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.

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.

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.

Structure and substrate specificity determinants of NfnB, a dinitroaniline herbicide-catabolizing nitroreductase from Sphingopyxis sp. strain HMH.

Nitroreductases are emerging as attractive bioremediation enzymes, with substrate promiscuity toward both natural and synthetic compounds. Recently, the nitroreductase NfnB from Sphingopyxis sp. strain HMH exhibited metabolic activity for dinitroaniline herbicides including butralin and pendimethalin, triggering the initial steps of their degradation and detoxification. However, the determinants of the specificity of NfnB for these herbicides are unknown. In this study, we performed structural and biochemical analyses of NfnB to decipher its substrate specificity. The homodimer NfnB is a member of the PnbA subgroup of the nitroreductase family. Each monomer displays a central α + ß fold for the core domain, with a protruding middle region and an extended C-terminal region. The protruding middle region of Val75-Tyr129 represents a structural extension that is a common feature to members of the PnbA subgroup and functions as an opening wall connecting the coenzyme FMN-binding site to the surface, therefore serving as a substrate binding site. We performed mutational, kinetic, and structural analyses of mutant enzymes and found that Tyr88 in the middle region plays a pivotal role in substrate specificity by determining the dimensions of the wall opening. The mutation of Tyr88 to phenylalanine or alanine caused significant changes in substrate selectivity toward bulkier dinitroaniline herbicides such as oryzalin and isopropalin without compromising its activity. These results provide a framework to modify the substrate specificity of nitroreductase in the PnbA subgroup, which has been a challenging issue for its biotechnological and bioremediation applications.

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.

Multifunctional cellulolytic auxiliary activity protein HcAA10-2 from Hahella chejuensis enhances enzymatic hydrolysis of crystalline cellulose.

The modular auxiliary activity (AA) family of proteins is believed to cause amorphogenesis in addition to oxidative cleavage of crystalline cellulose although the supporting evidence is limited. HcAA10-2 is a modular AA10 family protein (58 kDa) composed of a AA10 module and a family two carbohydrate binding module (CBM2), joined by a long stretch of 222 amino acids of unknown function. The protein was expressed in Escherichia coli and purified to homogeneity. Scanning electron microscopy and X-ray diffraction analysis of Avicel treated with HcAA10-2 provided evidence for the disruption of the cellulose microfibrils ("amorphogenesis") and reduction of the crystallinity index, resulting in a twofold increase of cellulase adsorption on the polysaccharide surface. HcAA10-2 exhibited weak endoglucanase-like activity toward soluble cellulose and cello-oligosaccharides with an optimum at pH 6.5 and 45 °C. HcAA10-2 catalyzed oxidative cleavage of crystalline cellulose released native and oxidized cello-oligosaccharides in the presence of copper and an electron donor such as ascorbic acid. Multiple sequence alignment indicated that His1, His109, and Phe197 in the AA10 module formed the conserved copper-binding site. The reducing sugar released from Avicel by the endoglucanase Cel5 and Celluclast accompanying HcAA10-2 was increased by four- and sixfold, respectively. Moreover, HcAA10-2 and Celluclast acted synergistically on pretreated wheat straw biomass resulting in a threefold increase in reducing sugar than Celluclast alone. Taken together, these results suggest that HcAA10-2 is a novel multifunctional modular AA10 protein possessing amorphogenesis, weak endoglucanase, and oxidative cleavage activities useful for efficient degradation of crystalline cellulose.

Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396.

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca(2+) greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.

Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates.

The chimeric proteins viz. CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A, are constructed by fusion of family 3, 4, and 30 cellulose binding modules (CBMs) to N-terminus of family 9 endoglucanase (Cel9A) from Alicyclobacillus acidocaldrious. The chimeric enzymes were successfully expressed in Escherichia coli and purified to homogeneity. The chimeric enzymes showed significant increase in Avicel (8-12 folds) and filter paper (7-10 folds) degradation activities compared to Cel9A endoglucanase. Computational protein modeling and simulation on the chimeric enzymes were applied to analyze the fused CBMs effect on the increased insoluble cellulosic substrates degradation activity. Thin layer chromatography analysis of the enzymatic hydrolysis products and distribution of reducing sugars between soluble and insoluble fractions indicated processive cleavage of insoluble cellulosic substrates by the chimeras. The fused CBMs played a critical accessory role for the Cel9A catalytic domain and changed its character to facilitate the processive cleavage of insoluble cellulosic substrates.