Protease Activity Research Articles

A novel approach to enhance high optically active L-lactate production from food waste by landfill leachate

The recycling of food waste (FW) through anaerobic fermentation into lactic acid (LA), with two isomers L-LA and D-LA, aligns with the principles of a bio-based circular economy. However, FW fermentation is often limited by competing pathways, acidification inhibition, and trace metals deficiency. This study investigates the introduction of landfill leachate, containing buffering agents (ammonia) and trace metals, into FW fermentation. Various dosages of landfill leachate, ranging from 90 (LN-90) to 450 mg/L (LN-450) based on inclusive ammonia calculation, were employed. Results showed that LA production peaked at 43.65 ± 0.57 g COD/L in LN-180 on day 6, with a high optical activity of L-LA at 92.40 ± 1.15 %. Fermentation pathway analysis revealed that landfill leachate amendment enhances hydrolysis (as evidenced by increased activity of amylase, α-glucosidase, and protease) and glycolysis (resulting in enhanced utilization of carbohydrates and glucose). The inclusive ammonia in leachate plays a crucial role as a buffer, maintaining optimal pH conditions (5–7), thereby reducing volatile fatty acid production and thus intensifying LA orientations. The increased activity of L-lactate dehydrogenase (L-LA generation) and decreased NAD-independent lactate dehydrogenase (LA consumption) in properly dosed leachate further explained the high accumulation of L-LA. Dominance of lactic acid bacteria, including Streptococcus, Enterococcus, Klebsiella, Bifidobacterium, Bavariicoccus, and Lacticaseibacillus, accounted for 91.08% (LN-90), while inhibitory effects were observed in LN-450 (4.45%). Functional gene analysis further supported the enhanced glycolysis, L-lactate dehydrogenase, and nitrogen assimilation. Finally, a network analysis indicates a beneficial effect on the genus Enterococcus and Klebsiella by landfill leachate addition. This study demonstrates the efficiency of utilizing landfill leachate to enhance LA recycling from FW fermentation, aligning with the concept of circular economy by transforming waste into valuable resources.

Enhancing cider quality through co-fermentation with acid protease and esterase-producing Metschnikowia species and Saccharomyces cerevisiae.

To date, cider production has primarily relied on Saccharomyces cerevisiae. Introducing novel non-Saccharomyces yeasts can enhance the diversity of cider properties. Among these, the Metschnikowia genus stands out for its ability to produce hydrolytic enzymes that may impact the sensorial and technological properties of cider. This study focused on evaluating the impact of three Metschnikowia species - Metschnikowia koreensis (Mk), M. reukaufii (Mr), and M. pulcherrima (Mp) - which exhibit acid protease and esterase activity, on the quality enhancement of cider. The research findings indicate that the overall quality of cider produced through co-fermentation with these species surpassed that of cider fermented with mono-fermentation of S. cerevisiae (Sc). The cider fermented with the Sc + Mk combination exhibited the lowest levels of harsh-tasting malic acid and higher levels of softer lactic acid. Sensory array analysis also demonstrated that the Sc + Mk fermented cider exhibited high sensor response values for compounds contributing to a complex overall olfactory composition and richness. Furthermore, the Sc + Mk fermented cider exhibited the highest total quantity and variety of volatile organic compounds (VOCs). Specifically, the concentrations of phenethyl alcohol, 3-methyl-1-butanol, ethyl octanoate, and decanoic acid were notably elevated in comparison with other groups. This study illustrates that Metschnikowia species, particularly M. koreensis, show significant potential as starters for cider due to their various technological properties, including acidity modulation, aroma enhancement, and color improvement. The findings of this study provide a foundation for improving cider quality by co-fermenting S. cerevisiae with innovative starter cultures. © 2024 Society of Chemical Industry.

Oleuropein enhances proteasomal activity and reduces mutant huntingtin-induced cytotoxicity.

Huntington's disease (HD) is a hereditary neurodegenerative disorder that primarily affects the striatum, a brain region responsible for movement control. The disease is characterized by the mutant huntingtin (mHtt) proteins with an extended polyQ stretch, which are prone to aggregation. These mHtt aggregates accumulate in neurons and are the primary cause of the neuropathology associated with HD. To date, no effective cure for HD has been developed. The immortalized STHdh Q111/Q111 striatal cell line, the mHtt-transfected wild-type STHdh Q7/Q7 striatal cell line, and N2a cells were used as Huntington's disease cell models. Flow cytometry was used to assess cellular reactive oxygen species and transfection efficiency. The CCK-8 assay was used to measure cell viability, while fluorescence microscopy was used to quantify aggregates. Immunoblotting analyses were used to evaluate the effects on protein expression. Polyphenols are natural antioxidants that offer neuroprotection in neurological disorders. In this study, we provide evidence that oleuropein, the primary polyphenol in olive leaves and olive oil, enhances cell viability in HD cell models, including. STHdh Q7/Q7 STHdh Q7/Q7 striatal cells, N2a cells ectopically expressing the truncated mHtt, and STHdh Q111/Q111 striatal cells expressing the full-length mHtt. Oleuropein effectively reduced both soluble and aggregated forms of mHtt protein in these HD model cells. Notably, the reduction of mHtt aggregates associated with oleuropein was linked to increased proteasome activity rather than changes in autophagic flux. Oleuropein seems to modulate proteasome activity through an unidentified pathway, as it did not affect the 20S proteasome catalytic β subunits, the proteasome regulator PA28γ, or multiple MAPK pathways. We demonstrated that oleuropein enhances the degradation of mHtt by increasing proteasomal protease activities and alleviates mHtt-induced cytotoxicity. Hence, we propose that oleuropein and potentially other polyphenols hold promise as a candidate for alleviating Huntington's disease.

Identification of High-Risk Lower Extremity Wounds Using Point-of-Care Test for Bacterial Protease Activity; A Single-Centre, Single-Blinded, Prospective Study.

Clinician observation is the mainstay to determine if wound infection is present, and focuses on presence of erythema, purulence, and odour. However, non-visible bacterial protease activity can delay wound healing and lead to complications. In this study, a point-of-care test to detect the presence of bacterial protease activity (BPA, tested with Woundchek Bacterial Status test) was appraised. A total of 130 patients with lower extremity wounds were recruited in vascular and podiatry clinics, and across two time-points 182 BPA tests were conducted subsequent to initial (blinded) clinician's wound appraisal. Clinical opinion ('no infection', 'possible' or 'definite' infection) and BPA result (negative or positive test) had a moderate Kendall's tau-c rank correlation coefficient of 0.32 (P < 0.001). Binary logistic regression analysis and principal component analysis showed that infection determined by clinical opinion was significantly associated with abovementioned clinical signs and a positive BPA test. However, a positive BPA result was also significantly linked with wound severity, such as number of lesions, chronicity and size. Throughout a 12-week follow-up period, median ulcer size was larger for wounds positive for BPA test at baseline (P 0.001) and week-12 (P 0.036; both Mann-Whitney U-test) respectively. As a pilot initiative, clinical staff were allowed to act on the BPA result if they wished; in 11 out of 71 test-positive cases (15%) this happened and antimicrobial dressing was applied instead of planned standard dressing. These results show that protease-releasing bacteria may be active in ulcers that do not (yet) exhibit hallmark signs of infection, and are associated with delayed healing. Targeted point-of-care testing for bacterial protease activity may have the potential to identify and enable pro-active (antimicrobial) management of these high-risk wounds.

Limosilactobacillus Regulating Microbial Communities to Overcome the Hydrolysis Bottleneck with Efficient One-Step Co-Production of H2 and CH4.

The efficient co-production of H2 and CH4 via anaerobic digestion (AD) requires separate stages, as it cannot yet be achieved in one step. Lactic acid bacteria (LAB) (Limosilactobacillus) release H2 and acetate by enhancing hydrolysis, potentially increasing CH4 production with simultaneous H2 accumulation. This study investigated the enhanced effect of one-step co-production of H2 and CH4 in AD by LAB and elucidated its enhancement mechanisms. The results showed that 236.3 times increase in H2 production and 7.1 times increase in CH4 production are achieved, resulting in profits of 469.39 USD. Model substrates lignocellulosic straw, sodium acetate, and H2 confirmes LAB work on the hydrolysis stage and subsequent sustainable volatile fatty acid production during the first 6 days of AD. In this stage, the enrichment of Limosilactobacillus carrying bglB and xynB, the glycolysis pathway, and the high activity of protease, acetate kinase, and [FeFe] hydrogenase, jointly achieved rapid acetate and H2 accumulation, driving hydrogenotrophic methanogenesis dominated. From day 7 to 24, with enriched Methanosarcina, and increased methenyltetrahydromethanopterin hydrogenase activity, continuously produced acetate led to the mainly acetoclastic methanogenesis shift from hydrogenotrophic methanogenesis. The power generation capacity of LAB-enhanced AD is 333.33 times that of China's 24,000m3 biogas plant.

Gossypol Inhibits Metastasis of Lung Cell Carcinoma by Reversing Epithelial to Mesenchymal Transition and Suppressing Proteases Activity.

Gossypol, a natural polyphenolic compound, possesses antivirus activity and induces cell death of different types of tumors. However, the efficacy of gossypol on lung carcinoma metastases and epithelial to mesenchymal transition remains unknown. The aim of the present work was to determine the cellular and molecular mechanism of the anti-cancer and anti-metastatic efficacies of gossypol on human lung carcinoma cells. Gossypol showed a marked suppression of the viability, motility, and invasion in H1299 and A549 cells. Zymography assay showed that gossypol was sufficient to suppress the activities of urokinase-type plasminogen activator and matrix metalloproteinase-2. Gossypol reversed TGF-β-induced epithelial to mesenchymal transition. Gossypol reduced vimentin, p-FAK, p-Src and p-paxillin. In vivo studies of gossypol were performed using subcutaneous inoculation and tail vein injection of A549 into immunodeficient BALB/c nude mice and severe combined immunodeficient mice.

A novel approach to produce soy protein isolate: Direct enzymatic hydrolysis of high-temperature soybean meal using proteinase produced by microbial solid-state fermentation

This study proposed a novel extraction method for soy protein isolate, which involved solid-state fermentation of high-temperature soybean meal. The proteinases secreted by microorganisms acted on the high-temperature soybean meal, making the SPI easier to extract. The study concludes that Bacillus amyloliquefaciens subsp. plantarum CICC 10265 could be used for solid-state fermentation of soybean meal, and the fermentation effect was good, with a yield of 41.91 % for SPI. Compared to the direct extraction of SPI from high-temperature soybean meal, the yield had increased by 130.19 %. Meanwhile, we also conducted research on the losses during the SPI extraction process. Through experiments, the study identified the patterns of protease activity changes and microbial colony growth during solid-state fermentation of soybean meal by Bacillus amyloliquefaciens subsp. plantarum CICC 10265. It was concluded that extracting SPI after 8 h of fermentation is more suitable. The experimental results indicated that the total amino acid content of SPI extracted from fermented soybean meal was 2.1 % higher compared to SPI extracted from low-temperature soybean meal. The extracted SPI also met the microbial standards.

Zootechnical Performance, Protease Activity and Proximate Composition of Nile Tilapia Fed Diets Containing Fish Silage Produced from Fish Waste

The present study was carried out to evaluate the impact of fish silage (FS) on the growth performances, protease enzyme activity and muscle compositions of Nile tilapia. In this regard, FS was produced from the waste parts of carp, pangus and tilapia and five iso-nitrogenous (containing 35% crude protein) diets were formulated in which fishmeal (FM) was used as the primary protein source in the control diet (0% FS, T1). The remaining four diets were formulated by replacing 25% (T2), 50% (T3), 75% (T4), and 100% (T5) FM protein with the FS. These diets were fed to tilapia at 8-6% of their body weight rearing them in glass tanks at the density of 15 fish/tank in triplicates. After fifty-days feeding trial, survival (%) of fish remained unaffected among the dietary treatments. Tilapia fed with 75% FS based diet showed improved growth and lower FCR, whereas those fed a diet with 100% FS exhibited lower growth rates. Notably, protease enzyme activity (mg/ml/min) increased with higher levels of FS inclusion. Increased crude protein content was observed in the fish fed the 75% FS diet. These findings underscore the potential of including FS up to 75% to enhance the growth performance of Nile tilapia.

In vitro study on efficacy of SKF7®, a Malaysian medicinal plant product against SARS-CoV-2

BackgroundIn early 2020, COVID-19 pandemic has mobilized researchers in finding new remedies including repurposing of medicinal plant products focusing on direct-acting antiviral and host-directed therapies. In this study, we performed an in vitro investigation on the standardized Marantodes pumilum extract (SKF7®) focusing on anti-SARS-CoV-2 and anti-inflammatory activities.MethodsAnti-SARS-CoV-2 potential of the SKF7® was evaluated in SARS-CoV-2-infected Vero E6 cells and SARS-CoV-2-infected A549 cells by cytopathic effect-based assay and RT-qPCR, respectively. Target based assays were performed on the SKF7® against the S1-ACE2 interaction and 3CL protease activities. Anti-inflammatory activity of the SKF7® was evaluated by nitric oxide inhibitory and TLR2/TLR4 receptor blocker assays.ResultsThe SKF7® inhibited wild-type Wuhan (EC50 of 21.99 µg/mL) and omicron (EC50 of 16.29 µg/mL) SARS-CoV-2 infections in Vero-E6 cells. The SKF7® also inhibited the wild-type SARS-CoV-2 infection in A549 cells (EC50 value of 6.31 µg/mL). The SKF7® prominently inhibited 3CL protease activity. The SKF7® inhibited the LPS induced-TLR4 response with the EC50 of 16.19 µg/mL.ConclusionsIn conclusion, our in vitro study highlighted anti-SARS-CoV-2 and anti-inflammatory potentials of the SKF7®. Future pre-clinical in vivo studies focusing on antiviral and immunomodulatory potentials of the SKF7® in affecting the COVID-19 pathogenesis are warranted.

Novel approach to exploring protease activity and targets in HIV-associated obstructive lung disease using combined proteomic-peptidomic analysis

BackgroundObstructive lung disease (OLD) is increasingly prevalent among persons living with HIV (PLWH). However, the role of proteases in HIV-associated OLD remains unclear.MethodsWe combined proteomics and peptidomics to comprehensively characterize protease activities. We combined mass spectrometry (MS) analysis on bronchoalveolar lavage fluid (BALF) peptides and proteins from PLWH with OLD (n = 25) and without OLD (n = 26) with a targeted Somascan aptamer-based proteomic approach to quantify individual proteases and assess their correlation with lung function. Endogenous peptidomics mapped peptides to native proteins to identify substrates of protease activity. Using the MEROPS database, we identified candidate proteases linked to peptide generation based on binding site affinities which were assessed via z-scores. We used t-tests to compare average forced expiratory volume in 1 s per predicted value (FEV1pp) between samples with and without detection of each cleaved protein and adjusted for multiple comparisons by controlling the false discovery rate (FDR).FindingsWe identified 101 proteases, of which 95 had functional network associations and 22 correlated with FEV1pp. These included cathepsins, metalloproteinases (MMP), caspases and neutrophil elastase. We discovered 31 proteins subject to proteolytic cleavage that associate with FEV1pp, with the top pathways involved in small ubiquitin-like modifier mediated modification (SUMOylation). Proteases linked to protein cleavage included neutrophil elastase, granzyme, and cathepsin D.InterpretationsIn HIV-associated OLD, a significant number of proteases are up-regulated, many of which are involved in protein degradation. These proteases degrade proteins involved in cell cycle and protein stability, thereby disrupting critical biological functions.

Superactivity of Bimetallic CuFeO2 Submicron Particles towards Selective Proteolysis, Depending on their Surface Hydroxyls.

Nano bimetallic oxides as nanoproteases have the great advantages in the heterogeneous hydrolysis of proteins. Here, we report that bimetallic delafossite CuFeO2 submicron particles (CuFeO2 SMPs) display a high protease activity towards selective cleavage of peptide bond involving hydrophobic residue at 25 °C. CuFeO2 SMPs have excellent regeneration performance with high structural stability. The strong Lewis acidity of Fe3+ and the strong nucleophilicity of Cu+ bound hydroxyl groups are both necessary for the high protease activity of CuFeO2 SMPs. Low-valent metal ion has a great advantage in that low-valent Cu+ bound hydroxyl has strong nucleophilicity, resulting in promotion of protein hydrolysis via high-efficient bimetallic catalysis. This study provides evidence that the protease activity of CuFeO2 SMPs depends on metal ion-bound hydroxyls on their surface. Our findings highlight that the valence of metal ions in artificial protease and their surface hydroxyls are two important factors that determine their catalytic efficiency.

Cathepsin C (dipeptidyl peptidase 1) inhibition in adults with bronchiectasis: AIRLEAF®, a Phase II randomised, double-blind, placebo-controlled, dose-finding study.

Bronchiectasis is characterised by uncontrolled neutrophil serine protease (NSP) activity. Cathepsin C (CatC; dipeptidyl peptidase 1) activates NSPs during neutrophil maturation. CatC inhibitors can potentially reduce neutrophil-mediated lung damage. This Phase II, randomised, double-blind, placebo-controlled trial (AIRLEAF®; NCT05238675) evaluated efficacy, safety and optimal dosing of BI 1291583, a novel, reversible CatC inhibitor, in adults with bronchiectasis.In total, 322 participants were randomised (2:1:1:2) to receive one of three oral doses of BI 1291583 (1 mg/2.5 mg/5 mg) or placebo for 24 to 48 weeks. A multiple comparison procedure and modelling approach was used to demonstrate a non-flat dose-response curve based on the time to first pulmonary exacerbation up to Week 48. In addition, efficacy of individual BI 1291583 doses was evaluated based on the frequency of exacerbations, severe exacerbations (fatal or leading to hospitalisation and/or intravenous antibiotic administration), lung function and quality of life.A significant dose-dependent benefit of BI 1291583 over placebo was established based on time to first exacerbation (shape: Emax; adjusted p-value: 0.0448). Treatment with BI 1291583 5 mg and 2.5 mg numerically reduced the risk of an exacerbation compared with placebo (hazard ratios: 0.71 and 0.66, 95% CIs 0.48-1.05 and 0.40-1.08; both p>0.05). BI 1291583 2.5 mg showed numerically better efficacy compared with 5 mg across several endpoints; 1 mg was similar to placebo. The safety profile of BI 1291583 was similar to placebo.Treatment with BI 1291583 resulted in a reduction in the risk of experiencing an exacerbation in adults with bronchiectasis.

Influence of raceway based biofloc system on the growth and physiological responses of Penaeus vannamei and GIF tilapia in a polyculture model – BFT aquaculture system

The present study investigates the efficiency of raceway-based biofloc technology for the production of Penaeus vannamei and Genetically Improved Farmed tilapia in a polyculture model. Six raceway tanks were used to explore the growth performance, digestive enzyme activities, bacterial count and histology of P. vannamei reared with GIF tilapia using raceway-based biofloc and clear water raceway culture systems. Each raceway (6.8 m x 2.2 m x 3 m) was stocked with 60 shrimp/m3 of P. vannamei (0.93±0.09 g), and 5 fish/m3 of GIF tilapia (0.42±0.01 g) and reared for 90 days. Biofloc, with a carbon: nitrogen ratio of 15:1, is developed and maintained using soyahull pellet powder as organic carbon source, with continuous aeration was provided. The present study found significantly higher weight gain of P. vannamei (16.09±0.26 g) and GIF tilapia (11.71±0.35 g) in raceway-based biofloc culture system. Survival of P. vannamei and GIF tilapia did not exhibit any significant variations between raceway-based biofloc and clear water raceway systems. Significantly higher digestive enzymes activities of protease (0.25±0.01 and 0.14±0.02 U/ mg protein/min), lipase (0.88±0.04 and 0.37±0.01 U/ mg protein/min) and amylase (0.008±0.00 and 0.0016±0.00 U/ mg protein/min) are recorded in P. vannamei and GIF tilapia in raceway-based biofloc culture system, respectively. Higher values of total heterotrophic bacteria (52.25±0.88×104 CFU/ml, 49.63±1.10 ×107 CFU/g and 53.12±0.44×107 CFU/g) and bacillus counts (76.50±0.46×102 CFU/ml, 155.62±0.89×102 CFU/g and 238.75±0.82×102 CFU/g) are recorded in culture water, P.vannamei gut and GIF tilapia gut in raceway based biofloc culture system, respectively. No histopathological changes are observed in the GIF tilapia (gut), P. vannamei (gut and hepatopancreas), but deformities such as congestion of the tips of few secondary lamellae are noticed in the GIF tilapia (gill) reared in the clear water raceway system. Therefore, the study suggests that polyculture of P. vannamei and GIF tilapia in a raceway-based biofloc system is advantageous over the clear water raceway system in terms of production augmentation with improved physiological conditions.

Study on the effect of ascorbic acid on the biosynthesis of pigment and citrinin in red yeast rice based on comparative transcriptomics.

Pigment is one of the most important metabolites in red yeast rice. However, citrinin may accumulate and cause quality security issues. In the present study, the effect of ascorbic acid (EAA) on the pigment and citrinin was studied, and the metabolic mechanism was discussed using comparative transcriptomics. The introduction of EAA increased the pigment by 58.2% and decreased citrinin by 65.4%. The acid protease activity, DPPH scavenging rate, and total reducing ability also increased by 18.7, 9.0, and 26.7%, respectively. Additionally, a total of 791 differentially expressed genes were identified, and 79 metabolic pathways were annotated, among which carbon metabolism, amino acid metabolism, and fatty acid metabolism were closely related to the biosynthesis of pigment and citrinin. Ethanol dehydrogenase (M pigC), oxidoreductase (M pigE), reductase (M pigH), and monooxygenase (M pigN) may be related to the increase of pigment. ctnC and pksCT contributed to the decline of citrinin.

Characterization of the extracellular proteases from Bacillus inaquosorum strain E1-8 and its application in the preparation of hydrolysates from plant and animal proteins with antioxidant, antifreeze and anti-browning properties.

Bacillus inaquosorum strains is widely recognized for their plant-growth-promoting and biocontrol capabilities, yet their roles in protease production remain unclear. The present study aimed to comprehensively assess the protease-producing performance of B. inaquosorum strain E1-8, at the same time as exploring the novel application of agricultural Bacillus proteases in the preparation of protein hydrolysates for fresh-cut fruits preservation. First, genomic sequencing revealed the diversity of E1-8 proteases, indicating 15 putative extracellular proteases. Subsequently, the fermentation conditions for E1-8 protease production were optimized, with sweet potato powder and soybean meal identified as the most suitable carbon and nitrogen sources, respectively, resulting in a maximum protease activity of 321.48 U mL-1. Upon culturing the strain under these optimized conditions, only an S8 family serine protease and an M48 family metalloprotease were revealed by secretomic analysis and protease inhibitor assays. Additionally, the optimal protease conditions for generating protein hydrolysates from soy, pea, fish and porcine proteins were determined. The molecular weight of the hydrolysates primarily ranged from 2000 to 180 Da, with a total of 17 amino acids identified. The application of these hydrolysates demonstrated a 2,2-diphenyl-1-picrylhydrazyl (i.e. DPPH) scavenging activity ranging from 58.64% to 84.12%, significantly reducing of the melting peaks and the freezing points. Furthermore, the browning index of apple slices stored at 4 °C decreased by 14.81% to 22.15% on the second day, and similar effects were observed in fresh-cut banana stored at 4 °C for 7 days. The protein hydrolysates obtained exhibit remarkable antioxidant, antifreeze and anti-browning properties for fresh-cut fruits. © 2024 Society of Chemical Industry.

Soil and root microbiome analysis and isolation of plant growth-promoting bacteria from hybrid buffaloberry (Shepherdia utahensis 'Torrey') across three locations.

The effects of climate change are becoming increasingly hazardous for our ecosystem. Climate resilient landscaping, which promotes the use of native plants, has the potential to simultaneously decrease the rate of climate change, enhance climate resilience, and combat biodiversity losses. Native plants and their associated microbiome form a holo-organism; interaction between plants and microbes is responsible for plants' growth and proper functioning. In this study, we were interested in exploring the soil and root microbiome composition associated with Shepherdia utahensis, a drought hardy plant proposed for low water use landscaping, which is the hybrid between two native hardy shrubs of Utah, S. rotudifolia and S. argentea. The bulk soil, rhizosphere, root, and nodule samples of the hybrid Shepherdia plants were collected from three locations in Utah: the Logan Campus, the Greenville farm, and the Kaysville farm. The microbial diversity analysis was conducted, and plant growth-promoting bacteria were isolated and characterized from the rhizosphere. The results suggest no difference in alpha diversity between the locations; however, the beta diversity analysis suggests the bacterial community composition of bulk soil and nodule samples are different between the locations. The taxonomic classification suggests Proteobacteria and Actinobacteriota are the dominant species in bulk soil and rhizosphere, and Actinobacteriota is solely found in root and nodule samples. However, the composition of the bacterial community was different among the locations. There was a great diversity in the genus composition in bulk soil and rhizosphere samples among the locations; however, Frankia was the dominant genus in root and nodule samples. Fifty-nine different bacteria were isolated from the rhizosphere and tested for seven plant growth-promoting (PGP) traits, such as the ability to fix nitrogen, phosphates solubilization, protease activity, siderophore, Indole Acetic Acid (IAA) and catalase production, and ability to use ACC as nitrogen source. All the isolates produced some amount of IAA. Thirty-one showed at least four PGP traits and belonged to Stenotrophomonas, Chryseobacterium, Massilia, Variovorax, and Pseudomonas. We shortlisted 10 isolates that showed all seven PGP traits and will be tested for plant growth promotion.

Interplanting potato with grapes improved yield and soil nutrients by optimizing the interactions of soil microorganisms and metabolites.

Interplanting crops is the best method to grow crops synergistically for better utilization of land and agro-resources. Grape (Vitis vinifera) and potato (Solanum tuberosum L.) have highly efficient agricultural planting systems in China, however, how soil physicochemical properties and soil microbial communities and metabolites affect the output of grape-potato interplanting remained unknown. In this study, we employed three planting patterns (CK: grape monocropping; YY: grape interplanted with potato (variety 'Favorita'); LS: grape interplanted with potato (variety 'Longshu7')) at two experimental sites i.e., the Huizhou (2022) site and the Qingyuan site (2023). The grape variety for all planting patterns was 'Sunshine Rose'. Soil samples (top 0-20 cm) at both sites were collected to observe the diversity of bacterial communities and soil metabolites. Our findings revealed that, compared with monocropping, the interplanted systems resulted in higher concentrations of total nitrogen, available phosphorus, and available potassium and enhanced the activities of acid phosphatase, urease, and protease. The potato root exudates also altered the relative abundance of Bacillus, Kaistobacter, and Streptomyces in the rhizosphere. Among the soil metabolites, lipids and organic acids showed the most significant changes. Notably, 13-L-hydroperoxylinoleic acid is the key differentially abundant metabolite involved in the regulation of linoleic acid metabolism pathways. The association analyses of the metabolome, microbiome, and soil physicochemical properties revealed that the interactions of microbes and metabolites resulted in differences in the soil nutrient content, whereas the interactions of 13-L-hydroperoxylinoleic acid and Firmicutes improved the soil nutrient levels and bacterial composition in the interplanting systems. In summary, our findings demonstrated that intercropping grapes with potato 'Favorita' was better with respect to improving soil nutrients, soil enzyme activity, the diversity of soil bacteria, and soil metabolites without causing adverse impacts on grape yield. Overall, this study explained the physiological mechanisms by which soil microorganisms and metabolites promote potato growth in grape interplanting and provided new perspectives for the utilization of soil resources in vineyards.

Bacillus cereus CGMCC 1.60196: a promising bacterial inoculant isolated from biological soil crusts for maize growth enhancement.

Soil microbial inoculants are widely recognized as an environmentally friendly strategy for promoting crop growth and increasing productivity. However, research on utilizing the microbial resources from desert biological soil crusts to enhance crop growth remains relatively unexplored. In the present work, a bacterial strain designated AC1-8 with high levels of amylase, protease, and cellulase activity was isolated from cyanobacterial crusts of the Tengger Desert and identified as Bacillus cereus (CGMCC 1.60196). The refinement of the fermentation parameters of B. cereus CGMCC 1.60196 determined that the most effective medium for biomass production was composed of 5 g/L glucose, 22 g/L yeast extract and 15 g/L MgSO4, and the optimal culture conditions were pH 6.0, temperature 37°C, inoculation quantity 3% and agitation speed 240 rpm. Furthermore, the utilization of B. cereus CGMCC 1.60196 has resulted in substantial improvements in various growth parameters of maize seedlings, including shoot length, shoot fresh and dry weights, root fresh and dry weights, and the contents of chlorophyll a, chlorophyll b, and total chlorophyll. The most pronounced growth promotion was observed at an application concentration of 1 × 109 CFU/m2. These results suggest that the novel B. cereus strain, isolated from cyanobacterial crusts, can be regarded as an exemplary biological agent for soil improvement, capable of enhancing soil conditions, promoting crop cultivation and supporting food production.

The Effect of the Lysine Acetylation Modification of ClpP on the Virulence of Vibrio alginolyticus.

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus.

Dose-response curves for the effects of Lactobacillus plantarum on growth performance, feed utilization, and health status of Litopenaeus vannamei shrimp. Optimizing the economic efficiency of supplementation

Abstract A 14-weeks feeding trial was conducted to employ polynomial regression analysis to establish the optimal dosage of Lactobacillus plantarum (Lac) for enhancing the growth performance, digestive enzyme activities, and blood biochemical, redox balance, and immunity response of Litopenaeus vannamei shrimp (initial body weight = 2.94 ± 0.03 g). A total of 240 healthy Litopenaeus vannamei shrimp were randomly distributed into four equal groups and were fed diets containing 0, 200, 400 and 800 mg Lac /kg diet respectively for 98 days. Increasing the levels of dietary Lac cubically improved growth performance and feed utilization ( p &lt;0.01), the optimal doses were established at 600 and 650 mg Lac/kg diet, respectively. Muscles thickness decreased significantly in all treated group compared to the control ( p &lt;0.05). The dietary treatment quadratic ally affected total protein ( p &lt;0.0001), tri-glycerides ( p &lt;0.0001), and cortisol ( p =0.0097), the optimal responses were observed at 650, 700, and 600 mg Lac/kg diet, respectively. Meanwhile the activities of liver enzymes (ALT and AST), the levels of blood urea and digestive enzymes (amylase and proteases) were cubically enhanced by the treatment, the optimal dosages were at 600 and 650 mg Lac/kg diet for liver enzymes, and urea concentration, respectively and at 650 and 700 mg Lac/kg diet for the activities of amylase and protease, respectively. With regards to redox balance, increasing the levels of Lac caused a quadratic decrease in the levels of malondialdehyde ( p =0.0398) and a cubic increase in the activities of superoxide dismutase ( p =0.0265), and catalase ( p =0.0163), the corresponding dose–response curves showed that the optimal dose was at 650 mg/kg diet. However, the levels of total antioxidant capacity were in a quadratic increase ( p =0.0372), maximizing at a level of 600 mg Lac / kg diet. Concerning the immunity response, both of lysozyme and IgM significantly affected by the dietary treatment ( p =0.0002 and 0.0001, respectively), maximizing at 600 and 650 mg Lac/kg diet, respectively. Dietary supplementation of Lac had significant and substantial impacts on the economic efficiency ( p &lt;0.0001). In conclusion, the dietary inclusion of 600-700 mg Lac /kg diet can be used as an effective and practical feeding strategy to enhanced growth performance, feed efficacy, redox balance and nonspecific immune responses in Litopenaeus vannamei shrimp.