SP-1, a Serine Protease from the Gut Microbiota, Influences Colitis and Drives Intestinal Dysbiosis in Mice

Increased protease activity has been linked to the pathogenesis of IBD. While most studies have been focusing on host proteases in gut inflammation, it remains unclear how to address the potential contribution of their bacterial counterparts. In the present study, we report a functional characterization of a newly identified serine protease, SP-1, from the human gut microbiota. The serine protease repertoire of gut Clostridium was first explored, and the specificity of SP-1 was analyzed using a combinatorial chemistry method. Combining in vitro analyses and a mouse model of colitis, we show that oral administration of recombinant bacteria secreting SP-1 (i) compromises the epithelial barrier, (ii) alters the microbial community, and (ii) exacerbates colitis. These findings suggest that gut microbial protease activity may constitute a valuable contributor to IBD and could, therefore, represent a promising target for the treatment of the disease.

Impact of Microbial Protease Enzyme and Dietary Crude Protein Levels on Growth and Nutrients Digestibility in Broilers over 15–28 Days

In this trial, a 3 × 2 factorial design with different dietary crude protein levels (CP, 17, 19 and 21%) and two levels of exogenous protease (0 and 30,000 IU/kg) was used. A total of 540 two-week old broilers (Ross-308) was randomly allocated to experimental diets over 15–28 days of age. The interaction between dietary protein levels and enzyme supplementation showed that body weight gain was significantly (p < 0.05) higher in birds fed CP-19 (1114.7 g) and CP-21 (1108.8 g) with enzymes supplementation. Feed intake was higher (p < 0.05) in broilers fed with CP-17 than CP-19 with supplementation of the protease enzyme. Results also revealed that the feed conversion ratio (FCR) was significantly (p < 0.05) improved in birds fed with CP-19 and CP-21 and protease supplementation. Total tract N retention was lower (p < 0.05) in birds fed CP-17 with no enzyme than the other dietary groups. Similarly, the gross energy (GE) was significantly (p < 0.05) lower in birds fed CP-17 with or without the protease enzyme. Abdominal fat was higher (p < 0.05) in CP-17 (0.96%) without the protease enzyme. It was concluded that a diet at 19% CP with the protease enzyme improved the performance and nutrient digestibility in broilers over 15–28 days.

Standardised Ileal Amino Acid Digestibility in Field Pea Seeds of Two Cultivars Differing in Flower Colour for Broiler Chickens: Effects of Bird Age and Microbial Protease

This study aimed to determine and compare standardised ileal digestibility (SID) coefficients of amino acids (AA) in raw seeds of the white-(WF) and the coloured-flowered (CF) field pea cultivar as sole sources of AA in the diets fed to broiler chickens aged 14 or 28 days. An additional purpose was to check the influence of exogenous protease added to pea-based assay diets on AA SID in birds at both ages. Each assay diet was offered to six replicate pens. On both sampling days, the contents from the lower half of the ileum were collected for determination of the apparent digestibility values. The SID coefficients were calculated using ileal endogenous AA losses determined from birds fed an N-free diet. Results indicated a substantial advantage of WF pea over CF pea as a source of digestible Lys, Met, Cys, His, Ile, Leu, Phe, Val, Asp and Glu for 14-day-old chickens. With the exception of methionine and cysteine, there was no significant difference between these two cultivars in the SID values of AA in 28-day-old birds. The protease increased SID of nutritionally essential AA from WF pea-based diet at both ages, and from CF pea-based diet in chickens aged 28 days. In conclusion, the SID coefficients of indispensable AA determined at 14 days of age in low-tannin WF peas are not applicable to the formulation of grower-type feeds containing seeds of CF cultivars.

Metaproteomics reveals potential signatures of disease-specific alterations in the gut microbial proteolytic events in inflammatory bowel disease

Background: Proteolysis regulation allows gut microbes to respond rapidly to dynamic intestinal environments by fast degradation of misfolded proteins and activation of regulatory proteins. However, alterations of gut microbial proteolytic signatures under complex disease status such as inflammatory bowel disease (IBD, including Crohn’s disease (CD) and ulcerative colitis (UC)) have not been investigated. Here, we explored the microbial proteolysis landscape using two public datasets containing 623 metaproteomes from 447 fecal and 176 mucosal luminal interface (MLI) samples from IBD patients and healthy individuals using a semi-tryptic peptide centric mining approach. Results: Fecal metaproteome revealed a global alteration of gut microbial proteolysis signatures in IBD, while MLI metaproteome demonstrated remarkable disease-specific and location-specific alterations at taxonomic, functional, and cleavage site motif levels. The functional alterations in IBD mainly involved microbial carbohydrate transport and metabolism, oxidative stress, cell motility, protein synthesis and maturation. Altered microbial proteolysis signatures of CD and UC mainly occurred in terminal ileum and descending colon, respectively. Microbial proteolysis patterns exhibited low correlations with β-diversity and moderate correlations with microbial protease transcriptome, respectively. Human protease inhibitors and immunoglobulins were mainly negatively associated with microbial proteolysis patterns, suggesting the inhibitory effects of these host factors on gut microbial proteolysis events. Conclusions: Our findings highlight the complex and diverse proteolytic alterations of gut microbiome in IBD, providing a unique layer of information beyond taxonomic and proteomic abundance.

Characteristics of Fish Protein Hydrolysate from Yellowstripe Scad (Selaroides leptolepis) Produced by a Local Microbial Protease

Fish protein hydrolysate (FPH) containing small protein or peptides and amino acids has a great attention related to the provision of high protein foods to overcome the problem of malnutrition. This research was purposed to prepare FPH from yellowstripe scad (Selaroides leptolepis) by using a local microbial protease from Bacillus subtilis BII-1. Hydrolysis process was done in a laboratory scale (500 g minced fish) at 55oC for 6 h. The liquid hydrolysate was then spray dried using whey protein and maltodextrin at a concentration of 20 and 30% for each filler. The treatment of whey protein powder produced FPHs with higher protein content (31.71-33.97% db) and slightly yellowish in color compared to maltodextrin (11.88-16.66% db). Their foaming capacity and stability were 20-100% and 15% in 5-10 min, respectively. However, FPHs prepared with maltodextrin had no foaming capacity. The hydrolysates from both treatments had low water and oil absorption with the value less than 3 mL/g hydrolysate. A trial on scaling up production using 30 kg fish, showed that optimization or adjustment should be carried out due to the high amount and high protein content of the residual products.

Hydrolyzation of duck meat protein using Bacillus cereus TD5B protease, pepsin, trypsin and their potency as an angiotensin converting enzyme inhibitor

This study was aimed to explore their potency of protein-bioactive of native ducks-meat after enzymatic hydrolysis by Bacillus-cereus TD5B-protease, Pepsin, and Trypsin as an angiotensin-converting enzyme (ACE) inhibitor. The samples: ducks-meats from 10 months age of male Mojosari and Magelang-Duck. The experiments: individually hydrolysis of meat-protein using protease-enzyme (0.1 % w/w) from Bacillus-cereus TD5B, Pepsin, or Trypsin. The observed parameters: protein concentration, protein molecular weight, ACE-inhibitor activity, and IC-value (IC50). Data of protein concentration were statistically analyzed using T-Test, while data of SDS-PAGE and ACE-inhibiting activity were analyzed descriptively. The results showed that soluble protein concentration increased due to the hydrolysis process, from 0.826±0.108 mg/mL to 1.050±0.197 mg/mL (Microbial-protease), 2.122±0.141 mg/mL (pepsin), 1.641±0.071 mg/mL (trypsin) for Mojosari-duck and 0.642±0.038 mg/mL to 1.171±0.534 mg/mL(Microbial-protease), 2.100±0.376 mg/mL(pepsin), 1.725±0.092 mg/mL(trypsin) for Magelang-duck. The SDS-PAGE pattern showed that there was a decrease of molecular weight of duck-meats due to the hydrolysis process, from the range of 196.53-43.88 kDa to the range of 71.35-10.12 kDa. Duck-meat protein hydrolysate had ACE-inhibiting activity 71.7%(Mojosari-Microbial-Protease) IC50 54μg/mL, 57%(Mojosari-Pepsin) IC50 151μg/mL, 75.8%(Mojosari-Trypsin) IC50 51μg/mL and 52.8%(Magelang-Microbial-Protease) IC50 83μg/mL, 78,5%(Magelang-Pepsin) IC50 85μg/mL, 83.9%(Magelang-Trypsin) IC50 22μg/mL. In conclusion, hydrolysate of Magelang duck-meat used Trypsin had better potency as an ACE-inhibitor.

E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance

Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Actinoallomurus A8, and its recombinant active form produced by Streptomyces lividans TK24. E40 is resistant to pepsin and trypsin, and active in the acidic pH range 3 to 6. E40 efficiently degrades the most immunogenic 33-mer as well as the whole gliadin proteins, as demonstrated by SDS-PAGE, HPLC, LC-MS/MS, and ELISA. T lymphocytes from duodenal biopsies of celiac patients showed a strongly reduced or absent release of IFN-γ when exposed to gluten digested with E40. Data in gastrointestinal simulated conditions suggest that no toxic peptides are freed during gluten digestion by E40 into the stomach to enter the small intestine, thus counteracting the intestinal inflammatory cascade to occur in CD patients. E40 is proposed as a novel candidate in Oral Enzymatic Therapy for the dietary management of gluten toxicity.