Genome-wide identification and characterization of members of the LEA gene family in Panax notoginseng and their transcriptional responses to dehydration of recalcitrant seeds.

BACKGROUND: Late embryogenesis abundant (LEA) proteins play an important role in dehydration process of seed maturation. The seeds of Panax notoginseng (Burkill) F. H. Chen are typically characterized with the recalcitrance and are highly sensitive to dehydration. However, it is not very well known about the role of LEA proteins in response to dehydration stress in P. notoginseng seeds. We will perform a genome-wide analysis of the LEA gene family and their transcriptional responses to dehydration stress in recalcitrant P. notoginseng seeds. RESULTS: In this study, 61 LEA genes were identified from the P. notoginseng genome, and they were renamed as PnoLEA. The PnoLEA genes were classified into seven subfamilies based on the phylogenetic relationships, gene structure and conserved domains. The PnoLEA genes family showed relatively few introns and was highly conserved. Unexpectedly, the LEA_6 subfamily was not found, and the LEA_2 subfamily contained 46 (75.4%) members. Within 19 pairs of fragment duplication events, among them 17 pairs were LEA_2 subfamily. In addition, the expression of the PnoLEA genes was obviously induced under dehydration stress, but the germination rate of P. notoginseng seeds decreased as the dehydration time prolonged. CONCLUSIONS: We found that the lack of the LEA_6 subfamily, the expansion of the LEA_2 subfamily and low transcriptional levels of most PnoLEA genes might be implicated in the recalcitrant formation of P. notoginseng seeds. LEA proteins are essential in the response to dehydration stress in recalcitrant seeds, but the protective effect of LEA protein is not efficient. These results could improve our understanding of the function of LEA proteins in the response of dehydration stress and their contributions to the formation of seed recalcitrance.

Exogenous gibberellic acid shortening after-ripening process and promoting seed germination in a medicinal plant Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F.H. Chen is an essential plant in the family of Araliaceae. Its seeds are classified as a type of morphophysiological dormancy (MPD), and are characterized by recalcitrance during the after-ripening process. However, it is not clear about the molecular mechanism on the after-ripening in recalcitrant seeds. RESULTS: In this study, exogenous supply of gibberellic acid (GA3) with different concentrations shortened after-ripening process and promoted the germination of P. notoginseng seeds. Among the identified plant hormone metabolites, exogenous GA3 results in an increased level of endogenous hormone GA3 through permeation. A total of 2971 and 9827 differentially expressed genes (DEGs) were identified in response to 50 mg L-1 GA3 (LG) and 500 mg L-1 GA3 (HG) treatment, respectively, and the plant hormone signal and related metabolic pathways regulated by GA3 was significantly enriched. Weighted gene co-expression network analysis (WGCNA) revealed that GA3 treatment enhances GA biosynthesis and accumulation, while inhibiting the gene expression related to ABA signal transduction. This effect was associated with higher expression of crucial seed embryo development and cell wall loosening genes, Leafy Contyledon1 (LEC1), Late Embryogenesis Abundant (LEA), expansins (EXP) and Pectinesterase (PME). CONCLUSIONS: Exogenous GA3 application promotes germination and shorts the after-ripening process of P. notoginseng seeds by increasing GA3 contents through permeation. Furthermore, the altered ratio of GA and ABA contributes to the development of the embryo, breaks the mechanical constraints of the seed coat and promotes the protrusion of the radicle in recalcitrant P. notoginseng seeds. These findings improve our knowledge of the contribution of GA to regulating the dormancy of MPD seeds during the after-ripening process, and provide new theoretical guidance for the application of recalcitrant seeds in agricultural production and storage.

The NMR studies of CMP inhibition of polysialylation.

The overexpression of polysialic acid (polySia) on neural cell adhesion molecules (NCAM) promotes hypersialylation, and thus benefits cancer cell migration and invasion. It has been proposed that the binding between the polysialyltransferase domain (PSTD) and CMP-Sia needs to be inhibited in order to block the effects of hypersialylation. In this study, CMP was confirmed to be a competitive inhibitor of polysialyltransferases (polySTs) in the presence of CMP-Sia and triSia (oligosialic acid trimer) based on the interactional features between molecules. The further NMR analysis suggested that polysialylation could be partially inhibited when CMP-Sia and polySia co-exist in solution. In addition, an unexpecting finding is that CMP-Sia plays a role in reducing the gathering extent of polySia chains on the PSTD, and may benefit for the inhibition of polysialylation. The findings in this study may provide new insight into the optimal design of the drug and inhibitor for cancer treatment.

The Discovery, Enzymatic Characterization and Functional Analysis of a Newly Isolated Chitinase from Marine-Derived Fungus Aspergillus fumigatus df347.

In order to discover a broad-specificity and high stability chitinase, a marine fungus, Aspergillus fumigatus df347, was identified in the sediments of mangrove wetlands in Qinzhou Bay, China. The chitinase gene (AfChi28) from A. fumigatus df347 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme AfChi28 was purified and characterized. AfChi28 is an acido-halotolerant- and temperature-resistant bifunctional enzyme with both endo- and exo-cleavage functions. Its enzymatic products are mainly GlcNAc, (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4. Na+, Mg2+, K+, Ca2+ and Tris at a concentration of 50 mM had a strong stimulatory effect on AfChi28. The crude enzyme and pure enzyme exhibited the highest specific activity of 0.737 mU/mg and 52.414 mU/mg towards colloidal chitin. The DxDxE motif at the end of strand ß5 and with Glu154 as the catalytic residue was verified by the AlphaFold2 prediction and sequence alignment of homologous proteins. Moreover, the results of molecular docking showed that molecular modeling of chitohexaose was shown to bind to AfChi28 in subsites -4 to +2 in the deep groove substrate-binding pocket. This study demonstrates that AfChi28 is a promising chitinase for the preparation of desirable chitin oligosaccharides, and provides a foundation for elucidating the catalytic mechanism of chitinases from marine fungi.

Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site-directed mutagenesis.

BACKGROUND: Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis. RESULTS: Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s-1 and 568.93 L mmol-1 s-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol-1 s-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference. CONCLUSIONS: The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.

Disaster Nursing Development in China and Other Countries: A Bibliometric Study.

PURPOSE: China is a country with frequent disasters, and nurses play indispensable roles in the disaster process. The Chinese disaster nursing specialty developed with several deficiencies. This study aimed to identify the limitations in the development of disaster nursing in China and to provide a reference for the future by comparing relevant studies between China and other countries. DESIGN: A systematic literature review was conducted in English and Chinese databases to identify disaster nursing articles published from January 1, 2000, to December 31, 2016. METHODS: This study followed the systematic literature collection tactic and bibliometric method. Basic information such as country, number of publications, and discussed disaster types were described through frequency distributions. Article themes were extracted and divided into the four phases of the International Council of Nurses Framework of Disaster Nursing Competencies. FINDINGS: 1,384 articles were included in the analysis, containing 781 written in Chinese and 603 written in English (with 56 of them written by Chinese researchers). The number of Chinese disaster nursing articles and other publications increased sharply between 2007 and 2009 but dropped significantly afterwards, while the total number of articles in other countries fluctuated, with a general upward trend. Compared to other countries, there were fewer research methods used and less focus on disaster prevention and preparedness in China, an imbalanced focus on disaster types, and a lack of focus on prevention, preparedness, and recovery phases. CONCLUSIONS: In China, there is a lack of stable development of disaster nursing research, a lack of study types, and less focus on disaster prevention, preparedness, and recovery. Varied study methods and an increased focus on disaster prevention and preparedness are required in the future. CLINICAL RELEVANCE: This study analyzed the deficiencies in Chinese disaster nursing, which led to recommendations and proposed directions for future studies and a clinical focus in this field, in compliance with the United Nations guidelines for disaster management.

[Assessment of Influence Detective Position Variability on Precision of Near Infrared Models for Soluble Solid Content of Watermelon].

Non-destructive detection for soluble solids content (SSC) is important to improve watermelon's internal quality, which attracts more and more attention from consumers. In order to realize the precise detection for SSC of mini watermelon's whole surface by using Near-infrared (NIR) spectroscopy and reduce the influence of detective position variability on the accuracy of NIR prediction model for SSC, the diffused transmission spectra and soluble solids content were collected from three different detective positions of 'jingxiu' watermelon, including the equator, calyx and stem. The prediction models of single detective position and mixed three detective positions for SSC were established with Partial least square (PLS). Successive projections algorithm (SPA) and competitive adaptive reweighted sampling (CARS) were adopted to select effective variables of NIR spectroscopy for SSC of watermelon as well. The results showed that the prediction model of mixed three detective positions was better than the model of single detective position. Meanwhile, 42 characteristic variables of NIR spectroscopy selected with CARS were used to establish PLS prediction model for SSC. The prediction model was simplified significantly and the prediction accuracy for SSC was improved greatly. The correlation coefficient of prediction (RP) and root mean square error of prediction (RMSEP) by CARS-PLS were 0.892, 0.684 °Brix for the equator, 0.905, 0.621 °Brix for the calyx, 0.899, 0.721 °Brix for the stem, respectively. However, the prediction result of SPA-PLS established by 19 characteristic wavelength variables of NIR spectroscopy was bad for the equator, calyx and stem detective positions. The correlation coefficient of prediction (RP) is less than 0.752 and root mean square error of prediction (RMSEP) is relatively high. It was proposed that the PLS prediction model established by mixed three different detective positions with effective characteristic wavelength variables selected by CARS can improve the prediction accuracy for SSC. And the CARS-PLS prediction model can achieve fast and precise detection for SSC of mini watermelon's whole surface. The influence of detective position variability on the accuracy of NIR prediction model could be reduced simultaneously. This paper could provide theoretical basis for calibrating NIR prediction model for SSC of mini watermelon. It also could provide reference for developing the portable and non-destructive detection equipment for soluble solids content of mini watermelon's whole surface.

Phase diagram and electronic indication of high-temperature superconductivity at 65 K in single-layer FeSe films.

The recent discovery of possible high-temperature superconductivity in single-layer FeSe films has generated significant experimental and theoretical interest. In both the cuprate and the iron-based high-temperature superconductors, superconductivity is induced by doping charge carriers into the parent compound to suppress the antiferromagnetic state. It is therefore important to establish whether the superconductivity observed in the single-layer sheets of FeSe--the essential building blocks of the Fe-based superconductors--is realized by undergoing a similar transition. Here we report the phase diagram for an FeSe monolayer grown on a SrTiO3 substrate, by tuning the charge carrier concentration over a wide range through an extensive annealing procedure. We identify two distinct phases that compete during the annealing process: the electronic structure of the phase at low doping (N phase) bears a clear resemblance to the antiferromagnetic parent compound of the Fe-based superconductors, whereas the superconducting phase (S phase) emerges with the increase in doping and the suppression of the N phase. By optimizing the carrier concentration, we observe strong indications of superconductivity with a transition temperature of 65±5 K. The wide tunability of the system across different phases makes the FeSe monolayer ideal for investigating not only the physics of superconductivity, but also for studying novel quantum phenomena more generally.

Optimization of medium composition for cis,cis-muconic acid production by a Pseudomonas sp. mutant using statistical methods.

cis,cis-Muconic acid (CCMA) is used as a platform chemical for the production of several high-value compounds. For this article, an optimization strategy has been used to optimize medium composition for CCMA production from fairly cheap benzoate by Pseudomonas sp. 1167. The effect of different concentrations of medium components on CCMA production was studied. CCMA yields obtained from Plackett-Burman design (PBD) showed wide variation (3.95-5.87 g/L), and the first-order model indicated that (NH(4))(2)SO(4) (P < 0.01) and K(2)HPO(4) · 3H(2)O (P < 0.02) were the significant components for CCMA production. Then the optimization was performed by steepest ascent design (SAD) and central composite design (CCD), and a validation experiment was conducted to verify the predicted value. The optimal medium composition was: 12 g/L sodium benzoate, 2.5 g/L sodium succinate, 0.7932 g/L (NH(4))(2)SO(4), 1.5612 g/L K(2)HPO(4) · 3H(2)O, 1.2 g/L MgSO(4) · 7H(2)O, 0.4 g/L yeast extract, 0.08 g/L FeCl(3) · 6H(2)O, and 0.08 g/L ethylenediamine tetraacetic acid (EDTA). Under these conditions, a maximum of 7.18 g/L CCMA was produced per 12 g/L benzoate with a highly efficient process within 11 hr and a molecular conversion yield of 61%. Altogether, our results provide valuable insights into nutritional supplementation of CCMA production by using statistical methods, which may benefit a cost-competitive industrial fed-batch fermentation process using a cheap substrate.

New advances in protein engineering for industrial applications: Key takeaways.

Recent advancements in protein/enzyme engineering have enabled the production of a diverse array of high-value compounds in microbial systems with the potential for industrial applications. The goal of this review is to articulate some of the most recent protein engineering advances in bacteria, yeast, and other microbial systems to produce valuable substances. These high-value substances include α-farnesene, vitamin B12, fumaric acid, linalool, glucaric acid, carminic acid, mycosporine-like amino acids, patchoulol, orcinol glucoside, d-lactic acid, keratinase, α-glucanotransferases, ß-glucosidase, seleno-methylselenocysteine, fatty acids, high-efficiency ß-glucosidase enzymes, cellulase, ß-carotene, physcion, and glucoamylase. Additionally, recent advances in enzyme engineering for enhancing thermostability will be discussed. These findings have the potential to revolutionize various industries, including biotechnology, food, pharmaceuticals, and biofuels.

Current status and emerging frontiers in enzyme engineering: An industrial perspective.

Protein engineering mechanisms can be an efficient approach to enhance the biochemical properties of various biocatalysts. Immobilization of biocatalysts and the introduction of new-to-nature chemical reactivities are also possible through the same mechanism. Discovering new protocols that enhance the catalytic active protein that possesses novelty in terms of being stable, active, and, stereoselectivity with functions could be identified as essential areas in terms of concurrent bioorganic chemistry (synergistic relationship between organic chemistry and biochemistry in the context of enzyme engineering). However, with our current level of knowledge about protein folding and its correlation with protein conformation and activities, it is almost impossible to design proteins with specific biological and physical properties. Hence, contemporary protein engineering typically involves reprogramming existing enzymes by mutagenesis to generate new phenotypes with desired properties. These processes ensure that limitations of naturally occurring enzymes are not encountered. For example, researchers have engineered cellulases and hemicellulases to withstand harsh conditions encountered during biomass pretreatment, such as high temperatures and acidic environments. By enhancing the activity and robustness of these enzymes, biofuel production becomes more economically viable and environmentally sustainable. Recent trends in enzyme engineering have enabled the development of tailored biocatalysts for pharmaceutical applications. For instance, researchers have engineered enzymes such as cytochrome P450s and amine oxidases to catalyze challenging reactions involved in drug synthesis. In addition to conventional methods, there has been an increasing application of machine learning techniques to identify patterns in data. These patterns are then used to predict protein structures, enhance enzyme solubility, stability, and function, forecast substrate specificity, and assist in rational protein design. In this review, we discussed recent trends in enzyme engineering to optimize the biochemical properties of various biocatalysts. Using examples relevant to biotechnology in engineering enzymes, we try to expatiate the significance of enzyme engineering with how these methods could be applied to optimize the biochemical properties of a naturally occurring enzyme.

Exogenous abscisic acid prolongs the dormancy of recalcitrant seed of Panax notoginseng.

The seeds of Panax notoginseng (Burk.) F. H. Chen are typically characterized by their recalcitrance and after-ripening process and exhibit a high water content at harvest as well as a high susceptibility to dehydration. Storage difficulty and the low germination of recalcitrant seeds of P. notoginseng are known to cause an obstacle to agricultural production. In this study, the ratio of embryo to endosperm (Em/En) in abscisic acid (ABA) treatments (1 mg·l-1 and 10 mg·l-1, LA and HA) was 53.64% and 52.34%, respectively, which were lower than those in control check (CK) (61.98%) at 30 days of the after-ripening process (DAR). A total of 83.67% of seeds germinated in the CK, 49% of seeds germinated in the LA treatment, and 37.33% of seeds germinated in the HA treatment at 60 DAR. The ABA, gibberellin (GA), and auxin (IAA) levels were increased in the HA treatment at 0 DAR, while the jasmonic acid (JA) levels were decreased. ABA, IAA, and JA were increased, but GA was decreased with HA treatment at 30 DAR. A total of 4,742, 16,531, and 890 differentially expressed genes (DEGs) were identified between the HA-treated and CK groups, respectively, along with obvious enrichment in the ABA-regulated plant hormone pathway and the mitogen-activated protein kinase (MAPK) signaling pathway. The expression of pyracbactin resistance-like (PYL) and SNF1-related protein kinase subfamily 2 (SnRK2s) increased in the ABA-treated groups, whereas the expression of type 2C protein phosphatase (PP2C) decreased, both of which are related to the ABA signaling pathway. As a result of the changes in expression of these genes, increased ABA signaling and suppressed GA signaling could inhibit the growth of the embryo and the expansion of developmental space. Furthermore, our results demonstrated that MAPK signaling cascades might be involved in the amplification of hormone signaling. Meanwhile, our study uncovered that the exogenous hormone ABA could inhibit embryonic development, promote dormancy, and delay germination in recalcitrant seeds. These findings reveal the critical role of ABA in regulating the dormancy of recalcitrant seeds, and thereby provide a new insight into recalcitrant seeds in agricultural production and storage.

Ferroptosis is a new therapeutic target for spinal cord injury.

Spinal cord injury is a serious traumatic disease. As Ferroptosis has been increasingly studied in recent years, it has been found to be closely related to the pathophysiological processes of spinal cord injury. Iron overload, reactive oxygen species accumulation, lipid peroxidation and glutamate accumulation associated with Ferroptosis are all present in spinal cord injury, and thus Ferroptosis is thought to be involved in the pathological processes secondary to spinal cord injury. This article highlights the relationship between Ferroptosis and spinal cord injury, lists substances that improve spinal cord injury by inhibiting Ferroptosis, and concludes with a discussion of the problems that may be encountered in the clinical translation of Ferroptosis inhibitors as a means of enabling their faster use in clinical treatment.

Effects of basic application of chlorocholine chloride combined with nitrogen fertilizer on nitrogen use of summer maize in North China Plain.

To clarify the effects of combined applications of chlorocholine chloride (CCC) and nitrogen fertilizer (CN) on nitrogen metabolism and nitrogen use efficiency of summer maize, we conducted a field experiment in Xinxiang experimental station of Chinese Academy of Agricultural Sciences in 2018 and 2019, with four nitrogen application rates (0, 62.5, 125 and 187.5 kg·hm-2), and two maize varieties of Jingnongke 728 (JNK728) and Zhongdan 909 (ZD909). The results showed that across the two years CN-CCC increased maize yield by 7.7% and 5.0% under the nitrogen application rates of 62.5 kg·hm-2 and 125 kg·hm-2, respectively. CN-CCC increased the contents of nitrate reductase, glutamine synthetase, glutamate synthetase and soluble protein, and finally promoted nitrogen metabolism. Under the low and middle nitrogen application conditions (62.5 kg·hm-2 and 125 kg·hm-2), plant nitrogen content of JNK728 and ZD909 increased by 17.6% and 30.3%, grain nitrogen content increased by 10.3% and 17.4%, nitrogen partial productivity, agronomic efficiency of applied nitrogen, recovery efficiency of applied nitrogen, nitrogen use efficiency increased by 10.0%, 15.7%, 23.3%, 24.8% and 5.7%, 15.0%, 49.9%, 71.7%, respectively. In conclusion, appropriate basic application of CN-CCC could enhance nitrogen metabolism, increase nitrogen use efficiency and grain yield of summer maize. Our results showed that CCC combined basic nitrogen application of 125 kg·hm-2 had the best effect.

Influence of arm position change from adduction to abduction on intracavitary electrocardiogram.

PURPOSE: The aim of this study is to evaluate the influence of arm movements from adduction to abduction on intracavitary electrocardiogram and the position of a catheter tip. METHODS: Overall, 192 peripherally inserted central catheter lines were placed under intracavitary electrocardiogram guidance and 188 of them were enrolled in the study. The catheter was first placed at a time point corresponding to the peak P wave with the arm in adduction. The arm was then abducted to 90° without changing catheter insertion length. During the procedure, basal electrocardiogram, intracavitary electrocardiogram, and radiographs with the arm in adduction and abduction were recorded. Amplitude wave changes and catheter movements were measured on electrocardiogram records and radiographs, respectively. RESULTS: In 188 cases, the P wave displayed typical changes, and 97.8% (184/188) catheters were successfully placed correctly. At the peak P wave, the amplitude of the peak P wave was 8.64 times greater than that of the basal P wave, and the P/R ratio was 0.61. When the arm was abducted to 90°, the amplitude of the P wave dropped to 57% of its peak, P/R decreased from 0.61 to 0.34, and the catheter tip moved cephalad 1.00 and 0.77 vertebral body units in male and female patients, respectively. CONCLUSION: Peripherally inserted central catheter moves toward the heart when the arm position changes from abduction to adduction. Peripherally inserted central catheter tip placement at the peak P wave with patient's arm in adduction is accurate and can prevent the catheter from advancing too low. R wave can function as a reference for observing P wave changes during peripherally inserted central catheter placement.

Physicochemical and antibacterial properties of fabricated ovalbumin-carvacrol gel nanoparticles.

The applications of carvacrol are limited due to its poor stability and water solubility, and high volatility; however, ovalbumin can be used to encapsulate hydrophobic molecules, improve their aqueous solubility, and reduce their volatility. In this study, we fabricated ovalbumin-carvacrol nanoparticles (OCGns) under different pH (2, 5, 7, and 9) conditions using a gel embedding method and investigated their physicochemical and antibacterial properties. Rheological experiments revealed that the G' of ovalbumin gels (OGs) prepared under different pH conditions were OG-2 > OG-7 > OG-9 > OG-5. Carvacrol addition reduced the tight structure of ovalbumin and carvacrol under pH 5 and 7 conditions, with hardness first decreasing and then increasing, but increasing under pH 2 and 9 conditions. Fluorescence and infrared spectroscopy indicated complex formation, with carvacrol increasing the average diameter of nanoparticles prepared at pH 2, 5, 7, and 9. Encapsulation reached 89.34 and 91.86% at pH 2 and 9, respectively; however, inhibition experiments revealed that the minimum inhibitory concentration of OCGn-2 against Gram-positive Bacillus cereus and Salmonella (0.08 and 0.16 mg mL-1, respectively) was lower than that of OCGn-9 (both 0.28 mg mL-1). Moreover, OCGn-2 possessed a better dense gel structure and a higher stability, encapsulation rate, and antibacterial activity, suggesting that pH affects gel microstructure and thus the encapsulation efficiency and bacteriostatic properties of the prepared nanoparticles. These results contribute to our knowledge of the design and fabrication of polymeric nanoparticle delivery systems for bioactive compounds with beneficial properties.

Influence of Calcium Ions on the Thermal Characteristics of α-amylase from Thermophilic Anoxybacillus sp. GXS-BL.

BACKGROUND: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotechnology. OBJECTIVE: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. METHODS: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. RESULTS: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α- helical contents increasing, and the transition from α-helices to ß-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. CONCLUSION: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

Simulated Protein Thermal Detection (SPTD) for Enzyme Thermostability Study and an Application Example for Pullulanase from Bacillus deramificans.

BACKGROUND: The relationship between protein structure and its bioactivity is one of the fundamental problems for protein engineering and pharmaceutical design. METHOD: A new method, called SPTD (Simulated Protein Thermal Detection), was proposed for studying and improving the thermal stability of enzymes. The method was based on the evidence observed by conducting the MD (Molecular Dynamics) simulation for all the atoms of an enzyme vibrating from the velocity at a room temperature (e.g., 25°C) to the desired working temperature (e.g., 65°C). According to the recorded MD trajectories and the coordinate deviations of the constituent residues under the two different temperatures, some new strategies have been found that are useful for both drug delivery and starch industry. CONCLUSION: The SPTD technique presented in this paper may become a very useful tool for pharmaceutical design and protein engineering.

A comparative study for the organic byproducts from hydrothermal carbonizations of sugarcane bagasse and its bio-refined components cellulose and lignin.

Sugarcane bagasse was refined into cellulose, hemicellulose, and lignin using an ethanol-based organosolv technique. The hydrothermal carbonization (HTC) reactions were applied for bagasse and its two components cellulose and lignin. Based on GC-MS analysis, 32 (13+19) organic byproducts were derived from cellulose and lignin, more than the 22 byproducts from bagasse. Particularly, more valuable catechol products were obtained from lignin with 56.8% share in the total GC-MS integral area, much higher than the 2.263% share in the GC-MS integral areas of bagasse. The organic byproducts from lignin make up more than half of the total mass of lignin, indicating that lignin is a chemical treasure storage. In general, bio-refinery and HTC are two effective techniques for the valorization of bagasse and other biomass materials from agriculture and forest industry. HTC could convert the inferior biomass to superior biofuel with higher energy quantity of combustion, at the same time many valuable organic byproducts are produced. Bio-refinery could promote the HTC reaction of biomass more effective. With the help of bio-refinery and HTC, bagasse and other biomass materials are not only the sustainable energy resource, but also the renewable and environment friendly chemical materials, the best alternatives for petroleum, coal and natural gas.

Fabrication and Characterization of Graphene Microcrystal Prepared from Lignin Refined from Sugarcane Bagasse.

Graphene microcrystal (GMC) is a type of glassy carbon fabricated from lignin, in which the microcrystals of graphene are chemically bonded by sp³ carbon atoms, forming a glass-like microcrystal structure. The lignin is refined from sugarcane bagasse using an ethanol-based organosolv technique which is used for the fabrication of GMC by two technical schemes: The pyrolysis reaction of lignin in a tubular furnace at atmospheric pressure; and the hydrothermal carbonization (HTC) of lignin at lower temperature, followed by pyrolysis at higher temperature. The existence of graphene nanofragments in GMC is proven by Raman spectra and XRD patterns; the ratio of sp² carbon atoms to sp³ carbon atoms is demonstrated by XPS spectra; and the microcrystal structure is observed in the high-resolution transmission electron microscope (HRTEM) images. Temperature and pressure have an important impact on the quality of GMC samples. With the elevation of temperature, the fraction of carbon increases, while the fraction of oxygen decreases, and the ratio of sp² to sp³ carbon atoms increases. In contrast to the pyrolysis techniques, the HTC technique needs lower temperatures because of the high vapor pressure of water. In general, with the help of biorefinery, the biomass material, lignin, is found to be qualified and sustainable material for the manufacture of GMC. Lignin acts as a renewable substitute for the traditional raw materials of glassy carbon, copolymer resins of phenol formaldehyde, and furfuryl alcohol-phenol.