Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

Enzymatic transformation of esculetin as a potent class of α-glucosidase inhibitors.

A rapid and simple enzymatic transformation of the representative coumarin esculetin (1) with polyphenol oxidase originating from Agaricus bisporus afforded five new oxidized metabolites, esculetinins A (2), B (3), C (4), D (5), and E (6), together with the known compound isoeuphorbetin (7). The structures of the oligomerized transformation products were established on the basis of spectroscopic interpretations. The esculetin oligomers 2 and 3 revealed highly enhanced inhibitory activities against α-glucosidase, with IC50 values of 0.7 ± 0.1 and 2.3 ± 0.3 µM, respectively, as compared to the original esculetin. Kinetic analysis also exhibited that the two new potent metabolites 2 and 3 have competitive modes of action.

Regio- and stereoselective biocatalytic hydration of fatty acids from waste cooking oils en route to hydroxy fatty acids and bio-based polyesters.

The development of biorefinery approaches is of great relevance for the sustainable production of valuable compounds. In accordance with circular economy principles, waste cooking oils (WCOs) are renewable resources and biorefinery feedstocks, which contribute to a reduced impact on the environment. Frequently, this waste is wrongly disposed of into municipal sewage systems, thereby creating problems for the environment and increasing treatment costs in wastewater treatment plants. In this study, regenerated WCOs, which were intended for the production of biofuels, were transformed through a chemo-enzymatic approach to produce hydroxy fatty acids, which were further used in polycondensation reaction for polyester production. Escherichia coli whole cell biocatalyst containing the recombinantly produced Elizabethkingia meningoseptica Oleate hydratase (Em_OhyA) was used for the biocatalytic hydration of crude WCOs-derived unsaturated free fatty acids for the production of hydroxy fatty acids. Further hydrogenation reaction and methylation of the crude mixture allowed the production of (R)- 10-hydroxystearic acid methyl ester that was further purified with a high purity (> 90%), at gram scale. The purified (R)- 10-hydroxystearic acid methyl ester was polymerized through a polycondensation reaction to produce the corresponding polyester. This work highlights the potential of waste products to obtain bio-based hydroxy fatty acids and polyesters through a biorefinery approach.

Structure and chemopreventive activity of fucoidans from the brown alga Alaria angusta.

Six fucoidan fractions were isolated from the brown alga Alaria angusta. Structures of enzymatic hydrolysis products of the fraction 1AaF2 (Fuc:Gal ~ 1:1; 33 % of sulfates) by fucanase from Wenyingzhuangia fucanilytica were studied by chemical and instrumental (NMR spectroscopy and mass-spectrometry) methods. It was shown that 1AaF2 consisted of two structurally different fucoidans: a sulfated 1,3;1,4-α-L-fucan and an enzyme-resistant sulfated and acetylated complex fucogalactan (Fuc:Gal ~ 1:2; 19 % of sulfates) 1AaF2_HMP containing extended 1,3-linked fucose and 1,3/1,4-linked galactose fragments (up to 5 residues). The fractions 1AaF2 and 1AaF2_HMP were a non-cytotoxic, possessed dose-dependent chemopreventive effect on EGF-induced neoplastic cell transformation of mouse normal epidermal JB6 Cl41 cells and inhibited the colony formation of human melanoma SK-MEL-28 cells.

Application of ß-Glucosidase in a Biphasic System for the Efficient Conversion of Polydatin to Resveratrol.

Resveratrol, an ingredient of traditional Chinese medicine, has beneficial effects on human health and huge potential for application in modern medicine. Polydatin is extracted from plants and then deglycosylated into resveratrol; enzymatic methods are preferred for this reaction. In this study, a ß-D-glucosidase from Sphingomonas showed high efficiency in transforming polydatin into resveratrol and was tolerant toward organic solvents. Applying this enzyme in a biphasic transformation system resulted in 95.3% conversion of 20% concentration crude polydatin to resveratrol in 4 h. We thus report a new method for high-efficiency, clean production of resveratrol.

Microbial valorization of lignin: Prospects and challenges.

Lignin is the world's second most prevalent biomaterial, but its effective value-added product valorization methods are still being developed. The most common preparation processes for converting lignin to platform chemicals and biofuels are fragmentation and depolymerization. Due to its structural diversity, fragmentation generally produces a variety of products, necessitating tedious separation and purifying methods to isolate the desired products. Bacterial-based techniques are commonly utilized for lignin fragmentation due to their high metabolitic activity. Recent advancements in lignin valorization utilizing bacteria, such as lignin decomposing microbes and major pathways involved that can breakdown lignin into various valuable products namely lipids, furfural, vanillin, polyhydroxybutyrate, poly lactic acid blends were discussed in this review. This review also covers the genetic and fermentation methodologies to enhance lignin decomposition, challenges and future trends of microbe based lignin valorization.

The biosynthesis of the cannabinoids.

Cannabis has been integral to Eurasian civilization for millennia, but a century of prohibition has limited investigation. With spreading legalization, science is pivoting to study the pharmacopeia of the cannabinoids, and a thorough understanding of their biosynthesis is required to engineer strains with specific cannabinoid profiles. This review surveys the biosynthesis and biochemistry of cannabinoids. The pathways and the enzymes' mechanisms of action are discussed as is the non-enzymatic decarboxylation of the cannabinoic acids. There are still many gaps in our knowledge about the biosynthesis of the cannabinoids, especially for the minor components, and this review highlights the tools and approaches that will be applied to generate an improved understanding and consequent access to these potentially biomedically-relevant materials.

Bio-based and cost effective method for phenolic compounds removal using cross-linked enzyme aggregates.

This work aimed at presenting a green method using a new source of peroxidase isolated from Raphanus sativus var. niger (RSVNP) in immobilized form, for the treatment of wastewater. To ensure stability and enzymatic activity in the biodegradation process, RSVNP was immobilized as a cross-linked enzyme aggregate (CLEAs). With more than 29% of recovered activity and 85% aggregation yield, acetone was selected as the best precipitating agent. The formed protein aggregates required 2% (v/v) of glutaraldehyde (GA) concentration and a ratio of 9:1 (v/v) enzyme (E) amount to cross-linker (E/GA). Compared to the free enzyme, RSVNP-CLEAs were found more chemically and thermally stable and exhibited good storage stability for more than 8 weeks. In addition, RSVNP-CLEAs were evaluated for their ability to remove phenol and p-cresol from aqueous solution by varying several operating conditions. A maximal yield (98%) of p-cresol conversion was recorded after 40 min; while 92% of phenol was degraded after 1 h duration time. The reusability of RSVNP-CLEAs was tested, displaying 71% degradation of phenol in the third batch carried out and more than 54% was achieved for p-cresol after four successive reuses in the presence of hydrogen peroxide at 2 mM concentration.

Optimizing the sulfation-modification system for scale preparation of chondroitin sulfate A.

Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees.

Bi-enzymatic virus-like bionanoreactors for the transformation of endocrine disruptor compounds.

Endocrine disruptor compounds (EDCs) are pollutants able to alter both hormone synthesis and their regulation in animals and humans, thus, EDCs represent a risk for public health and for the environment. Cytochrome P450 enzymes (CYPs) are involved in the detoxification of a wide range of compounds, and it has been established that these enzymes produce the initial biotransformation of many EDCs. In this work, a bionanoreactor based on the encapsulation of an enhanced peroxygenase CYPBM321B3 inside the capsid of bacteriophage P22 virus-like particles (VLPs) was designed and characterized. VLPs were functionalized with glucose oxidase to generate in situ hydrogen peroxide necessary to activate the transformation of bisphenol A, nonylphenol, 17ß-estradiol, triclosan, and resorcinol. Catalytic parameters, as well as the chemical nature of reaction products are presented. The enzymatic nanoreactors showed specific activities varying from 0.175 to 0.456 min-1 in the transformation of these EDCs, which are equivalent to 22-77% of the activity obtained with free CYP. The capacity to transform structurally diverse compounds, easy production and glucose fueled catalytic activity make these enzymatic nanoreactors an interesting platform for enzyme delivery in the biomedical field.

Whole-cell biocatalytic, enzymatic and green chemistry methods for the production of resveratrol and its derivatives.

Resveratrol and the biosynthetically related stilbenes are plant secondary metabolites with diverse pharmacological effects. The versatile functions of these compounds in plant defense mechanisms as phytoalexins on one hand, and in human health as potential pharmaceutical agents on the other, have attracted lots of interest in recent years to understand their biosynthetic pathways and their biological properties. Because of difficulties in obtaining resveratrol and its glucosylated derivatives as well as oligomeric forms in sufficient amounts for evaluation of their activity by plant sourcing or total synthesis, biotechnology may provide a competitive approach for the large-scale and low cost production of biologically active stilbenes. Additionally, one major limitation in the use of resveratrol and related aglycone derivatives as therapeutic agents is associated with their inherent poor aqueous solubility and low bioavailability. This article examines approaches for the synthesis of potential pharmacologically resveratrol derivatives in vivo by exploiting whole microorganisms, enzymatic and biocatalytic approaches allowing their full utilization for medicine, food and cosmetic applications. These methods also have the advantage of enabling the one-step production of stilbene compounds, compared to the time-consuming and environmentally unfriendly procedures used for their total synthesis or their extraction from plants. Increasing the desired products yield and biological activity through glucosylation (ß-D-glucosides versus α-D-glucosides) and oligomerization methodologies of resveratrol including green chemistry methods in organic solvent-free media are discussed as well.

Biodegradation mechanism of polyesters by hydrolase from Rhodopseudomonas palustris: An in silico approach.

Massively used plastics have caused worldwide environmental concerns. Polyesters like polylactic acid (PLA) are one of the mostly used plastics due to its excellent physical and chemical properties and low-cost advantages. It is critical to develop the elimination and recycle techniques for polyesters. Experimental studies have shown that a hydrolase RPA1511 isolated from Rhodopseudomonas palustris can efficiently depolymerize polylactic acid (PLA) into oligomers and monomers. It was also active against emulsified aliphatic polymers as well as multipurpose soluble ester monomers (α-naphthyl ester and p-nitrophenyl ester). In the present study, molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area method were applied to screen all amino acids from hydrolase RPA1511 and identify the most important amino acids during substrate binding. Seven substrates were considered: PLA (dimer and tetramer), polycaprolactone, butylene succinate, 1-naphthyl acetate, 2-naphthyl formate, p-nitrophenyl acetate. The results highlighted the importance of amino acids like Tyr139, Tyr213, Arg259, Thr46. Subsequent quantum mechanics/molecular mechanics calculations were also performed to determine the detailed degradation mechanism of hydrolase RPA1511 toward PLA and explore the role of the active site residues during catalysis. The results demonstrated that degradation involves two elementary steps: enzyme acylation and PLA hydrolysis. The corresponding Boltzmann average barriers are 20.40 kcal/mol and 14.45 kcal/mol. The electrostatic influence analysis of 15 amino acids on the rate-determining step indicated that amino acids His114, Trp219 and Ala273 facilitate the reaction while the Arg244 suppresses the reaction which may serve as future mutation studies to enhance the enzymatic efficiency.

Biotransformation of short-chain chlorinated paraffins (SCCPs) with LinA2: A HCH and HBCD converting bacterial dehydrohalogenase.

Short-chain chlorinated paraffins (SCCPs) are polyhalogenated hydrocarbons as are hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs). They all have been classified as persistent organic pollutants (POPs) under the UN Stockholm Convention. Per se such compounds are transformed slowly in the environment, transported over long distances and accumulate in biota. Several Sphingomonadacea strains isolated from HCH dump sites have evolved to express enzymes that can transform HCHs and HBCDs. We hypothesized that LinA2, a dehydrohalogenase expressed in such bacteria, may also transform CPs to chlorinated olefins (COs). Three mixtures of penta- to deca-chlorinated undecanes (C11), dodecanes (C12) and tridecanes (C13) were exposed to LinA2. High-resolution full-scan mass spectra (R∼8'000) of CPs and COs were obtained applying a soft ionization method, enhancing chloride-adduct [M+Cl]- formation. A mathematical deconvolution procedure was used to separate interfering spectra to verify that LinA2 indeed catalyzed the conversion of CPs to COs. About 20-40% of the material was transformed in 24 h, about 50-70% was converted in 200 h. A bimodal first-order kinetic model could describe transformations of reactive and persistent CPs. Under the given conditions reactive CPs (τ1/2 = 1.4-6.9 h) were converted 30 to 190-times faster than the persistent ones (τ1/2 = 150-260 h). Proportions of persistent isomers (pp) varied from 60 to 80%. Lower chlorinated homologues contained higher proportions of persistent isomers. In conclusion, SCCP mixtures contain both, material that is readily converted by LinA2, and persistent material that is not or only slowly transformed.

Biotransformation of organic micropollutants by anaerobic sludge enzymes.

Biotransformation of organic micropollutants (OMPs) in wastewater treatment plants ultimately depends on the enzymatic activities developed in each biological process. However, few research efforts have been made to clarify and identify the role of enzymes on the removal of OMPs, which is an essential knowledge to determine the biotransformation potential of treatment technologies. Therefore, the purpose of the present study was to investigate the enzymatic transformation of 35 OMPs under anaerobic conditions, which have been even less studied than aerobic systems. Initially, 13 OMPs were identified to be significantly biotransformed (>20%) by anaerobic sludge obtained from a full-scale anaerobic digester, predestining them as potential targets of anaerobic enzymes. Native enzymes were extracted from this anaerobic sludge to perform transformation assays with the OMPs. In addition, the effect of detergents to recover membrane enzymes, as well as the effects of cofactors and inhibitors to promote and suppress specific enzymatic activities were evaluated. In total, it was possible to recover enzymatic activities towards 10 out of these 13 target OMPs (acetyl-sulfamethoxazole and its transformation product sulfamethoxazole, acetaminophen, atenolol, clarithromycin, citalopram, climbazole, erythromycin, and terbutryn, venlafaxine) as well as towards 8 non-target OMPs (diclofenac, iopamidol, acyclovir, acesulfame, and 4 different hydroxylated metabolites of carbamazepine). Some enzymatic activities likely involved in the anaerobic biotransformation of these OMPs were identified. Thereby, this study is a starting point to unravel the still enigmatic biotransformation of OMPs in wastewater treatment systems.

Radiosensitizing effect of the fucoidan from brown alga Fucus evanescens and its derivative in human cancer cells.

Fucoidan from brown alga Fucus evanescens and its product of enzymatic hydrolysis have precisely established structure and possess significant biological activities. The aim of present study was to determine radiosensitizing activity of fucoidan from brown alga F. evanescens and its derivative in human melanoma, breast adenocarcinoma, and colorectal carcinoma cell lines and elucidate mechanism of their action. The fucoidan from F. evanescens and its derivative had a comparable radiosensitizing activity and increased the inhibiting effect of X-ray radiation on proliferation and colony formation of human cancer cells, with significant inhibition of melanoma cells. The molecular mechanism of this action was associated with the induction of apoptosis by activating the initiator and effector caspases, suppressing the expression of the anti-apoptotic protein, and enhancing the fragmentation of DNA. The obtained data confirm the prospects of using fucoidan's derivative in combination with radiation therapy for the improvement of the schemes of cancer therapy.

Cloning and expression of the sucrose phosphorylase gene in Bacillus subtilis and synthesis of kojibiose using the recombinant enzyme.

BACKGROUND: Kojibiose as a prebiotic and inhibitor of α-glucosidase exhibits potential for a wide range of applications in the food and medicine fields; however, large-scale separation and extraction of kojibiose from nature is difficult. Sucrose phosphorylase (SPase) can be used for the production of kojibiose, and currently, SPase is only heterologously expressed in E. coli, making it unsuitable for use in the food industry. However, Bacillus subtilis is generally considered to be a safe organism potentially useful for SPase expression. RESULTS: Here, for the first time, we heterologously expressed Bifidobacterium adolescentis SPase in a food-grade B. subtilis strain. The results showed that SPase was efficiently secreted into the extracellular medium in the absence of a signal peptide. After culturing the recombinant strain in a 3-L bioreactor, crude SPase yield and activity reached 7.5 g/L and 5.3 U/mL, respectively, the highest levels reported to date. The optimal reaction conditions for kojibiose synthesis catalyzed by recombinant SPase were as follows: 0.5 M sucrose, 0.5 M glucose, 0.02 Uenzyme/mgall_substrates, pH 7.0, 50 °C, and 30 h. Furthermore, the substrate-conversion rate reached 40.01%, with kojibiose accounting for 104.45 g/L and selectivity for kojibiose production at 97%. CONCLUSIONS: Here, we successfully expressed SPase in B. subtilis in the absence of a signal peptide and demonstrated its secretion into the extracellular medium. Our results indicated high levels of recombinant enzyme expression, with a substrate-conversion rate of 40.01%. These results provide a basis for large-scale preparation of kojibiose by the recombinant SPase.

Enzymatic Activation of the Emerging Drug Resveratrol.

The plant originated stilbene "resveratrol" (3,4',5-trans-trihydroxystilbene) is well known for its diverse health benefits including anti-tumor, anti-inflammatory, anti-microbial, and anti-oxidant properties. Besides a significant amount of reports on different aspects of its application as prodrug in the last 50 years, still, a strategy leading to the production of the active drug is missing. The aim of this work was to evaluate the enzymatic activation of prodrug resveratrol to the effective drug piceatannol, without engaging expensive cofactors. Five different heme proteins were analyzed for the transformation of resveratrol. Kinetic parameters of resveratrol transformation and analysis of the transformed products were conducted through HPLC and GC-MS. Effect of pH and organic solvent on the transformation process had also been evaluated. Among all tested heme proteins, only a variant of cytochrome P450BM3 from Bacillus megaterium (CYPBM3F87A) was found suitable for piceatannol production. The most suitable pH for the reaction conditions was 8.5, while organic solvents did not show any effect on transformation. For resveratrol transformation, the turnover rate (k cat) was 21.7 (± 0.6) min-1, the affinity constant (K M) showed a value of 55.7 (± 16.7) µM for a catalytic efficiency (k cat/K M) of 389 min-1 mM-1. GC-MS analysis showed that the only product from resveratrol transformation by cytochrome P450BM3 is the biologically active piceatannol. The enzymatic transformation of resveratrol, an emerging compound with medical interest, to active product piceatannol by a variant of cytochrome P450BM3 in the absence of expensive NADPH cofactor is demonstrated. This enzymatic process is economically attractive and can be scaled up to cover the increasing medical demand for piceatannol.

Recent advances in production of 5-aminolevulinic acid using biological strategies.

5-Aminolevulinic acid (5-ALA) is the precursor for the biosynthesis of tetrapyrrole compounds and has broad applications in the medical and agricultural fields. Because of the disadvantages of chemical synthesis methods, microbial production of 5-ALA has drawn intensive attention and has been regarded as an alternative in the last years, especially with the rapid development of metabolic engineering and synthetic biology. In this mini-review, recent advances on the application and microbial production of 5-ALA using novel biological approaches (such as whole-cell enzymatic-transformation, metabolic pathway engineering and cell-free process) are described and discussed in detail. In addition, the challenges and prospects of synthetic biology are discussed.

Mass spectrometry based in vitro assay investigations on the transformation of pharmaceutical compounds by oxidative enzymes.

The ubiquitous presence of trace organic chemicals in wastewater and surface water leads to a growing demand for novel removal technologies. The use of isolated enzymes has been shown to possess the capability for a targeted application but requires a clearer mechanistic understanding. In this study, the potential of peroxidase from horseradish (HRP) and laccase from Pleurotus ostreatus (LccPO) to transform selected trace organic chemicals was studied using mass spectrometry (MS)-based in vitro enzyme assays. Conversion by HRP appeared to be more efficient compared to LccPO. Diclofenac (DCF) and sotalol (STL) were completely transformed by HRP after 4 h and immediate conversion was observed for acetaminophen (APAP). During treatment with LccPO, 60% of DCF was still detectable after 24 h and no conversion was found for STL. APAP was completely transformed after 20 min. Sulfamethoxazole (SMX), carbamazepine (CBZ), ibuprofen (IBP) and naproxen (NAP) were insusceptible to enzymatic conversion. In pharmaceutical mixtures, HRP exhibited a preference for DCF and APAP and the generally less efficient conversion of STL was enhanced in presence of APAP. Transformation product pattern after treatment with HRP revealed polymerization products for DCF while STL showed cleavage reactions. DCF product formation shifted towards a proposed dimeric iminoquinone product in presence of APAP whereas a generally less pronounced product formation in mixtures was observed for STL. In conclusion, the enzymatic treatment approach worked selectively and efficiently for a few pharmaceuticals. However, for application the investigation and possibly immobilization of multiplex enzymes being able to transform diverse chemical structures is recommended.

Enzymatic transformation of ginsenosides in Korean Red Ginseng (Panax ginseng Meyer) extract prepared by Spezyme and Optidex.

BACKGROUND: In this study, we examined the effects of various enzymes on chemical conversions of ginsenosides in ginseng extract prepared by amylases. METHODS: Rapidase, Econase CE, Viscozyme, Ultraflo L, and Cytolase PCL5 were used for secondary enzymatic hydrolysis after amylase treatment of ginseng extract, and ginsenoside contents, skin permeability, and chemical compositions including total sugar, acidic polysaccharide, and polyphenols were determined on the hydrolyzed ginseng extract. RESULTS: Rapidase treatment significantly elevated total ginsenoside contents compared with the control (p < 0.05). In particular, deglycosylated ginsenosides including Rg3, which are known as bioactive compounds, were significantly increased after Rapidase treatment (p < 0.05). The Rapidase-treated group also increased the skin permeability of polyphenols compared with the control, showing the highest level of total sugar content among the enzyme treatment groups. CONCLUSION: This result showed that Rapidase induced the conversion of ginsenoside glycosides to aglycones. Meanwhile, Cytolase PCL5 and Econase treatments led to a significant increase of uronic acid (acidic polysaccharide) level. Taken together, our data showed that the treatments of enzymes including Rapidase are useful for the conversion and increase of ginsenosides in ginseng extracts or products.