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 K and k for 2,6-DMP were of 200 μM and 23 s, 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 Cu and Mn 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-β-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.

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Organisms inhabiting tidal mixing-front zones in shallow temperate seas are subjected to large semidiurnal temperature fluctuations in summer. The ability to optimize energy acquisition to this episodic thermal oscillation may determine the survival, growth and development of these ectotherms. We compared the physiological and molecular responses of Haliotis discus hannai cultivated in suspended cages to fluctuating or stable temperature conditions. Several physiological indicators (respiration, excretion rates and O:N) were measured in both conditions, and alterations in the proteome during thermal fluctuations were assessed. No summer mortality was observed in abalone cultivated in fluctuating temperatures compared with that at stable high temperatures. Metabolic rates increased sharply during stable warm summer conditions and fluctuated in accordance with short-term temperature fluctuations (20–26 °C). Ammonia excretion rates during acute responses were comparable in both conditions. When abalone were exposed to fluctuating temperatures, enzyme activities were downregulated and structure-related protein expression was upregulated compared with that at an acclimation temperature (26 °C), highlighting that exposure to low temperatures during fluctuations alters molecular processes. Our results reveal that modulation of physiological traits and protein expression during semidiurnal thermal fluctuations may buffer abalone from the lethal consequences of extreme temperatures in summer.

In this study, we systematically evaluated the factors affecting the ionization efficiency of paper spray ionization (PSI), such as electric field, solvent supply rate, and paper thickness and hydrophobicity. The observed paper spray plume was classified into three modes: single cone-jet, multi-jet, and rim-jet modes. With the increase in the spraying voltage, the spray plume appeared in order of single cone-jet, multi-jet, and rim-jet modes. The rim-jet mode exhibited the lowest standard deviation and high ionization efficiency among the three spray modes. The main parameter determining the spray mode was the charge density of the droplets generated by paper spray, which depends on the electric field and solvent supply rate. A thicker paper reduced the electric repulsion between the jets and lowered the threshold voltage to reach the rim-jet mode. Lowering the solvent supply rate caused mode transitions from the single cone-jet to the rim-jet, possibly due to the increased droplet charge density. The hydrophobic modification on a paper substrate led to a different ionization mechanism or electrostatic spray ionization at low applied voltages.

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