Alkaline Protease Activity Research Articles

Effects of water temperature on growth, health and digestive processes of the thicklip grey mullet Chelon labrosus

This study aimed to identify the effects of water temperature on the rearing of the omnivorous teleost Chelon labrosus and to determine the optimal temperature for the culture of this interesting candidate for the diversification of European aquaculture. The fish (initial weight 26.81 ± 6.2 g) were held at four water temperatures (18, 22, 26 and 30 °C) in triplicate for 90 days in 100 L recirculating tanks (n = 14 / tank). The effects of said temperatures were assessed on growth, intestinal health and the digestive processes of the fish. At 18 °C, energy consumption was low, leading to a high accumulation of reserves and slow growth, a typical overwintering strategy. The best growth results were obtained at 22 °C, followed closely by 26 °C, which resulted in low energy reserves. The fact that the fish at 26 °C grew less than the ones at 22 °C while having similar energy consumption, suggested that this temperature was close to the end of the optimal range, but no signs of stress were detected. However, at 30 °C the fish had lower energy reserves but grew significantly less, which can be considered indicative of stress. Health impairment at this temperature was confirmed by the epithelial lesions found in this group. The growth and energy availability of the different groups is discussed in the light of the activities of α-amylase, pepsin and alkaline proteases. As an integration of all these results, a quadratic regression model resulting from growth values (SGR) in relation to water temperature was performed, and it allowed predicting an actual optimal water temperature of 22.8 °C for growth of C. labrosus. This is the first assessment of the optimal water temperature for the culture of C. labrosus, and it will be highly valuable for the development of its intensive culture.

Variability of soil enzyme activities and nutrients with forest gap renewal interacting with soil depths in degraded karst forests

Forest gaps serve as crucial starting points for forest self-renewal. Within these gaps, soil enzymes play an essential role in driving nutrient transformation and species renewal. However, how forest gap renewal regulates soil fertility through soil enzyme across various soil depths remains unclear. This investigation delved into the dynamics of soil enzymes and nutrients within karst forest gaps at four developmental age stages: Infancy (<10 years), Youth (10–20 years), Middlescence (20–30 years), and Elder (>30 years) across different soil depths (0–10 cm, 10–20 cm, and 20–30 cm). The study determined the contents of CNP and the activities of soil enzymes such as polyphenol oxidase, catalase, protease, and alkaline phosphatase. Two-way ANOVA was employed to assess the variations in gap ages and soil depths, along with their interaction. Redundancy analysis (RDA) was used to reveal the correlations between soil enzyme activities and CNP fertility indicators. The results indicated that gap age significantly influenced the activities of alkaline phosphatase, polyphenol oxidase, and protease, as well as the contents of total and available nitrogen, phosphorus, and organic carbon, alongside the ratios of N/P, C/N, and C/P. Catalase activity, however, did not vary significantly with gap age, although soil depth influenced these parameters, excluding total phosphorus and the C/N ratio. Notably, in the 0–10 cm soil layer, alkaline phosphatase activity, total nitrogen, available nitrogen, and organic carbon contents significantly increased with gap age increase, followed by a significant decrease after the middlescence stage. In the 10–20 cm and 20–30 cm soil layers, Alkaline phosphatase and polyphenol oxidase activities, along with N/P and C/N ratios initially decreased and then increased with gap age increase. During the Infancy, Youth, and Middlescence stages, Polyphenol oxidase and protease activities, CNP contents, as well as N/P and C/P ratios generally decreased with soil depth increase or after an increase in the 10–20 cm soil layer. RDA highlighted the variational positive and negative correlations between soil enzyme activities and CNP fertility indicators across soil depths during forest gap renewal, with explanatory rates ranging from 88.05 % to 94.67 % across soil layers. Overall, this study suggests that forest gap renewal significantly influences soil enzymes, CNP content and stoichiometry, modifying soil CNP content through stoichiometric regulation across different soil depths, and ultimately sustaining soil fertility via soil enzymes.

The effect of various dietary MUFA content and n-6/n-3 ratios on rainbow trout (Oncorhynchus mykiss) fed on a whole plant protein based diet: growth performance, digestive enzymes activity, intestinal morphometry and fatty acids profile of muscle and liver.

The effect of diets with various dietary MUFA content and n-6/n-3 ratios, diets 1 (55.33, 1.00), 2 (25.30, 1.00), 3 (55.13, 2.05), 4 (24.92, 2.03), 5 (54.94, 8.06) and 6 (24.91, 8.06) and a control diet with fish oil (FO), diet 7 (33.60, 5.97), was studied on growth and digestive physiology of rainbow trout, Oncorhynchus mykiss. Juveniles, 14.65g ± 0.17, were fed the experimental diets for eight weeks. Those fish fed diet 1 (55.33, 1.00) had the lowest growth and nutritional indices, while fish fed diet 6 (24.91, 8.06) possessed the highest indices. However, fish fed D7 (33.60, 5.97) was intermediate in this regard. Proximate body composition results revealed no significant differences among various experimental groups (P > 0.05). Fish fed on diet 1 possessed the highest alkaline protease activity, while fish raised on diet 2 showed the lowest enzyme activity (P < 0.05). Fish fed on diets 1, 3 and 4 had the highest lipase activity (P > 0.05). Amylase activity and intestinal parameters did not differ among groups (P > 0.05). MUFA contents of liver and muscle reflected the MUFA contents of the diets; however, liver PUFA contents was not affected by dietary PUFA contents (P < 0.05). However, 18:3n-3 contents of liver decreased as dietary n-6/n-3 ratio increased (P < 0.05). Furthermore, the liver 20:4n-6 contents considerably increased as dietary n-6/n-3 increased to 8.06 (P < 0.05). Muscle 22:6n-3 content of groups 1 to 6 was lower than group 7 (P < 0.05). In conclusion, rainbow trout was considerably capable of de novo synthesis of 22:6n-3. Meanwhile, increasing dietary n-6/n-3 ratio to 8.06 decreased the muscle 22:6n-3 content which requires further considerations regarding fish immune competence and nutritional quality for human consumption.

Intra-cohort cannibalism and aggressive behavior in tropical gar (Atractosteus tropicus) larvae: Effects of stocking density and starvation

Cannibalism in early fish developmental stages is a biological phenomenon affecting aquaculture, so seeding density and feeding schemes play a fundamental role in its optimal control. The objective of this research was to evaluate two initial stocking densities, 0.7 larvae/L (D7) and 1.4 larvae/L (D14), which were fed (F) and not fed (NF) in tropical gar (Atractosteus tropicus) larvae on growth, attacks, survival, and digestive enzyme activity, digestive morphology, and oxidative enzymes and aggressiveness gene expression. A total of 420 larvae (0.022 ± 0.003 g; 1.52 ± 0.10 cm) were placed in 70 L tanks connected to a recirculation system. Treatment F larvae were fed a formulated food. D14F larvae had the highest final weight, total length, and specific growth rate (SGR) (p < 0.05). The highest survival percentage was in D7F larvae (46.19 ± 2.74) (p < 0.05). The highest frequency of attacks occurred in D14F larvae (26.66 ± 0.82) (p < 0.05). The coefficient of variation (CV) and size heterogeneity (SH) were higher in D7F larvae (13.56 ± 1.80; 0.81 ± 0.10) (p < 0.05). The F larvae started attacks at 8 days after hatching (DAH), while the NF larvae showed it at 10 DAH. The difference in weight between the attacker and the attacked was 16.44 ± 9.75% and in total length 12.28 ± 4.57% (p < 0.05). Acid and alkaline protease, trypsin, and lipase activities presented differences between both factors, as well as the interaction between them. D14F larvae presented the highest Glutathione peroxidase (GPX) activity (p < 0.05), while D7-NF larvae registered the highest catalase (CAT) activity (p < 0.05). Superoxide dismutase (SOD) activities only showed differences between F and NF larvae (p < 0.05). The liver presented the highest percentage of area with melanomacrophage centers (MMC) in D7-NF larvae, and hepatocytes with the highest area were recorded in D14F larvae (p < 0.05). In gut, the highest enterocytes were recorded in D14F larvae (p < 0.05). cat, cck, and gpx expression did not show difference between feeding regimes. The expression of sod1 and sod2 in D7F and D14F treatments presented the highest expressions (p < 0.05). D7F larvae presented higher expression of avp1, crh, htr1, sstr1, th and tph1 genes (p < 0.05). Our results show that A. tropicus larvae D14F presented the highest growth and morphometric parameters and decreased CV and SH, which shows a more homogeneous growth. Regarding cannibalism, fed larvae (D7F, D14F) presented cannibalism in a greater proportion than unfed larvae regardless of density, however, it is necessary to analyze other physiological and social factors that could be related to the aggressiveness and cannibalism of the species.

Mineral Components, Organic Matter Quality and Soil Enzymatic Activity under the Influence of Differentiated Farmyard Manure and Nitrogen Fertilisation

Research was carried out on the impact of long-term use of cattle manure (30 t ha−1 FYM) and various doses of N (0, 40, 60 and 120 t ha−1) in the form of ammonium nitrate on the following soil parameters: salinity, hydrolytic acidity, total exchangeable base cations, cation exchange capacity, degree of base saturation of the sorption complex, total organic carbon and total nitrogen content, dissolved organic matter, fractional composition of organic matter and content of bioavailable macroelements: phosphorus, potassium and magnesium in the soil of a multi-year static field experiment. The activities of dehydrogenases, catalase, alkaline and acid phosphatase and proteases were also tested. A significant effect of FYM and N fertilisation on the content of bioavailable macroelements was found. The application of manure at a dose of 30 t ha−1 mitigated the negative effects of the application of N at a dose of 120 kg ha−1. A higher content of total organic carbon (8.42 g kg−1) and humic acid fraction (1761 mg kg−1) and higher values of the CHA/CFA ratio (0.79; parameters that are indicators of soil quality) were found in the soil fertilised with manure compared to the soil without manure added (TOC—7.00 g kg−1; CHAs—1285 mg kg−1; CHAs/CFAs 0.66). The activity of the tested enzymes was also significantly determined by the applied fertilisation. Enzyme activity was highest in the soil to which manure had been applied. Nitrogen fertilisation varied in its impact on the activity of enzymes according to the specifics of each enzyme. The content of humic acids and CHA/CFA values correlated positively with the content of soil minerals and the activity of dehydrogenases, catalase, alkaline and acid phosphatase and GMea and TEI indices. Dehydrogenases and acid phosphatase can be considered enzymes that take part in transforming organic matter towards the formation of FAs.

Biodegradations of three different rank coals by a newly isolated bacterium Bacillus sp. XK1

Biodegradation of coal was closely related to coal rank. In this study, three kinds of coals whose coal ranks from high to low were Zhundong coal (ZDC), Hongshaquan coal (HSQC) and Heishan coal (HSC) were degraded by Bacillus sp. XK1 isolated from mine water. The degradation experiment results indicated that the degradation rate increased with the decrease of coal rank, and the highest degradation rate of HSC (low-rank coal) was 78.2 %, which was three times higher than that of ZDC (high-rank coal). The enzyme activity experiment showed that the alkaline protease and lignin peroxidase secreted by Bacillus sp. XK1 were key substances for coal biodegradation, and the alkaline protease activity and lignin peroxidase activity were all the highest in the presence of HSC, which were beneficial for coal biodegradation. The adsorption experiment found that HSC was more favorable to contact with Bacillus sp. XK1 than ZDC and HSQC. Furthermore, biodegradation products analysis suggested that with the decrease of coal rank, the contents of long chain alkanes in liquid products increased, among which the highest content of long chain alkane in liquid product of HSC was 86.98 %. The above experimental results demonstrated that biodegradation of low rank coal was an effective pathway.

Growth, survival, digestive activity and respiratory metabolism of Octopus maya juveniles fed with prepared diets.

The effects of two prepared feeds were tested on growth, survival, enzymatic activity, nutritive reserves in the digestive gland and oxygen consumption of Octopus maya juveniles. For the first time, a semihumid paste (HD, control) and a dry diet, in pelleted form (PD, experimental) with the same formulation were used for this species. The experiment lasted for 42 days. Results indicate that growth rates were similar for both diets (p > 0.05); however, survival (70%) was higher with the PD compared to the HD (48%) (p < 0.05). The performance index was higher for octopuses fed the PD (p < 0.05). No differences in acid proteases activity were observed. However, a higher activity of alkaline proteases in the octopuses fed the PD was observed (p < 0.05). Ingestion rate was higher for octopuses fed the PD. Routine energy inversion was similar in both treatments (p > 0.05). A greater energy inversion was observed in octopuses fed the PD, whose active metabolism was double compared to the octopuses fed the HD. Results showed that the PD promoted similar growth compared to the HD diet but favored survival, and a greater investment in the active metabolism, reflected in the apparent heat increase.

Changes in growth, morphology, and levels of digestive enzymes and growth-related hormones in early ontogeny of black scraper, Thamnaconus modestus

IntroductionThe black scraper, Thamnaconus modestus, is a highly valued marine fish species, but its output has gradually decreased in recent years, which may be due to its low survival rate during early ontogenesis.MethodsTherefore, in this study, we assessed the changes in growth, morphology, digestive enzymes, and hormone levels in T. modestus from 0–60 days post-hatching (dph) and revealed growth turning points by morphological measurement and determination of digestive enzyme activities and hormone levels. We found that ontogenesis could be divided into the larval (0–20 dph) and juvenile (20–60 dph) stages. Acid and alkaline protease activity significantly increased and decreased, respectively, from 12–25 dph, likely due to the development of stomach and gastric glands. Acid phosphatase levels significantly increased at 0 and 4 dph, which may be related to the regulation of metabolism and immune protection. A sharp increase in alkaline phosphatase levels at 20 and 25 dph was observed and was likely due to the development of the brush border membrane of enterocytes. The amylase level was significantly higher at 25, 30, and 35 dph, possibly due to better digestion and absorption during the transition from consuming Artemia to compound feed. In newly hatched larvae, the level of thyroid hormones triiodothyronine (T3) and thyroxine (T4) gradually increased and peaked at 35 dph, highlighting the importance of these hormones during the development of T. modestus. Growth hormone (GH) levels first increased from 0–8 dph, with a plateau at 8–20 dph, and then increased at 25–30–35 dph. For insulin-like growth factor 1 (IGF-1), a significant increase with a subsequent plateau was observed between 8 and 20 dph, followed by a substantial decrease between 30 and 35 dph. These results suggest that the regulating functions of GH and IGF-1 are synchronised. Digestive enzyme activity and hormone levels of abnormal fry at 30 dph were lower than those of normal fish, highlighting the importance of specific hormones, especially T4 and IGF-1, in the development of T. modestus.

Digestive proteases of Morelet’s crocodile (Crocodylus moreletii) in three life stages

Introduction: Morelet’s crocodile (Crocodylus moreletii) is a species distributed in the Mexican southeast and threatened due to multiple pressures. Objective: To characterize the digestive proteases in the acid phase (stomach) and alkaline phase (intestine) of three life stages of C. moreletii in captivity (hatchling, juvenile, and adult). Methods: Total alkaline and acid protease activities were quantified using casein and haemoglobin as substrates. Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were quantified using synthetic substrates. Protease profiles were analysed by SDS-PAGE and Native-PAGE. Results: The specific activity of acid and alkaline proteases showed differences between the three stages, finding the highest activity in the juveniles. Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were higher in hatchlings. There were differences in optimum pH and temperature of acid and alkaline proteases, trypsin, and leucine aminopeptidase between the three stages, demonstrating the diversification of the enzymes according to different stages, as well as the presence of specific isoforms in each stage of C. moreletii. The acid phase zymogram showed four bands with pepsin-like acid activity in the hatchling and juvenile crocodile, while in the adult only two of the four bands were detected. The alkaline zymogram showed that the hatchling had the highest number of activity bands compared to the other stages, corresponding to the high specific activity reported in the alkaline phase. Conclusions: Digestive proteases of Morelet’s crocodile differ in their biochemical characteristics and the number of proteases between hatchling, juvenile, and adult. This could help in the future design of balanced diets as well to the sustainable management and production of this species.

Magnesium oxide nanoparticles reduce clubroot by regulating plant defense response and rhizosphere microbial community of tumorous stem mustard (Brassica juncea var. tumida).

Clubroot, caused by Plasmodiophora brassicae, is a major disease that significantly impairs the yield of cruciferous crops and causes significant economic losses across the globe. The prevention of clubroot, especially in tumorous stem mustard (without resistant varieties), are is limited and primarily relies on fungicides. Engineered nanoparticles have opened up new avenues for the management of plant diseases, but there is no report on their application in the prevention of clubroot. The results showed that the control efficacy of 500 mg/L MgO NPs against clubroot was 54.92%. However, when the concentration was increased to 1,500 and 2,500 mg/L, there was no significant change in the control effect. Compared with CK, the average fresh and dry weight of the aerial part of plants treated with MgO NPs increased by 392.83 and 240.81%, respectively. Compared with the F1000 treatment, increases were observed in the content of soil available phosphorus (+16.72%), potassium (+9.82%), exchangeable magnesium (+24.20%), and water-soluble magnesium (+20.64%) in the 1,500 mg/L MgO NPs treatment. The enzyme-linked immune sorbent assay (ELISA) results showed that the application of MgO NPs significantly increased soil peroxidase (POD, +52.69%), alkaline protease (AP, +41.21%), alkaline phosphatase (ALP, +79.26%), urease (+52.69%), and sucrase (+56.88%) activities; And also increased plant L-phenylalanine ammonla-lyase (PAL, +70.49%), polyphenol oxidase (PPO, +36.77%), POD (+38.30%), guaiacol peroxidase (POX, +55.46%) activities and salicylic acid (SA, +59.86%) content. However, soil and plant catalase (CAT, -27.22 and - 19.89%, respectively), and plant super oxidase dismutase (SOD, -36.33%) activities were significantly decreased after the application of MgO NPs. The metagenomic sequencing analysis showed that the MgO NPs treatments significantly improved the α-diversity of the rhizosphere soil microbial community. The relative abundance of beneficial bacteria genera in the rhizosphere soil, including Pseudomonas, Sphingopyxis, Acidovorax, Variovorax, and Bosea, was significantly increased. Soil metabolic functions, such as oxidative phosphorylation (ko00190), carbon fixation pathways in prokaryotes (ko00720), indole alkaloid biosynthesis (ko00901), and biosynthesis of various antibiotics (ko00998) were significantly enriched. These results suggested that MgO NPs might control clubroot by promoting the transformation and utilization of soil nutrients, stimulating plant defense responses, and enriching soil beneficial bacteria.

Evaluation of extracellular alkaline proteases from Bacillus for environmentally friendly detergent additives

Growing demand for environmentally friendly and sustainable enzyme solutions drives markets for alkaline proteases across various industries. Alkaline proteases produced by different Bacillus strains exhibit unique properties and versatility. Production of alkaline proteases from Bacillus amyloliquefaciens TBRC 2902, B. siamensis TBRC 1180, B. subtilis TBRC 6663, and B. velezensis TBRC 7773 was optimized by adjusting production media, considering carbon and nitrogen sources to enhance extracellular enzyme activities. Optimized extracellular protease activity was achieved using tapioca starch for TBRC 2902 and TBRC 1180, and soluble starch for TBRC 7773, while both carbon sources were optimal for TBRC 6663. Skim milk was an equally effective nitrogen source for TBRC 2902, TBRC 1180, and TBRC 7773, whereas soytone was as effective as yeast extract for TBRC 6663. Inorganic nitrogen sources, such as diammonium hydrogen phosphate for TBRC 2902 and potassium nitrate for TBRC 1180, enhanced alkaline protease activity by 10-20%. The protease from B. siamensis TBRC 1180 exhibited optimal pH and temperature values of 9.0 and 60°C, respectively, while the others had optimal values of 8.0 and 50°C. All enzymes tolerated non-ionic surfactants, retaining over 40% activity after 24 hours exposure to Triton X-100, Tween-20, and Tween-80, indicating their potential as detergent additives.

Enhanced anaerobic digestion of human feces by ferrous hydroxyl complex (FHC): Stress factors alleviation and microbial resistance improvement

Anaerobic digestion (AD) offers a reliable strategy for resource recovery from source-separated human feces (HF), but is limited by a disproportionate carbon/nitrogen (C/N) ratio. Ferrous hydroxyl complex (FHC) was first introduced into the HF-AD system to mediate methanogenesis. Mono-digestion of undiluted HF was inhibited by high levels of volatile fatty acids (VFAs), ammonia, and hydrogen sulfide (H2S). FHC addition at optimum dosage (500–1000 mg/L) increased the cumulative methane (CH4) yield by 22.7%, enhanced the peak value of daily CH4 production by 60.5%, and shortened the lag phase by 24.7%. H2S concentration in biogas was also greatly decreased by FHC via precipitation. FHC mainly facilitated the hydrolysis, acidification, and methanogenesis processes. The production and transformation of VFAs were optimized in the presence of FHC, thus relieving acid stress. FHC elevated the activities of alkaline protease, cellulase, and acetate kinase by 32.3%, 18.2%, and 30.3%, respectively. Microbial analysis revealed that hydrogenotrophic methanogens prevailed in mono-digestion at high HF loading but were weakened after FHC addition. FHC also enriched Methanosarcina, thereby expanding the methanogenesis pathway and improving the resistance to ammonia stress. This work would contribute to improving the methanogenic performance and resource utilization for HF anaerobic digestion.

The fermentation optimization for alkaline protease production by Bacillus subtilis BS-QR-052.

Proteases exhibit a wide range of applications, and among them, alkaline proteases have become a prominent area of research due to their stability in highly alkaline environments. To optimize the production yield and activity of alkaline proteases, researchers are continuously exploring different fermentation conditions and culture medium components. In this paper, the fermentation conditions of the alkaline protease (EC 3.4.21.14) production by Bacillus subtilis BS-QR-052 were optimized, and the effect of different nutrition and fermentation conditions was investigated. Based on the single-variable experiments, the Plackett-Burman design was used to explore the significant factors, and then the optimized fermentation conditions, as well as the interaction between these factors, were evaluated by response surface methodology through the Box-Behnken design. The results showed that 1.03% corn syrup powder, 0.05% MgSO4, 8.02% inoculation volume, 1:1.22 vvm airflow rate, as well as 0.5% corn starch, 0.05% MnSO4, 180 rpm agitation speed, 36°C fermentation temperature, 8.0 initial pH and 96 h incubation time were predicted to be the optimal fermentation conditions. The alkaline protease enzyme activity was estimated to be approximately 1787.91 U/mL, whereas subsequent experimental validation confirmed it reached 1780.03 U/mL, while that of 500 L scale-up fermentation reached 1798.33 U/mL. This study optimized the fermentation conditions for alkaline protease production by B. subtilis through systematic experimental design and data analysis, and the activity of the alkaline protease increased to 300.72% of its original level. The established model for predicting alkaline protease activity was validated, achieving significantly higher levels of enzymatic activity. The findings provide valuable references for further enhancing the yield and activity of alkaline protease, thereby holding substantial practical significance and economic benefits for industrial applications.

Sustainable utilization of feather waste to produce alkaline serine protease by genetically engineered Bacillus amyloliquefaciens

In this study, the efficient production of alkaline serine protease from engineered Bacillus amyloliquefaciens was achieved using feather meal as nitrogen sources. By screening of signal peptides library, a recombinant strain HZ12/SP2 with alkaline protease activity of 399.00 U/mL was constructed, which was 357% higher than the control strain HZ12/SP0. Furthermore, the alkaline protease activity was increased by 47%, up to 586.73 U/mL by deletion of the genes pssA, clsA, and sigF. Finally, the feather meal was used as nitrogen sources, and the strain ΔFCP/SP2 produced a maximum protease activity of 688.42 U/mL, which was 580% higher than the strain HZ12/SP0. In conclusion, an efficient alkaline serine protease-expression strain was constructed, which realized the valuable reuse of feather waste as nitrogen sources.

Effects of Dietary Sodium Propionate on Growth, Digestive Enzyme Activity, and Expression of Immune System Genes in Juveniles of Tropical Gar (Atractosteus tropicus)

We determined the effects of sodium propionate (SP) added to the diets of Atractosteus tropicus juveniles with respect to the growth, survival, digestive enzyme activity, and expression of genes that are associated with the immune system. Five treatments (0, 0.5, 1.0, 1.5, and 2.0%) were evaluated in triplicate on 180 fish (3.65 ± 0.12 g) distributed among 15 (70 L) tanks. The juveniles were fed five times a day with 5% feed in relation to the biomass of the organism. The treatment with 0.5% SP showed a final weight value of 25.7 ± 4.5 g, absolute weight of gain (AWG) of 21.93 ± 4.39 g, and specific growth rate (SGR) of 3.1 ± 0.26. Treatments with 1.5 and 2.0% SP showed the highest survival (91.6%). The control group (0%) showed a greater activity of lipases. There was a tendency that the highest activity of alkaline proteases and chymotrypsin occurred in the 0 and 0.5% treatments. The maximum relative expression of the genes ocln, muc2, and nod2 occurred in the 1.5% treatment. The inclusion of SP in the diet of A. tropicus juveniles could benefit the activity of some digestive enzymes as well as the expression of genes related to the function of the intestinal barrier, therefore benefitting the survival of the organisms.

Quantification of thermo-halotolerant alkaline protease activity derived from Bacillus licheniformis strains isolated from extreme environments in Morocco

Quantification of thermo-halotolerant alkaline protease activity derived from Bacillus licheniformis strains isolated from extreme environments in Morocco

Impact of residual antibiotics on microbial decomposition of livestock manures in Eutric Regosol: Implications for sustainable nutrient recycling and soil carbon sequestration

The land application of livestock manure has been widely acknowledged as a beneficial approach for nutrient recycling and environmental protection. However, the impact of residual antibiotics, a common contaminant of manure, on the degradation of organic compounds and nutrient release in Eutric Regosol is not well understood. Here, we studied, how oxytetracycline (OTC) and ciprofloxacin (CIP) affect the decomposition, microbial community structure, extracellular enzyme activities and nutrient release from cattle and pig manure using litterbag incubation experiments. Results showed that OTC and CIP greatly inhibited livestock manure decomposition, causing a decreased rate of carbon (28%–87%), nitrogen (15%–44%) and phosphorus (26%–43%) release. The relative abundance of gram-negative (G-) bacteria was reduced by 4.0%–13% while fungi increased by 7.0%–71% during a 28-day incubation period. Co-occurrence network analysis showed that antibiotic exposure disrupted microbial interactions, particularly among G- bacteria, G+ bacteria, and actinomycetes. These changes in microbial community structure and function resulted in decreased activity of urease, β-1,4-N-acetyl-glucosaminidase, alkaline protease, chitinase, and catalase, causing reduced decomposition and nutrient release in cattle and pig manures. These findings advance our understanding of decomposition and nutrient recycling from manure-contaminated antibiotics, which will help facilitate sustainable agricultural production and soil carbon sequestration.

Mycolytic effect and underlying mechanism of Chaetomium subaffine LB‐1 on phytopathogenic fungi via morphological, physio‐biochemical and transcriptomic analysis

AbstractChaetomium subaffine LB‐1 is a biocontrol strain screened by our laboratory. Mycolytic effect and its action mechanism was researched in this study with Botrytis cinerea and Alternaria solani as test phytopathogenic fungi. Microscopic observation found that hyphae of the test fungi exhibited vacuolated and flocculent when cultured in LB‐1 culture filtrate for 48 h. Physio‐biochemical detection indicated that hyphae of the test fungi displayed red fluorescence with propidium iodide (PI) staining when cultured in LB‐1 culture filtrate 24 h later; conductivity and the leakage value of the extracellular fluid of the test fungi were significantly higher (p ≤ .05) than corresponding control fluid of test fungi cultured in potato dextrose broth (PDB), but the malondialdehyde (MDA) contents in hyphae of the test fungi have not significant difference (p ≤ .05) between treatment and control. Colorimetric determination showed that the activities of alkaline protease, neutral protease, acidic protease and β‐l,3‐glucanase in LB‐1 culture filtrate were significant. In RNA‐seq analysis, 3644 differentially expressed genes (DEGs) were found in 6 days shaking‐cultured LB‐1 as compared to that of 0 days shaking‐cultured, of which 1756 were up‐regulated and 1888 were down‐regulated. In significantly up‐regulated DEGs, 12 were annotated as cell wall hydrolase, 13 as enzyme of antibiotic substance production and 18 as membrane transporter. The above results indicated that C. subaffine LB‐1 might exert mycolytic effect through altering the permeability of cell membrane and the integrity of cell wall of phytopathogenic fungi, and this altering effect was closely related with cell wall hydrolase, antifungal substance and membrane transporter.

Purification and Characterization of Alkaline Protease Isolated from Cotton (Gossypium hirsutum) Seeds

Proteases are widely utilized both in physiological and commercial fields such as medicine, food, detergent, and leather. Plant-originated proteases play a significant role in several biomedical fields due to their easy accessibility and activity. Pakistan is an agro-based country and can be an ideal place for the isolation of industrially important proteases from plant sources such as cotton, which is the main crop and frequently available and low cost. Purification of protease was carried out by fractionation with two-fold acetone, ethanol, methanol and various concentrations (40-80%) of ammonium sulphate. The precipitates formed were collected after centrifugation and dialyzed for 24 hours against universal buffer pH 7.0 and was centrifuged in a cooled refrigerated. The dialyzed sample was loaded on Sephadex G–100 gel column. The fractions of the samples were collected and their absorbance of protein was monitored at 280 nm. The homogeneity of the purified enzyme was checked by SDS gel electrophoresis The purified protease enzyme has optimum activity at 30°C and pH 8.0 when casein was used as substrate. The Km and Vmax values of purified cotton seed's alkaline protease activity was recorded as 0.03M and 17 μmol/minute respectively. Protease activity was increased by the addition of cysteine but inhibited by Iodoacetic acid and β-Mercaptoethanol and decreased with some metal ions. These characteristics of the purified enzyme allowed classifying it as a cysteine protease. In conclusion, this study suggests that the alkaline protease enzyme is the best choice for commercial use

Genomic Characteristics and Comparative Genomics Analysis of the Endophytic Fungus Paraphoma chrysanthemicola DS-84 Isolated from Codonopsis pilosula Root.

Paraphoma chrysanthemicola is a newly identified endophytic fungus. The focus of most studies on P. chrysanthemicola has been on its isolation, identification and effects on plants. However, the limited genomic information is a barrier to further research. Therefore, in addition to studying the morphological and physiological characteristics of P. chrysanthemicola, we sequenced its genome and compared it with that of Paraphoma sp. The results showed that sucrose, peptone and calcium phosphate were suitable sources of carbon, nitrogen and phosphorus for this strain. The activities of amylase, cellulase, chitosanase, lipase and alkaline protease were also detected. Sequencing analysis revealed that the genome of P. chrysanthemicola was 44.1 Mb, with a scaffold N50 of 36.1 Mb and 37,077 protein-coding genes. Gene Ontology (GO) annotation showed that mannose-modified glycosylation was predominant in monosaccharide utilisation. The percentage of glycoside hydrolase (GH) modules was the highest in the carbohydrate-active enzymes database (CAZy) analysis. Secondary metabolite-associated gene cluster analysis identified melanin, dimethylcoprogen and phyllostictine A biosynthetic gene clusters (>60% similarity). The results indicated that P. chrysanthemicola had a mannose preference in monosaccharide utilisation and that melanin, dimethylcoprogen and phyllostictine A were important secondary metabolites for P. chrysanthemicola as an endophytic fungus.