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AbstractProtein engineering is an effective method to improve the thermal stability of biological macromolecules. Cytidine monophosphate kinase (CMK) is a key enzyme involved in nucleotide metabolism and participates in the production of cytidine triphosphate. It has previously been confirmed that CMK shows low thermal stability. This study aimed to enhance the thermal stability of CMK. Herein, we first analyzed native CMK using PoPMuSiC. Thereafter, we constructed single point mutants (Q30G, P62R, E85I, D148W and K165L) and multipoint mutants of CMK with higher thermal stability using site-directed mutagenesis. The thermal stability of the mutant Q30G/E85I/K165L was observed to be 1.2-fold higher than that of the wild type. The mutants were then allowed to catalyse 3′-sialyllactose (3′-SL) synthesis. After 51.72 mmol/L N-acetylneuraminic acid (sialic acid) was consumed in a 25 mL reaction system and 50.67 mmol/L 3′-SL was obtained, a decrease of 2 h was recorded in the reaction time, whereas the yield rate increased by 33.6%. The results suggest that the mutant Q30G/E85I/K165L can improve the thermal stability of CMK and its catalytic efficiency for 3′-SL synthesis. These findings are crucial for various applications involving enzyme conversion.

The Lower Paleozoic reservoir in the Micangshan tectonic belt is a new shale gas exploration area with excellent potential. However, the black shale’s sedimentary environment and tectonic setting and their effects on pore-scale fluid flow have not been thoroughly elucidated and urgently require further research. A large amount of trace and rare earth element data of black shale in this area was tested to analyze the sediment’s provenance, tectonic setting, and coupling process, and nuclear magnetic resonance was applied for fluid flow ability determination. The characteristics of trace elements and rare earth elements in shale samples were drilled from the Upper Sinian Doushantuo Formation, the Lower Cambrian Niutitang Formation, and the Lower Silurian Longmaxi Formation in the Micangshan tectonic belt. The results reveal that the provenance of the shales was dominated by island arc volcanic rocks and some ancient fine-grained sediments. The provenances of shale in the Micangshan, Huijunba, and Zhenba areas may have a strong genetic relationship with the igneous rocks from the nearest Hannan massif. The provenance of the Niutitang shale in the Ningqiang area may comprise sediments derived from the Longmenshan continental rift system. The provenance of the shale tends to transition from the island arc to the continental margin from the depositional stage of the Doushantuo Formation to the Longmaxi Formation. The Micangshan tectonic belt inherited the tectonic evolution of the South China block, North China block, and Qinling, and those blocks provided the igneous provenance of the shale by island arcs, which were formed due to weathering caused by the process of subduction and collision orogeny. The movable fluid distribution of the Longmaxi Formation and Niutitang Formation is relatively uniform, and excessive pursuit of large amount of movable fluid will be counterproductive to the shale reservoir. The proportion of the cracks was generally less than 3% except for one sample from the Doushantuo Formation with 12.06%. Based on the centrifugation force in this research, the low fluid flow limits were less than 1.47, and there are strong positive relationships between movable fluid saturation and saturated porosity. In this study, the coupling relationship between the material source and pore scale flow is discussed for the first time, and the results can provide theoretical references for local provenance analysis and tectonic movement.

For the interference problems in frequency sharing and geographical coexistence between 5G base stations and the adjacent frequency systems such as satellite earth stations, the different situations consisting of the interferences to 5G base stations and the interferences to other stations were analyzed, and the solutions to improve interference positioning efficiency including analyzing the same frequency signal using real-time spectrum function, identifying the signal using 5G NR demodulation function, and locating the interference source combined with same-frequency multiple radio source positioning method were presented.

2'-Fucosyllactose (2'-FL) is one of the most important oligosaccharides in human milk. In this study, a glucose–glycerol mixed carbon source and the deletion of the genes arcA and iclR were attempted for the first time in engineered Escherichia coli to improve 2'-FL production. The highest 2'-FL titre of 5.04 g/L, almost 1.4 times higher than that in the control strain, was obtained in the arcA mutant strain using shake-flask culture. In addition, the arcA mutant showed a significantly reduced accumulation of acetate. However, the deletion of iclR and the double deletion of arcA/iclR provided no benefit in terms of 2'-FL biosynthesis. Overall, these results suggest that deletion of arcA could efficiently increase the 2'-FL yield in E. coli and could lay a foundation for more efficient and larger-scale production. Moreover, in shake-flask culture under optimal conditions, the 2'-FL titer reached a maximum value of 7.14 g/L.

In this study, CS-NeuAc hydrogel was prepared from chitosan (CS) and N-acetylneuraminic acid (NeuAc) using cyclic freeze/thaw under 1-ethyl-3-(3-dimethylaminopropyl)-carbodiiminde/N-hydroxysuccinimide catalysis. Fourier transformed infrared spectroscopy, X-ray diffraction, and scanning electron microscopy analyses confirmed the successful synthesis of CS-NeuAc hydrogel. CS-NeuAc hydrogels were found to be equipped with suitable swelling behaviour, porosity, water evaporation rate, degradation rate, and good mechanical properties compared to CS. The swelling rate, water evaporation rate, and degradation rate of prepared hydrogel with a CS/NeuAc molar ratio of 10:1 were 710.7 ± 28.6%, 78.2 ± 1.6%, and 58.6 ± 1.5%, respectively. Moreover, CS-NeuAc hydrogel exhibited excellent biocompatibility. The evaluation of CS-NeuAc hydrogels’ antibacterial activity confirmed that they serve as effective antibacterial materials against Escherichia coli and Staphylococcus aureus with antibacterial rates above 95% and 92%, respectively, and the minimum inhibitory concentrations was above 400 μg/ml. In conclusion, CS-NeuAc hydrogel can be a candidate for biological antibacterial materials.

The probiotic Weizmannia coagulans (W. coagulans) BC2000 can increase the abundance of intestinal transforming ellagic acid (EA) bacteria and inhibit metabolic disorders caused by hyperlipidemia by activating liver autophagy. This study aimed to investigate the inhibitory effects of W. coagulans BC2000 and EA on hyperlipidemia-induced cholesterol metabolism disorders. C57BL/6J mice (n = 10 in each group) were fed a low-fat diet, high-fat diet (HFD), HFD supplemented with EA, HFD supplemented with EA and W. coagulans BC77, HFD supplemented with EA, and W. coagulans BC2000. EA and W. coagulans BC2000 supplementation prevented HFD-induced hypercholesterolemia and promoted fecal cholesterol excretion. Transcriptome analysis showed that primary bile acid biosynthesis in the liver was significantly activated by EA and W. coagulans BC2000 treatments. EA and W. coagulans BC2000 treatment also significantly increased the intestinal Eggerthellaceae abundance and the liver EA metabolites, iso-urolithin A, Urolithin A, and Urolithin B. Therefore, W. coagulans BC2000 supplementation promoted the intestinal transformation of EA, which led to the upregulation of liver bile synthesis, thus preventing hypercholesterolemia.

Levilactobacillus brevis strains can be isolated from traditional Chinese pickles and used as the starter cultures to improve the nutritional profiles of fermented juices. Three L. brevis strains (LBG-29, LBG-24, LBD–14) that produce high levels of gamma-aminobutyric acid (GABA; >300 mg/L) were isolated from traditional Chinese pickles. The strains showed tolerance to low pH and high bile salts and exhibited safety in vitro. Litchi juice was fermented using each strain at 37 °C for 48 h. The litchi juice was determined to be a good substrate for fermentation as the process enhanced its functional profile. Overall, cell vitality increased (above 8.7 log10 CFU/mL), the antioxidant activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion-reducing antioxidant power (FRAP) were significantly increased, and the antioxidant capacity of the 2,2′-amino-di(3-ethyl-benzothiazoline sulphonic acid-6)ammonium salt (ABTS) was decreased. There was also a significant increase in the GABA and acetic acid content after LBG-29 and LBG-24 fermentation. It was thus determined that the LBG-29 and LBG-24 strains could be used to improve beverage functionality and aid in the development of new products. This is the first report of litchi fermentation using L. brevis as a starter culture. Further research is required to elucidate the functional benefits for the human body and the nutritional and functional properties during its shelf life.

The Micangshan-Dabashan tectonic belt, located in the southern Qinling-Dabie Orogen near the northeastern Tibetan Plateau, is a crucial area for understanding the processes and mechanisms of orogenesis. Previous studies have been focused on the cooling process via thermochronology and the mechanism and process of basement uplift have been investigated. However, the coupling process of basement exhumation and sedimentary cap cooling is unclear. The tectono-thermal history constrained by the detrital apatite fission track (AFT) results could provide valuable information for understanding crustal evolution and the coupling process. In this study, we provided new detrital AFT thermochronology results from the Micangshan-Dabashan tectonic belt and obtained nine high-quality tectono-thermal models revealing the Meso-Cenozoic cooling histories. The AFT ages and lengths suggest that the cooling events in the Micangshan area were gradual from north (N) to south (S) and different uplift occurred on both sides of Micangshan massif. The cooling in Dabashan tectonic zone was gradual from northeast (NS) to southwest (SW). The thermal histories show that a relatively rapid cooling since ca. 160 Ma occurred in the Micangshan-Dabashan tectonic belt, which was a response to the event of Qinling orogenic belt entered the intracontinental orogenic deformation. This cooling event may relate to the northeastward dextral compression of the Yangtze Block. The sedimentary cap of Cambriano-Ordovician strata responded positively to this rapid cooling event and entered the PAZ since ca. 63 Ma. The deep buried samples may be limited affected by climate and water erosion and the accelerated cooling was not obvious in the Late Cenozoic. Collectively, the cooling processes of basement and sedimentary cap in Micangshan-Dabashan tectonic belt were inconsistent. The uplift of the sedimentary area is not completely consistent with that of the basement under thrust and nappe action. The rigid basement was not always continuous and rapidly uplifted or mainly showed as lateral migration in a certain stage because of the different intensities and modes of thrust and nappe action, and the plastic sedimentary strata rapidly uplifted due to intense folding deformation.

(1) Background: Ellagic acid (EA) acts as a product of gut microbiota transformation to prevent insulin resistance, which is limited by high-fat diet (HFD)-induced dysbiosis. The aim of this study was to investigate the synergistic effects and mechanisms of supplementation with the probiotic Weizmannia coagulans (W. coagulans) on the prevention of insulin resistance by EA; (2) Methods: C57BL/6J mice were divided into five groups (n = 10/group): low-fat-diet group, high-fat-diet group, EA intervention group, EA + W. coagulans BC77 group, and EA + W. coagulans BC2000 group; (3) Result: W. coagulans BC2000 showed a synergistic effect on EA’s lowering insulin resistance index and inhibiting high-fat diet-induced endotoxemia. The combined effect of BC2000 and EA activated the autophagy pathway in the mouse liver, a urolithin-like effect. This was associated with altered β-diversity of gut microbiota and increased Eggerthellaceae, a potential EA-converting family. Ellagic acid treatment alone and the combined use of ellagic acid and W. coagulans BC77 failed to activate the hepatic autophagy pathway; (4) Conclusions: W. coagulans BC2000 can assist EA in its role of preventing insulin resistance. This study provides a basis for the development of EA-rich functional food supplemented with W. coagulans BC2000.

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

The objective of this study was to investigate the stability and degradation kinetics of N-acetylneuraminic acid (Neu5Ac). The pH of the solution strongly influenced the stability of Neu5Ac, which was more stable at neutral pH and low temperatures. Here, we provide detailed information on the degradation kinetics of Neu5Ac at different pH values (1.0, 2.0, 11.0 and 12.0) and temperatures (60, 70, 80 and 90 °C). The study of the degradation of Neu5Ac under strongly acidic conditions (pH 1.0–2.0) is highly pertinent for the hydrolysis of polysialic acid. The degradation kinetics of alkaline deacetylation were also studied. Neu5Ac was highly stable at pH 3.0–10.0, even at high temperature, but the addition of H2O2 greatly reduced its stability at pH 5.0, 7.0 and 9.0. Although Neu5Ac has a number of applications in products of everyday life, there are no reports of rigorous shelf-life studies. This research provides kinetic data that can be used to predict product shelf lives at different temperatures and pH values.

Purpose: To determine the effect of sufentanil on myocardial apoptosis in rats with myocardial ischemia-reperfusion injury (MIRI). Methods: Fifty Sprague Dawley rats were randomly assigned to five groups: sham, model, low-dose, moderate-dose, and high-dose. The groups, except sham, underwent ligation of the left anterior descending coronary artery to establish the MIRI model. The low, moderate, and high-dose groups received intraperitoneal injections of sufentanil at different concentrations. Cardiac function, serum LDH, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) content were evaluated. The mRNA expression levels of apoptosis genes and protein levels of p38 and p-p38 were assessed in myocardial tissues using various methods while apoptosis was assessed by TUNEL assay. Results: Compared to sham group, the model group exhibited significant decrease in fractional shortening (FS) and ejection fraction (EF), increase in CK activity, LDH, and MDA contents, lower SOD activity. Model group also showed increase in mRNA levels of B-cell lymphoma-2 (Bcl-2) and caspase-3, higher apoptosis, significant increase in protein levels of p38 and p-p38, and higher level of myocardial apoptosis (p < 0.05). High-dose group demonstrated significant increase in FS and EF, decrease in LDH content and CK activity, lower MDA content, higher SOD activity, decrease in mRNA levels of Bcl-2 and caspase-3, lower apoptosis, decrease in protein levels of p38 and p-p38, and lower level of myocardial apoptosis (p < 0.05), when compared with model group. Conclusion: High-dose sufentanil reduces myocardial apoptosis and improves cardiac function, and thus can potentially be developed as a cardioprotective agent. [ABSTRACT FROM AUTHOR]

The potential of Phanerochaete chrysosporium in ethanol fermentation was evaluated. During the initial submerged cultivation, 1.76 g/L ethanol was obtained using glucose as substrate. After mutation, the ethanol concentration of an alcohol dehydrogenase (ADH) mutant reached 5.02 g/L. Both base transition and nine-base frame shift mutation occurred in the ADH gene of the mutant, changing the secondary and tertiary structures of ADH, as well as increasing the ADH activity during cultivation. Moreover, P. chrysosporium converted corn stalk to ethanol by a one-step process. After statistical optimizations, 0.26 g/g•substrate of ethanol yield was obtained on day 10. During the fermentation, the maximum lignin peroxidase, Mn-dependent peroxidase, and cellulase activities were 29.0 U/L, 256.5 U/L, and 40 U/mL, respectively, thus explaining why the fungus directly ferments corn stalk to ethanol. This study is the first report of the conversion of corn stalk without pretreatment to ethanol using a white-rot fungus.

Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3′-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3′-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3′-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3′-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3′-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3′-SL.

Mortierella alpina (M. alpina) is well known for arachidonic acid (ARA) production. However, low efficiency and unstableness are long existed problems for industrial production of ARA by M. alpina due to the lack of online regulations. The aim of the present work is to develop an online-regulation strategy for efficient and stable ARA production in industry. The strategy was developed in 50 L fermenters and then applied in a 200 m3 fermenter. Results indicated that yeast extract (YE) highly increased cell growth in shake flask, it was then used in bioreactor fermentation by various feeding strategies. Feeding YE to control respiratory quotient (RQ) at 1.1 during 0–48 h and at 1.5 during 48–160 h, dry cell weight, and ARA titer reached 53.1 and 11.49 g/L in 50 L fermenter, which were increased by 79.4 and 36.9% as compared to that without YE feeding, respectively. Then, the online RQ-feedback strategy was applied in 200 m3 bioreactor fermentation and an average ARA titer of 16.82 g/L was obtained from 12 batches, which was 41.0% higher than the control batches. This is the first report on successful application of online RQ-feedback control of YE in ARA production, especially in an industrial scale of 200 m3 fermentation. It could be applied to other industrial production of microbial oil by oleaginous microorganisms.

The enzymatic hydrolysis of food waste by commercially available enzymes and the subsequent ethanol fermentation of the hydrolysates by Saccharomyces cerecisiae H058 were studied in this work. The optimum batch enzymatic conditions were found to be saccharification pH of 4.5, temperature of 55!, glucoamylase concentration of 120 u/g, α-amylase concentration of 10 u/g, solid-liquid ratio of 1: 0.75 (w/w). Fed batch hydrolysis process was started with a solid-liquid ratio of 1: 1 (w/w), with solid food waste added at time lapse of 2 h to get a final solid-liquid ratio of 1: 0.5 (w/w). After 4 h of reaction, the reducing sugar concentration reached 194.43 g/L with a enzymatic digestibility of 93.12%. Further fermentation of the batch and fed batch enzymatic hydrolysates, which contained reducing sugar concentration of 131.41 and 194.43 g/L respectively, was performed using Saccharomyces cerevisiae H058, 62.93 and 90.72 g/L ethanol was obtained within 48 h.

Arachidonic acid (AA), an essential fatty acid in human body, fermented by Mortierella alpina I49-N18 was investigated in a shake-flask, and a 50-ton fermentor. In order to optimize the culture conditions, the effects of temperature, initial pH, culture time, carbon and nitrogen sources were studied. Furthermore, the way of adding sugar during fermentation was evaluated in a 50-ton fermentor. Under the optimum culture conditions, arachidonic acid produced in shake-flask and 50-ton fermentor was 4.55 and 5.11 g/L media, respectively. It was shown that the highest percentage of AA in lipids in shake-flask and 50-ton fermentor reached 70.20 and 53.01 %, respectively. Gas chromatography/mass spectrometry tests showed that the oil contained 80 % of polyunsaturated fatty acids such as arachidonic acid, γ-linolenic acid, and linoleic acid.