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.

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.

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.

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.

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.

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.

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.

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.

Cold-Adapted Glutathione S-Transferases from Antarctic Psychrophilic Bacterium Halomonas sp. ANT108: Heterologous Expression, Characterization, and Oxidative Resistance.

Glutathione S-transferases are one of the most important antioxidant enzymes to protect against oxidative damage induced by reactive oxygen species. In this study, a novel gst gene, designated as hsgst, was derived from Antarctic sea ice bacterium Halomonas sp. ANT108 and expressed in Escherichia coli (E. coli) BL21. The hsgst gene was 603 bp in length and encoded a protein of 200 amino acids. Compared with the mesophilic EcGST, homology modeling indicated HsGST had some structural characteristics of cold-adapted enzymes, such as higher frequency of glycine residues, lower frequency of proline and arginine residues, and reduced electrostatic interactions, which might be in relation to the high catalytic efficiency at low temperature. The recombinant HsGST (rHsGST) was purified to apparent homogeneity with Ni-affinity chromatography and its biochemical properties were investigated. The specific activity of the purified rHsGST was 254.20 nmol/min/mg. The optimum temperature and pH of enzyme were 25 °C and 7.5, respectively. Most importantly, rHsGST retained 41.67% of its maximal activity at 0 °C. 2.0 M NaCl and 0.2% H2O2 had no effect on the enzyme activity. Moreover, rHsGST exhibited its protective effects against oxidative stresses in E. coli cells. Due to its high catalytic efficiency and oxidative resistance at low temperature, rHsGST may be a potential candidate as antioxidant in low temperature health foods.

Sensitive and selective detection of Cu2+ ions based on fluorescent Ag nanoparticles synthesized by R-phycoerythrin from marine algae Porphyra yezoensis.

In this study, using a natural and green protein R-phycoerythrin (R-PE) extracted from marine Porphyra yezoensis as the stabilizer and reducer, silver nanoparticles (AgNPs) were synthesized. Based on this, a highly sensitive and selective method for the detection of Cu2+ ions was developed using R-PE-AgNPs as fluorescent probe. The interactions between R-PE-AgNPs and Cu2+ ions were systematically characterized by fluorescence spectroscopy, transmission electron microscopy (TEM), elemental mapping and Fourier transform infrared (FTIR). It was found that Cu2+ ions could cause aggregation of the R-PE-AgNPs, accompanied by the greatly increased particle size. Importantly, the method offered a wide linear detection range from 0 µM to 100.0 µM with a detection limit of 0.0190 µM. Moreover, the proposed method was successfully applied to analyze Cu2+ ions in tap water and lake water samples, acquiring satisfactory recovery between 91.6% and 102.2%. Such a green, fast and cost-effective fluorimetric method of the R-PE-AgNPs probe has great potential for tracing Cu2+ ions in diverse aqueous media.

A Novel Cold-Adapted and Salt-Tolerant RNase R from Antarctic Sea-Ice Bacterium Psychrobacter sp. ANT206.

A novel RNase R, psrnr, was cloned from the Antarctic bacterium Psychrobacter sp. ANT206 and expressed in Escherichia coli (E. coli). A bioinformatics analysis of the psrnr gene revealed that it contained an open reading frame of 2313 bp and encoded a protein (PsRNR) of 770 amino acids. Homology modeling indicated that PsRNR had reduced hydrogen bonds and salt bridges, which might be the main reason for the catalytic efficiency at low temperatures. A site directed mutation exhibited that His 667 in the active site was absolutely crucial for the enzyme catalysis. The recombinant PsRNR (rPsRNR) showed maximum activity at 30 °C and had thermal instability, suggesting that rPsRNR was a cold-adapted enzyme. Interestingly, rPsRNR displayed remarkable salt tolerance, remaining stable at 0.5-3.0 M NaCl. Furthermore, rPsRNR had a higher kcat value, contributing to its efficient catalytic activity at a low temperature. Overall, cold-adapted RNase R in this study was an excellent candidate for antimicrobial treatment.

A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178.

l-tert-leucine and its derivatives are useful as pharmaceutical active ingredients, in which leucine dehydrogenase (LeuDH) is the key enzyme in their enzymatic conversions. In the present study, a novel cold-adapted LeuDH, psleudh, was cloned from psychrotrophic bacteria Pseudoalteromonas sp. ANT178, which was isolated from Antarctic sea-ice. Bioinformatics analysis of the gene psleudh showed that the gene was 1209 bp in length and coded for a 42.6 kDa protein containing 402 amino acids. PsLeuDH had conserved Phe binding site and NAD⁺ binding site, and belonged to a member of the Glu/Leu/Phe/Val dehydrogenase family. Homology modeling analysis results suggested that PsLeuDH exhibited more glycine residues, reduced proline residues, and arginine residues, which might be responsible for its catalytic efficiency at low temperature. The recombinant PsLeuDH (rPsLeuDH) was purified a major band with the high specific activity of 275.13 U/mg using a Ni-NTA affinity chromatography. The optimum temperature and pH for rPsLeuDH activity were 30 °C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 °C. Moreover, the activity of rPsLeuDH was the highest in the presence of 2.0 M NaCl. Substrate specificity and kinetic studies of rPsLeuDH demonstrated that l-leucine was the most suitable substrate, and the catalytic activity at low temperatures was ensured by maintaining a high kcat value. The results of the current study would provide insight into Antarctic sea-ice bacterium LeuDH, and the unique properties of rPsLeuDH make it a promising candidate as a biocatalyst in medical and pharmaceutical industries.

Cloning, expression and biochemical characterization of recombinant superoxide dismutase from Antarctic psychrophilic bacterium Pseudoalteromonas sp. ANT506.

In this study, a superoxide dismutase gene (PsSOD) from Pseudoalteromonas sp. ANT506 was cloned and over expressed in Escherichia coli. The PsSOD has an open reading frame of 582 bp with a putative product of 193 amino acid residue and an estimated molecular size of 21.4 kDa. His-tagged PsSOD was subsequently purified 12.6-fold by Ni-affinity chromatography and the yield of 22.9%. The characterization of the purified rPsSOD exhibited maximum activity at 30 °C and pH 8.0. The enzyme exhibited 13.9% activity at 0 °C and had high-thermo lability at higher than 50 °C. rPsSOD exhibited well capability to 2.5 M NaCl (62.4%). These results indicated that rPsSOD exhibited special catalytic properties.

Biodegradation of polystyrene (PS) and polypropylene (PP) by deep-sea psychrophilic bacteria of Pseudoalteromonas in accompany with simultaneous release of microplastics and nanoplastics.

Plastics dumped in the environment are fragmented into microplastics by various factors (UV, weathering, mechanical abrasion, animal chewing, etc.). However, little is known about plastic fragmentation and degradation mediated by deep-sea microflora. To obtain deep-sea bacteria that can degrade plastics, we enriched in situ for 1 year in the Western Pacific using PS as a carbon source. Subsequently, two deep-sea prevalent bacteria of the genus Pseudoalteromonas (Pseudoalteromonas lipolytica and Pseudoalteromonas tetraodonis) were isolated after 6 months enrichment in the laboratory under low temperature (15 °C). Both showed the ability to degrade polystyrene (PS) and polypropylene (PP), and biodegradation accelerated the generation of micro- and nanoplastics. Plastic biodegradation was evidenced by the formation of carboxyl and carboxylic acid groups, heat resistance decrease and plastic weight loss. After 80 days incubation at 15 °C, the microplastic concentration of PS and PP could be up to 1.94 × 107/L and 5.83 × 107/L, respectively, and the proportion of nanoplastics (< 1 µm) could be up to 65.8 % and 73.6 %. The film weight loss were 5.4 % and 4.5 % of the PS films, and 2.3 % and 1.8 % of the PP films by P. lipolytica and P. tetraodonis, respectively; thus after discounting the weight loss of microplastics, the only 3.9 % and 2.8 % of the PS films, and 1.3 % and 0.7 % of the PP films, respectively, were truly degraded by the two bacteria respectively after 80 days of incubation. This study highlights the role of Pseudoalteromonas in fragmentation and degradation of plastics in cold dark pelagic deep sea.

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.

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.

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.

Molecular cloning, expression, purification and characterization of thioredoxin from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178.

Thioredoxin (Trx) is a highly conserved and multi-functional protein that plays a pivotal role in maintaining the redox state of the cell and in protecting the cell against oxidative stress. Trx gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178 was cloned and expressed as soluble protein in Escherichia coli (designated as PsTrx). Trx gene consisted of an open reading frame of 324-bp nucleotides encoding a protein of 108 amino acids with a calculated molecular mass of 11.88 kDa. The deduced protein included the conserved Cys-Gly-Pro-Cys active-site sequence. After purification by a single step Ni-NTA affinity chromatography, recombinant PsTrx with a high specific activity of 96.67 U/mg was obtained. The purified PsTrx had an optimal temperature and pH of 25 °C and 7.0, respectively, and showed about 55 % of the residual catalytic activity even at 0-10 °C. It had high tolerance to a wide range of NaCl concentrations (0-2 M NaCl) and was stable in the presence of H2O2. This research suggested that PsTrx displayed unique catalytic properties.

Optimization of cold-active lipase production from psychrophilic bacterium Moritella sp. 2-5-10-1 by statistical experimental methods.

Statistical experimental designs were applied to optimize cold-active lipase production by the psychrophilic bacterium Moritella sp. 2-5-10-1. First, a Plackett-Burmen design (PBD) was used to evaluate the significant effects of various fermentation parameters. The results indicated that soybean meal, temperature, and Tween-80 had significant influences on lipase production. The levels of these variables were optimized subsequently using central composite design (CCD). A quadratic regression model of cold-active lipase production was built, and verification experiments confirmed its validity. On subsequent scale-up in a 10-L bioreactor using optimized conditions, cold-active lipase production (30.56 U/mL) was obtained. The results clearly indicated that the model was adequate even on a large scale. To our knowledge, this is the first report of statistical optimization of cold-active lipase production by a psychrophilic bacterium.

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.