Magnetite-driven Bio-Fenton degradation of chloroacetanilide herbicides by a newly isolated hydrogen peroxide producing bacterium Desemzia sp. strain C1.

The efficiency of the Fenton reaction is markedly contingent upon the operational pH related to iron solubility. Therefore, a heterogeneous Fenton reaction has been developed to function at neutral pH. In the present study, the Bio-Fenton reaction was carried out using magnetite (Fe(II)Fe(III)2O4) and H2O2 generated by a newly isolated H2O2-producing bacterium, Desemzia sp. strain C1 at pH 6.8 to degrade chloroacetanilide herbicides. The optimal conditions for an efficient Bio-Fenton reaction were 10 mM of lactate, 0.5% (w/v) of magnetite, and resting-cells (O.D.600 = 1) of strain C1. During the Bio-Fenton reaction, 1.8-2.0 mM of H2O2 was generated by strain C1 and promptly consumed by the Fenton reaction with magnetite, maintaining stable pH conditions. Approximately, 40-50% of the herbicides underwent oxidation through non-specific reactions of •OH, leading to dealkylation, dechlorination, and hydroxylation via hydrogen atom abstraction. These findings will contribute to advancing the Bio-Fenton system for non-specific oxidative degradation of diverse organic pollutants under in-situ environmental conditions with bacteria producing high amount of H2O2 and magnetite under a neutral pH condition.

Fungal biodegradation of poly(butylene adipate-co-terephthalate)-polylactic acid-thermoplastic starch based commercial bio-plastic film at ambient conditions.

Microbial biodegradation of commercially available poly(butylene adipate-co-terephthalate)-polylactic acid-thermoplastic starch based bio-plastic has been pursued at high temperatures exceeding 55 °C. Herein, we first reported three newly isolated fungal strains from farmland soil samples of Republic of Korea namely, Pyrenochaetopsis sp. strain K2, Staphylotrichum sp. S2-1, and Humicola sp. strain S2-3 were capable of degrading a commercial bio-plastic film with degradation rates of 9.5, 8.6, and 12.2%, respectively after 3 months incubation at ambient conditions. Scanning electron microscopy (SEM) analyses showed that bio-plastic film was extensively fragmented with severe cracking on the surface structure after incubation with isolated fungal strains. X-ray diffraction (XRD) analysis also revealed that high crystallinity of the commercial bio-plastic film was significantly decreased after degradation by fungal strains. Liquid chromatography-mass spectrometry (LC-MS) analyses of the fungal culture supernatants containing the bio-plastic film showed the peaks for adipic acid, terephthalic acid (TPA), and terephthalate-butylene (TB) as major metabolites, suggesting cleavage of ester bonds and accumulation of TPA. Furthermore, a consortium of fungal strain K2 with TPA degrading bacterium Pigmentiphaga sp. strain P3-2 isolated from the same sampling site exhibited faster degradation rate of the bio-plastic film within 1 month of incubation with achieving complete biodegradation of accumulated TPA. We assume that the extracellular lipase activity presented in the fungal cultures could hydrolyze the ester bonds of PBAT component of bio-plastic film. Taken together, the fungal and bacterial consortium investigated herein could be beneficial for efficient biodegradation of the commercial bio-plastic film at ambient conditions.

Production of Four Flavonoid C-Glucosides in Escherichia coli.

Flavonoid C-glucosides, which are found in several plant families, are characterized by several biological properties, including antioxidant, anticancer, anti-inflammatory, neuroprotective, hepatoprotective, cardioprotective, antibacterial, antihyperalgesic, antiviral, and antinociceptive activities. The biosynthetic pathway of flavonoid C-glucosides in plants has been elucidated. In the present study, a pathway was introduced to Escherichia coli to synthesize four flavonoid C-glucosides, namely, isovitexin, vitexin, kaempferol 6-C-glucoside, and kaempferol 8-C-glucoside. A five- or six-step metabolic pathway for synthesizing flavonoid aglycones from tyrosine was constructed and two regioselective flavonoid C-glycosyltransferases from Wasabia japonica (WjGT1) and Trollius chinensis (TcCGT) were used. Additionally, the best shikimate gene module construct was selected to maximize the titer of each C-glucoside flavonoid. Isovitexin (30.2 mg/L), vitexin (93.9 mg/L), kaempferol 6-C-glucoside (14.4 mg/L), and kaempferol 8-C-glucoside (38.6 mg/L) were synthesized using these approaches. The flavonoid C-glucosides synthesized in this study provide a basis for investigating and unraveling their novel biological properties.

Improvement of thermoalkaliphilic laccase (CtLac) by a directed evolution and application to lignin degradation.

Thermoalkaliphilic laccase (CtLac) from the Caldalkalibacillus thermarum strain TA2.A1 has advantageous properties with potential industrial applications, such as high enzyme activity and stability at 70 °C and pH 8.0. In the present study, a directed evolution approach using a combination of random and site-directed mutagenesis was adopted to enhance the laccase activity of CtLac. Spectrophotometric assay and real-time oxygen measurement techniques were employed to compare and evaluate the enzyme activity among mutants. V243 was targeted for site-directed mutagenesis based on library screening. V243D showed a 25-35% higher laccase activity than wild-type CtLac in the spectrophotometric assay and oxygen consumption measurement results. V243D also showed higher catalytic efficiency than wild-type CtLac with decreased Km and increased kcat values. In addition, V243D enhanced oxidative degradation of the lignin model compound, guaiacylglycerol-ß-guaiacyl ether (GGGE), by 10% and produced a 5-30% increase in high-value aldehydes than wild-type CtLac under optimal enzymatic conditions (i.e., 70 °C and pH 8.0). Considering the lack of protein structural information, we used the directed evolution approach to predict Val at the 243 position of CtLac as one of the critical amino acids contributing to the catalytic efficiency of the enzyme. Moreover, it found that the real-time oxygen measurement technique could overcome the limitations of the spectrophotometric assay, and apply to evaluate oxidase activity in mutagenesis research. KEY POINTS: • CtLac was engineered for enhanced laccase activity through directed evolution approach • V243D showed higher catalytic efficiency (kcat/Km) than wild-type CtLac • V243D produced higher amounts of high-value aldehydes from rice straw than wild-type CtLac.

Analysis of phylogenetic markers for classification of a hydrogen peroxide producing Streptococcus oralis isolated from saliva by a newly devised differential medium.

Hydrogen peroxide (H2O2) is produced by alpha-hemolytic streptococci in aerobic conditions. However, the suitable method for detection of H2O2-producing streptococci in oral microbiota has not been setup. Here we show that o-dianisidine dye and horseradish peroxidase were useful in tryptic soy agar medium to detect and isolate H2O2-producing bacteria with the detection limit of one target colony in > 106 colony-forming units. As a proof, we isolated the strain HP01 (KCTC 21190) from a saliva sample using the medium and analyzed its characteristics. Further tests showed that the strain HP01 belongs to Streptococcus oralis in the Mitis group and characteristically forms short-chain streptococcal cells with a high capacity of acid tolerance and biofilm formation. The genome analysis revealed divergence of the strain HP01 from the type strains of S. oralis. They showed distinctive phylogenetic distances in their ROS-scavenging proteins, including superoxide dismutase SodA, thioredoxin TrxA, thioredoxin reductase TrxB, thioredoxin-like protein YtpP, and glutaredoxin-like protein NrdH, as well as a large number of antimicrobial resistance genes and horizontally transferred genes. The concatenated ROS-scavenging protein sequence can be used to identify and evaluate Streptococcus species and subspecies based on phylogenetic analysis.

Draft Genome Sequence of Desemzia sp. Strain C1, Producing Hydrogen Peroxide, Isolated from Oil-Contaminated Soil.

Here, we report the draft genome sequence of Desemzia sp. strain C1, which was isolated from oil-contaminated soil in South Korea and produces hydrogen peroxide (H2O2). The genome of Desemzia sp. strain C1 contains genes encoding various oxidases involved in H2O2 production and resistance to oxidative stress.

Non-specific degradation of chloroacetanilide herbicides by glucose oxidase supported Bio-Fenton reaction.

Bio-Fenton reaction supported by glucose oxidase (GOx) for producing H2O2 was applied to degrade persistent chloroacetanilide herbicides in the presence of Fe (Ⅲ)-citrate at pH 5.5. There were pH decrease to 4.3, the production of 8 mM H2O2 and simultaneous consumption to produce •OH radicals which non-specifically degraded the herbicides. The degradation rates followed the order acetochlor ≈ alachlor ≈ metolachlor > propachlor ≈ butachlor with the degradation percent of 72.8%, 73.4%, 74.0%, 47.4%, and 43.8%, respectively. During the Bio-Fenton degradation, alachlor was dechlorinated and filtered into catechol via the production of intermediates formed through a series of hydrogen atom abstraction and hydrogen oxide radical addition reactions. The current Bio-Fenton reaction leading to the production of •OH radicals could be applied for non-specific oxidative degradation to various persistent organic pollutants under in-situ environmental conditions, considering diverse microbial metabolic systems able to continuously supply H2O2 with ubiquitous Fe(II) and Fe(III) and citrate.

Degradation of sulfonated polyethylene by a bio-photo-fenton approach using glucose oxidase immobilized on titanium dioxide.

Polyethylene (PE) plastics are highly recalcitrant and resistant to photo-oxidative degradation due to its chemically inert backbone structure. We applied two novel reactions such as, Bio-Fenton reaction using glucose oxidase (GOx) enzyme alone and Bio-Photo-Fenton reaction using GOx immobilized on TiO2 nanoparticles (TiO2-GOx) under UV radiation, for (bio)degradation of pre-activated PE with sulfonation (SPE). From both the reactions, GC-MS analyses identified small organic acids such as, acetic acid and butanoic acid as a major metabolites released from SPE. In the presence of UV radiation, 21 fold and 17 fold higher amounts of acetic acid (4.78 mM) and butanoic acid (0.17 mM) were released from SPE after 6 h of reaction using TiO2-GOx than free GOx, which released 0.22 mM and 0.01 mM of acetic acid and butanoic acid, respectively. Our results suggest that (bio)degradation and valorization of naturally weathered and oxidized PE using combined reactions of biochemistry, photochemistry and Fenton chemistry could be possible.

A novel pathway for initial biotransformation of dinitroaniline herbicide butralin from a newly isolated bacterium Sphingopyxis sp. strain HMH.

Butralin (N-sec- Butyl-4-tert-butyl-2,6-dinitroaniline) is a highly persistent dinitroaniline herbicide frequently detected in the environment. In this study, butralin-degrading soil bacterium, Sphingopyxis sp. strain HMH was isolated from agricultural soil samples. Based on whole genome sequence analysis of the strain HMH, the gene encoding a nitroreductase NfnB was identified and expressed in Escherichia coli (E. coli), and protein was purified to homogeneity. NfnB is a flavin-nitroreductase, found to be a functional tetramer, composed of subunit molecular mass of 25 kDa. The metabolites from butralin degradation by strain HMH and purified NfnB were identified using ultra performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS), and a novel mechanism of butralin degradation was proposed. NfnB selectively nitro-reduced butralin into N- (sec-Butyl)-4-(tert-butyl)-6-nitrobenzene- 1,2-diamine, followed by formation of 5-(tert-Butyl)-3 -nitrobenzene-1,2-diamine and butanone by N- dealkylation through possible hydroxylation reaction onto the carbon linked amine of the N-(sec-Butyl) moiety. In our study, we could not detect the hydroxylated product 2-(2-Amino-4-tert-butyl-6-nitro- phenylamino)-butan-2-ol) (carbinolamine), instead its Schiff base product (E)-2-(Butan-2-yildeneamino)-5- (tert-butyl)-3-nitroaniline was detected. The release of butanone was further confirmed by derivatization with 2,4- dinitrophenylhydrazine (DNPH) followed by MS analysis. In conclusion, this study explores a novel multi-functional flavin- nitroreductase family enzyme NfnB, catalyzing unique and sequential nitroreduction and N-dealkylation through oxidative hydroxylation of dinitroaniline herbicide butralin.

Characterization of a novel thermostable carboxylesterase from thermoalkaliphilic bacterium Bacillus thermocloaceae.

A novel thermostable carboxylesterase (Est5250) of thermoalkaliphilic bacterium Bacillus thermocloaceae was heterologously expressed in Escherichia coli and its biochemical properties were investigated. Est5250 showed optimum esterase activity at 60 °C and pH 8.0. The enzyme was highly thermostable at 60 °C, interestingly, the thermostability was enhanced in the presence of Ca2+, retaining more than 60% of its original activity after 12 h of pre-incubation. Est5250 was active in the presence of 1% (v/v) of organic solvents and 0.1% (v/v) of non-ionic detergents. The enzyme activity was significantly enhanced up to 167% and 159% in the presence of 2-mercaptoethanol and dithiothreitol, respectively. Est5250 showed high substrate specificity for short-chain p-nitrophenyl-esters. Kinetic constants, Km and kcat, for p-nitrophenyl-acetate were 185.8 µM and 186.6 s-1, respectively. Est5250 showed outstanding thermostability and tolerance to various organic solvents under thermoalkaliphilic conditions, suggesting that it would be a highly suitable biocatalyst for various biotechnological applications. Abbreviations: B. thermocloaceae sp.: Bacillus thermocloaceae; E. coli: Escherichia coli; NP: nitrophenyl; DMSO: dimethyl sulfoxide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMF: dimethyl formamide; EGTA: ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid; CTAB: cetrimonium bromide; PMSF: phenylmethylsulfonyl fluoride; DEPC: diethyl pyrocarbonate; 2-ME: 2-mercaptoethanol; DTT: dithiothreitol.

Thermoalkaliphilic laccase treatment for enhanced production of high-value benzaldehyde chemicals from lignin.

Enzymatic conversion of lignin into high-value chemicals is a key step in sustainable and eco-friendly development of lignin valorization strategies. In the present study, a novel thermoalkaliphilic laccase, CtLac, from Caldalkalibacillus thermarum strain TA2.A1 was tested for the depolymerization of lignin and the production of value-added chemicals, using three different lignocellulosic biomass, organosolv lignin (OSL), and Kraft lignin. Seven valuable lignin monomers were identified from the CtLac-treated samples using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Remarkably, increases of 22.0%, 65.6%, and 27.3% of p-hydroxybenzaldehyde and increases of 111.1%, 93.5%, and 238.1% of vanillin were observed from rice straw, corn stover, and reed, respectively. Comparative analysis of lignin monomers released from rice straw, using Trametes versicolor laccase (TvL) and CtLac indicated efficient depolymerization of lignin by CtLac. CtLac treatment resulted in 2.3 fold and 5.6 fold, and 1.9 fold and 2.8 fold higher amounts of p-hydroxybenzaldehyde and vanillin from OSL and Kraft lignin, respectively, compared to CtLac-treated rice straw samples after 12 h reaction. OSL was the best substrate for the production of benzaldehyde chemicals using CtLac treatment. The results demonstrated potential application of bacterial laccase CtLac for valorization of biomass lignin into high-value benzaldehyde chemicals under thermoalkaliphilic conditions.

A novel laccase from thermoalkaliphilic bacterium Caldalkalibacillus thermarum strain TA2.A1 able to catalyze dimerization of a lignin model compound.

In the present study, the gene encoding a multicopper oxidase, more precisely a laccase from the thermoalkaliphilic aerobic bacterium Caldalkalibacillus thermarum strain TA2.A1 (CtLac), was cloned and expressed in Escherichia coli. CtLac is a monomeric protein with a molecular weight of 57 kDa as determined by native polyacrylamide gel electrophoresis. The optimum pH and temperature for 2,6-dimethoxyphenol (2,6-DMP) oxidation were 8.0 and 70 °C, respectively. The kinetic constants Km and kcat for 2,6-DMP were of 200 µM and 23 s-1, respectively. The enzyme was highly thermostable at 80 °C and retained more than 80% of its activity after 24 h preincubation under thermoalkaliphilic conditions. Remarkably, it showed a half-life of about 12 h at 90 °C. The enzyme activity was significantly enhanced by Cu2+ and Mn2+ and was not affected in the presence of most of the other metal ions. CtLac activity was stimulated in the presence of halides, organic solvents, and surfactants. Furthermore, the activity of CtLac on a dimeric lignin model compound, guaiacylglycerol-ß-guaiacyl ether (GGGE) was investigated. Liquid chromatography-mass spectrometry analysis indicated that CtLac catalyzes dimerization of GGGE to form a C5-C5 biphenyl tetramer. The stability and activity of CtLac characterized herein under thermoalkaliphilic conditions make it a highly suitable biocatalyst for various biotechnological and industrial applications.