An efficient preparation process of sisal fibers via the specialized retting microorganisms: Based on the ideal combination of degumming-related enzymes for the effective removal of non-cellulosic macromolecules.

Bioaugmentation retting with the specialized pectinolytic and xylanolytic microorganisms can accelerate the removal of non-cellulosic macromolecules around plant fibers, thus shortening retting time and facilitating fiber quality. Currently, few specialized microorganisms have been explored for the retting of sisal fibers. The present study excavated the retting fungi including Aspergillus micronesiensis HD 3-6, Penicillium citrinum HD 3-12-3, and Cladosporium sp. HD 4-13 from the region-specific soil samples of planting sisal, and investigated their bioaugmentation retting effects on raw sisal leaves. Results showed that combination of the three fungi achieved the most excellent degumming efficiency (13.69 % of residual gum in sisal fibers) and the highest fiber yield (4.47 %). Furthermore, this fungi combination had the ideal enzymatic hydrolysis features with high activities of pectinase, xylanase and mannanase whereas a low activity of cellulase during the whole retting process, thus endowing the prepared sisal fibers with the lowest mass percentage of non-cellulosic macromolecules (9.76 wt%) and the highest cellulose content (89.23 wt%). SEM and FT-IR analysis further verified that the non-cellulosic substances around sisal fibers were efficiently removed. In summary, the consortia of the three fungi achieved ideal degumming-related enzymes for the removal of non-cellulosic macromolecules, thus acquiring the efficient preparation of sisal fibers.

Chitin Deacetylase from Bacillus aryabhattai TCI-16: Heterologous Expression, Characterization, and Deacetylation Performance.

Chitin deacetylase (CDA) removes the acetyl group from the chitin molecule to generate chitosan in a uniform, high-quality deacetylation pattern. Herein, BaCDA was a novel CDA discovered from our previously isolated Bacillus aryabhattai strain TCI-16, which was excavated from mangrove soil. The gene BaCDA was cloned and overexpressed in Escherichia coli BL21 (DE3) to facilitate its subsequent purification. The purified recombinant protein BaCDA was obtained at a concentration of about 1.2 mg/mL after Ni2+ affinity chromatography. The molecular weight of BaCDA was around 28 kDa according to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. In addition, BaCDA exhibited a significant deacetylation effect on colloidal chitin, and the deacetylation degree was measured from the initial 25.69 to 69.23% by Fourier transform infrared (FT-IR) spectroscopy. Scanning electron microscopy (SEM) observation showed that the surface of colloidal chitin after enzymatic digestion was rough, the crystal fibers disappeared, and the chitin structure was loose and porous with grooves. The results of electrospray ionization mass spectrometry (ESI-MS) showed that BaCDA had full-deacetylation activity against (GlcNAc)4. Molecular docking revealed that BaCDA had an open active pocket capable of binding to the GlcNAc unit. This study not only provides a novel enzymatic resource for the green and efficient application of chitin but also helps to deepen the understanding of the catalytic mechanism of CDA.

Pretreatment of shrimp shells with an acidic deep eutectic solvent system for chitin extraction and its enhanced performance as a carrier for immobilized lipase.

Existing methods for chitin extraction usually produce substantial waste, which poses ecological hazards. Natural deep eutectic solvent (NADES) offers a promising one-step pretreatment alternative, replacing the resource-intensive demineralization (DM) and deproteinization (DP) process. Hence, in this study, the influence of various acidic NADES, on achieving a simplified one-step DM and DP in the chitin extraction process was investigated. The study yielded chitin with 87.73 % purity, and microstructural analysis showed that NADES pretreatment minimally affected chitin quality without deacetylation. In addition, chitin extracted using choline chloride-oxalic acid as a carrier displayed excellent performance in the immobilization of Geobacillus thermocatenulatus lipase 2 (GTL2) because of obvious Ca2+ activation effect. This process contributed to enhancement of immobilized enzyme activity. The immobilized GTL2 showed excellent hydrolytic capabilities, with its highest activity reaching 547.80 ± 20.62 U/mg, significantly better than the five commercial lipases that exhibited <40 % of the enzyme activity. Furthermore, the hydrolytic capacity of immobilized GTL2 was notably high for 4-nitrophenyl butyrate, measuring 935.47 ± 51.60 U/mg. This study provided a constructive approach for the one-step pretreatment of shrimp shells with organic acid-based NADES to isolate and purify chitin and its potential application as an immobilized carrier to enhance enzyme activity.

Co-production of surfactin and fengycin by Bacillus subtilis BBW1542 isolated from marine sediment: a promising biocontrol agent against foodborne pathogens.

Pathogenic bacteria contaminations and related diseases in food industries is an urgent issue to solve. The present study aimed to explore natural food biopreservatives from microorganisms. Using dilution-plate method, a strain BBW1542 with antimicrobial activities against various foodborne pathogenic bacteria was isolated from the seabed silt of Beibu Gulf, which was identified as Bacillus subtilis by the morphological observation and 16S rDNA sequences. The antimicrobial substances of B. subtilis BBW1542 exhibited an excellent stability under cool/heat treatment, UV irradiation, acid/alkali treatment, and protease hydrolysis. The genome sequencing analysis and antiSMASH prediction indicated that B. subtilis BBW1542 contained the gene cluster encoding lipopeptides and bacteriocin subtilosin A. MALDI-TOF-MS analysis showed that the lipopeptides from B. subtilis BBW1542 contained C14 and C15 surfactin homologues, together with fengycin homologues of C18 fengycin A/C16 fengycin B and C19 fengycin A/C17 fengycin B. In silico analysis showed that an eight-gene (sboA-albABCDEFG) operon was involved in the biosynthesis of subtilosin A in B. subtilis BBW1542, and the encoded subtilosin A presented an evident closed-loop structure containing 35 amino acids with a molecular weight of 3425.94 Da. Overall, the antagonistic B. subtilis BBW1542 displayed significant resource value and offered a promising alternative in development of food biopreservation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05864-3.

Structural and enzymatic characterization of a novel metallo-serine keratinase KerJY-23.

KerJY-23 was a novel keratinase from feather-degrading Ectobacillus sp. JY-23, but its enzymatic characterization and structure are still unclear. In this study, the KerJY-23 was obtained by heterologous expression in Escherichia coli BL21(DE3), and enzymatic properties indicated that KerJY-23 was optimal at 60 °C and pH 9.0 and could be promoted by divalent metal ions or reducing agents. Furthermore, KerJY-23 had a broad substrate specificity towards casein, soluble keratin, and expanded feather powder, but its in vitro degradation against chicken feathers required an additional reducing agent. Homology modeling indicated that KerJY-23 contained a highly conserved zinc-binding HELTH motif and a His-Asp-Ser catalytic triad that belonged to the typical characteristics of M4-family metallo-keratinase and serine-keratinase, respectively. Molecular docking revealed that KerJY-23 achieved a reinforced binding on feather keratin via abundant hydrogen bonding interactions. This work not only deepened understanding of the novel and interesting metallo-serine keratinase KerJY-23, but also provided a theoretical basis for realizing the efficient use of waste feather keratin.

Effective biodegradation on chicken feather by the recombinant KerJY-23 Bacillus subtilis WB600: A synergistic process coupled by disulfide reductase and keratinase.

Keratin wastes are abundantly available but rich in hard-degrading fibrous proteins, and the keratinase-producing microorganisms have gained significant attention due to their biodegradation ability against keratinous materials. In order to improve the degradation efficiency of feather keratins, the keratinase gene (kerJY-23) from our previously isolated feather-degrading Ectobacillus sp. JY-23 was overexpressed in Bacillus subtilis WB600 strain. The recombinant KerJY-23 strain degraded chicken feathers rapidly within 48 h, during which the activities of disulfide reductase and keratinase KerJY-23 were sharply increased, and the free amino acids especially the essential phenylalanine and tyrosine were significantly accumulated in feather hydrolysate. The results of structural characterizations including scanning electron microscopy, Fourier transform infrared spectrum, X-ray diffraction, and X-ray photoelectron spectroscopy, demonstrated that the feather microstructure together with the polypeptide bonds and SS bonds in feather keratins were attacked and destroyed by the recombinant KerJY-23 strain. Therefore, the recombinant KerJY-23 strain contributed to feather degradation through the synergistic action of the secreted disulfide reductase to break the SS bonds and keratinase (KerJY-23) to hydrolyze the polypeptide bonds in keratins. This study offers a new insight into the underlying mechanism of keratin degradation, and provides a potential recombinant strain for the valorization of keratin wastes.

Isolation of a novel feather-degrading Ectobacillus sp. JY-23 strain and characterization of a new keratinase in the M4 metalloprotease family.

Microbial keratinases have prominent potential in biotransformation of recalcitrant keratin substrates to value-added products which has made keratinases a research focus in the past decades. In this study, an efficient feather-degrading bacterium was isolated and identified as a novel species in Ectobacillus genus and designated as Ectobacillus sp. JY-23. The degradation characteristics analysis revealed that Ectobacillus sp. JY-23 could utilize chicken feathers (0.4% w/v) as the sole nutrient source and degraded 92.95% of feathers in 72 h. A significant increase in sulfite and free sulfydryl group content detected in the feather hydrolysate (culture supernatant) indicated efficient reduction of disulfide bonds, which inferred that the degradation mechanism of isolated strain was a synergetic action of sulfitolysis and proteolysis. Moreover, abundant amino acids were also detected, among which proline and glycine were the predominant free amino acids. Then, the keratinase of Ectobacillus sp. JY-23 was mined and Y1_15990 was identified as the keratinase encoding gene of Ectobacillus sp. JY-23 and designated as kerJY-23. Escherichia coli strain overexpressing kerJY-23 degraded chicken feathers in 48 h. Finally, bioinformatics prediction of KerJY-23 demonstrated that it belonged to the M4 metalloprotease family, which was a third keratinase member in this family. KerJY-23 showed low sequence identity to the other two keratinase members, indicating the novelty of KerJY-23. Overall, this study presents a novel feather-degrading bacterium and a new keratinase in the M4 metalloprotease family with remarkable potential in feather keratin valorization.

A marine lipopeptides-producing Bacillus amyloliquefaciens HY2-1 with a broad-spectrum antifungal and antibacterial activity and its fermentation kinetics study.

The antagonistic Bacillus amyloliquefaciens HY2-1 was a marine microbiology that was isolated previously from the seabed silt of Beibu Gulf in China by dual culture with Penicillium digitatum. As a continuous study, the present work focused on evaluating the antimicrobial activity, identifying the produced active components, and revealing the fermentation characteristics of B. amyloliquefaciens HY2-1, respectively. It was found that B. amyloliquefaciens HY2-1 exhibited a broad-spectrum antimicrobial activity against the tested seven phytopathogenic fungi and five pathogenic bacteria by producing Bacillus lipopeptides such as fengycin A (C14 to C19 homologues) and surfactin (C14 and C15 homologues). Morphological observation of P. digitatum under light microscope, scanning electron microscopy, transmission electron microscopy, and fluorescence microscope inferred that B. amyloliquefaciens exerted the antagonistic activity by damaging the fungal cell membrane, thus inhibiting the mycelium growth and sporification of phytopathogenic fungi. As a marine microbiology, our results showed that B. amyloliquefaciens could survive and metabolize even at the culture condition with 110 g/L of NaCl concentration, and the produced antimicrobial compounds exhibited excellent thermostability and acid-alkali tolerance. The dynamic models were further constructed to theoretically analyze the fermentation process of B. amyloliquefaciens HY2-1, suggesting that the synthesis of antimicrobial compounds was coupled with both cell growth and cell biomass. In conclusion, the marine lipopeptides-producing B. amyloliquefaciens HY2-1 showed a promising prospect to be explored as a biocontrol agent for plant disease control of crops and postharvest preservation of fruits and vegetables, especially due to its outstanding stress resistance and the broad-spectrum and effective antagonist on various phytopathogenic fungi.

Liquid hot water pretreatment combined with high-solids enzymatic hydrolysis and fed-batch fermentation for succinic acid sustainable processed from sugarcane bagasse.

In order to sustainable process of bio-succinic acid (SA), response surface methodology (RSM) was applied to optimize liquid hot water pretreatment pretreatment of sugarcane bagasse (SCB), followed by high-solids enzymatic hydrolysis of pretreated residual that without washing, then the hydrolysates and partial pretreatment liquid were used as carbon sources for SA fermentation. Results showed that the highest sugars yield could be achieved at pretreatment conditions of temperature 186 °C, time 25 min and solid-to-liquid ratio 0.08; enzymatic digestion the pretreated residuals at 20 % (w/v) solid content via enzymes reconstruction and fed-batch strategy, the obtained sugars reached to 121 g/L; by controlling the nutrition and conditions of the fermentation process, most of the C5 and C6 sugars in the hydrolysate and pretreatment liquid were converted into SA with a conversion rate high to 280 mg/g SCB. This study can provide a novel clue for clean and efficient biorefining of chemicals.

Nitrogen Removal Characteristics of a Marine Denitrifying Pseudomonas stutzeri BBW831 and a Simplified Strategy for Improving the Denitrification Performance Under Stressful Conditions.

A marine aerobic denitrifying bacterium was isolated and identified as Pseudomonas stutzeri BBW831 from the seabed silt of Beibu Gulf in China. According to the genome analysis, P. stutzeri BBW831 possessed a total of 14 genes (narG, narH, narI, narJ, napA, napB, nirB, nirD, nirS, norB, norC, norD, norQ, and nosZ) responsible for fully functional enzymes (nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase) involved in the complete aerobic denitrification pathway, suggesting that it had the potential for reducing nitrate to the final N2. Denitrification results showed that P. stutzeri BBW831 exhibited efficient nitrogen removal characteristics. Within 12 h, the NO3--N removal efficiency and rate reached 94.64% and 13.09 mg·L-1·h-1 under 166.10 ± 3.75 mg/L NO3--N as the sole nitrogen source, and removal efficiency of the mixed nitrogen (50.50 ± 0.55, 62.28 ± 0.74, and 64.26 ± 0.90 mg/L of initial NH4+-N, NO3--N, and NO2--N, respectively) was nearly 100%. Furthermore, a simplified strategy, by augmenting the inoculation biomass, was developed for promoting the nitrogen removal performance under high levels of NO2--N and salinity. As a result, the removal efficiency of the initial NO2--N up to approximately 130 mg/L reached 99.46% within 8 h, and the NO3--N removal efficiency achieved at 59.46% under the NaCl concentration even up to 50 g/L. The C/N ratio of 10 with organic acid salt such as trisodium citrate and sodium acetate as the carbon source was most conducive for cell growth and nitrogen removal by P. stutzeri BBW831, respectively. In conclusion, the marine P. stutzeri BBW831 contained the functional genes responsible for a complete aerobic denitrification pathway (NO3--N → NO2--N → NO → N2O → N2), and had great potential for the practical treatment of high-salinity nitrogenous mariculture wastewater.

Root cell wall remodeling: A way for exopolysaccharides to mitigate cadmium toxicity in rice seedling.

Exopolysaccharides (EPS) are macromolecules with environment beneficial properties. Currently, numerous studies focus on the absorption of heavy metals by EPS, but less attention has been paid to the effects of EPS on the plants. This study explored the effects of EPS from Lactobacillus plantarum LPC-1 on the structure and function of cell walls in rice seedling roots under cadmium (Cd) stress. The results showed that EPS could regulate the remodeling process of the cell walls of rice roots. EPS affects the synthesis efficiency and the content of the substances that made up the cell wall, and thus plays an essential role in limiting the uptake and transport of Cd in rice root. Furthermore, EPS could induce plant resistance to heavy metals by regulating the lignin biosynthesis pathway in rice roots. Finally, the cell wall remodeling induced by EPS likely contributes to plant stress responses by activating the reactive oxygen species (ROS) signaling.

Exopolysaccharides from Lactobacillus plantarum reduces cadmium uptake and mitigates cadmium toxicity in rice seedlings.

Exopolysaccharides (EPSs) can be used as effective exogenous substances to alleviate the toxic effect of cadmium (Cd) on rice and other crops, thus improving plant growth characteristics under stress conditions, and reducing the accumulation of Cd in grains, but the underlying mechanism is still unclear. In the present work, the effects of EPSs from Lactobacillus plantarum on the efficiency of Cd absorption and distribution in rice seedlings under Cd stress were investigated. The results revealed that growth of rice seedlings was severely inhibited by exposure to Cd, resulting in the decrease of plant height, leaf length and biomass. This inhibition phenomenon was alleviated by the addition of EPSs from L. plantarum LPC-1. The underlying mechanism might be that EPSs could facilitate the accumulation efficiency of Cd in rice roots and reduce the transportation rate of Cd from root to leaves, therefore decreasing the Cd content in leaves. Further research showed that Cd contents in the cell wall fraction of the rice seedling root were increased by the addition of EPSs, while the proportions of Cd in the cell organelle and cell soluble component were reduced. Application of EPSs promotes the proportion of pectate- and protein- integrated Cd in rice roots. While the content of water-soluble Cd, which is more toxic to plants, decreased continuously both in roots and leaves. Our study clearly confirmed the positive effects of EPSs on alleviating Cd toxicity and decreasing Cd translocation in rice above-ground parts. Furthermore, the subcellular distribution and chemical forms of Cd in different rice seedlings parts were also affected by the addition of EPSs, which might be an important potential mechanism for EPSs in respect of alleviating Cd toxicity for rice. These findings provided a foundation for the application of exogenous substances on improving the growth performance of crops under heavy metal stress.

Isolation, characterization, and genome sequencing of a novel chitin deacetylase producing Bacillus aryabhattai TCI-16.

Chitin deacetylase (CDA) is a chitin degradation enzyme that catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues, playing an important role in the high-value utilization of waste chitin. The shells of shrimp and crab are rich in chitin, and mangroves are usually recognized as an active habitat to shrimp and crab. In the present study, a CDA-producing bacterium, strain TCI-16, was isolated and screened from the mangrove soil. Strain TCI-16 was identified and named as Bacillus aryabhattai TCI-16, and the maximum CDA activity in fermentation broth reached 120.35 ± 2.40 U/mL at 36 h of cultivation. Furthermore, the complete genome analysis of B. aryabhattai TCI-16 revealed the chitin-degrading enzyme system at genetic level, in which a total of 13 putative genes were associated with carbohydrate esterase 4 (CE4) family enzymes, including one gene coding CDA, seven genes encoding polysaccharide deacetylases, and five genes encoding peptidoglycan-N-acetyl glucosamine deacetylases. Amino acid sequence analysis showed that the predicted CDA of B. aryabhattai TCI-16 was composed of 236 amino acid residues with a molecular weight of 27.3 kDa, which possessed a conserved CDA active like the known CDAs. However, the CDA of B. aryabhattai TCI-16 showed low homology (approximately 30%) with other microbial CDAs, and its phylogenetic tree belonged to a separate clade in bacteria, suggesting a high probability in structural novelty. In conclusion, the present study indicated that the novel CDA produced by B. aryabhattai TCI-16 might be a promising option for bioconversion of chitin to the value-added chitosan.

Dynamics of the Bacterial Community's Soil During the In-Situ Degradation Process of Waste Chicken Feathers.

Bacteria play an important role in the biodegradation of feather waste. The exploration of the related microbial community structure and diversity is essential to improve the performance of feather waste treatment processes. In the present work, an in-situ soil sampled from a poultry farm was directly used to simulate and accelerate the natural degradation processes of feather waste under laboratory conditions, in which the dynamics of the microbial communities was further analyzed by Illumina HiSeq high-throughput 16S rRNA gene sequencing. Biochemical factors, including pH, feather degradation rate and soluble protein content were also determined in this study. The biochemical results showed that the in-situ soil exhibited an effective degradability on chicken feathers, and the degradation rate of feathers reached 57.95 ± 3.09% at 120 h of cultivation. Meanwhile, soluble protein content and pH reached 33.62 ± 1.45 mg/mL 8.99 ± 0.08, respectively. The results of bacterial diversity analysis showed that bacterial community structure and composition significantly varied in each phase of degradation. Additionally, the bacteria system with feather degradability might consist of Bacillus, Chryseobacterium, Lysobacter, Brevibacillus, and Stenotrophomonas genera. This system may include the following key pathways: carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, membrane transport, replication and repair, translation, signal transduction and energy metabolism. Moreover, the bacterial communities may occur community succession during the degradation processes of chicken feathers. In summary, the present work provided valuable insights into the understanding of microbial community and metabolic functions for feather degradation, although the in-situ biodegradation process was conducted under laboratory conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00996-6.

Revealing the Promoting Effect of Betaine on Vitamin B12 Biosynthetic Pathway of Pseudomonas denitrificans by Using a Proteomics Analysis.

BACKGROUND: Our previous comparative metabolomics research revealed that betaine (N,N,Ntrimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. OBJECTIVES: Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. MATERIALS AND METHODS: By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine in addition to P. denitrificans fermentation medium. RESULTS: The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, precorrin-2 C(20)- methyltransferase, precorrin-8X methylmutase, and precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the latestage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. CONCLUSION: The present study demonstrated clearly that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

Alkaline hydrogen peroxide pretreatment combined with bio-additives to boost high-solids enzymatic hydrolysis of sugarcane bagasse for succinic acid processing.

Alkaline hydrogen peroxide (AHP) pretreatment of sugarcane bagasse (SCB) at mild conditions was optimized with response surface methodology (RSM), then enzymatic hydrolysis was performed at high-solids substrate loading (30 %, w/v), followed by fed-batch fermentation to convert the fermentable sugars into succinic acid (SA). Results showed the AHP pretreatment conditions of H2O2 concentration 5.5 % (v/v), solid-to-liquid ratio 0.08, pretreatment temperature 65 °C and time 5 h could achieve the highest sugar yield (74.3 %); both additives and fed-batch strategy were favored to boost enzymatic hydrolysis, the concentration and yield of total sugars reached to 195 g/L and 70 % with cellulase dosage of only 6 FPU/g dry biomass (DM); all glucose and xylose could be utilized after fed-batch fermentation, and the obtained concentration and yield of SA reached 41.4 g/L and 63.8 %. In summary, a SA conversion rate high to 0.29 g/g SCB raw material could be achieved via the developed process.

Discovery and evaluation on the antibacterial and cytotoxic activities of a novel antifungalmycin N2 produced from Streptomyces sp. strain N2.

Antifungalmycin N2 (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) was a novel metabolite produced from Streptomyces sp. strain N2, and the present study aimed to evaluate its antibacterial and cytotoxic properties. By using Oxford cup method, the obtained results revealed that antifungalmycin N2 exhibited a significant antibacterial activity against the pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Micrococcus kristinae, especially the Gram-positive S. aureus. Meanwhile, the MTT assay showed that antifungalmycin N2 could exert a marked inhibitory action on tumor cell lines, such as the cell lines of BEL-7402 (human hepatocellular carcinoma), Hela (human cervical carcinoma), HCT116 (human colon cancer), and SW620 (human colon cancer). And the IC50 values antifungalmycin N2 against the above cell lines ranged from 11.23 to 15.37 µg/mL. In conclusion, the antibacterial and cytotoxic activities suggested that the novel antifungalmycin N2 was a promising active structure to be developed as new drug for treating infectious diseases and cancers.

Antifungal Action of Antifungalmycin N2 Against Rhizoctonia solani by Disrupting Cell Membrane and Inhibiting Succinate Dehydrogenase.

Antifungalmycin N2 (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) was a novel structural antifungal metabolite produced by Streptomyces sp. strain N2. Our previous study reported that the antagonistic interaction between antifungalmycin N2 and Rhizoctonia solani was accompanied by an oxidative stress in R. solani cell, indicating a probable damage occurred in the cell membranes and mitochondria. To verify this, the present study focused on investigating the effects of antifungalmycin N2 on the structure and function of cell membranes and mitochondria of R. solani. Morphological observations in transmission electron microscopy and fluorescence microscope showed that cell membranes of R. solani were damaged, and its cytoplasmic organelles were disorganized when treated with antifungalmycin N2. Meanwhile, the kinetics of membrane-related physiological and biochemical parameters, such as the increased malondialdehyde level, dropped ergosterol formation, and enhanced electrical conductivity in R. solani mycelia, further confirmed that antifungalmycin N2 would disrupt the cell membrane structure and function. More significantly, antifungalmycin N2 had a significantly inhibitory effect on the succinate dehydrogenase (SDH) activity of R. solani, and indicated that the mode and site of action of antifungalmycin N2 against R. solani might be similar to the existing succinate dehydrogenase inhibitors fungicides by binding in the ubiquinone-binding site. In conclusion, the above results demonstrated that the mode and site of action of antifungalmycin N2 targeted to cell membrane and SDH of R. solani, thus exerting the antifungal activity by damaging cell membrane structure and function, together with inhibiting the SDH activity.

Structural characterization and induced copper stress resistance in rice of exopolysaccharides from Lactobacillus plantarum LPC-1.

Excessive accumulation of copper could decrease growth and quality of crops, and little information was currently available on the role of exopolysaccharides (EPS) from Lactobacillus plantarum in inducing copper stress resistance in plants. The main objective of this work was to purify and characterize the EPS produced by our isolated L. plantarum LPC-1, and evaluate its potential protection for rice against copper stress. Firstly, two fractions (EPS-1 and EPS-2) were separated and purified from L. plantarum LPC-1 by DEAE-52 cellulose and Sephadex G-100 cellulose column chromatography. According to the further scanning electron microscope (SEM), ultraviolet-visible (UV), fourier-transform infrared spectroscopy (FTIR) spectroscopy and gas chromatography (GC) analyses, it was observed that EPS-1 and EPS-2 were heteropolysaccharides that were composed of mannose and glucose with molar ratio of 2.40:15.01 and 3.02:11.63, respectively. Additionally, the two fractions possessed considerable antioxidant activities, and EPS-1 had a stronger antioxidant activity than EPS-2 in vitro. Furthermore, exogenous addition of EPS significantly alleviated the toxic effects by copper on rice seedlings. In conclusion, this study provided evidence of the EPS-mediated reduction of copper toxicity in rice seedlings at physiological and biochemical levels, suggesting that EPS could be considered as novel and effective plant immune inducers in crops.

Antagonistic activity and mechanism of an isolated Streptomyces corchorusii stain AUH-1 against phytopathogenic fungi.

The various diseases that occur during the growth of plants usually cause a significant reduction in production and quality of agricultural products. Actinomycetes, especially Streptomyces spp., become a valuable biological control resource due to their preponderant abilities to produce various secondary metabolites with novel structure and remarkable biological activity. The present work aimed to isolate an effective antagonistic actinomycete against various soilborne phytopathogenic fungi. By dual culture with Fusarium oxysporum f. sp. niveum, an antagonistic actinomycete named Streptomyces corchorusii stain AUH-1 was screened out from 26 soil samples. The in vitro bioassay results showed that S. corchorusii stain AUH-1 had a broad-spectrum antagonistic activity against a range of fungal plant pathogens, such as F. oxysporum f. sp. niveum, Phytophthora parasitica var. nicotianae, Rhizoctonia solani, P. capsica, Botryosphaeria dothidea, F. oxysporum f. sp. vasinfectum, Verticillium dahliae, and F. oxysporum f. sp. cucumerinum. According to the morphological observations in scanning electron microscopy (SEM) and fluorescence microscope (FM), it was found that the cell membranes of F. oxysporum f. sp. niveum were damaged when treated with the antifungal metabolite form S. corchorusii stain AUH-1. Meanwhile, the dropped ergosterol formation and increased malondialdehyde levels further confirmed that S. corchorusii strain AUH-1 exerted its antagonistic activity against F. oxysporum f. sp. niveum via damaging the structure and function of cell membranes. In conclusion, S. corchorusii strain AUH-1 showed a promising prospect for the development of biological agent, especially due to its broad-spectrum and effective antagonist on various soil-borne plant pathogens.