Enzymes Trypsin Research Articles

Food availability modulates the combined effects of ocean acidification and warming on fish growth

When organisms are unable to feed ad libitum they may be more susceptible to negative effects of environmental stressors such as ocean acidification and warming (OAW). We reared sea bass (Dicentrarchus labrax) at 15 or 20 °C and at ambient or high PCO2 (650 versus 1750 µatm PCO2; pH = 8.1 or 7.6) at ad libitum feeding and observed no discernible effect of PCO2 on the size-at-age of juveniles after 277 (20 °C) and 367 (15 °C) days. Feeding trials were then conducted including a restricted ration (25% ad libitum). At 15 °C, growth rate increased with ration but was unaffected by PCO2. At 20 °C, acidification and warming acted antagonistically and low feeding level enhanced PCO2 effects. Differences in growth were not merely a consequence of lower food intake but also linked to changes in digestive efficiency. The specific activity of digestive enzymes (amylase, trypsin, phosphatase alkaline and aminopeptidase N) at 20 °C was lower at the higher PCO2 level. Our study highlights the importance of incorporating restricted feeding into experimental designs examining OAW and suggests that ad libitum feeding used in the majority of the studies to date may not have been suitable to detect impacts of ecological significance.

Purification and partial characterization of a novel bacteriocin produced by bacteriocinogenic Lactobacillus fermentum BZ532 isolated from Chinese fermented cereal beverage (Bozai)

In current study, a novel bacteriocin LF-BZ532 was produced by Lactobacillus fermentum BZ532 that was isolated from Chinese fermented cereal drink (locally called bozai) and identified by 16s rDNA sequencing. The bacteriocin LF-BZ532 was purified by ammonium sulfate precipitation, SP-Sepharose cation exchange chromatography and AKTA purifier RP-HPLC, in succession. Then purified product was subjected to MALDI-TOF MS and designated its molecular mass was 1105.563 Da. Bacteriocin LF-BZ532 was found to have broad antimicrobial spectrum against both Gram-positive and Gram-negative bacteria, including anti-listerial and anti-pseudomonas activity. It was significantly thermostable with 88.19% and 56.98% residual activity at 100 and 121ᴼC (30, 20 min) respectively. Moreover, it was considerably stable at a range of pH (2–8) but all the activity was eliminated against proteolytic (Proteinase K, pepsin and trypsin) enzymes, suggested its peptide nature. Based on above premise, it is proposed that purified LF-BZ532 and its producing strain Lactobacillus fermentum BZ532 significantly proved as potent candidates to be use as a bio-preservative in food industry.

Physicochemical properties of oci fish joruk (Rastrelliger kanagurta) and the antioxidant activity of joruk hydrolysate

Joruk is a traditional fishery product made by spontaneous fermentation process using lactic acid bacteria by salting method, followed by fermentation process. Hydrolysis of proteins with the pepsin and a combination of pepsin and trypsin enzymes by in vitro can produce bioactive peptides. The aims of this study were to investigate the change of soluble protein and lactic acid content in oci fish joruk (Rastrelliger kanagurta) and to determine the influence of the enzyme hydrolysis on the antioxidant activity of joruk. The raw material used in this highest study was the oci fish obtained from Gorontalo, Indonesia. The produced joruk was added with the concentration of palm sugar 10%, 20% and 30% then fermented for 8 days, 10 days and 12 days. Protein hydrolysis using the pepsin enzyme and a combination of pepsin and trypsin enzyme. The results showed that the addition of palm sugar and fermentation time can reduce soluble protein and increase the lactic acid content. The highest antioxidant activity was found in joruk hydrolysate that hydrolyzed by pepsin enzyme with the addition of 30% palm sugar and 12 days fermentation, with a value of 5.28%.

The effect of dietary sericite on growth performance, digestive enzymes activity, gut microbiota and haematological parameters of Nile tilapia, Oreochromis niloticus (L.) fingerlings

A feeding trial was conducted to assess the effect of dietary supplementation of sericite (silicate clay) on growth performance, digestive enzymes activity, immune parameters and gut microbiota, of Nile tilapia, Oreochromis niloticus (L.) fingerlings. Five isonitrogenous diets were formulated each diet supplemented with different levels of sericite 0 (control), 2.5, 5, 7.5 and 10 g/kg diet. After 70 days of feeding trial, supplemental sericite were quadratically improved the final body weight (FBW) (P = 0.023), weight gain (WG) (P = 0.012), specific growth rate (SGR) (P = 0.023) and feed conversion ratio (FCR) P = 0.023) and protein efficiency ratio (PER) (quadratic, P = 0.045). However, the relationship between FCR and sericite levels was expressed by a broken-line model with an identified optimal breakpoint of 6.3 g/kg of sericite inclusion in the diets. Additionally, significant quadratic increase in alkaline phosphatase (ALP), amylase, trypsin, chymotrypsin and lipase enzymes were detected (P = 0.026; P = 0.023; P = 0.013; P = 0.045; P = 0.023) as the level of sericite increased in the diet. Furthermore, dietary sericite levels exhibited linear decreased in the total count bacteria, E. coli and Enterobacteriaceae of stomach and gut of experimental fish (linear, P = 0.032; P = 0.024; P = 0.035; P = 0.023; P = 0.012; P = 0.039, respectively). There was no effect of dietary sericite levels on hemoglobin (Hb), hematocrit (Htc) and red blood cells (RBCs), but a quadratic trend was observed in white blood cells (WBCs), monocytes and lymphocytes of fish (quadratic, P = 0.036; P = 0.013; P = 0.034), respectively. Increasing dietary sericite levels did not affect alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin and globulin but quadratically increased total protein (quadratic, P = 0.012) and IgM exhibited linear trend (linear, P = 0.045). Results in this study indicate that the addition of sericite as feed additive enhanced the growth, feed utilization, digestive enzyme activities, blood profile and gut biota of Nile tilapia.

Prevention of protein and lipid oxidation in freeze-thawed bighead carp (Hypophthalmichthys nobilis) fillets using silver carp (Hypophthalmichthys molitrix) fin hydrolysates

Conversion of aquatic by-products into value-added food ingredients has gained great interest worldwide, and enzymatic hydrolysis is one of the most accepted methods for the recovery of by-product proteins. In our study, fins from silver carp (Hypophthalmichthys molitrix) were hydrolyzed by four enzymes (trypsin, alcalase, papain, and neutrase) and the antioxidant activity of the derived fin hydrolysates was evaluated both in vitro and in freeze-thawed bighead carp (Hypophthalmichthys nobilis) fillets. The results showed that fin hydrolysates by trypsin and alcalase exhibited strong in vitro scavenging activity against 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals and chelating activity to ferrous ions. Freeze-thaw-induced protein oxidation (the formation of carbonyls and disulfide bonds) and degradation (the loss of Ca2+-ATPase activity) were significantly (P < 0.05) inhibited by fin hydrolysates. In addition, fin hydrolysates reduced lipid oxidation as evidenced by decreased free fatty acid content, peroxide value, thiobarbituric acid reactive substances (TBARS), and fluorescent compounds after multiple freeze-thaw cycles. Therefore, fin hydrolysates by trypsin and alcalase could be potential natural antioxidants in preservation of fish fillets.

Sustained and improved enzymatic activity of trypsin immobilized in the Langmuir Blodgett film of DPPC: A rapid enzyme sensor for the detection of Azocasein

Immobilizing enzymes on biomimicking monolayer ultrathin films of phospholipids exhibit enhanced enzymatic activities (EAs) useful for biosensing applications. Preparation of such substrates with enzyme Trypsin (TRYP) immobilized in dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix with enhanced and sustained enzymatic activity is a real challenge, as the isoelectric point of TRYP is reported to exist at pH ~ 10.5. This paper reports for the first time a facile procedure to fabricate smart substrates of TRYP entrapped in the DPPC monolayer using Langmuir–Blodgett (LB) technique. The incorporation of TRYP into DPPC monolayer at the air–water interface has been demonstrated from adsorption kinetics (π – t), room temperature surface pressure–area (π – A) isotherm plot and compressibility (β – π) studies. Circular dichroism (CD) and Grazing angle IR (GAIR) spectra signify changes in the secondary structure of incubated TRYP when immobilized in DPPC monolayer. Improved and sustained EA of TRYP upon its incubation on the organized monolayer LB films of DPPC has been observed. The reason behind such improvement in the EA of immobilized TRYP has also been suggested. The as fabricated substrates of immobilized TRYP can be used to sense soy milk, soy yoghurt, mayonnaise, meringue etc. using Azocasein as the protease substrate.

Evaluation of the Inhibitory Effect of a Medicinal Herb &amp;lt;i&amp;gt;Phyllanthus amarus&amp;lt;/i&amp;gt; on the Activity of &amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt;-Amylase, Pepsin and Trypsin

The medical herb Phyllanthus amarus play a crucial role in indigenous medicine. Therapeutically, these plants’ extract acts as potential players that inhibits several digestive enzymes that are relevant to the management of Peptic ulcers and Diabetes Mellitus, which occur due to the overproduction of such enzymes. Evaluation of inhibitory effect of this extract was carried out against Pepsin, α-amylase, Trypsin enzymes along with the effect of thermal stability and ammonium sulphate precipitation on these inhibitory assays. P. amarus leave’s extract with different concentration gradients were used in this research analysis. Results obtained along with the literature analysis revealed photochemical compounds such as polyphenols causes inhibitory nature in the extract. Maximal percentage of inhibition of amylase, pepsin and trypsin were found to be 71% (0.32 mg/ml), 85% (0.08 mg/ml) and 87% (1.28 mg/ml) respectively. In thermal stability assay the maximum percentage of inhibition for amylase, pepsin and trypsin was observed at 30% (80°C), 68% (4°C) and 5% (37°C). Enzymes inhibitory assays on ammonium sulphate precipitation elicited maximum percentage of inhibition for amylase, pepsin and trypsin as 42% (at 45% of (NH4)2SO4), 58% (at 15% of (NH4)2SO4) and 40% (at 30% of (NH4)2SO4) respectively. This research concluded that Phyllanthus amarus leave extracts are potential inhibitors of α-amylase, pepsin and trypsin enzymes. Ammonium sulphate precipitation was helpful to purify the polyphenols the active compounds to a good extend. Also, thermal stability was helpful to check the stability of these active photochemical compounds present in the extract. Thus, P. amarus is an effective inhibitor to be used as supplements in the disease management.

Alternative “Green” Antimicrobial Agents Obtained by Selective Sorption from Lactobacillus plantarum Culture

Abstract According to the world health organization report from September 2016, the development of pathogenic bacteria resistance to antimicrobial drugs is one of the most important problems of the modern medicine. In this regard, the urgent task is the search for alternative antibiotics for the treatment of bacterial infections. One approach to solving this problem is obtaining antimicrobial compounds synthesized by probiotic lactic acid bacteria. The probiotic strain of Lactobacillus plantarum 8P-A3, was chosen to study its antimicrobial action. This strain produces at least two bacteriocins – plantaricin EF and plantaricin NC8. The chromatographic isolation of peptide fractions from the supernatant was carried out using a polymer sorbent based on methacrylic acid and ethyleneglycol dimethacrylate. Optimal parameters for chromatographic process were determined. It is shown that all the target biologically active substances were bound with the sorbent in sorption at acidic pH values. Elution was performed in isocratic mode. The antimicrobial activity of the obtained peptide fractions against indicator culture was determined by turbidimetric method. During incubation process, the turbidity of the microbial suspension was determined by measuring the optical density at λ = 600 nm. It is revealed that the fraction obtained at rinse by eluent with pH 8 has the maximum inhibitory ability. Сhromatomass-spectrometry analysis of the peptide fraction was carried out using Shimadzu LCMS-8040.The antimicrobial activity of the fraction is comparable to the action of ampicillin against gram-negative bacteria Escherichia coli. To confirm the peptide nature of the antimicrobial activity of the fraction, an indicator culture was incubated with the fraction treated with proteolytic enzymes (trypsin). It is determined the fraction can be stored at −18 °C and saves antimicrobial properties after defrosting.

Effect of Different Treatments on Antioxidative Stability of the Scallop Protein Hydrolysates

Effects of different treatments on the antioxidant activity of scallop protein hydrolysates (SPH) were evaluated using DPPH radical scavenging activity and reducing power. Results showed that the antioxidant activity of SPH had good heating-resistance from 25°C to 65°C. The antioxidant activity of SPH could retain under acidic environment, but rapidly reduced under alkaline conditions. Addition of D-galactose, D-xylose, and D-fructose at 65°C could increase the antioxidant activity of SPH, but no such effect was not observed at this temperature. With the increase of storage time, the antioxidant activity of SPH gradually decreased. Moreover, pepsin digestion treatment slightly reduced the antioxidant activity of SPH, and further trypsin and mixed enzyme (trypsin + chymotrypsin) digestion significantly reduced this activity (p 0.05). In conclusion, SPH may be used as food ingredients or food supplements in different food fields.

Extraction of crude collagen from &lt;em&gt;Pterygoplichthys pardalis&lt;/em&gt; skin and determination of functional properties of its hydrolysates

Collagen has a great demand and fish skin is a safe alternative source of collagen. Pterygoplichthys pardalis is an endemic, freshwater fish with no economic benefit. The study was conducted to extract crude collagen from the skin of P. pardalis using a simple method and identify the functional properties of its hydrolysates. Acid and pepsin soluble collagens were extracted from skin using citric acid (CA) and ethylene diamine tetraacetic acid (EDTA). Selected crude collagens were hydrolysed using pepsin, protease and trypsin enzymes (1:100). Extracted crude collagens and best hydrolysates were selected using 8 and 15% SDS-PAGE. Hydrolysates were analysed for antioxidant, Fe(II) chelating and antibacterial activities. CA treatment showed significantly (P&lt;0.05) higher yield than EDTA treatment. Antioxidant properties, metal chelation and antibacterial activities were not significantly (P&gt;0.05) different. Hydrolysates showed significantly (P&lt;0.05) higher functional properties than crude collagen in both treatments. These results conclude that collagen hydrolysates from P. pardalis have good antioxidant, metal chelating and antibacterial properties.

Uterine inflammatory conditions in dairy cows: Diagnosis and treatment

The diagnosis of inflammatory conditions in the uterus depends primarily on the severity of the inflammation process and associated clinical symptoms. Because of the period of occurrence and characteristic signs, severe lesions in the uterus, such as metritis puerperalis, are not difficult to diagnose and do not require the use of diagnostic methods other than clinical examinations and, if necessary, additional tests to determine the general condition of the animals. However, in this case, it is important to precisely identify the pathogenic factor and to determine its susceptibility to antibiotics in order to define further treatment. Endometritis is the biggest diagnostic problem, especially its subclinical form, which can be diagnosed only by additional cytological or histopathological tests. These tests, although available, are not routinely performed in Poland at this time. The latest diagnostic methods, such as immunohistochemical tests or flow cytometry, have provided many previously unknown data, for example, on the action of the local resistance mechanisms in the uterus. Since the immune system of the uterus is not fully known, the above tests may improve the understanding of its activity or may help modulate its functions in the future as a potential preventative measure or treatment for inflammatory conditions of the uterus. The treatment of inflammatory conditions in dairy cows is currently based on two basic methods: intrauterine administration of antibiotics and intramuscular injections of prostaglandin-based preparations. The main goal of the treatment is to improve the fertility of cows in the herd, and since these methods are often ineffective, there have been attempts to treat endometritis with iodine-based preparations and preparations containing enzymes (trypsin, chymotrypsin and papain). They did not, however, improve the fertility of cows. The administration of non-steroid anti-inflammatory drugs to eliminate the inflammatory condition of the uterus and to prevent early deaths of embryos at the time of the greatest risk of embryo loss did not increase the number of pregnancies in cows either. Literature data suggest that immunomodulation and the use of probiotics may be useful in the prevention and treatment of endometritis.

Investigating Monoliths (Vinyl Azlactone-co-Ethylene Dimethacrylate) as a Support for Enzymes and Drugs, for Proteomics and Drug-Target Studies.

Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 μm inner diameter (ID) × 160 mm, 180 μm ID × 110 mm or 250 μm ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules (i.e., proteases or drugs), as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a “fit-for-all” material. We also discuss issues related to the repeatability of monolith-preparations.

Extraction, Purification, and Characterization of Trypsin Obtained from the Digestive System of Yellowfin Seabream (Acanthopagrus latus).

The development of the marine aquaculture industry has led to the generation of significant amounts of fish wastes. Marine farm wastes exert adverse effects on the surrounding area of the cages. On the other hand, wastes of fish and other aquatic animals are regarded as major sources of valuable natural bioactive compounds, including enzymes, proteins, bioactive peptides, oil, amino acids, collagen, gelatin, calcium, biopolymers, and water-soluble minerals. To investigate the potential of marine fish waste, the whole digestive system of yellowfin seabream (Acanthopagrus latus) was extracted for extraction and identification of trypsin enzyme. Fish (179.93&amp;plusmn;93.67 g; 184&amp;plusmn;28.17 cm) were caught from the Persian Gulf and stored at -20 &amp;deg;C. Yellowfin seabream were dissected and their whole digestive systems were removed. Samples were thoroughly washed with distilled water and purified through defatting using acetone and ammonium sulfate precipitation. The following issues were assessed: the total and specific activity of trypsin, protein determination, molecular weight, enzyme activity and stability in different pH values and temperatures. The obtained results indicated that specific activity and protein content of trypsin enzyme were 4.4 U and 3.4 mg/ml, respectively. The molecular weight of 23 kDa was reported for trypsin using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. Maximum activity and stability of trypsin were observed at 60&amp;deg;C and 45&amp;deg;C, respectively. Trypsin demonstrated maximum activity and stability at a pH value of 8.0. In general, the results of the current study suggested that trypsin extracted from the digestive system of yellowfin seabream has considerable potential for industrial applications, such as the food industry, owing to its characteristics and stability under alkaline conditions.

Dual Insecticidal Effects of Adenanthera pavonina Kunitz-Type Inhibitor on Plodia interpunctella is Mediated by Digestive Enzymes Inhibition and Chitin-Binding Properties.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

Layer‐by‐layer self‐assembly of multifunctional enzymatic UF membranes

ABSTRACTIn order to eliminate membrane fouling and to ensure enzymatic hydrolysis of urea, multifunctional biocatalytic membranes were prepared by using urease (URE) and trypsin (TRY) enzymes on the sulfonated polysulfone (SPSf) ultrafiltration membrane via layer‐by‐layer (LbL) self‐assembly method. The membrane architecture consisted of multilayer assembly with TRY and URE enzymes as the outer layer and inner sandwiched layer, respectively. Polyethyleneimine (PEI) and alginate (ALG) were used as cationic and anionic polyelectrolytes. Sulfonation and PEI deposition were successfully accomplished as confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy analysis, contact angle measurements, staining with toluidine blue and Congo red dyes and dead‐end filtration experiments. A characteristic value of SPSf membrane with a high water permeability (1000 L/m2.h.bar) and 95% bovine serum albumin (BSA) rejection was observed. In static conditions, URE activities of SPSf2‐PEI‐URE membrane were not affected by BSA fouling, while TRY immobilizations with increased concentrations (SPSf2‐PEI‐URE‐PEI‐ALG‐TRY) significantly lowered the activity of URE. In dynamic conditions, each deposited layer exhibited individual resistance to flow that can be considered as irreversible fouling and caused 90% of flux decline for the SPSf2‐PEI‐URE‐PEI‐ALG‐TRY membrane assembly. The recovery of the initial flux for the multilayered membrane at the end of six fouling and washing cycles was observed 85%. Moreover, at the end of 5 cycles, 78% of the URE initial activity of the multilayered membrane was preserved. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48750.

Analysis of protein profiles and peptides during in vitro gastrointestinal digestion of four Chinese dry-cured hams

In this study, the in vitro digestion with pepsin and trypsin enzymes was applied to mimic the gastrointestinal digestion of dry-cured hams from different producing districts of China. The analysis of digestibility and particle size revealed that the digestion characteristics of different dry-cured hams were significantly different (p < 0.05). During the gel electrophoresis analysis, protein profiles were characterized by analyzing 14 and 9 proteins generated during pepsin and pepsin/trypsin digestion. The relative intensities of protein bands could be used to distinguish different dry-cured hams based on principal component analysis (PCA) and linear discriminant analysis (LDA). According to the analysis of peptide profiles, more than 50% of peptides were distributed in the molecular weight ranging from 1500 to 2500 Da, and two-step digestion had little effect on peptide generation due to the cleavage site specificity of pepsin and trypsin. This study provided evidences that it was possible to identify producing districts of different dry-cured hams based on digested characteristics.

Response of intestine microbiota, digestion, and immunity in Pacific white shrimp Litopenaeus vannamei to dietary succinate

Succinate (SA) is the precursor of propionate that influence the intestine physiological function of animals. In this study, we investigated the microbiome, digestive, and immune responses in the intestines of Pacific white shrimp Litopenaeus vannamei fed diets containing different levels of SA: 0 g/kg (Control), 2.5 g/kg (SA1), 5.0 g/kg (SA2), and 10.0 g/kg (SA3) for 56 days. The results showed that dietary SA improved the intestine mucosa morphology, increased the short-chain fatty acids contents, and altered the intestine microbiota composition. Specifically, SA increased the abundances of Proteobacteria and decreased the abundance of Bacteroidetes. The beneficial bacteria including Ruegeria, Lutimonas, Mameliella, and Sphingomonas were enriched in response to dietary SA; these genus might be involved in nutrient metabolism and the degradation of toxic compounds and pollutants. Additionally, dietary SA also improved the activity of digestive enzymes (amylase, lipase, trypsin, and pepsin) and immune enzymes (total antioxidant capacity, phenoloxidase, total nitric oxide synthase, and nitric oxide), and the expression of immune-related genes (prophenoloxidase, anti-lipopolysaccharide factor, lysozyme, heat shock protein 70, thioredoxin, mucin-1, mucin-2, mucin-5 AC, mucin-5B, and mucin-19) in the intestine of the shrimp. These results revealed that dietary SA had a positive effect on the intestine health of L. vannamei by modulating the microbial composition and SCFA contents, and enhancing the digestive and immune response in the shrimp intestine.

Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization

Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5′-4,4′-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 μM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.

Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6.

Kallikrein‐related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high‐throughput screen within the European Lead Factory program. Structure‐guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)‐3‐(4‐carbamimidoylphenyl)‐N‐((S)‐1‐(naphthalen‐1‐yl)propyl)‐2‐oxooxazolidine‐5‐carboxamide) and 34 ((5R)‐3‐(6‐carbamimidoylpyridin‐3‐yl)‐N‐((1S)‐1‐(naphthalen‐1‐yl)propyl)‐2‐oxooxazolidine‐5‐carboxamide) have single‐digit nanomolar potency against KLK6, with over 25‐fold and 100‐fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6‐dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.

Trypsin polymorphism and modulation in Penaeus vannamei (Boone, 1931): a review

The trypsin enzyme of white shrimp Penaeus vannamei is a polymorphic molecule, which longest isolated and sequenced cDNA encoded a pre-proenzyme of 255 amino acids. Three of the described sequences are translated in the digestive gland, and SDS-PAGE detects the isoforms. The three isoforms named C, B and A are distributed by an individual to originate three trypsin phenotypes (CBA, CB and CA) that have already been isolated and characterized by defining theirs biochemical and kinetical differences. The CBA phenotypes exerted higher hydrolytic capabilities than the others. The trypsin phenotypes are inherited in a Mendelian fashion, and external or internal factors do not modulate them. In commercial hatcheries in Sinaloa, Mexico, the most abundant phenotype was CBA, followed by CB and CA. The following two hypotheses may explain this finding: phenotype CA is not chosen throughout the selection process of breeders because they do not fit the anatomical characteristics desired by professionals in aquaculture or it is possible that shrimps with the CA phenotype die during the early stages of development. Two experimental designs were developed trying to explain such hypotheses and, in both experiments, CA phenotype frequency declined to zero when shrimps reached around 5 g of body weight. So far, no evidence explains the mortality of juvenile shrimps with phenotype CA. Research is needed to explain why CA phenotypes disappeared, how the survival of this phenotype can be improved, and how to produce shrimps with the most hydrolytic capability in support of the shrimp aquaculture industry.