Characteristics of cold-adapted carbonic anhydrase and efficient carbon dioxide capture based on cell surface display technology.

Carbonic anhydrase (CA) is currently under investigation because of its potential to capture CO2. A novel N-domain of ice nucleoproteins (INPN)-mediated surface display technique was developed to produce CA with low-temperature capture CO2 based on the mining and characterization of Colwellia sp. CA (CsCA) with cold-adapted enzyme structural features and catalytic properties. CsCA and INPN were effectively integrated into the outer membrane of the cell as fusion proteins. Throughout the display process, the integrity of the membrane of engineered bacteria BL21/INPN-CsCA was maintained. Notably, the study affirmed positive applicability, wherein 94 % activity persisted after 5 d at 15 °C, and 73 % of the activity was regained after 5 cycles of CO2 capture. BL21/INPN-CsCA displayed a high CO2 capture capacity of 52 mg of CaCO3/mg of whole-cell biocatalysts during CO2 mineralization at 25 °C. Therefore, the CsCA functional cell surface display technology could contribute significantly to environmentally friendly CO2 capture.

Noninvasive detection of diabetes mellitus based on skin fluorescence and diffuse reflectance spectroscopy.

There is an urgent need for a mass population screening tool for diabetes. Skin tissue contains a large number of endogenous fluorophores and physiological parameter markers related to diabetes. We built an excitation-emission spectrum measurement system with the excited light sources of 365, 395, 415, 430, and 455 nm to extract skin characteristics. The modeling experiment was carried out to design and verify the accuracy of the recovery of tissue intrinsic discrete three-dimensional fluorescence spectrum. Blood oxygen modeling experiment results indicated the accuracy of the physiological parameter extraction algorithm based on the diffuse reflectance spectrum. A community population cohort study was carried out. The tissue-reduced scattering coefficient and scattering power of the diabetes were significantly higher than normal control groups. The Gaussian multi-peak fitting was performed on each excitation-emission spectrum of the subject. A total of 63 fluorescence features containing information such as Gaussian spectral curve intensity, central wavelength position, and variance were obtained from each person. Logistic regression was used to construct the diabetes screening model. The results showed that the area under the receiver operating characteristic curve of the model for predicting diabetes was 0.816, indicating a high diagnostic value. As a rapid and non-invasive detection method, it is expected to have high clinical value.

Continuous generation and release of microplastics and nanoplastics from polystyrene by plastic-degrading marine bacteria.

Plastic waste released into the environments breaks down into microplastics due to weathering, ultraviolet (UV) radiation, mechanical abrasion, and animal grazing. However, little is known about the plastic fragmentation mediated by microbial degradation. Marine plastic-degrading bacteria may have a double-edged effect in removing plastics. In this study, two ubiquitous marine bacteria, Alcanivorax xenomutans and Halomonas titanicae, were confirmed to degrade polystyrene (PS) and lead to microplastic and nanoplastic generation. Biodegradation occurred during bacterial growth with PS as the sole energy source, and the formation of carboxyl and carboxylic acid groups, decreased heat resistance, generation of PS metabolic intermediates in cultures, and plastic weight loss were observed. The generation of microplastics was dynamic alongside PS biodegradation. The size of the released microplastics gradually changed from microsized plastics on the first day (1344 nm and 1480 nm, respectively) to nanoplastics on the 30th day (614 nm and 496 nm, respectively) by the two tested strains. The peak release from PS films reached 6.29 × 106 particles/L and 7.64 × 106 particles/L from degradation by A. xenomutans (Day 10) and H. titanicae (Day 5), respectively. Quantification revealed that 1.3% and 1.9% of PS was retained in the form of micro- and nanoplastics, while 4.5% and 1.9% were mineralized by A. xenomutans and H. titanicae at the end of incubation, respectively. This highlights the negative effects of microbial degradation, which results in the continuous release of numerous microplastics, especially nanoplastics, as a notable secondary pollution into marine ecosystems. Their fates in the vast aquatic system and their impact on marine lives are noted for further study.

Highly efficient low-temperature biodegradation of polyethylene microplastics by using cold-active laccase cell-surface display system.

To eliminate efficiency restriction of polyethylene microplastics low-temperature biodegradation, a novel InaKN-mediated Escherichia coli surface display platform for cold-active degrading laccase PsLAC production was developed. Display efficiency of 88.0% for engineering bacteria BL21/pET-InaKN-PsLAC was verified via subcellular extraction and protease accessibility, exhibiting an activity load of 29.6 U/mg. Cell growth and membrane integrity revealed BL21/pET-InaKN-PsLAC maintained stable growth and intact membrane structure during the display process. The favorable applicability was confirmed, with 50.0% activity remaining in 4 days at 15 °C, and 39.0% activity recovery retention after 15 batches of activity substrate oxidation reactions. Moreover, BL21/pET-InaKN-PsLAC possessed high polyethylene low-temperature depolymerizing capacity. Bioremediation experiments proved that the degradation rate was 48.0% within 48 h at 15 °C, and reached 66.0% after 144 h. Collectively, cold-active PsLAC functional surface display technology and its significant contributions to polyethylene microplastics low-temperature degradation constitute an effective improvement strategy for biomanufacturing and microplastics cold remediation.

Three-phase partitioning, a recyclable method for the purification of R-phycoerythrin from a red algae Porphyra yezoensis.

In this study, R-phycoerythrin (R-PE) was isolated and characterized from Porphyra yezoensis by three-phase partitioning (TPP) method. The effects of temperature, time, pH, salt saturation, and volume ratio on the purity and recovery rate of R-PE were studied. The optimum extraction conditions were determined as follows: salt saturation of 70%, temperature of 25 °C, time of 45 min, pH of 7.0, and volume ratio of 1:1. Under the optimal extraction conditions, the purity of R-PE was 3.90. The results of SDS-PAGE showed that R-PE has three bands at 23 kDa, 22 kDa, and 18 kDa, corresponding to its α, ß, γ subunits. The structure and optical activity of R-PE did not change before and after purification based on ultraviolet, infrared, and fluorescence spectra. In addition, the purity and recovery rate of R-PE extracted by tert-butanol were evaluated. The results showed that the extraction performance of tert-butanol for R-PE remained unchanged in three recoveries. These show that TPP is an efficient, green, and recyclable extraction technology.

Study of fluorescence spectroscopy and molecular mechanisms for the interaction of Hg2+ ions and R-phycoerythrin from marine algae (Porphyra yezoensis).

Heavy metal is a worldwide hazardous material, and many efforts were made to detect them sensitively and selectively. R-phycoerythrin (R-PE), a marine fluorescent protein, is abundant in red algae and participates in photosynthesis. In this work, the fluorescence spectroscopy and molecular mechanism of Hg2+ ions and R-PE were further explored through fluorescence spectrum measurements, time-resolved fluorescence lifetimes, peak fitting of Fourier transform infrared spectroscopy, and molecular docking simulation in this study. It was proved by fluorescent spectrum measurements that Hg2+ ions could lead to static fluorescence quenching. Besides, the interaction was a spontaneous and exothermic process driven by hydrogen bond and Van der Waals (VDW) force. Importantly, Hg2+ ions bound to 78LYS and 82CYS on the α chain and 73CYS and 82CYS on the ß chain, which resulted in the structural changes of the peptide chain and affected the secondary structure contents of R-PE. This study further explained the effect of Hg2+ ions on marine fluorescent protein R-PE.

Characteristics and polyethylene biodegradation function of a novel cold-adapted bacterial laccase from Antarctic sea ice psychrophile Psychrobacter sp. NJ228.

Biotreatment of polyethylene (PE) waste is an emerging topic in environmental remediation; in particular, the degrading enzymes requires further exploration. This study described a novel cold-adapted laccase (PsLAC) from an Antarctic psychrophile and characterized its PE-degradation ability. Homology modeling revealed that PsLAC possessed a typical bacterial laccase catalytic structure and unique cold adaptation structural characteristics such as few hydrogen bonds. Recombinant PsLAC (rPsLAC) retained 54.3% residual activity at 0 â„ƒ and presented increased Km values at low temperatures and a relatively high kcat value (42.65 s-1). Collectively, these factors help resist cold stress. rPsLAC possessed substantial salt tolerance at 1.5 M NaCl, with 119.80% activity, and Cu2+ enhanced its activity to 127.10%. PE-degradation experiments indicated that 13.2% weight was lost, and the water contact angle was decreased to 74.6°. Polar functional groups such as carbonyl and carboxyl groups on PE surface were detected in Fourier transform infrared spectroscopy; X-ray diffraction exhibited that crystallinity reduced by 25%. Enormous damage to PE surface and interior was observed via scanning electron microscopy. Overall, PsLAC, with its unique cold-adapted catalytic structure and biochemical characteristics, could supplement the diversity of sources and properties of bacterial laccases and ensure PE-degradation with a novel cold-adapted enzyme resource.

Wavelength and frequency optimization in spatial frequency domain imaging for two-layer tissue.

Spatial frequency domain imaging is a non-contact, wide-field, fast-diffusion optical imaging technique, which in principle uses steady-state spatially modulated light to irradiate biological tissue, reconstruct two-dimensional or three-dimensional tissue optical characteristic map through optical transmission model, and further quantify the spatial distribution of tissue physiological parameters by multispectral imaging technique. The selection of light source wavelength and light field spatial modulation frequency is directly related to the accuracy of tissue optical properties and tissue physiological parameters extraction. For improvement of the measurement accuracy of optical properties and physiological parameters in the two-layer tissue, a multispectral spatial frequency domain imaging system is built based on liquid crystal tunable filter, and a data mapping table of spatially resolved diffuse reflectance and optical properties of two-layer tissue is established based on scaling Monte Carlo method. Combined with the dispersion effect and window effect of light-tissue interaction, the study applies numerical simulation to optimize the wavelength in the 650-850 nm range with spectral resolution of 10 nm. In order to minimize the uncertainty of the optical properties, Cramér-Rao bound is used to optimize the optical field spatial modulation frequency by transmitting the uncertainty of optical properties. The results showed that in order to realize the detection of melanin, oxyhemoglobin, deoxyhemoglobin, water and other physiological parameters in two-layer tissue, the best wavelength combination was determined as 720, 730, 760 and 810 nm according to the condition number. The findings of the Cramér-Rao bound analysis reveal that the uncertainty of optical characteristics for the frequency combinations [0, 0.3] mm-1, [0, 0.2] mm-1, and [0, 0.1] mm-1 increases successively. Under the optimal combination of wavelength and frequency, the diffuse reflectance of the gradient gray-scale plate measured by the multi-spectral spatial frequency domain imaging system is linearly correlated with the calibration value. The error between the measured liquid phantom absorption coefficient and the collimation projection system based on colorimetric dish is less than 2%. The experimental results of human brachial artery occlusion indicate that under the optimal wavelength combination, the change of the second layer absorption coefficient captured by the three frequency combinations decreases in turn, so as the change of oxygen saturation.

A novel fluorescent digitonin derivative for non-invasive skin cholesterol detection: potential application in atherosclerosis screening.

There is a great demand for the rapid and non-invasive atherosclerosis screening method. Cholesterol content in the epidermis of the skin is an early biomarker for atherosclerosis. Risk assessment of atherosclerosis can be achieved by measuring cholesterol in the epidermis. Here, we synthesised a new fluorescent digitonin derivative (FDD) for the non-invasive detection of skin cholesterol. The results of fluorescence spectroscopy studies indicated that the probe exhibited desirable selectivity for cholesterol. The proof-of-concept preclinical study confirmed that FDD can detect different concentrations of skin cholesterol; patients diagnosed with atherosclerotic cardiovascular disease and the at-risk atherosclerosis group exhibited higher skin cholesterol content than the normal group. The area under the ROC curve for distinguishing the normal/disease group was 0.9228 (95% confidence interval, 0.8938 to 0.9518), and the area under the ROC curve for distinguishing the normal/risk group was 0.9422 (95% confidence interval, 0.9178 to 0.9665). We anticipate that this non-invasive skin cholesterol test may be used as a risk assessment tool for atherosclerosis screening in a large population for further examination and intervention in high-risk populations.

Molecular cloning, characterization, and homology modeling of serine hydroxymethyltransferase from psychrophilic bacterium Psychrobacter sp.

Serine hydroxymethyltransferase (SHMT) plays a significant role in the synthesis of l-serine, purine, and thymidylate, which could be extensively applied in the treatment of cancers and the development of antibiotics. In this study, cloned from Psychrobacter sp. ANT206, a novel cold-adapted SHMT gene (psshmt, 1257 bp) encoding a protein of 418 amino acids was expressed in Escherichia coli. The homology modeling result revealed that PsSHMT owned fewer Proline (Pro) residues and hydrogen bonds compared with its homologs from mesophilic E. coli and thermophilic Geobacillus stearothermophilus. In addition, the molecular weight of the purified recombinant PsSHMT (rPsSHMT) was identified to be 45 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, approximately. The enzymatic characteristics of the cold-adapted rPsSHMT displayed that its optimum temperature and pH were 30°C and 7.5, respectively, and its enzymatic activity could be inhibited by Cu2+ , significantly. rPsSHMT also showed a high kcat value and low ΔG at low temperatures. Furthermore, arginine (Arg) could affect the activity of rPsSHMT and be vital to its active sites. The results of this study reflected that these characteristics of the cold-adapted rPsSHMT made it a remarkable candidate that could be utilized in multiple industrial fields under low temperatures.

Evaluation and analysis of the toxicity of mercury (Hg2+) to allophycocyanin from Spirulina platensis in vitro.

As a global environmental pollution problem, heavy metal pollution has brought great harm to human beings. In this work, we studied the toxicity of Hg2+ on allophycocyanin (APC) at the molecular level. Firstly, APC was extracted and purified from Spirulina platensis mud and its purity (A650/A280) reached 3.75. In addition, the fluorescence intensity of APC decreased with increasing Hg2+ concentration from 0 to 5 × 10-6 mol L-1. The theoretical calculation and experimental results showed that the fluorescence quenching of APC by Hg2+ was static and had a good linear relationship. Moreover, the UV-Vis spectra of APC showed a significant decrease at 200 nm and 650 nm with the increase of Hg2+ concentration from 0 to 5×10-6 mol L-1, and a red-shift at 200 nm, which indicated that Hg2+ not only affected the structure of APC but also affected the light absorption and photosynthetic function of APC. Furthermore, the results of molecular simulation demonstrate that Hg2+ combinations with the Met77, Cys81 in the α chain and the Arg77, Cys81 in the ß chain, which interact between the peptide chain and the chromophore, and Hg2+ forms a Hg-S bond with -SH. This study provides new insights into the structure and how Hg2+ effect the optical properties of APC.

Immobilization of psychrophile Psychrobacter sp. ANT206 onto novel reusable magnetic nanoparticles and its application for nitro-aromatic compounds biodegradation under low temperature.

Efficient biodegradation may offer a solution for the treatment of nitro-aromatic compounds (NACs) with toxicity, mutagenicity and persistence in the environment. In this study, dopamine (DA) functionalized magnetic nanoparticles with biocompatibility and hydrophilicity were synthesized and utilized for the immobilization of nitro-aromatic compounds degrading psychrophile Psychrobacter sp. ANT206 harboring the cold-adapted nitroreductase. The prepared nanocarriers were characterized using multiple methods. The highest immobilization yield of cells immobilized by Fe3O4@SiO2@DA was 90.67% under the optimized conditions of 10 °C, pH 7.5, 2 h and cell/support 1.2 mg/mg, and the activity recovery was 89.41%. In addition, the obtained immobilized cells displayed excellent salinity stability and reusability. Moreover, immobilized P. sp. ANT206 strains showed remarkable biodegradation capability on nitrobenzene and p-nitrophenol. This study introduced those novel Fe3O4@SiO2@DA nanoparticles could be applied as ideal and low-cost nanocarriers for the immobilization of cells and large-scale bioremediation of hazardous NACs with perspective applications under low temperature.

Glutaredoxin Interacts with GR and AhpC to Enhance Low-Temperature Tolerance of Antarctic Psychrophile Psychrobacter sp. ANT206.

Glutaredoxin (Grx) is an important oxidoreductase to maintain the redox homoeostasis of cells. In our previous study, cold-adapted Grx from Psychrobacter sp. ANT206 (PsGrx) has been characterized. Here, we constructed an in-frame deletion mutant of psgrx (Δpsgrx). Mutant Δpsgrx was more sensitive to low temperature, demonstrating that psgrx was conducive to the growth of ANT206. Mutant Δpsgrx also had more malondialdehyde (MDA) and protein carbonylation content, suggesting that PsGrx could play a part in the regulation of tolerance against low temperature. A yeast two-hybrid system was adopted to screen interacting proteins of 26 components. Furthermore, two target proteins, glutathione reductase (GR) and alkyl hydroperoxide reductase subunit C (AhpC), were regulated by PsGrx under low temperature, and the interactions were confirmed via bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). Moreover, PsGrx could enhance GR activity. trxR expression in Δpsgrx, Δahpc, and ANT206 were illustrated 3.7, 2.4, and 10-fold more than mutant Δpsgrx Δahpc, indicating that PsGrx might increase the expression of trxR by interacting with AhpC. In conclusion, PsGrx may participate in glutathione metabolism and ROS-scavenging by regulating GR and AhpC to protect the growth of ANT206. These findings preliminarily suggest the role of PsGrx in the regulation of oxidative stress, which could improve the low-temperature tolerance of ANT206.

Removal and Fouling Influence of Microplastics in Fertilizer Driven Forward Osmosis for Wastewater Reclamation.

Insufficient removal of microplastics (MPs) and nanoplastics (NPs) may exert negative effects on the environment and human health during wastewater reclamation. The fertilizer-driven forward osmosis (FDFO) is an emerging potential technology to generate high-quality water for irrigation of hydroponic systems. In this study, the removal of MPs/NPs by the FDFO process together with their impact on FDFO membrane fouling was investigated, due to FDFO's low molecular weight cut-off and energy requirement by using fertilizer as draw solution. Plastic particles with two different sizes (100 nm and 1 µm) and extracellular polymers released by real wastewater bacteria were utilized as model compounds for FDFO performance comparison. Results show that FDFO membrane system could generate high-quality irrigation water with only fertilizer, completely removing extracellular polymers, MPs and NPs from wastewater. It was found that the MPs and NPs themselves do not cause a significant membrane fouling. Moreover, it could help to reduce the membrane fouling caused by extracellular substances. That is probably because MPs and NPs helped to form a loose and porous fouling layer. Therefore, the FDFO process could be a long-term stable (low fouling) process for the reclamation of wastewater with high-quality requirements.

Development of a non-invasive method for skin cholesterol detection: pre-clinical assessment in atherosclerosis screening.

BACKGROUND: Establishing a high-accuracy and non-invasive method is essential for evaluating cardiovascular disease. Skin cholesterol is a novel marker for assessing the risk of atherosclerosis and can be used as an independent risk factor of early assessment of atherosclerotic risk. METHODS: We propose a non-invasive skin cholesterol detection method based on absorption spectroscopy. Detection reagents specifically bind to skin cholesterol and react with indicator to produce colored products, the skin cholesterol content can be obtained through absorption spectrum information on colored products detected by non-invasive technology. Gas chromatography is used to measure cholesterol extracted from the skin to verify the accuracy and reliability of the non-invasive test method. A total of 342 subjects were divided into normal group (n = 115), disease group (n = 110) and risk group (n = 117). All subjects underwent non-invasive skin cholesterol test. The diagnostic accuracy of the measured value was analyzed by receiver-operating characteristic (ROC) curve. RESULTS: The proposed method is able to identify porcine skin containing gradient concentration of cholesterol. The values measured by non-invasive detection method were significantly correlated with gas chromatography measured results (r = 0.9074, n = 73, p < 0.001). Bland-Altman bias was - 72.78 ± 20.03 with 95% limits of agreement - 112.05 to - 33.51, falling within the prespecified clinically non-significant range. We further evaluated the method of patients with atherosclerosis and risk population as well as normal group, patients and risk atherosclerosis group exhibited higher skin cholesterol content than normal group (all P < 0.001). The area under the ROC curve for distinguishing Normal/Disease group was 0.8642 (95% confidence interval, 0.8138 to 0.9146), meanwhile, the area under the ROC curve for distinguishing Normal/Risk group was 0.8534 (95% confidence interval, 0.8034 to 0.9034). CONCLUSIONS: The method demonstrated its capability of detecting different concentration of skin cholesterol. This non-invasive skin cholesterol detection system may potentially be used as a risk assessment tool for atherosclerosis screening, especially for a large population.

Cloning, Expression, Characterization, and Antioxidant Protection of Glutaredoxin3 From Psychrophilic Bacterium Psychrobacter sp. ANT206.

Glutaredoxins (Grxs) are proteins that catalyze the glutathione (GSH)-dependent reduction of protein disulfides. In this study, a Grx-related gene (264 bp), encoding a Ps-Grx3, was cloned from Psychrobacter sp. ANT206. Sequence analysis indicated the presence of the active site motif CPYC in this protein. Homology modeling showed that Ps-Grx3 had fewer hydrogen bonds and salt bridges, as well as a lower Arg/(Arg + Lys) ratio than its mesophilic homologs, indicative of an improved catalytic ability at low temperatures. Site-directed mutagenesis demonstrated that the Cys13, Pro14, and Cys16 sites were essential for the catalytic activity of Ps-Grx3, while circular dichroism (CD) spectroscopy confirmed that point mutations in these amino acid residues led to the loss or reduction of enzyme activity. Furthermore, analysis of the biochemical properties of Ps-Grx3 showed that the optimum temperature of this enzyme was 25 °C. Importantly, Ps-Grx3 was more sensitive to tBHP and CHP than to H2O2, and retained approximately 40% activity even when the H2O2 concentration was increased to 1 mm Regarding substrate specificity, Ps-Grx3 had a higher affinity for HED, L-cystine, and DHA than for S-sulfocysteine and BSA. We also investigated the DNA-protective ability of Ps-Grx3 using the pUC19 plasmid, and found that Ps-Grx3 could protect supercoiled DNA from oxidation-induced damage at 15°C for 1.5 h. This study provides new insights into the structure and catalytic activity of a cold-adapted Grx3.

The elucidation of the biodegradation of nitrobenzene and p-nitrophenol of nitroreductase from Antarctic psychrophile Psychrobacter sp. ANT206 under low temperature.

Psychrobacter is one important typical strain in the Antarctic environment. In our previous study, Psychrobacter sp. ANT206 from Antarctica with novel cold-adapted nitroreductase (PsNTR) could biodegrade nitrobenzene and p-nitrophenol in low temperature environment. In this study, the in-frame deletion mutant of psntr (Δpsntr-ANT206) that displayed well genetic stability and kanamycin resistance stability was constructed using allelic replacement method. Additionally, Δpsntr-ANT206 was more sensitive to nitrobenzene and p-nitrophenol in the comparison of heat and hyperosmolarity, suggesting that psntr gene participated in the regulation of the tolerance against nitro-aromatic compounds (NACs). Further analysis was conducted by integrated gas chromatography-mass spectrometry (GC-MS), and several metabolites were identified. Among them, ethylbenzene, L-Alanine, citric acid, aniline, 4-aminophenol and other metabolites were different between the wild-type strain and Δpsntr-ANT206 under nitrobenzene and p-nitrophenol stress at different time periods under low temperature, respectively. These data could increase the knowledge of the construction of deletion mutant strains and biodegradation mechanism of NACs of typical strains Psychrobacter from Antarctica, which would also provide the basis of the molecular technique on the regulation of bioremediation of the contaminants under low temperature in the future.

Efficient Purification of R-phycoerythrin from Marine Algae (Porphyra yezoensis) Based on a Deep Eutectic Solvents Aqueous Two-Phase System.

R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in Porphyra yezoensis with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A565/A280) of R-PE was 3.825, and the yield was 69.99% (w/w) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K2HPO4 (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.

Purification, biochemical characterization and DNA protection against oxidative damage of a novel recombinant superoxide dismutase from psychrophilic bacterium Halomonas sp. ANT108.

A novel superoxide dismutase (referred hereafter to as HsSOD) from the psychrophilic bacterium Halomonas sp. ANT108 was purified and characterized. Escherichia coli (E. coli) was selected as the expression host. After recombinant HsSOD (rHsSOD) was purified, the specific activity was determined to be 213.47 U/mg with a purification ratio of approximately 3.61-fold. SDS-PAGE results demonstrated that rHsSOD has the molecular weight of 31.3 kDa, and type identification revealed that it belongs to Cu/Zn SOD. The optimum activity of rHsSOD was at 35 °C and 28% of its maximum activity remained at 0 °C. Further enzymatic assays indicated that rHsSOD exhibited thermal instability with a half-life of 20 min at 60 °C. Moreover, Cu2+ and Zn2+ significantly promoted rHsSOD activity. The values of Km and Vmax were 0.33 mM and 476.19 U/mg, respectively. Interestingly, rHsSOD could avoid DNA strand breakage formed by metal-catalyzed oxidation, demonstrating its antioxidant capacity. To summarize, the results suggested that rHsSOD has relatively high catalytic efficiency and oxidation resistance at low temperatures.

A New Cold-Adapted and Salt-Tolerant Glutathione Reductase from Antarctic Psychrophilic Bacterium Psychrobacter sp. and Its Resistance to Oxidation.

A new glutathione reductase gene (psgr) coding for glutathione reductase (GR) from an Antarctic bacterium was cloned and overexpressed into Escherichia coli (E. coli). A sequence analysis revealed that PsGR is a protein consisting of 451 amino acids, and homology modeling demonstrated that PsGR has fewer hydrogen bonds and salt bridges, which might lead to improved conformational flexibility at low temperatures. PsGR possesses the flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding motifs. Recombinant PsGR (rPsGR) was purified using Ni-NTA affinity chromatography and was found to have a molecular mass of approximately 53.5 kDa. rPsGR was found to be optimally active at 25 °C and a pH of 7.5. It was found to be a cold-adapted enzyme, with approximately 42% of its optimal activity remaining at 0 °C. Moreover, rPsGR was most active in 1.0 M NaCl and 62.5% of its full activity remained in 3.0 M NaCl, demonstrating its high salt tolerance. Furthermore, rPsGR was found to have a higher substrate affinity for NADPH than for GSSG (oxidized glutathione). rPsGR provided protection against peroxide (H2O2)-induced oxidative stress in recombinant cells, and displayed potential application as an antioxidant protein. The results of the present study provide a sound basis for the study of the structural characteristics and catalytic characterization of cold-adapted GR.