Glutaraldehyde-crosslinked Rhizopus oryzae whole cells show improved catalytic performance in alkene epoxidation.

BACKGROUND: Existing methods for alkene epoxidation are based on lipase-catalysed perhydrolysis. However, the inactivation of the expensive lipase enzyme is problematic for enzymatic epoxidation at large scales due to the use of hydrogen peroxide and peracids at high concentrations in the reaction. The immobilisation of whole cells appears to be a promising approach to alleviate this problem. RESULTS: A green oxidation system containing hydrogen peroxide, Na3C6H5O7, an acyl donor, and glutaraldehyde (GA)-crosslinked cells of Rhizopus oryzae was developed for the epoxidation of alkenes. GA-crosslinked cells of Rhizopus oryzae were adopted as a biocatalyst into the epoxidation system. A variety of alkenes were oxidised with this system, with a 56-95% analytical yield of the corresponding epoxides. The catalytic performance of the crosslinked treated cells was substantially improved compared to that of the untreated cells and the initial reaction rate increased from 126.71 to 234.72 mmol/L/h, retaining 83% yields even after four batches of reactions. The addition of 3.5 mmol Na3C6H5O7 not only acts as an acid-trapping reagent to eliminate the negative effect of the carboxylic acid on the alkene oxide but also forms a saturated salt solution with the aqueous phase, affecting the concentration of H2O2 in the three phases and thus the epoxidation reaction. Organic solvents with a logP value > 0.68 were good at producing hydroxy peracids; however, this method is only suitable for oxidation in a two-liquid phase. CONCLUSIONS: Compared with other lipase biocatalysts, the GA-crosslinked whole-cell biocatalyst is inexpensive, readily available, and highly stable. Therefore, it can be considered promising for industrial applications.

Improving efficiency and reducing enzyme inactivation during lipase-mediated epoxidation of α-pinene in a double-phase reaction system.

Chemoenzymatic epoxidation of olefin mediated by lipase is a green and environmentally friendly alternative process. However, the mass transfer barrier and lipase deactivation caused by the traditional organic-water biphasic reaction system have always been the focus of researchers' attention. To overcome these issues, we investigated the effects of reaction temperature and two important substrates (H2O2 and acyl donor) on the epoxidation reaction and interfacial mass transfer. As a result, we determined the optimal reaction conditions: a temperature of 30 °C, 30 wt-% H2O2 as the oxygen source, and 1 M lauric acid as the oxygen carrier. Additionally, by simulating the conditions of shaking flask reactions, we designed a batch reactor and added a metal mesh to effectively block the direct contact between high-concentration hydrogen peroxide and the enzyme. Under these optimal conditions, the epoxidation reaction was carried out for 5 h, and the product yield reached a maximum of 93.2%. Furthermore, after seven repetitive experiments, the lipase still maintained a relative activity of 51.2%.

The trade-offs and synergies of the ecological-production-living functions of grassland in the Qilian mountains by ecological priority.

Grassland degradation has caused increasingly prominent conflict between ecological environment conservation and socioeconomic development in the Qilian Mountains, China. How to effectively trade-offs and synergies to ecological and socioeconomic is essential to achieving the sustainable development of the grassland ecosystem. However, few studies have addressed the trade-offs and synergies of grassland ecosystem services in terms of coupling the natural ecosystem and the socioeconomic system. Therefore, we constructed an index of the analyzed trade-offs and synergies of grassland ecosystem services from the perspective of "ecological-production-living" functions (EPLFs) and analyzed the spatial-temporal characteristics of grassland EPLF trade-off and synergy relationships based on the data from the implementation of three conservation policies in the Qilian Mountains from 2003 to 2020. The results showed evident spatial and temporal differentiation of the grassland EPLFs. The ecological function was consistent with the production function, trending upward initially and then decreasing. The living function showed a trend of continuous increase. The spatial pattern of grassland EPLFs showed that the northwest and southeast were more active than the middle of the Qilian Mountains, and the regional gradient difference was apparent. The trade-off and synergy relationships of grassland EPLFs have obvious spatial correlations as well; spatial differences were evident under different conservation policies. With national park construction, the synergistic relationship gradually weakened and the trade-off relationship gradually strengthened. These results suggest that the policy of ecological priority increased trade-offs and reduced synergies among EPLFs was not conducive to coupling and coordinating grassland EPLFs for development in the Qilian Mountains. Our study also demonstrates that maintaining moderate grassland grazing pressure and the appropriate number of herdsmen is crucial to sustainably develop the grassland ecosystem in the Qilian Mountains, and further research into coupling mechanisms for grassland EPLFs is needed to reduce trade-offs and increase synergies with grassland ecosystem services.

Pyrene derivative-functionalized mesoporous silica-Cu2+ hybrid ensemble for fluorescence "turn-on" detection of H2S and logic gate application in aqueous media.

The ensemble system PyH-SBA-15-Cu2+ was obtained via coordination interaction of pyrene derivative-functionalized mesoporous SBA-15 and Cu2+, and applied for the selective and sensitive detection of H2S over pH 6.0-12.0 in aqueous media. The sensing strategy was designed on the basis of the H2S-induced dissolution of Cu2+ from PyH-SBA-15-Cu2+. Cu2+ has good binding affinity to N atoms in PyH-SBA-15; therefore, the organic-inorganic hybrid ensemble PyH-SBA-15-Cu2+ was formed, which is nonfluorescent in aqueous solution because of the Cu2+-promoted emission quenching of PyH-SBA-15. The addition of H2S induces the dissolution of PyH-SBA-15-Cu2+ by the formation of stable CuS, thereby producing fluorescence revival of PyH-SBA-15. The correlative "turn-on" fluorescence signals of this ensemble system are linearly proportional to [H2S] in the concentration region of 0-1.0 × 10-4 M, showing a low detection limit of 3.7 × 10-7 M. Other common anions do not induce distinct fluorescence changes. When using the fluorescence intensity signal changes of PyH-SBA-15 as outputs and Cu2+ and S2- as inputs, PyH-SBA-15 can act as an XNOR logic gate.

Preparation of PVDF/CdS/Bi2WO6/ZnO hybrid membrane with enhanced visible-light photocatalytic activity for degrading nitrite in water.

In this work, a visible light-driven ternary heterojunction photocatalyst, CdS/Bi2WO6/ZnO, was synthesized using hydrothermal, ultrasonic dispersion, and deposition precipitation methods. The results show that photocatalysts with flower-like heterostructures were obtained, which could efficiently separate electron-hole pairs, and the photocatalytic activity was thereby significantly enhanced. Furthermore, CdS/Bi2WO6/ZnO and polyvinylidene fluoride (PVDF) were used to fabricate hybrid membranes via a phase-conversion method. The samples were characterized using SEM, TEM, EDX, XRD, DRS, XPS, PL, and N2 adsorption-desorption isotherms, and the transient photocurrent response. The photocatalytic activity of the hybrid membrane was evaluated, and 92.58% of the nitrite was converted into non-toxic substances within 4 h under simulated sunlight irradiation. This result indicated that the photocatalyst exhibited a good photocatalytic activity after immobilization. The possible mechanism was elucidated by studying the product during the photocatalytic degradation, and the effects of different pH values, electron scavengers, and hole scavengers on the photocatalytic performance were further investigated.

Biotransformation of the azo dye reactive orange 16 by Aspergillus flavus A5P1: Performance, genetic background, pathway, and mechanism.

This study reports the strain Aspergillus flavus A5P1 (A5P1), which is with the capable of degrading the azo dye reactive orange 16 (RO16). The mechanism of RO16 degradation by A5P1 was elucidated through genomic analysis, enzymatic analysis, degradation pathway analysis and oxidative stress analysis. Strain A5P1 exhibited aerobic degradation of RO16, with optimal degradation at an initial pH of 3.0. Genomic analysis indicates that strain A5P1 possesses the potential for acid tolerance and degradation of azo dye. Enzymatic analysis, combined with degradation product analysis, demonstrated that extracellular laccase, intracellular lignin peroxidase, and intracellular quinone reductase were likely key enzymes in the RO16 degradation process. Oxidative stress analysis revealed that cell stress responses may participate in the RO16 biotransformation process. The results indicated that the biotransformation of RO16 may involves biological processes such as transmembrane transport of RO16, cometabolism of the strain with RO16, and cell stress responses. These findings shed light on the biodegradation of RO16 by A5P1, indicating A5P1's potential for environmental remediation.

Substrate Characterization for Hydrolysis of Multiple Types of Aromatic Esters by Promiscuous Aminopeptidases.

Synthetic aromatic esters, widely employed in agriculture, food, and chemical industries, have become emerging environmental pollutants due to their strong hydrophobicity and poor bioavailability. This study attempted to address this issue by extracellularly expressing the promiscuous aminopeptidase (Aps) from Pseudomonas aeruginosa GF31 in B. subtilis, achieving an impressive enzyme activity of 13.7 U/mg. Notably, we have demonstrated, for the first time, the Aps-mediated degradation of diverse aromatic esters, including but not limited to pyrethroids, phthalates, and parabens. A biochemical characterization of Aps reveals its esterase properties and a broader spectrum of substrate profiles. The degradation rates of p-nitrobenzene esters (p-NB) with different side chain structures vary under the action of Aps, showing a preference for substrates with relatively longer alkyl side chains. The structure-dependent degradability aligns well with the binding energies between Aps and p-NB. Molecular docking and enzyme-substrate interaction elucidate that hydrogen bonding, hydrophobic interactions, and π-π stacking collectively stabilize the enzyme-substrate conformation, promoting substrate hydrolysis. These findings provide new insights into the enzymatic degradation of aromatic ester pollutants, laying a foundation for the further development and modification of promiscuous enzymes.

Complete mitochondrial genome of Angelica dahurica and its implications on evolutionary analysis of complex mitochondrial genome architecture in Apiaceae.

Angelica dahurica is a kind of Chinese traditional herbs with economic and ornament value, widely distributed in China. Despite its significance, there have been limited comprehensive investigations on the genome of A. dahurica, particularly regarding mitochondrial genomes. To investigate the conversion between mitochondrial genome and chloroplast genome, a complete and circular mitochondrial genome was assembled using Oxford Nanopore Technologies (ONT) long reads. The mitochondrial genome of A. dahurica had a length of 228,315 base pairs (bp) with 45.06% GC content. The mitochondrial genome encodes 56 genes, including 34 protein-coding genes, 19 tRNA genes and 3 rRNA genes. Moreover, we discovered that 9 homologous large fragments between chloroplast genome and mitochondrial genome based on sequence similarity. This is the first report for A. dahurica mitochondrial genome, which could provide an insight for communication between plastid genome, and also give a reference genome for medicinal plants within the Angelica family.

Enzymatic synthesis of oleic acid-based epoxy monomer for the production of value added polymers.

An oleic acid-based epoxy monomer was synthesized by reacting 2-hydroxyethyl acrylate with epoxy stearic acid and an immobilized lipase. NMR, electrospray ionization mass spectrometry and gel permeation chromatography were used to characterize the intermediates and products. 2-(Acryloyloxy) ethyl epoxy stearate was synthesized with a yield of 87 % w/w. After free radical polymerization, epoxy stearic acid-grafted epoxy polymer with molecular weight of 15,150 g/mol was obtained; the final yield was 81 % w/w.

Efficient antibacterial and dye adsorption by novel fish scale silver biochar composite gel.

A silver-loaded carbon-chitosan-polyvinyl alcohol gel (C/CTS/PVA) was designed for suppressing microbial growth and dye adsorption. The antibacterial test results showed that C/CTS/PVA gel had a good antibacterial ability against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The inhibition rate in water was 100 %, and the antibacterial rate remained above 95 % within 35 days after preparation. The tight spatial structure provided by the adhesive effect of PVA and CTS effectively prevented water loss and enhanced the stability of the gel. The adsorption curves of the gel were fitted by establishing the pseudo-first order and pseudo-second order kinetic models. The adsorption curves were more consistent with the pseudo-second-order kinetic model. The best adsorption effect for Malachite green was 128.12 mg/g. C/CTS/PVA gel had a remarkable adsorption effect on Malachite green, Congo red, Methyl orange, and Methylene blue. In general, C/CTS/PVA gels have great potential for the treatment of sewage in the future.

[Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering].

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

Rapid and simple colorimetric assay for screening angiotensin I-converting enzyme inhibitors.

CONTEXT: Angiotensin-converting enzyme (ACE) is one of the main regulators of blood pressure through its action on the renin-angiotensin system. ACE inhibitory peptides from natural materials inhibit ACE activity and have considerable importance as antihypertensive agents. OBJECTIVE: A new chromogenic reaction method for determining hippuric acid (HA) and angiotensin I-converting enzyme (ACE) inhibitor activity was developed. MATERIALS AND METHODS: This method is based on the reaction of HA with p-dimethylaminobenzaldehyde in the presence of quinoline, acetate, and acetic anhydride. The red-orange formation product in the reaction has a stable absorbance in the visible region and it was determined at 478 nm. The assay conditions were optimized and by using an ACE concentration of 12 mU/mL in enzymatic reaction, the method was applied to monitor the IC(50) values (the concentration of inhibitor required to inhibit 50% of the ACE activity) for captopril and Saurida elongata (Synodontidae) muscle protein hydrolyzate. RESULTS: With the proposed method, IC(50) values for captopril and Saurida elongata muscle protein hydrolyzate were determined as 0.0123 µM and 0.1648 mg/mL, respectively. Those results correspond to the IC(50) values of 0.0109 µM and 0.1820 mg/mL obtained by high-performance liquid chromatography (HPLC) method. DISCUSSION AND CONCLUSION: The proposed method is rapid, accurate, reproducible and convenient, and suitable for screening ACE inhibitor peptides from food materials while it does not require HA extraction from the components of the ACE activity assay reaction.

[Research on partial least squares for determination of impurities in the presence of high concentration of matrix by ICP-AES].

A method for detecting trace impurities in high concentration matrix by ICP-AES based on partial least squares (PLS) was established. The research showed that PLS could effectively correct the interference caused by high level of matrix concentration error and could withstand higher concentrations of matrix than multicomponent spectral fitting (MSF). When the mass ratios of matrix to impurities were from 1 000 : 1 to 20 000 : 1, the recoveries of standard addition were between 95% and 105% by PLS. For the system in which interference effect has nonlinear correlation with the matrix concentrations, the prediction accuracy of normal PLS method was poor, but it can be improved greatly by using LIN-PPLS, which was based on matrix transformation of sample concentration. The contents of Co, Pb and Ga in stream sediment (GBW07312) were detected by MSF, PLS and LIN-PPLS respectively. The results showed that the prediction accuracy of LIN-PPLS was better than PLS, and the prediction accuracy of PLS was better than MSF.

A New Source of Diterpene Lactones From Andrographis paniculata (Burm. f.) Nees-Two Endophytic Fungi of Colletotrichum sp. With Antibacterial and Antioxidant Activities.

Endophytic fungi of medicinal plants are abundant, and their metabolites often have antioxidant, antibacterial, and antitumor effects and can produce secondary metabolites identical or similar to those of their hosts, which can mitigate the problem of insufficient supply of medicinal plants. In this study, we screened endophytic fungi for strains that produce the same diterpene lactones as Andrographis paniculata based on their biological activity. Firstly, the dominant group of endophytic fungi of Andrographis paniculata was screened and pathogenicity was studied using Koch's rule. Secondly, DPPH, ABTS, OH, PTIO radical scavenging, and FRAP assays were used to detect the antioxidant activity of the extracellular extracts of the strains, and total phenol and total flavonoid contents of the strains with high antioxidant capacity were determined. S. aureus, B. subtilis, E. coli, and P. aeruginosa were used to determine the antibacterial activity of the mycelial extracts of the strains. Finally, the secondary metabolites of the mycelial extracts of the strains were examined by high-performance liquid chromatography. The results showed that 32 strains of Andrographis paniculata were relatively isolated > 70% and non-pathogenic. Extracellular extracts of strains AP-1 and AP-4 showed vigorous antioxidant activity, and AP-4, AP-12, AP-47, and AP-48 showed antibacterial activity against four strains of bacteria. The HPLC results indicated that the mycelial extracts of AP-4 and AP-12 contained diterpene lactones. The two endophytic fungi were recognized as Colletotrichum sp. The study successfully obtained diterpene lactones from the endophytic fungus of Andrographis paniculata and confirmed the feasibility of using endophytic fungal strains to produce active substances consistent with the host. It was also useful for exploring endophytic fungi and medicinal plants. The relationship provides theoretical guidance.

Enzymatic synthesis of L-lactic acid from carbon dioxide and ethanol with an inherent cofactor regeneration cycle.

Efficient conversion of carbon dioxide is of great interests to today's endeavors in controlling greenhouse gas emission. A multienzyme catalytic system that uses carbon dioxide and ethanol to produce L-lactate was demonstrated in this work, thereby providing a novel reaction route to convert bio-based ethanol to an important building block for synthesis biodegradable polymers. The synthetic route has a unique internal cofactor regeneration cycle, eliminating the need of additional chemical or energy for cofactor regeneration. Lactate was successfully synthesized with 41% of ethanol converted in a batch reaction, while a turnover number of 2.2 day⁻¹ was reached for cofactor regeneration in a reaction with continuous feeding of ethanol. A kinetic model developed based on reaction kinetic parameters determined separately for each reaction step predicted well the reaction rates and yields of the multienzyme reaction system.

Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(-)-mandelic acid production.

A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His6-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V(max) and K(m) parameters were determined to be 27.9 µmol min⁻¹ mg⁻¹ protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(-)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(-)-mandelic acid.

Simultaneous leaching of multiple heavy metals from a soil column by extracellular polymeric substances of Aspergillus tubingensis F12.

The use of the biological agents for leaching heavy metals from contaminated soils is a very promising method that is both efficient and eco-friendly. In this study, a fungus Aspergillus tubingensis F12 was reported to possess a strong adsorption capacity for various heavy metal ions and shown to adsorb 90.8% Pb, 68.4% Zn, 64.5% Cr, 13.1% Cu, 12.9% Ni, and 6.9% Cd in aqueous solution. As extracellular polymeric substance (EPS) was found to play a leading role in the adsorption of metal ions, we applied EPS as a leaching agent to simultaneously remove six metals from soil in a column leaching experiment. The flow rate, initial solution pH, initial EPS concentration, and ionic strength were investigated using response surface methodology. The minimum and maximum metal leaching capacities were determined to be 0.089 mg/g and 3.703 mg/g, respectively. Verified by Fourier transform infrared spectroscopy, scanning electron microscope energy dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy, we made the preliminary deductions that ion exchange determines the leaching capacity limit and that biosorption plays a large role in reaching that limit. Additionally, the redox behaviour of Cu produced more carboxyl groups, which increased the adsorption of heavy metals. The ecological impact of this method was also examined; we found that the influences of leaching with EPS on soil properties and microbial community structure were slight. Therefore, the reported leaching process might have application prospects for metal removal from soil.

[Advances in quantification of lentiviral vectors].

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.

Impacts of red mud on lignin depolymerization and humic substance formation mediated by laccase-producing bacterial community during composting.

The aim of this study was to investigate the impacts of red mud on lignin degradation, humic substance formation and laccase-producing bacterial community in composting to better improve composting performances. The results indicated that the organic matter contents of final compost products in the treatment group with red mud (T) decreased by 25.74%, which was more than the control group without red mud (CK) (12.09%). The final lignin degradation ratio and humic substance concentration of the T were 18.67% and 22.80% higher than that of the CK, respectively. The final C/N values of compost in the CK and T were 11.32 and 10.66, respectively, which were both less than 15, suggesting that compost reached maturity. Redundancy analysis showed that temperature was the main factors driving the variation of laccase-producing bacterial community. Pearson analysis suggested that Pseudomonas, Phenylobacterium, and Caulobacter were the most significantly correlated with lignin degradation and humification in the T.

Efficient decomposition of lignocellulose and improved composting performances driven by thermally activated persulfate based on metagenomics analysis.

In this study, fresh dairy manure and bagasse pith were used as raw materials to study the effect of potassium persulfate in the aerobic composting process. The influence of sulfate radical anion (SO4-·) generated by thermally activated persulfate on physicochemical parameters, lignocellulose degradation, humic substance (HS) formation, microbial community succession, and carbohydrate-active enzymes (CAZymes) composition were assessed during composting. Experimental results showed that the degradation rates of cellulose, hemicellulose and lignin in the treatment group with potassium persulfate (PS) (61.47%, 74.63%, 73.1%) were higher than that in blank control group (CK) (59.98%, 71.47%, 70.89%), respectively. Additionally, persulfate additive promoted dynamic variation of dissolved organic matter (DOM) and accelerated the formation of HS. Furthermore, metagenomics analysis revealed that persulfate changed the structure of the microbial community, and the relative abundances of Actinobacteria and Proteobacteria increased by 17.64% and 34.09% in PS, whereas 12.09% and 29.96% in CK. Glycoside hydrolases (GHs) and auxiliary activities (AAs) families were crucial to degrade lignocellulose, and their abundances were more in PS. Redundancy analysis (RDA) manifested that Actinobacteria and Proteobacteria were closely associated with lignocellulosic degradation. In brief, persulfate could accelerate the degradation of organic components, promote the formation of HS, optimize the structure of microbial community, and improve the compost quality.