably due to PLGA hydrophobicity and dimensions and to the production of specific antibodies against PEG. In conclusion, the PEGylated MWCNTs at low degree seemed the most biocompatible nanocarrier for adsorbed DAAO, preserving its anticancer activity and forming a bio-corona able to reduce both defensive responses and blood clearance.

D-enantiomers of amino acids (D-AAs) are only present in low amounts in nature, frequently at trace levels, and for this reason, their biological function was undervalued for a long time. In the past 25 years, the improvements in analytical methods, such as gas chromatography, HPLC, and capillary electrophoresis, allowed to detect D-AAs in foodstuffs and biological samples and to attribute them specific biological functions in mammals. These methods are time-consuming, expensive, and not suitable for online application; however, life science investigations and industrial applications require rapid and selective determination of D-AAs, as only biosensors can offer. In the present review, we provide a status update concerning biosensors for detecting and quantifying D-AAs and their applications for safety and quality of foods, human health, and neurological research. The review reports the main challenges in the field, such as selectivity, in order to distinguish the different D-AAs present in a solution, the simultaneous assay of both L- and D-AAs, the production of implantable devices, and surface-scanning biosensors. These innovative tools will push future research aimed at investigating the neurological role of D-AAs, a vibrant field that is growing at an accelerating pace.

In nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these “unnatural” molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-enzymatic processes representing in vitro pathways of alternative/exchangeable enzymes that allow the generation of an artificial metabolism for D-AAs synthetic purposes.

Enantiomerically pure α-amino acids are compounds of primary interest for the fine chemical, pharmaceutical, and agrochemical sectors. Amino acid oxidases are used for resolving D,L-amino acids in biocatalysis. We recently demonstrated that L-amino acid deaminase from Proteus myxofaciens (PmaLAAD) shows peculiar features for biotechnological applications, such as a high production level as soluble protein in Escherichia coli and a stable binding with the flavin cofactor. Since L-amino acid deaminases are membrane-bound enzymes, previous applications were mainly based on the use of cell-based methods. Now, taking advantage of the broad substrate specificity of PmaLAAD, a number of natural and synthetic L-amino acids were fully converted by the purified enzyme into the corresponding α-keto acids: the fastest conversion was obtained for 4-nitrophenylalanine. Analogously, starting from racemic solutions, the full resolution (ee > 99%) was also achieved. Notably, D,L-1-naphthylalanine was resolved either into the D- or the L-enantiomer by using PmaLAAD or the D-amino acid oxidase variant having a glycine at position 213, respectively, and was fully deracemized when the two enzymes were used jointly. Moreover, the complete stereoinversion of L-4-nitrophenylalanine was achieved using PmaLAAD and a small molar excess of borane tert-butylamine complex. Taken together, recombinant PmaLAAD represents a L-specific amino acid deaminase suitable for producing the pure enantiomers of several natural and synthetic amino acids or the corresponding keto acids, compounds of biotechnological or pharmaceutical relevance.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein (HvRNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, HvRNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of HvRNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. HvRNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released HvRNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.

Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor and non-motor symptoms of Parkinson's Disease (PD), and to L-DOPA-induced dyskinesia. Besides the main agonist L-glutamate, two other amino acids in the atypical D-configuration, D-serine and D-aspartate, activate NMDARs. In the present work, we investigated the effect of dopamine depletion on D-amino acids metabolism in the brain of MPTP-lesioned Macaca mulatta, and in the serum and cerebrospinal fluid of PD patients. We found that MPTP treatment increases D-aspartate and D-serine in the monkey putamen while L-DOPA rescues both D-amino acids levels. Conversely, dopaminergic denervation is associated with selective D-serine reduction in the substantia nigra. Such decrease suggests that the beneficial effect of D-serine adjuvant therapy previously reported in PD patients may derive from the normalization of endogenous D-serine levels and consequent improvement of nigrostriatal hypoglutamatergic transmission at glycine binding site. We also found reduced D-serine concentration in the cerebrospinal fluid of L-DOPA-free PD patients. These results further confirm the existence of deep interaction between dopaminergic and glutamatergic neurotransmission in PD and disclose a possible direct influence of D-amino acids variations in the changes of NMDAR transmission occurring under dopamine denervation and L-DOPA therapy. Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor and non-motor symptoms of Parkinson’s Disease (PD), and to L-DOPA-induced dyskinesia. Besides the main agonist L-glutamate, two other amino acids in the atypical D-configuration, D-serine and D-aspartate, activate NMDARs. In the present work, we investigated the effect of dopamine depletion on D-amino acids metabolism in the brain of MPTP-lesioned Macaca mulatta, and in the serum and cerebrospinal fluid of PD patients. We found that MPTP treatment increases D-aspartate and D-serine in the monkey putamen while L-DOPA rescues both D-amino acids levels. Conversely, dopaminergic denervation is associated with selective D-serine reduction in the substantia nigra. Such decrease suggests that the beneficial effect of D-serine adjuvant therapy previously reported in PD patients may derive from the normalization of endogenous D-serine levels and consequent improvement of nigrostriatal hypoglutamatergic transmission at glycine binding site. We also found reduced D-serine concentration in the cerebrospinal fluid of L-DOPA-free PD patients. These results further confirm the existence of deep interaction between dopaminergic and glutamatergic neurotransmission in PD and disclose a possible direct influence of D-amino acids variations in the changes of NMDAR transmission occurring under dopamine denervation and L-DOPA therapy.

Among nanocelluloses, bacterial nanocellulose (BNC) has proven to be a promising candidate in a range of biomedical applications, from topical wound dressings to tissue-engineering scaffolds. Chemical modifications and incorporation of bioactive molecules have been obtained, further increasing the potential of BNC. This study describes the incorporation of vancomycin and ciprofloxacin in BNC and in modified BNC to afford bioactive BNCs suitable for topical wound dressings and tissue-engineering scaffolds. BNC was modified by grafting glycidylmethacrylate (GMA) and further cross-linking with ethylene glycol dimethacrylate (EGDMA) with the formation of stable C&ndash, C bonds though a radical Fenton-type process that involves generation of cellulose carbon centred radicals scavenged by methacrylate structures. The average molar substitution degree MS (MS = methacrylate residue per glucose unit, measured by Fourier transform infrared (FT&ndash, IR) analysis) can be modulated in a large range from 0.1 up to 3. BNC-GMA, BNC-EGDMA and BNC-GMA-EGDMA maintain the hydrogel status until MS reaches the value of 1. The mechanical stress resistance increase of BNC-GMA and BNC-GMA-EGDMA of MS around 0.8 with respect to BNC suggests that they can be preferred to BNC for tissue-engineering scaffolds in cases where the resistance plays a crucial role. BNC, BNC-GMA, BNC-EGDMA and BNC-GMA-EGDMA were loaded with vancomycin (VC) and ciprofloxacin (CP) and submitted to release experiments. BNC-GMA-EGDMA of high substitution degree (0.7&ndash, 1) hold up to 50 percentage of the loaded vancomycin and ciprofloxacin amount, suggesting that they can be further investigated for long-term antimicrobial activity. Furthermore, they were not colonized by Staphylococcus aureus (S.A.) and Klebsiella pneumonia (K.P.). Grafting and cross-linking BNC modification emerges from our results as a good choice to improve the BNC potential in biomedical applications like topical wound dressings and tissue-engineering scaffolds.

In some environments, a number of crops, notably maize and nuts can be contaminated by aflatoxin B

With the only exception of glycine, all amino acids exist in two specular structures which are mirror images of each other, called D-(dextro) and L-(levo) enantiomers. During evolution, L-amino acids were preferred for protein synthesis and main metabolism; however, the D-amino acids (D-AAs) acquired different and specific functions in different organisms (from playing a structural role in the peptidoglycan of the bacterial cell wall to modulating neurotransmission in mammalian brain). With the advent of sophisticated and sensitive analytical techniques, it was established during the past few decades that many foods contain considerable amounts of D-AAs: we consume more than 100 mg of D-AAs every day. D-AAs are present in a variety of foodstuffs, where they fulfill a relevant role in producing differences in taste and flavor and in their antimicrobial and antiaging properties from the corresponding L-enantiomers. In this review, we report on the derivation of D-AAs in foods, mainly originating from the starting materials, fermentation processes, racemization during food processing, or contamination. We then focus on leading-edge methods to identify and quantify D-AAs in foods. Finally, current knowledge concerning the effect of D-AAs on the nutritional state and human health is summarized, highlighting some positive and negative effects. Notwithstanding recent progress in D-AA research, the relationships between presence and nutritional value of D-AAs in foods represent a main scientific issue with interesting economic impact in the near future.

In some environments, a number of crops, notably maize and nuts can be contaminated by aflatoxin B1 and related compounds resulting from the growth of aflatoxin-producing Aspergilli. Fungal peroxidases have been shown to degrade a number of mycotoxins, including aflatoxin B1 (AFB1). Therefore, the purpose of this study was to investigate the in vitro enzymatic degradation AFB1 by a recombinant type B dye decolorizing peroxidase (Rh_DypB). Analysis of the reaction products by HPLC-MS analysis showed that under optimized conditions AFB1 was efficiently transformed by Rh_DypB, reaching a maximum of 96% conversion after 4 days of reaction at 25 °C. Based on high resolution mass spectrometry analysis, AFB1 was demonstrated to be quantitatively converted to AFQ1, a compound with a significantly lower toxicity. A number of low molecular mass compounds were also present in the final reaction mixture in small quantities. The results presented in this study are promising for a possible application of the enzyme Rh_DypB for aflatoxin reduction in feed.

Peroxidases are well-known biocatalysts produced by all organisms, especially microorganisms, and used in a number of biotechnological applications. The enzyme DypB from the lignin-degrading bacterium Rhodococcus jostii was recently shown to degrade solvent-obtained fractions of a Kraft lignin. In order to promote the practical use, the N246A variant of DypB, named Rh_DypB, was overexpressed in E. coli using a designed synthetic gene: by employing optimized conditions, the enzyme was fully produced as folded holoenzyme, thus avoiding the need for a further time-consuming and expensive reconstitution step. By a single chromatographic purification step, > 100 mg enzyme/L fermentation broth with a > 90% purity was produced. Rh_DypB shows a classical peroxidase activity which is significantly increased by adding Mn2+ ions: kinetic parameters for H2O2, Mn2+, ABTS, and 2,6-DMP were determined. The recombinant enzyme shows a good thermostability (melting temperature of 63–65 °C), is stable at pH 6–7, and maintains a large part of the starting activity following incubation for 24 h at 25–37 °C. Rh_DypB activity is not affected by 1 M NaCl, 10% DMSO, and 5% Tween-80, i.e., compounds used for dye decolorization or lignin-solubilization processes. The enzyme shows broad dye-decolorization activity, especially in the presence of Mn2+, oxidizes various aromatic monomers from lignin, and cleaves the guaiacylglycerol-β-guaiacyl ether (GGE), i.e., the Cα-Cβ bond of the dimeric lignin model molecule of β-O-4 linkages. Under optimized conditions, 2 mM GGE was fully cleaved by recombinant Rh_DypB, generating guaiacol in only 10 min, at a rate of 12.5 μmol/min mg enzyme.

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine ?-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.

pLG72 is a small, primate-specific protein of 153 amino acids. It is the product of the G72 gene, expressed in testis, spinal cord, and brain. The presence of G72 transcript and pLG72 has recurrently been called into question, however G72 mRNA and pLG72 protein levels were higher in blood and brain of patients with schizophrenia than in healthy controls. On the one hand, the SNP rs2391191 corresponding to the R30K substitution in pLG72 was genetically linked to schizophrenia, reduced thickness of the brain cortex in schizophrenia-affected individuals, and altered memory function. Various lines of evidence indicated that pLG72 is a mitochondrial protein, specifically an extrinsic protein bound on the outer membrane. Over the years, pLG72 was proposed to be involved in different functions: (a) overexpression induces mitochondria fragmentation, increasing the numbers of shorter and more mobile ones which could be delivered faster to regions of intense growth and facilitating the dendritic complexity; (b) it might induce oxidative stress by interacting with methionine-R-sulfoxide reductase B2; and (c) it binds and modulates the activity of FMN-containing oxidoreductase of the respiratory complex I. The main role of this protein, however, is related to its binding to the human flavoenzyme D-amino acid oxidase (hDAAO), i.e., the main catabolic enzyme for D-enantiomer of serine. This D-amino acid is a main endogenous coagonist of the N-methyl-D-aspartate type glutamate receptor (NMDAR) involved in main functions such as synaptic plasticity, learning, memory, and excitotoxicity. For this work, we reviewed the recent literature concerning the hDAAO-pLG72 interaction, focusing on the molecular details of the interaction, the effect of hDAAO function and stability, and the cellular effects, especially on D-serine concentration. The main effects related to the pathological R30K substitution are also reported. We have highlighted the gaps in our knowledge of this human protein as well as the relevance of clarifying the molecular details of hDAAO-pLG72 interaction in order to design molecules to modulate hDAAO activity/stability and thus NMDAR function acting at the D-serine cellular level.

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the second-generation antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit

Cephalosporins currently constitute the most widely prescribed class of antibiotics and are used to treat diseases caused by both Gram-positive and Gram-negative bacteria. Cephalosporins contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C (CephC). The 7-ACA nucleus is not sufficiently potent for clinical use; however, a series of highly effective antibiotic agents could be produced by modifying the side chains linked to the 7-ACA nucleus. The industrial production of higher-generation semi-synthetic cephalosporins starts from 7-ACA, which is obtained by deacylation of the naturally occurring antibiotic CephC. CephC can be converted to 7-ACA either chemically or enzymatically using D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase. Both these methods show limitation, including the production of toxic waste products (chemical process) and the expense (the enzymatic one). In order to circumvent these problems, attempts have been undertaken to design a single-step means of enzymatically converting CephC to 7-ACA in the course of the past 10 years. The most suitable approach is represented by engineering the activity of a known glutaryl-7-aminocephalosporanic acid acylase such that it will bind and deacylate CephC more preferentially over glutaryl-7-aminocephalosporanic acid. Here, we describe the state of the art in the production of an effective and specific CephC acylase.

Graphical abstract Treatment of 50 μM RBBR with 60 μg of N. gerenzanensis peroxidase preparation: the dye decolorizing activity was clearly observed., Highlights • Actinomycetes represent an attractive source of ligninolytic enzymes. • 43 actinomycetes were screened for laccase and peroxidase activities. • The novel species N. gerenzanensis produces a valuable bacterial peroxidase activity. • The dye-decolorizing activity paves the way for an industrial use of this peroxidase., Degradation of lignin constitutes a key step in processing biomass to become useful monomers but it remains challenging. Compared to fungi, bacteria are much less characterized with respect to their lignin metabolism, although it is reported that many soil bacteria, especially actinomycetes, attack and solubilize lignin. In this work, we screened 43 filamentous actinomycetes by assaying their activity on chemically different substrates including a soluble and semi-degraded lignin derivative (known as alkali lignin or Kraft lignin), and we discovered a novel and valuable peroxidase activity produced by the recently classified actinomycete Nonomuraea gerenzanensis. Compared to known fungal manganese and versatile peroxidases, the stability of N. gerenzanensis peroxidase activity at alkaline pHs and its thermostability are significantly higher. From a kinetic point of view, N. gerenzanensis peroxidase activity shows a Km for H2O2 similar to that of Phanerochaete chrysosporium and Bjerkandera enzymes and a lower affinity for Mn2+, whereas it differs from the six Pleurotus ostreatus manganese peroxidase isoenzymes described in the literature. Additionally, N. gerenzanensis peroxidase shows a remarkable dye-decolorizing activity that expands its substrate range and paves the way for an industrial use of this enzyme. These results confirm that by exploring new bacterial diversity, we may be able to discover and exploit alternative biological tools putatively involved in lignin modification and degradation.

Enzymatic lignin degradation represents a key challenge for integrated biorefineries. Notwithstanding the rich content in aromatic compounds, lignin's complex structure has hampered identification of an effective and cost-efficient enzymatic procedure to transform it into less complex product families. Advancements in enzymatically modifying or degrading lignin require a simple and reliable analytical method to quickly screen diverse lignin samples by employing different enzymes and conditions. Here, we report on a novel, rapid, and economic colorimetric assay for lignin oxidation based on the reaction of 2,4-dinitrophenylhydrazine with the carbonyl groups generated by enzymatic oxidation. The assay was validated on monomeric and dimeric lignin model compounds by comparison with HPLC analysis. The colorimetric method was used to investigate the activity of ten laccases and eight peroxidases on three technical lignins under different experimental conditions (e.g., by altering pH and mediator used). The colorimetric method was also coupled to a size-exclusion chromatographic separation of the lignin sample obtained after the enzymatic treatment in order to verify whether the enzymatic treatment resulted in lignin depolymerization, too. On the basis of this novel procedure, appropriate enzymatic treatments can now be identified to generate valuable lignin product streams.

Evidence is accumulating that oxygen access in proteins is guided and controlled. We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoprotein D-amino acid oxidase (DAAO) that have a high affinity for dioxygen and are in close proximity to the flavin. With the goal of enhancing the reactivity of DAAO with oxygen, we have performed site-saturation mutagenesis at three positions that flank the putative oxygen channels and high-affinity sites. The most interesting variants at positions 50, 201 and 225 were identified by a screening procedure at low oxygen concentration. The biochemical properties of these variants have been studied and compared with those of wild-type DAAO, with emphasis on the reactivity of the reduced enzyme species with dioxygen. The substitutions at positions 50 and 225 do not enhance this reaction, but mainly affect the protein conformation and stability. However, the T201L variant shows an up to a threefold increase in the rate constant for reaction of O(2) with reduced flavin, together with a fivefold decrease in the K(m) for dioxygen. This effect was not observed when a valine is located at position 201, and is thus attributed to a specific alteration in the micro-environment of one high-affinity site for dioxygen (site B) close to the flavin that plays an important role in the storage of oxygen. The increase in O(2) reactivity observed for T201L DAAO is of great interest for designing new flavoenzymes for biotechnological applications.

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O(2) in the protein framework of the flavoprotein D-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O(2) leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O(2) high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O(2) affinity. In this variant, the reactivity of the reduced enzyme with O(2) is decreased >or=100-fold and the turnover number approximately 1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H(+) from the product imino acid =NH(2)(+) group bound on the flavin Re-side to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.

D-amino acid oxidase (DAAO) has recently become of interest as a biocatalyst for industrial applications and for therapeutic treatments. It has been used in gene-directed enzyme prodrug therapies, in which its production of H2O2 in tumor cells can be regulated by administration of substrate. This approach is limited by the locally low O2 concentration and the high K(m) for this substrate. Using the directed evolution approach, one DAAO mutant was identified that has increased activity at low O2 and D-Ala concentrations and a 10-fold lower K(m) for O2. We report on the mechanism of this DAAO variant and on its cytotoxicity towards various mammalian cancer cell lines. The higher activity observed at low O2 and D-Ala concentrations results from a combination of modifications of specific kinetic steps, each being of small magnitude. These results highlight the potential in vivo applicability of this evolved mutant DAAO for tumor therapy.

In recent years, by investigating bacterial degradation of lignin, interesting enzymatic activities aimed at breaking specific linkages were identified. In this work we focused on the tetrahydrofolate (THF)-dependent O-demethylase LigM from the bacterium Sphingobium sp. strain SYK-6 which converts vanillic acid to protocatechuic acid (PCA). The recombinant LigM was overexpressed in E. coli up to 80 mg L−1 fermentation broth, and the main kinetic parameters on the best substrate vanillic acid (kcat = 0.19 s−1 and Km = 54 μM) and the substrate specificity were identified. The substrate preference was rationalized using a three-dimensional model of LigM structure in complex with THF, which also allowed us to propose a reaction mechanism. LigM efficiently converts vanillic acid into PCA but the reaction requires a 10-fold molar excess of the THF cofactor. In order to limit the cofactor consumption, the plant methionine synthase MetE enzyme was also overexpressed in E. coli and used in combination with LigM. Under optimized conditions, the dual enzyme system produced 5 mM PCA using 0.1 mM THF only, a 500-fold decrease in the cofactor:substrate molar ratio compared to the mono-enzyme process. This represents the first regeneration method for THF in a biocatalytic process.

The beta-O-4 aryl ether linkages represent about 50% of all ethers in various lignins. At least three enzymatic steps are required to break them down: a NAD(+)-dependent C-alpha dehydrogenase (such as LigD and L), a glutathione lyase that releases guaiacol (i.e., a beta-etherase such as LigE and F), and a glutathione-dependent lyase (i.e., LigG). In this work the LigD, L, E, F, and G from Sphingobium sp. SYK-6 were overexpressed in E. coli and purified with high yields. After characterizing the stability and kinetic properties of LigD and L on the lignin model compound GGE (1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol) and the thermostability of all five recombinant Lig enzymes, the experimental conditions for GGE bioconversion could be optimized (i.e., pH 9.0, 25 degrees C, approximate to 0.1 mg mL(-1) of each enzyme, and 0.5 mM racemic substrate). Under optimal conditions, and by recycling NADH using the L-lactate dehydrogenase-pyruvate system, GGE was fully converted into the final products 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one and guaiacol in

A number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redox-sensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nano-sized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers.

Laccases from different sources are employed in a number of biotechnological processes, each characterized by specific reaction constraints and thus requiring an enzyme with suitable properties. In order to avoid the bias generated by different assay methodologies, in this work we investigated the main properties of ten laccases from fungi and bacteria under identical conditions. As a general rule, the optimal activity was apparent at pH 3-4 and was lost at pH≥7.0 (all laccases were stable at pH≥7.0); enzymes active at neutral pH values were also identified. For all tested laccases, activity increased with temperature up to 80°C and stability was good at 25°C. Interestingly, laccases insensitive to high salt concentration were identified, this favoring their use in treating waste waters. Indeed, bacterial laccases retained a significant activity in the presence of DMSO (up to 40% final concentration) and of surfactants, suggesting that they can be applied in lignin degradation processes requiring solvents. The available laccases are versatile and satisfy requirements related to different processes. Notably, the recombinant laccase from Bacillus licheniformis favorably compares with the tested enzymes, indicating that it is well suited for different biotechnological applications.

L-Aspartate oxidase from the thermophilic archaebacterium Sulfolobus tokodaii (StLASPO) catalyzes the stereoselective oxidative deamination of L-aspartate to yield oxaloacetate, ammonia and hydrogen peroxide. The recombinant flavoenzyme shows distinctive features that make it attractive for biotechnological applications (it is highly thermostable, it is stable in a broad pH range, it tightly binds the FAD cofactor and it shows a low K-m for dioxygen). In order to set up an efficient and economically feasible bioconversion process, in this work, we investigated the immobilization of this novel biocatalyst. The best results in terms of immobilization yield have been obtained when StLASPO was immobilized on the amino support Relizyme (TM) HA403/S R after activation with glutaraldehyde and on the epoxy support SEPABEADS (R) EC-EP/S (in both cases, through the free amino groups of the enzyme), as well as prepared as cross-linked enzyme aggregates (CLEA). By using the Relizyme (TM) HA403/S R support, as well as by the CLEA preparation procedure, full immobilization in terms of enzymatic activity was obtained. Immobilized StLASPO was used for the resolution of racemic solutions of 50 mM D, L-aspartate achieving full resolution in

A main goal of green biotechnology is to reduce our dependence on fossil reserves and to increase the use of renewable materials. For this, lignocellulose, which is composed of cellulose, hemicellulose and lignin, represents the most promising feedstock. The latter is a complex aromatic heteropolymer formed by radical polymerization of guaiacyl, syringyl, and p-hydroxyphenyl units linked by β-aryl ether linkages, biphenyl bonds and heterocyclic linkages. Accordingly, lignin appears to be a potentially valuable renewable aromatic chemical, thus representing a main pillar in future biorefinery. The resistance of lignin to breakdown is the main bottleneck in this process, although a variety of white-rot fungi, as well as bacteria, have been reported to degrade lignin by employing different enzymes and catabolic pathways. Here, recent investigations have expanded the range of natural biocatalysts involved in lignin degradation/modification and significant progress related to enzyme engineering and recombinant expression has been made. The present review is focused primarily on recent trends in ligninolytic green biotechnology to suggest the potential (industrial) application of ligninolytic enzymes. Future perspectives could include synergy between natural enzymes from different sources (as well as those obtained by protein engineering) and other pretreatment methods that may be required for optimal results in enzyme-based, environmentally friendly, technologies.

The main industrial process for producing 7-aminocephalosporanic acid (7-ACA), a precursor of semi-synthetic cephalosporin antibiotics, is the two-step enzymatic route based on D-amino acid oxidase and glutaryl acylase working in separate reactors. Taking advantage of the recently developed variants of cephalosporin C acylase from Pseudomonas N176 (named VAC) and the optimized recombinant overproduction of the two enzymes, we set up a one-pot system to directly convert cephalosporin C into 7-ACA. We optimized the process by identifying the most favorable operational conditions, substrate, and enzyme concentrations. Among the VAC variants employed, our results indicated that HS-HS-F72βR VAC is the best choice because of the high activity on both substrates (glutaryl-7-ACA and cephalosporin C) and the absence of substrate and product inhibition effects. Under optimized conditions and by adding further aliquots of the biocatalysts, >98% of cephalosporin C was converted, yielding 7-ACA as the main reaction product (oxo-7-ACA was below the detection level and glutaryl-7-ACA was

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O(2) in the protein framework of the flavoprotein D-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O(2) leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O(2) high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O(2) affinity. In this variant, the reactivity of the reduced enzyme with O(2) is decreasedor=100-fold and the turnover number approximately 1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H(+) from the product imino acid =NH(2)(+) group bound on the flavin Re-side to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and D-isomer of amino acids to yield the corresponding alpha-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3- phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the k(cat)/K(m) ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the alpha 2-alpha 3 loop (comprising residues 50-60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg(54) could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide.

Determination of the D-amino acid content in foods and in biological samples is a very important task. In order to achieve this goal we developed a biosensor employing the flavoenzyme D-amino acid oxidase from the yeast Rhodotorula gracilis. To produce a device in which the D-amino acid composition does not alter the results, both the wild-type and a number of mutants obtained by rational design and directed evolution approaches were used. An analysis of D-amino acid oxidase mutants activity on D-amino acid mixtures containing various ratios of neutral, acidic, and basic substrates identified the Amberzyme-immobilized T60A/Q144R/K152E and M213G mutants as the best choice: their response shows an only limited dependence on the solution composition when at least 20% of the D-amino acid is made up of D-alanine (standard error is approximately 5-9%). This is the first report, to our knowledge, demonstrating that the entire D-amino acid content can be determined by using a screen-printed electrode amperometric biosensor, with a detection limit of 0.25 mM and a mean response time of 10-15 min. The D-amino acid assay based on R. gracilis DAAO-biosensor is inexpensive, simple to perform, and rapid: the D-amino acid concentration of a variety of biological samples can be investigated using this assay.

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an similar to 90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-aminocephalosporanic acid.