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.

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.

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.

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.

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.

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.