Acid Protease Research Articles

Characterization of collagen in salmon nasal cartilage: enzymatic solubilization with fungal acid protease

Characterization of collagen in salmon nasal cartilage: enzymatic solubilization with fungal acid protease

Citrus pomace co-fermentation improved its protein and amino acids by Bacillus amyloliquefaciens and Candida utilis

High-value utilization of citrus pomace (CP) is of great significance to the sustainable development of the citrus industry. Co-fermentation of CP was investigated using the autochthonous probiotic bacteria, Bacillus amyloliquefaciens BF2 and Candida utilis GIM 2.9. Crude protein, soluble protein, and small peptide content of CP increased by 40.01%, 923.53%, and 626.67% after fermentation, respectively. Essential amino acids increased from 28.51% to 47.04% of total free amino acids. Leucine level reached 4.04 mg/g DW, increasing by 1293.10%, and produced histidine, valine, cysteine, and tyrosine. Scanning electron microscopy results showed that BF2 and GIM 2.9 could effectively degrade and utilize the CP fibers. Enzymolysis co-fermentation (ECF) system secreted higher levels of non-starch polysaccharide enzymes and proteases than co-fermentation (CF) system. Acid protease activity was positively correlated with soluble protein and free amino acid contents. BF2 possessed 63 essential amino acid-biosynthesis genes, nearly twice as many as that of non-essential amino acids. Eight key enzyme genes were identified for leucine biosynthesis in the BF2 genome, suggesting that BF2 played a key role in leucine biosynthesis. Our results provide a potential method for the high-value utilization of CP as a protein feed additive or dietary supplement.

"Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.

Effects of metamifop on ammonia production and metabolism of Monopterus albus

The use of herbicides is believed to have an impact on the metabolism, physiology and biochemistry of fish. In this study, we studied the effects of metamifop on the production and metabolism of Monopterus. albus living in the water. According to the semi-lethal concentration of metamifop for 96 h, four MET concentration groups (0.2-, 0.4-, 0.6- and 0.8 mg L−1) were set up for 96 h exposure test. The ammonia discharge rate decreased, hemolymph ammonia content increased significantly, and hemolymph urea nitrogen content decreased at all time periods of metamifop exposure. In liver, the protein content decreased, the neutral protease content increased significantly (p < 0.01), amino acid content increased, and ATP content increased significantly (p < 0.01). In brain, the protein content increased, the activity of acid protease, neutral protease and alkaline protease all decreased, amino acid content decreased significantly (p < 0.01), and the content of ATP decreased. Glutamic-pyruvic transaminase (GPT) activity did not change in liver but decreased in brain. Glutamine synthetase (GS) activity decreased in liver and increased in brain. Glutaminase (GLS) activity decreased in liver and increased in brain. In conclusion, the liver and brain tissues of M. albus react differently to MET exposure. The liver mainly synthesizes energy through hydrolyzed protein, while the brain mainly synthesizes protein. Amino acids produced by protein hydrolysis cannot be converted to alanine for storage, and the degraded amino acids lead to the elevation of endogenous ammonia. MET inhibits the removal of ammonia from M. albus. Only liver tissue can detoxify the eel by converting ammonia into glutamine. Brain should have to tolerate high levels of endogenous ammonia.

Transcriptomic profiling revealed important roles of amino acid metabolism in fruiting body formation at different ripening times in Hypsizygus marmoreus.

Hypsizygus marmoreus is an industrial mushroom that is widely cultivated in East Asia. Its long postripening stage before fruiting severely limits its industrialized production. Five different mycelial ripening times (30, 50, 70, 90, and 100 d) were chosen and primordia (30P, 50P, 70P, 90P, and 110P) were collected for comparative transcriptomic analyses. The corresponding substrates (30F, 50F, 70F, 90F, and 110F) were used for nutrient content and enzyme activity determination. In pairwise comparisons between 110P and other primordia, a total of 1,194, 977, 773, and 697 differentially expressed genes (DEGs) were identified in 30P_110P, 50P_110P, 70P_110P, and 90P_110P, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG) functional enrichment analyses revealed that the DEGs were mainly associated with amino acid metabolism, and lipid and carbohydrate metabolism pathways. Tyrosine, tryptophan, phenylalanine and histidine metabolism were enriched in all groups. Among the main carbon nutrients, the contents of cellulose and hemicellulose were high, and the lignin content decreased with the extension of the ripening time. Laccase had the highest activity, and acid protease activity decreased with the extension of the ripening time. The highly enrichment for amino acid metabolic pathways in primordia reveals that these pathways are essential for fruiting body formation in H. marmoreus, and these results will provide a basis for the optimization of its cultivation.

A Strategy for Rapid Acquisition of the β-D-Fructofuranosidase Gene through Chemical Synthesis and New Function of Its Encoded Enzyme to Improve Gel Properties during Yogurt Processing.

A chemical gene synthesis strategy was developed in order to obtain β-D-fructofuranosidase, and a novel gene, AlFFase3, was characterized from Aspergillus luchuensis and expressed in Escherichia coli. The recombinant protein was purified, showing a molecular mass of 68.0 kDa on SDS-PAGE, and displaying a specific activity towards sucrose of up to 771.2 U mg-1, indicating its exceptional enzymatic capacity. AlFFase3 exhibited stability between pH 5.5 and 7.5, with maximal activity at pH 6.5 and 40 °C. Impressively, AlFFase3, as a soluble protein, was resistant to digestion by various common proteases, including Flavourzyme, acidic protease, pepsin, neutral protease, Proteinase K, alkaline proteinase, and trypsin. AlFFase3 also demonstrated significant transfructosylation activity, with a yield of various fructooligosaccharides up to 67%, higher than almost all other reports. Furthermore, we demonstrated that the addition of AlFFase3 enhanced the growth of probiotics in yogurt, thereby increasing its nutritional value. AlFFase3 also improved the formation of yogurt gel, reducing the gel formation time and lowering the elasticity while increasing its viscosity, thereby improving the palatability of yogurt and reducing production costs.

Enhancement of sodium salty taste modulate by protease-hydrolyzed Gum Arabic

Salt reduction strategies in liquid foods remains a significant challenge. The oral perception of Na+ is strongly dependent on its salivary penetration and mucosal adsorption. To understand the mechanism of Na+ saltiness enhancement modulated by polysaccharide, protease hydrolysis (trypsin and acid protease) was used to modify the molecular conformation of Gum Arabic (GA), a highly branched polyanionic polysaccharide. The salivary permeation, mucosal retention in mucin layer, and salty taste of Na+ were investigated. Saltiness-enhanced effect of GA is strongly influenced by its molecular conformation. Compared to native GA, protease-hydrolyzed GA exhibited a higher saltiness-enhanced effect due to more rapid Na+ diffusion in saliva and stronger mucosal retention. Protease-hydrolyzed GA, especially acid protease-hydrolyzed GA (GA-AcPr), exhibited lower zeta potential (from original −22.5 to −27.96) and stronger Na+ loading capacity. The obvious decreases in molecular weight (Mw) and the radius of gyration (Rg) were observed for trypsin-hydrolyzed GA (GA-Trypsin), which may be responsible for the higher Na+ diffusion effect of GA-saliva solution due to decreased molecule twine around each other. GA-AcPr exhibited looser and highly branched conformation, as evidenced by a more pronounced "U-shaped" curve and higher Rg/Rh. Moreover, Quartz crystal microbalance with dissipation (QCM-D) results verified stronger affinity between GA-AcPr and mucin, indicating that the saltiness-enhanced effect of GA-AcPr may be mainly achieved by increasing adhesive retention of Na+. These results would be useful to better understand the structure-function relationship of polysaccharides and the taste perception of Na+.

Development of Ratiometric Fluorescence Sensing Molecule for Caspase-14, a Key Enzyme of Epidermis Metabolism

Skin is the vastest organ harboring principle functions to protect from external stimuli and internal moisture desorption. The loss of functions might bring about diseases and senescence and well maintained metabolisms are recognized as crucial points to prevent those situations. But due to complexities of skin tissues, there would be some difficulties in progress of elucidation for skin metabolisms in detail. Various differentiated tissues layered in order can form dermis and epidermis to exfoliate as scurf in the top layer finally. Caspase-14 categorized in cysteine-aspartic acid protease (Caspase) family that has been identified as key enzymes to promote programmed cell deaths by sequential activations is known to be specifically expressed in the upper tissues and involved in the differentiation, but not verified to have relationship with final exfoliation processes at the time of cell death. We then developed Fluorescence Resonance Energy Transfer (FRET) based sensing molecules to monitor Caspase-14 activity quantitatively. We utilized Green Fluorescent Protein (GFP) and organic fluorescent dyes because FRET phenomena could be achieved under condition with regulated interactions between at least two fluorescent molecules. The elaborately designed fluorescent complexes could render quantifiable sensing over fluorescent background but be simply introduced into cells via endocytic mechanisms to detect intracellular enzymatic activities alive. In this study, we demonstrated Caspase-14 activation during apoptosis induction in cultured normal epidermal cells that suggested possibilities of Caspase-14 contribution to programmed cell deaths.

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

Comparative Analysis of the Alkaline Proteolytic Enzymes of Yarrowia Clade Species and Their Putative Applications

Proteolytic enzymes are commercially valuable and have multiple applications in various industrial sectors. The most studied proteolytic enzymes produced by Yarrowia lipolytica, extracellular alkaline protease (Aep) and extracellular acid protease (Axp), were shown to be good candidates for different biotechnological applications. In this study, we performed a comprehensive analysis of the alkaline proteolytic enzymes of Yarrowia clade species, including phylogenetic studies, synteny analysis, and protease production and application. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we reconstructed the evolutionary scenario of the XPR2 gene for species of the Yarrowia clade. Furthermore, except for the proteolytic activity of the analyzed Yarrowia clade strains, the brewers' spent grain (BSG) was used as a substrate to obtain protein hydrolysates with antioxidant activity. For each culture, the degree of hydrolysis was calculated. The most efficient protein hydrolysis was observed in the cultures of Y. lipolytica, Y. galli, and Y. alimentaria. In contrast, the best results obtained using the 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) method were observed for the culture medium after the growth of Y. divulgata, Y. galli, and Y. lipolytica on BSG.

Effect of kidney bean extract on phytohaemagglutinin activity, functional properties, and the estimated glycaemic index in‐vitro

SummaryThis study aimed to decrease the activity of phytohaemagglutinins and kidney bean α‐amylase activity in some selected stable foods. Kidney bean α‐amylase inhibitors may inhibit mammalian α‐amylase, reducing the estimated glycaemic index (eGI). The effect of temperature, pH and protease pretreatment on PHA activity in the crude extract was investigated. Besides, the inhibitory effect of kidney bean extract on α‐amylase activity and properties was assessed. Results showed that the PHA activity was decreased to 6.97% from the aqueous extract of kidney beans, while the α‐AI activity was retained by 85.89% by acid protease treatment. The extract's α‐amylase inhibitory activity remained at 87.37% following simulation in a gastric environment, whereas no effect was observed in a simulated intestinal environment. Adding 3% of the extract decreased the eGI of bread, rice, steamed bread, and steamed corn bread from 65.00, 71.65, 68.17, and 63.41 to 30.69, 39.51, 22.30, and 19.69, respectively. The processing method of crude kidney bean extract examined in this study has significant reference value for the development of kidney bean α‐amylase inhibitors and can enhance the intensive processing level and industrial development value of kidney beans worldwide. This discovery suggests that kidney bean extracts can be used to develop foods with a low GI.

New insights into the microalgal culture using kitchen waste: Enzyme pretreatment and mixed microalgae self-flocculation

With the development of the catering industry, the output of kitchen waste increases significantly. To respond to the carbon neutral policy, this study proposed the technology of combining kitchen waste treatment and microalgae culture. The kitchen waste was pretreated by enzymatic hydrolysis, and the optimal composition and proportion of compound enzymes were investigated. The results showed that acid protease: α-amylase: glucoamylase: cellulase = 50:1:10:1 (enzyme activity ratio) was chosen as the most suitable compound enzymes according to the growth and water purification effect of Scenedesmus obliquus in the hydrolysate. To optimize the microalgae biomass accumulation especially high-value substances, microalgae harvesting, and water purification effects, mixed culture of microalgae was performed. Chlorella sp. HQ and Scenedesmus sp. LX1 were the best combination, in which the contents of lipid, polysaccharide, and protein were 8.78 ± 4.48%, 2.11 ± 0.14%, and 8.38 ± 0.12%, respectively. After combination, the flocculation rate reached up to 63.75 ± 1.08%, and the removal rates of chemical oxygen demand, total nitrogen, and total phosphorus were 88.54 ± 0.58%, 90.97 ± 4.73%, and 88.81 ± 4.75%, respectively. The above results indicated that the enzymatic hydrolysate of kitchen waste had a promising application prospect in microalgae culture, and self-flocculation of mixed microalgae had the potential to recover biomass.

Characterization of Aspartic Proteases from Paracoccidioides brasiliensis and Their Role in Fungal Thermo-Dimorphism

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and is caused by fungi from the Paracoccidioides genus. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. Proteases play an important role in the host invasion process and immune modulation in many pathogenic microorganisms. Aspartyl proteases are virulence factors in many human fungal pathogens that play an important role in the host invasion process morphogenesis, cellular function, immunity, and nutrition. In the present study, we characterized the modulation of acid proteases from Paracoccidioides brasiliensis. We detected four aspartyl proteases in P. brasiliensis with high homology to aspartic protease from Saccharomyces cerevisiae Pep4. Furthermore, we demonstrated that Pepstatin A can inhibit dimorphic switching (mycelium→yeast) in P. brasiliensis. In addition, these genes were modulated during thermo-dimorphism (M→Y transition) in the presence or absence of carbon and nitrogen sources and during growth at pH 4 during 24 and 48 h. We also observed that P. brasiliensis increase the secretion of aspartic proteases when cultivated at pH 4, and these acid proteases cleave BSA, collagen, and hemoglobin. These data suggest that aspartyl proteases are modulated by environmental conditions and during fungal thermo-dimorphism. Thus, this work brings new possibilities for studying the role of aspartyl proteases in the host-pathogen relationship and P. brasiliensis biology.

Application of the novel manta-ray foraging algorithm to optimize acidic peptidase production in solid-state fermentation using binary agro-industrial waste

Peptidases, which constitute about 20% of the global enzyme market, have found applications in detergent, food and pharmaceutical industries, and could be produced on a large scale using low-cost agro-industrial waste. An acidophilic Bacillus cereus strain produced acidic peptidase on binary-agro-industrial waste comprising yam peels and fish processing waste at pH 4.5 with high catalytic activity. A five-variable central composite rotatable design of a response surface methodology was used to model bioprocess conditions for improved peptidase production in solid-state fermentation. Data generated was leveraged as the basis for applying the novel Manta-ray foraging optimization-linked feed-forward artificial neural network to predict bioprocess conditions optimally. Results obtained from the optimization experiments revealed a significant coefficient of determination of 0.9885 with low-performance error. The bioprocess predicted a peptidase activity of 1035.32 U/mL under optimized conditions set as 54.8 g/100 g yam peels, 23.85 g/100 g fish waste, 0.31 g/100 g CaCl2, 47.54% (v/w) moisture content, and pH 2. Peptidase activity was improved 5-fold, and was stable for 240 min between pH 2.5 and 3.5. Michaelis-Menten kinetics revealed a Km of 0.119 mM and a catalytic efficiency of 45462.19 mM−1 min−1. The bioprocess holds promise for sustainable enzyme-driven applications.

Accelerated Accumulation of γ-Aminobutyric Acid and Modifications on Its Metabolic Pathways in Black Rice Grains by Germination under Cold Stress

Germination can increase γ-aminobutyric acid (GABA) accumulation in grains, but the combined effects of germination and other external stress on rice grains have been little studied. In this investigation, enhanced accumulation of GABA and modification of its metabolic pathways in black rice grains were investigated during germination under cold stress. The combination of cold stress and germination resulted in a greater accumulation of GABA than germination alone. The treatment of cold stress at 0 °C for 1 h and germination for 72 h induced a maximum GABA content of 195.64 mg/100 g, 51.54% higher compared to the control, which was superior to any other treatment. We modified the metabolism of the GABA shunt to the orientation of GABA synthesis, in which the activity of glutamic acid decarboxylase and protease were stimulated. The total content of free amino acid indicated an upward trend as germination prolonged. The degradation of polyamines was partly promoted due to elevated diamine oxidase and polyamine oxidase activity, but the activity of amino-aldehyde dehydrogenase for the direct synthesis of GABA in the pathway was suppressed. The result implied that the GABA shunt might play a major role in enhancing GABA accumulation induced by cold stress and germination rather than the polyamines degradation pathway. This investigation provides a practical reference for GABA accumulation by germination under cold stress and a theoretical basis for the possible mechanism underlying the accelerating action.

Microbial driving mechanism of soil conditioner on reducing cadmium uptake by rice and improving soil environment

Soil conditioners can reduce the transfer of heavy metal ions from the soil to crops and play a crucial role in safe food production. However, it remains unclear how soil conditioners reduce cadmium (Cd) accumulation in rice (Oryza sativa L.). In this study, five soil conditioners (U-, N-, T-, L-, and C-type) commonly used in China were selected, and the effects of soil conditioners on bacterial and fungal community diversity, soil enzyme activity, soil pH, and organic matter content were investigated through field experiments in two sites. The Cd content of rice was significantly reduced by 32.4–55.1 % in site I and 16.0–33.3 % in site II under the five soil conditioner treatments. The decrease in Cd content was related to the increase in soil pH, organic matter content, and Cd fixation-related flora caused by soil conditioner application. U-, N- and C-type conditioners increased soil pH, inducing an increase in the relative abundances of microbial populations related to Cd sorption and sequestration (Firmicutes, Nitrospirota, Actinobacteriota, and Methylomirabilota). U-, T-, and L-type conditioners increased soil organic matter, causing an increase in the relative abundance of microbial flora associated with macromolecule degradation, including bacteria (Acidobacteriota and Chloroflexi) and fungi (Ascomycota and Basidiomycota). Conditioner application reduced the availability of soil Cd; indirectly promoted the increased abundance of beneficial soil bacteria; and increased the activities of invertase, urease, dehydrogenase, catalase, and acid protease. In conclusion, soil conditioner reduces the effective soil Cd by adjusting soil pH and organic matter content, increasing Cd-passivating flora and Cd-tolerant flora. Soil conditioner also increases the abundance of other beneficial flora, enhances soil enzyme activity, promotes the microecological balance of soil under Cd stress, and finally reduces Cd accumulation in rice.

Assessing the Fate of Superparamagnetic Iron Oxide Nanoparticles Carrying Usnic Acid as Chemical Cargo on the Soil Microbial Community.

In the present study we evaluate the effect of superparamagnetic iron oxide nanoparticles (SPIONs) carrying usnic acid (UA) as chemical cargo on the soil microbial community in a dystrophic red latosol (oxysol). Herein, 500 ppm UA or SPIONs-framework carrying UA were diluted in sterile ultrapure deionized water and applied by hand sprayer on the top of the soil. The experiment was conducted in a growth chamber at 25 °C, with a relative humidity of 80% and a 16 h/8 h light-dark cycle (600 lx light intensity) for 30 days. Sterile ultrapure deionized water was used as the negative control; uncapped and oleic acid (OA) capped SPIONs were also tested to assess their potential effects. Magnetic nanostructures were synthesized by a coprecipitation method and characterized by scanning and transmission electron microscopy (SEM and TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), zeta potential, hydrodynamic diameter, magnetic measurements, and release kinetics of chemical cargo. Uncapped and OA-capped SPIONs did not significantly affect soil microbial community. Our results showed an impairment in the soil microbial community exposed to free UA, leading to a general decrease in negative effects on soil-based parameters when bioactive was loaded into the nanoscale magnetic carrier. Besides, compared to control, the free UA caused a significant decrease in microbial biomass C (39%), on the activity of acid protease (59%), and acid phosphatase (23%) enzymes, respectively. Free UA also reduced eukaryotic 18S rRNA gene abundance, suggesting a major impact on fungi. Our findings indicate that SPIONs as bioherbicide nanocarriers can reduce the negative impacts on soil. Therefore, nanoenabled biocides may improve agricultural productivity, which is important for food security due to the need of increasing food production.

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation

BackgroundAdding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied.ResultsIn our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0–5.0 and 30–60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below.ConclusionsIn this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.

Inulin Supplementation in Diets for Tropical Gar (Atractosteus tropicus) Larvae: Effects on Growth, Survival, and Digestive and Antioxidant Enzyme Activities

The effect of adding inulin to balanced diets for tropical gar (Atractosteus tropicus) larvae on growth, survival, digestive enzyme activity, and antioxidant activity was evaluated. The diets were supplemented with 0.5, 1.0, 1.5, 2.0, and 2.5% inulin in addition to a control diet (0% inulin). A total of 1800 larvae of A. tropicus distributed in 18 tanks were used; the larvae were fed five times a day (8:00, 11:00, 13:00, 15:00, and 18:00) with Artemia nauplii from the absorption of the yolk (from 3–7 days after hatching, DAH) up to 10 DAH, which was mixed with the experimental feeds from 8–11 DAH (co-feeding) and exclusively with the balanced diets from 12 DAH to 21 DAH. Larvae fed the control diet (0% inulin) had the highest growth in weight and length, followed by fish fed the 2.5 and 2.0% inulin inclusions. However, survival showed that the fish fed with the inclusion of 2.5% inulin had the highest percentage (34.7%) compared to the rest of the treatments. On the other hand, the highest digestive enzymatic activities (acid and alkaline proteases, amylase, and lipase) were recorded in the larvae fed with 2 and 2.5% inulin. Likewise, catalase (CAT) and superoxide dismutase (SOD) activities were higher in larvae fed the control diet with 0% inulin. Supplementation of 2.0% to 2.5% inulin in the diet is recommended for A. tropicus larvae as it improves survival and digestive enzyme activity during this early stage of life.

Combined Effects of Dietary Probiotic Lactobacillus sp. and Prebiotic Glycyrrhizic Acid on Growth Performance, Feed Utilization and Digestive Enzymes Activities of Nile Tilapia (Oreochromis niloticus) Juveniles

The present study investigated the effects of dietary commercial probiotic Lactobacillus sp. and commercial prebiotic glycyrrhizic acid on the growth performance, feed utilization and digestive enzymes activities of Nile tilapia. Seven diets with or without probiotics and prebiotics supple-mentation namely Control (without probiotics and prebiotics), P10, P20 and P30 (Control + Pro-aqua) and V2, V4 and V6 (Control +Viusid) were fed to juvenile Nile tilapia (5.22±0.05 g) for 60 days in triplicate tanks (30 fish per tank). All fish were fed ad libitum three times a day for 60 days. At the end of the experiment, the weight gain and specific growth rate of the fish fed with probiotic content group was found to be higher than the other groups (P&lt;0.05). In the experiment, there was no change in the nutritional value of tilapia fed with a diet containing all feeding groups (P&gt;0.05). On the other hand, significant changes were observed in alkaline and acid protease enzyme activities depending on the days (P&lt;0.05). Especially, after the first 15-day period of the experiments, increases in both supplemented groups enzyme activities were determined. The increase in enzyme activity in the probiotic supplemented groups showed higher activity than the prebiotic administered groups (P&lt;0.05). It can be concluded that probiotic can act as a growth promoter and increase digestive enzymes activity in tilapia.