Maximum Protease Activity Research Articles

Potential of the Liquid Fermentation of Fishery Waste by Paenibacillus elgii for Metalloprotease Production

This study attempted to use fishery processing wastes to produce protease by Paenibacillus elgii TKU051. Of the tested wastes, tuna head powder (THP) was found to be the most effective carbon and nitrogen (C/N) source, and the optimal conditions were as follows: 0.811% THP, 0.052% K2HPO4, 0.073% MgSO4, initial pH of 8.96, incubation temperature of 31.4 °C, and incubation time of 3.092 days to achieve the maximum protease activity of 2.635 ± 0.124 U/mL. A protease with a molecular weight of 29 kDa was purified and biochemically characterized. Liquid chromatography with tandem mass spectrometry analysis revealed an amino acid sequence of STVHYSTR of P. elgii TKU051 protease, suggesting that the enzyme may belong to the M4 family of metalloproteases. The optimal activity of the enzyme was achieved at 60 °C and pH 8. P. elgii TKU051 protease was strongly inhibited by ethylenediaminetetraacetic acid and 1,10-phenanthroline, indicating its precise metalloprotease property. P. elgii TKU051 protease displayed the activity toward casein and raw fishery wastes such as tuna heads, tuna viscera, shrimp heads, and squid pens. Finally, the purified P. elgii TKU051 protease could improve the free-radical scavenging activity of fishery wastes. In short, P. elgii TKU051 has potential application in eco-friendly approaches to efficiently convert fishery wastes to metalloprotease.

Production, Optimization, and Partial Purification of Alkali-Thermotolerant Proteases from Newly Isolated Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12

Proteases that can remain active under extreme conditions such as high temperature, pH, and salt concentration are widely applicable in the commercial sector. The majority of the proteases are rendered useless under harsh conditions in industries. Therefore, there is a need to search for new proteases that can tolerate and function in harsh conditions, thus improving their commercial value. In this study, 142 bacterial isolates were isolated from diverse alkaline soil habitats. The two highest protease-producing bacterial isolates were identified as Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12, respectively, based on 16S rRNA sequencing. Optimal protease production was detected at pH 8, 37 °C, 48 h, 5% (w/v) NaCl for Bacillus subtilis S1 (99.8 U/mL) and pH 9, 37 °C, 72 h, 10% (w/v) NaCl for Bacillus amyloliquefaciens KSM12 (94.6 U/mL). The molecular weight of these partially purified proteases was then assessed on SDS-PAGE (17 kDa for Bacillus subtilis S1 and 65 kDa for Bacillus amyloliquefaciens KSM12), respectively. The maximum protease activity for Bacillus subtilis S1 was detected at pH 8, 40 °C, and for Bacillus amyloliquefaciens KSM12 at pH 9, 60 °C. These results suggest that the proteases secreted by Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12 are suitable for industries working in a highly alkaline environment.

Apparent digestibility coefficients of carbohydrates and enzymes activity in major carps in response to Chromium Chloride Hexahydrate

This study investigated the effect of carbohydrate-based corn diets with chromium chloride hexahydrate supplementation on nutrient digestibility and enzyme activity in major carps (Cirrhinus mrigala, Catla catla, and Labeo rohita) for a period of 90 days. One control (T1) and five experimental diets (T2, T3, T4, T5, T6) were prepared by using gelatinized and non-gelatinized corn with the combination of various levels (0.0, 0.6, and 0.8 mg/kg) of CrCl3H12O6. After acclimatization with a control diet (T1) (32% CP), twenty fingerlings of these species were randomly stocked in eighteen digestibility tanks, each with one replicate. At the end of the trial, results showed that gelatinized corn with chromium supplementation significantly enhanced (P<0.05) digestibility of nutrients and enzyme activities (amylase and protease) in liver and gut in all treated groups. Crude protein (76.05±0.54), crude lipid (84.53±0.46), carbohydrate (69.15±2.19), and gross energy (76.92±0.74) showed the highest apparent digestibility in Labeo rohita as compared to Catla catla and Cirrhinus mrigala. While highest amylase (liver:3.27±0.126 U/ml and gut: 1.29±0.045 U/ml) and protease activity (liver:1.22±0.116 U/ml and gut 3.42±0.087 U/ml) was recorded in Catla catla subsequently followed by Labeo rohita and Cirrhinus mrigala. Among the treatments, highest amylase activity was observed in the corn gelatinized group at 0.6 mg/kg chromium level, while maximum protease activity was observed at 0.8 mg/kg of chromium level (gelatinized group). Comparison among gelatinized and non-gelatinized corn diets suggested that gelatinization in fish feed performed best as a carbohydrate energy source with chromium supplementation at an appropriate level (0.6 mg/kg) for three major carps as compared to non-gelatinized corn.

Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease.

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15µg of protein/mg and specific protease activity of 3.87 ± 0.52mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50°C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072μM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589mM and Vmax 1.909mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.

ISOLATION, CHARACTERIZATION AND EXPLORATION OF THE BIOLOGICAL ACTIVITY OF ENDOPHYTIC BACTERIA FROM MEDICINAL PLANTS

Bacterial endophytes are known to reside inside tissues of plants and can form a variety of different relationships including symbiotic, mutualistic, commensalistic and ammensalism. There has been increasing evidence, that endophytic bacteria can stimulus plant growth significantly by the production of phytohormones analogous to PGPR activity. In the present study, leaf explants of 6 medicinally important plants viz. Abrus precatorius (AP), Aegle marmelos (AM), Aloe barbadensis (AB), Annona squamosal (AS), Azadirachta indica (AI) and Oscimum sanctum (OS) were used for isolation and characterization of bacterial endophytes via morphological, biochemical examinations. Total 21 endophytes were isolated on Luria-Bertani (LB) and Nutrient agar isolation medium. Isolates were further screened for the enzymatic activity (amylase, protease, cellulase and lipase), and antifungal potential against plant fungal pathogen Aspergillus niger. Endophytic bacteria isolates from Aegle marmelos, Aloe barbadensis, and Azadirachta indica showed maximum amylase, protease, cellulase, and lipase activities in addition to antifungal activities. The screened endophytic isolates will be useful as a possible source of novel bioactive metabolites and potential biopesticides and biofertilizers.

A Taguchi design approach for the enhancement of a detergent‐biocompatible alkaline thermostable protease production by Streptomyces mutabilis strain TN‐X30

AbstractThe ability of microorganisms to grow at high temperature, alkaline pH, and high salinity makes them an attractive target for enzyme‐production with several industrial applications. One strain TN‐X30 has been selected as protease producer and identified as Streptomyces mutabilis after a phenotypic and molecular study. Its production of protease was improved using Taguchi L27 design. The strategy was carried out to identify the optimum levels and the interaction of the screened factors. Following this step, maximum protease activity (10,895 U/ml) was achieved after 6‐days of incubation. The TN‐X30 protease activity had an optimum of pH and temperature of 10 and 65°C, respectively. Thermodynamic parameters at 60°C were enthalpy 14.26 kJ/mol, entropy −220 J/mol/K, and Gibbs free energy 90.53 kJ/mol. TN‐X30 protease production displayed a 16‐fold increase reaching 175,000 U/ml in a 100‐L fermentor. Furthermore, the lyophilization in presence of sorbitol enhanced the stability of the TN‐X30 protease which remained active at 75% after 24‐months of storage. The lyophilized TN‐X30 protease exhibited exceptional stability indexes in presence of some known commercialized detergent components as NEODOL® 25‐7, Dehydol® LT 7, Na2 CMC, Galaxy LAS, Galaxy LES 70, Galaxy 110, Galaxy CAPB Plus, and Sulfacid K. The lyophilized enzyme also displayed high stability with respect to both solid and liquid detergents. Finally, TN‐X30 protease exhibited remarkable destaining of blood, egg, and chocolate stained cloth pieces. These findings may promote TN‐X30 protease for use as bioadditive in detergent formulation, thereby reducing environmental chemical threat.

Cuticle degrading enzyme production by some isolates of the entomopathogenic fungus

Biochemical characterisation with different entomopathogenic fungal isolates showed that the maximum protease activity was recorded by Bb112 (0.98 U ml−1). Ma SBI (27.02 U ml−1) and Bb112 (26.18 U ml−1) and were on par in chitinase activity. Subtilisin (Pr1) activity was more with Bb112 (3.25 U ml−1) and Ma SBI (3.11 U ml−1).

Effects of Temperature and pH on the Stability of Protease Produced by Alcaligenes faecalis P2

Background: Bacterial proteases represent a group of very important industrial enzymes. They are involved in the hydrolysis of peptide bond found in protein. Industrial application of bacterial proteases has been limited by low yield and instability at biotechnological process conditions. This study was design to investigate the effect of temperature and pH on the stability of protease produced by Alcaligene faecalis strain P2.
 Methodology: Protease-producing bacteria were isolated from beans effluent-impacted soil and screened for protease production on Casein agar plate. Protease assay was carried out following standard method for protease determination and the stability of the protease produced was investigated over temperature range of 20 to 90 oC and pH range of 3 to 12. The protease-producing bacterium was identified using its molecular characteristics.
 Results: Protease assay result showed that the amount of tyrosine released by one unit of the crude enzyme was 0.176 µmol/mL resulting in 0.19 U/mL protease activity. Stability studies showed that the protease had wide spectrum of pH and temperature stability. The protease was most stable at pH 9 and temperature of 40 oC after 30 min. Maximum protease activity of 1.259 U/mL was recorded with pH 9 after 30 min. The protease-producing bacterium was classified as Alcaligene faecalis P2 based on phylogenetic analysis of its 16S gene analysis. The sequences have been submitted to GenBank under the accession number MZ477004.
 Conclusion: This study therefore has demonstrated that beans effluent-impacted soil harbours protease producing bacteria with high industrial potentials. In addition, the study revealed that the protease produced in this study can retain its activity over wide temperature and pH.

Agro-Industrial Wastes: A Substrate for Multi-Enzymes Production by Cryphonectria parasitica

This study aims to produce a mix of enzymes through Solid State Fermentation (SSF) of raw materials. Four different, easily available, agro-industrial wastes were evaluated as SSF substrates for enzymes production by Cryphonectria parasitica (Murr.) Barr. environmental strains named CpA, CpB2, CpC4, and CpC7. Among the tested wastes, organic wheat bran for human use and wheat bran for animal feed better supports C. parasitica growth and protease production without any supplements. SDS-PAGE analyses highlighted the presence of three bands corresponding to an extracellular laccase (77 kDa), to the endothiapepsin (37 kDa), and to a carboxylesterase (60.6 kDa). Protease, laccase, and esterase activities by C. parasitica in SSF were evaluated for 15 days, showing the maximum protease activity at day 9 (3955.6 AU/gsf,). Conversely, the best laccase and esterase production was achieved after 15 days. The C. parasitica hypovirulent CpC4 strain showed the highest laccase and esterase activity (93.8 AU/gsf and 2.5 U/gsf, respectively). These results suggest the feasibility of a large-scale production of industrially relevant enzymes by C. parasitica strains in SSF process on low value materials.

IN VITRO EVALUATION OF FUNGICIDAL ACTIVITY OF BROMELAIN FROM ANANAS COMOSUS AGAINST FUNGAL PHYTOPATHOGENS INFECTING MUSA PARADISIACA

Bromelain from Ananas comosus is a mixture of proteolytic enzymes which are cysteine protease inhibitors containing thiol endopeptidases employed as a phytomedical compound. The present study focussed on extraction and characterization of bromelain and to determine its protein concentration and protease enzyme activity. The effect of temperature and pH on the extracted enzyme was also determined. The extracted enzyme was determined for its molecular weight by SDS and characterized for the presence of their functional groups by FT-IR spectroscopy. The purified bromelain of stem showed highest concentration of protein with maximum protease activity when compared to that of fruit bromelain and on characterization by FT-IR was found to contain thiol containing amino groups. The extracted and characterized bromelain was treated against isolated fungal pathogens of Musa paradisiaca. The isolated bromelain was assessed for their enzyme activity and protein concentration in vitro against the isolated fungal spores and determined for its activity. The determined two-way ANOVA indicated that there were significant differences at different concentrations after incubation at varying time period and the effective concentration and time were determined through Turkey post-hoc analysis which showed stem bromelain possess more effective antifungal activity in vitro against fungal phytopathogens of Musa paradisiaca.

Effect of dietary lipid levels on growth, body composition, and enzyme activities of larvae of butter catfish, Ompok bimaculatus (Actinopterygii: Siluriformes: Siluridae)

The Indian butter catfish, Ompok bimaculatus (Bloch, 1794), is a high-value catfish that has gained immense consumer preference in South-East Asia. However, information on the nutritional requirements of this species is scanty. Hence, an experiment was conducted to evaluate the effects of varying dietary lipid levels on growth, body composition, and activities of digestive and metabolic enzymes in larvae. Three isonitrogenous (40% crude protein) diets were formulated by supplementing fish and vegetable oil (1:1) at 4.5% (D1), 7% (D2), and 9.5% (D3) levels (containing crude lipid 5.7%, 8.0%, and 10.45%, respectively in diets D1–D3) to a fish meal- and oilcake-based formulated diet. Experimental diets were fed to butter catfish larvae (0.15 ± 0.01 g) in triplicate groups for a period of 42 days. Proximate compositions of the experimental diets, as well as fish carcass, were analyzed using standard procedures (AOAC 2005). Digestive and metabolic enzyme activities were analyzed at the completion of the experiment by standard methodology. Butter catfish larvae fed the diet D2 (8% crude lipid) resulted in the best performance in terms of weight gain (final weight 1.40 ± 0.07 g), net weight gain (1.31 ± 0.06 g), specific growth rate (5.50 ± 0.05% · day−1), and protein efficiency ratio (2.39 ± 0.17). The highest lipid deposition (2.90 ± 0.12%) in the carcass was also recorded in fish reared on diet D2. The final weight, net weight gain, protein efficiency ratio, and specific growth rate were significantly (P < 0.05) higher in D2 having 8% lipid. Moisture and lipid contents of the whole body were significantly (P < 0.05) higher in larvae fed diet D2. Amylase activity in fish significantly (P < 0.05) decreased with increasing dietary lipid levels. The maximum alkaline protease, pepsin, and lipase activities were noticed in the larvae fed diet D2. Progressive decrease in liver glucose-6-phosphate dehydrogenase activities and significant increase (P < 0.05) in the activities of neoglucogenic enzymes (glucose-6-phosphatase and fructose-1,6-bis phosphatase) were noticed with an increase in dietary lipid levels. Significantly lower (P < 0.05) activities of LDH, ALT, and AST were recorded in the group fed diet D2. Results of the study indicated that 8% crude lipid in the diet could assure optimum growth and survival of butter catfish larvae during early development. An appraisal on growth, body composition, and digestive as well as metabolic function in the butter catfish larvae recorded in the study might provide some important information to consider application of formulated diets for the larviculture of Ompok bimaculatus.

Effect of dietary lipid levels on growth, body composition, and enzyme activities of larvae of butter catfish, Ompok bimaculatus (Actinopterygii: Siluriformes: Siluridae)

The Indian butter catfish, Ompok bimaculatus (Bloch, 1794), is a high-value catfish that has gained immense consumer preference in South-East Asia. However, information on the nutritional requirements of this species is scanty. Hence, an experiment was conducted to evaluate the effects of varying dietary lipid levels on growth, body composition, and activities of digestive and metabolic enzymes in larvae. Three isonitrogenous (40% crude protein) diets were formulated by supplementing fish and vegetable oil (1:1) at 4.5% (D1), 7% (D2), and 9.5% (D3) levels (containing crude lipid 5.7%, 8.0%, and 10.45%, respectively in diets D1–D3) to a fish meal- and oilcake-based formulated diet. Experimental diets were fed to butter catfish larvae (0.15 ± 0.01 g) in triplicate groups for a period of 42 days. Proximate compositions of the experimental diets, as well as fish carcass, were analyzed using standard procedures (AOAC 2005). Digestive and metabolic enzyme activities were analyzed at the completion of the experiment by standard methodology. Butter catfish larvae fed the diet D2 (8% crude lipid) resulted in the best performance in terms of weight gain (final weight 1.40 ± 0.07 g), net weight gain (1.31 ± 0.06 g), specific growth rate (5.50 ± 0.05% · day−1), and protein efficiency ratio (2.39 ± 0.17). The highest lipid deposition (2.90 ± 0.12%) in the carcass was also recorded in fish reared on diet D2. The final weight, net weight gain, protein efficiency ratio, and specific growth rate were significantly (P < 0.05) higher in D2 having 8% lipid. Moisture and lipid contents of the whole body were significantly (P < 0.05) higher in larvae fed diet D2. Amylase activity in fish significantly (P < 0.05) decreased with increasing dietary lipid levels. The maximum alkaline protease, pepsin, and lipase activities were noticed in the larvae fed diet D2. Progressive decrease in liver glucose-6-phosphate dehydrogenase activities and significant increase (P < 0.05) in the activities of neoglucogenic enzymes (glucose-6-phosphatase and fructose-1,6-bis phosphatase) were noticed with an increase in dietary lipid levels. Significantly lower (P < 0.05) activities of LDH, ALT, and AST were recorded in the group fed diet D2. Results of the study indicated that 8% crude lipid in the diet could assure optimum growth and survival of butter catfish larvae during early development. An appraisal on growth, body composition, and digestive as well as metabolic function in the butter catfish larvae recorded in the study might provide some important information to consider application of formulated diets for the larviculture of Ompok bimaculatus.

Use of a Sequential Fermentation Method for the Production of Aspergillus tamarii URM4634 Protease and a Kinetic/Thermodynamic Study of the Enzyme

Microbial proteases are commonly produced by submerged (SmF) or solid-state fermentation (SSF), whose combination results in an unconventional method, called sequential fermentation (SF), which has already been used only to produce cellulolytic enzymes. In this context, the aim of the present study was the development of a novel SF method for protease production using wheat bran as a substrate. Moreover, the kinetic and thermodynamic parameters of azocasein hydrolysis were estimated, thus providing a greater understanding of the catalytic reaction. In SF, an approximately 9-fold increase in protease activity was observed compared to the conventional SmF method. Optimization of glucose concentration and medium volume by statistical means allowed us to achieve a maximum protease activity of 180.17 U mL−1. The obtained enzyme had an optimum pH and temperature of 7.0 and 50 °C, respectively. Kinetic and thermodynamic parameters highlighted that such a neutral protease is satisfactorily thermostable at 50 °C, a temperature commonly used in many applications in the food industry. The results obtained suggested not only that SF could be a promising alternative to produce proteases, but also that it could be adapted to produce several other enzymes.

Impact assessment of Karanja deoiled cake and sundried biogas slurry as a mixed substrate on the nematicidal potential of Purpureocillium lilacinum

The present study explored the feasibility of waste biomass viz., Karanja (Pongamia pinnata) deoiled cake (KDC), and sundried biogas slurry (BGS) as a mixed substrate to develop Purpureocillium lilacinum 6029 based fungal formulation against plant-parasitic nematode, Meloidogyne incognita. The fungus cultured on KDC-BGS in a 60/40 ratio showed maximum egg mass inhibition (96.8%) and protease activity (376.65 U/g) along with superior colonization ability (100% colonization of egg mass on the third day). Incidentally, P. lilacinum spores obtained from the traditional substrate (wheat) inhibited only 68.72% egg mass hatching with a comparatively poor colonization rate construing the linkage between the nature of substrate and virulence of the fungus. Bioefficacy studies on tomato plants in the greenhouse revealed that the formulation with KDC-BGS as a substrate was 1.19 times more effective than the wheat-based formulation in controlling nematodes. Enhanced physical-biochemical parameters of tomato plants treated with the bioformulation further substantiated KDC-BGS as a reliable and economical substrate for P. lilacinum.

Enhancing food waste biodegradation rate in a food waste biodigester with the synergistic action of hydrolase-producing Bacillus paralicheniformis GRA2 and Bacillus velezensis TAP5 co-culture inoculation.

Food waste (FW) minimization at the source by using food waste biodigester (FWBs) has a vast potential to lower down the impact of increasing organic fraction in municipal solid waste generation. To this end, this research sought to check the performance of locally isolated hydrolase-producing bacteria (HPB) to improve food waste biodegradation rate. Two under-explored HPB identified as Bacillus paralicheniformis GRA2 and Bacillus velezensis TAP5 were able to produce maximum amylase, cellulase, protease and lipase activities, and demonstrated a significant hydrolase synergy in co-culture fermentation. In vitro biodegradation analysis of both autoclaved and non-autoclaved FW revealed that the HPB inoculation was effective to degrade total solids (>62%), protein (>19%), total fat (>51), total sugar (>86%), reducing sugar (>38%) and starch (>50%) after 8-day incubation. All co-culture treatments were recorded superior to the respective monocultures and the uninoculated control. The results of FW biodegradation using batch-biodigester trial indicated that the 1500 mL and 1000 mL inoculum size of HPB inoculant reached a plateau on the 4th day, with gross biodegradation percentage (GBP) of >85% as compared to control (66.4%). The 1000 mL inoculum was sufficient to achieve the maximum GBP (>90%) of FW after an 8-day biodigestion in a FWB.

Effect of Rhamnolipids on Pathogenicity Characteristics of Microorganisms in Organic Compost

Background: Rhamnolipids are hydrophilic glycolipids, often classified as biosurfactants. They are produced by different bacterial species. Rhamnolipids are extensively studied in biological research because of their interesting features like antimicrobial, antifungal, and antiviral activities. Materials and Methods: To study the antimicrobial effect of rhamnolipid, we conducted a crosssectional study on the eight different pathogenic bacterial strains from November 2019 to June 2020. These bacterial strains were isolated from the organic compost. Both disk diffusion and broth microdilution methods were used to evaluate the inhibitory effects of rhamnolipids on these pathogenic bacteria. Also, protease and amylase enzyme activities were evaluated in these eight bacterial isolates. Results: For Bacillus stearothermophilus, within the area of 31.5 mm, no growth was observed, hence proving the inhibitory effect of rhamnolipid. After calculating the minimum inhibitory (MIC) and minimum lethal concentrations (MLC) for each bacterial strain, it has been found that the studied bacteria were more susceptible to rhamnolipids than most of the antibiotics. Strains were also quantified for their enzymatic activity of proteases and amylases. The bacterial strains of Bacillus stearothermophilus, Brevibacillus brevis, Bacillus licheniformis, and Bordetella petrii showed maximum protease activity. Whereas Escherichia coli, Enterococcus faecalis, Enterobacter aerogenes, and Pseudomonas aeruginosa showed high amylase activity. Conclusion: Rhamnolipids can be used as a potential antimicrobial agent for treatment of infections.

Characterization of digestive enzymes during early ontogeny of white Snook (Centropomus viridis).

The white snook (Centropomus viridis) is a species with great aquaculture potential, therefore, the present study aims two objectives; 1) to characterize digestive enzymatic activity during larval development and; 2) characterize the diversity and functionality of digestive enzymes in an early juvenile stage of C. viridis. On first feeding, larvae were 2.75 ± 0.09 mm total length and grew to 24.35 ± 4.35 mm after 42 days. The specific activity of acid proteases was detected with low activity from day 1 (Days after hatching, DAH) to day 21, and increased at 23 DAH, reaching maximum activity at 40 DAH. Meanwhile, digestive enzymatic activity of alkaline proteases was observed from hatching to day 40. From hatching until 40 DAH, the presence of alkaline proteases, trypsin, chymotrypsin, leucine aminopeptidase, α-amylase and lipase were detected with different activity patterns. Total and specific activity of digestive enzymes on juveniles showed higher activity of acid than alkaline proteases, while α-amylase had a higher activity than lipases. The maximum activity of acid proteases was observed at 40 °C and at pH 2.0, while digestive alkaline proteases showed maximum activity at 60 °C. α-amylases and lipases showed their maximum activities at 50 °C. The maximum alkaline protease and α-amylase activities were found at pH 7 to 9. The enzymatic activity of juvenile's acid proteases was highly inhibited by Pepstatin A (96%), however the activity of alkaline proteases incubated with PHEN, TLCK, SBTI, PMFS, and OVO showed reductions of 51.29, 30.16, 27.98, 27.26, and 24.68% respectively. Lipase activity was totally inhibited with Ebelactone B, and highly inhibited by Ebelactone A, Orlistat, 1% SDS, and PMFS (96.12, 77.16, 64.22, and 51.29%, respectively). Seven bands (102.6, 65.7, 58.9, 40.6, 27.4, 21.5, and 15.8 kDa) resolved by protein electrophoresis were detected with alkaline protease activity, identified as serine proteases and metalloproteases, and three bands (57, 43.9, and 32.6 kDa) with lipase-like activity were also identified. These results show that C. viridis ontogeny entails an indirect development of its digestive system with a functional stomach on day 23, and characterized by increases in pepsin activity. Indeed, white snook ontogeny shows a similar developmental trajectory to other carnivorous marine fish, showing a diversity of alkaline proteases that work in a wide range of pH and temperatures. Thus, the identification of these enzymes provides advantages to formulate white snook aqua-feeds.

Seaweed extracts as prospective plant growth bio-stimulant and salinity stress alleviator for Vigna sinensis and Zea mays

The objective of this investigation was to evaluate the impact of seed priming with seaweed liquid extracts (SLEs) prepared from three Egyptian seaweeds (Ulva fasciata, Cystoseira compressa, and Laurencia obtusa) in a concentration of 20 g L-1 on seed germination and seedling growth of Vigna sinensis and Zea mays as well as the effectiveness of these extracts in ameliorating salinity stress. Laurancia extract (LLE) induced the maximum positive response in germination as well as producing the maximum increases in all seedling morphological parameters with percent of response 87.14% for hypocotyl growth of V. sinensis and 85.71% coleoptile elongation of Z. mays. The maximum amylase and protease activities were recorded with LLE priming. Ulva, Cystoseira, Laurencia liquid extract consortium (UCLLE) induced the highest promotion effect for both V. sinensis and Z. mays giving significant increments for all growth criteria, dry biomass 0.301 and 0.438 g seedling-1, Chl a 18.98 and 13.79 mg g-1 FW, total carbohydrates 460.88 and 518.14 g seedling-1, protein 219.72 and 207.49 g seedling-1 respectively. Seeds primed with UCLLE consortium exhibited remarkable tolerance for salinity stress, meanwhile the highest ameliorating effect was pronounced with 10% SW supplementation. Priming with SLEs enhances the growth of either V. sinensis or Z. mays under salinity stress, whereas UCLLE consortium induced significant increases in biomass 0.231 and 0.379 g seedling-1, respectively. Seed priming with SLEs enhanced guaiacol peroxidase activity giving the highest value with UCLLE consortium conjugated with 25% SW (12.47 and 13.05 μmol min-1 g-1 FW), respectively. Application of Canonical Corresponding analysis (bi-plot) demonstrates a significant correlation between variables of SLEs components and germination indices of the two plants concluding total nitrogen, total phosphorous, potassium, ascorbic acid, and amino acids besides carbohydrate content. Therefore, results maintain the concept of SLEs as agricultural biocatalyst which could be practiced as prospective biostimulants for being feasible, sustainable, eco-friendly, and effective in improving salinity tolerance for some plants.

Extracellular neutral protease from Arthrospira platensis: Production, optimization and partial characterization

Proteases are industrially important catalysts. They belong to a complex family of enzymes that perform highly focused proteolysis functions. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. In the present study, a novel extracellular neutral protease produced from Arthrospira platensis was detected and characterized. Its proteolytic activity was strongly activated by β-mercaptoethanol, 5,5-dithio-bis-(2-nitrobenzoic acid) and highly inhibited by Hg2+ and Zn2+ metal ions which support the fact that the studied protease belongs to the cysteine protease family. Using statistical modelling methodology, the logistic model has been selected to predict A. platensis growth-kinetic values. The optimal culture conditions for neutral protease production were found using Box-Behnken Design. The maximum experimental protease activities (159.79 U/mL) was achieved after 13 days of culture in an optimized Zarrouk medium containing 0.625 g/L NaCl, 0.625 g/L K2HPO4 and set on 9.5 initial pH.The extracellular protease of A. platensis can easily be used in the food industry for its important activity at neutral pH and its low production cost since it is a valuation of the residual culture medium after biomass recovery.

Influence of graded level of dietary protein with equated level of limiting amino acids on growth, feed utilization, body indices and nutritive profile of snubnose pompano, Trachinotus blochii (Lacepede, 1801) reared in low saline water

An experiment was conducted to delineate the effect of varying level of dietary protein with balanced level of limiting amino acids such as lysine and methionine on growth, feed utilization, body indices and digestive enzymology of snubnose pompano, Trachinotus blochii reared in low saline water. There were four experimental diets formulated with 350 (CP350), 400 (CP400), 450 (CP450) and 500 g/kg (CP500) crude protein and each with 100 g/kg crude fat and were assigned to four treatments, CP350, CP400, CP450 and CP500 respectively. Totally eighty four fingerlings with the body weight in the range of 16−18 g were randomly distributed in four treatment groups with each of three replicates. Fish were fed to apparent satiation twice daily with the assigned experimental diet. At the end of 13 weeks of feeding trial, weight gain percentage (WG%) and specific growth rate (SGR) of fish fed with CP400 and CP450 diets were significantly higher than those of fish fed with CP350 and CP500 diets. Growth rate on metabolic body weight (GRMBW), daily growth coefficient (DGC) and average daily growth (ADG) were higher in CP400 group and lower in CP350 group. The viscerosomatic index (VSI), hepatosomatic index (HSI), gastrosomatic index (GSI) and intra-peritoneal fat ratio (IPFR) showed an increasing trend as the crude protein content of the experimental diet increases and the maximum value was witnessed in CP500 treatment. Crude lipid and protein content of whole body tissue was found highest in CP400 and lowest level was noticed in CP350 group. The maximum intestinal protease activity was noticed in CP400 group. In conclusion, analysis of second degree polynomial regression revealed that the optimum dietary protein level could be in the range of 395.3–426.7 g/kg for maximum growth of juvenile snubnose pompano.