A Novel Gelatinase from Marine Flocculibacter collagenilyticus SM1988: Characterization and Potential Application in Collagen Oligopeptide-Rich Hydrolysate Preparation

Although the S8 family in the MEROPS database contains many peptidases, only a few S8 peptidases have been applied in the preparation of bioactive oligopeptides. Bovine bone collagen is a good source for preparing collagen oligopeptides, but has been so far rarely applied in collagen peptide preparation. Here, we characterized a novel S8 gelatinase, Aa2_1884, from marine bacterium Flocculibacter collagenilyticus SM1988T, and evaluated its potential application in the preparation of collagen oligopeptides from bovine bone collagen. Aa2_1884 is a multimodular S8 peptidase with a distinct domain architecture from other reported peptidases. The recombinant Aa2_1884 over-expressed in Escherichia coli showed high activity toward gelatin and denatured collagens, but no activity toward natural collagens, indicating that Aa2_1884 is a gelatinase. To evaluate the potential of Aa2_1884 in the preparation of collagen oligopeptides from bovine bone collagen, three enzymatic hydrolysis parameters, hydrolysis temperature, hydrolysis time and enzyme-substrate ratio (E/S), were optimized by single factor experiments, and the optimal hydrolysis conditions were determined to be reaction at 60 ℃ for 3 h with an E/S of 400 U/g. Under these conditions, the hydrolysis efficiency of bovine bone collagen by Aa2_1884 reached 95.3%. The resultant hydrolysate contained 97.8% peptides, in which peptides with a molecular weight lower than 1000 Da and 500 Da accounted for 55.1% and 39.5%, respectively, indicating that the hydrolysate was rich in oligopeptides. These results indicate that Aa2_1884 likely has a promising potential application in the preparation of collagen oligopeptide-rich hydrolysate from bovine bone collagen, which may provide a feasible way for the high-value utilization of bovine bone collagen.

Bioinformatic mapping of a more precise Aspergillus niger degradome

Aspergillus niger has the ability to produce a large variety of proteases, which are of particular importance for protein digestion, intracellular protein turnover, cell signaling, flavour development, extracellular matrix remodeling and microbial defense. However, the A. niger degradome (the full repertoire of peptidases encoded by the A. niger genome) available is not accurate and comprehensive. Herein, we have utilized annotations of A. niger proteases in AspGD, JGI, and version 12.2 MEROPS database to compile an index of at least 232 putative proteases that are distributed into the 71 families/subfamilies and 26 clans of the 6 known catalytic classes, which represents ~ 1.64% of the 14,165 putative A. niger protein content. The composition of the A. niger degradome comprises ~ 7.3% aspartic, ~ 2.2% glutamic, ~ 6.0% threonine, ~ 17.7% cysteine, ~ 31.0% serine, and ~ 35.8% metallopeptidases. One hundred and two proteases have been reassigned into the above six classes, while the active sites and/or metal-binding residues of 110 proteases were recharacterized. The probable physiological functions and active site architectures of these peptidases were also investigated. This work provides a more precise overview of the complete degradome of A. niger, which will no doubt constitute a valuable resource and starting point for further experimental studies on the biochemical characterization and physiological roles of these proteases.

Recombinant metalloprotease as a perspective enzyme for meat tenderization

Peptidase family M9 (MEROPS database) is true collagenases and contains bacterial collagenases from Vibrio and Clostridium. One of the producers of M9A subfamily peptidase is Aeromonas salmonicida (locus - ASA_3723). The aim of the study was production of recombinant metallopeptidase Aeromonas salmonicida by transformation Pichia pastoris for further meat tenderization. Laboratory amounts of recombinant peptidase were obtained and test evaluation of enzyme activity was performed. Recombinant peptidase broke the peptide bond «Pro-Leu-Gly-Met-Trp-Ser-Arg» (one of the collagen chains, (Mw = 846.06)). The concentration of the substrate (peptide) after 180 min was 2 – fold decrease as compared with control. The maximum shear force of heat-treated samples had a 1.27 – fold decrease as compared with the control. As a result of histological studies of beef shank samples, the specific effect of the supernatant on the structure of connective tissue was established. Muscle fibers have not changed. The recombinant enzyme could be used for the meat tenderization.

Metallopeptidases ofToxoplasma gondii:in silicoidentification and gene expression

Metallopeptidases are a family of proteins with domains that remain highly conserved throughout evolution. These hydrolases require divalent metal cation(s) to activate the water molecule in order to carry out their catalytic action on peptide bonds by nucleophilic attack. Metallopeptidases from parasitic protozoa, includingToxoplasma, are investigated because of their crucial role in parasite biology. In the present study, we screened theT. gondiidatabase using PFAM motifs specific for metallopeptidases in association with the MEROPS peptidase Database (release 10.0). In all, 49 genes encoding proteins with metallopeptidase signatures were identified in theToxoplasmagenome. An Interpro Search enabled us to uncover their domain/motif organization, and orthologs with the highest similarity by BLAST were used for annotation. These 49 Toxoplasmametallopeptidases clustered into 15 families described in the MEROPS database. Experimental expression analysis of their genes in the tachyzoite stage revealed transcription for all genes studied. Further research on the role of these peptidases should increase our knowledge of basicToxoplasmabiology and provide opportunities to identify novel therapeutic targets. This type of study would also open a path towards the comparative biology of apicomplexans.

Crystal structure analysis of dipeptidyl amino peptidase from P. mexicana WO24.

Dipeptidyl aminopeptidase (DAP or DPP, EC 3.4.14) catalyses the removal of dipeptides from the amino termini of peptides and proteins. In microorganisms, we have reported the identification, purification, and characterization of DAP BI, DAP BII, DAP BIII, and DAP IV (bacterial DPP4), POP fromPseudoxanthomonas mexicanaWO24, and demonstrated that DAP BI, DAP BIII, DAP IV and POP belong to the POP family and they are classified into the clan SC, family S9 in the MEROPS database. On the basis of the enzymological data we obtained, we proposed that bacterial DAPs should be classified in a manner different from that of mammalian DPPs, except for the DAP IV. The DAP IV liberates dipeptides from the free amino terminus and has a specificity for both proline and hydroxyproline residues in the penultimate position of peptides. Here, we report the first structure of the bacterial DPP IV (P. mexicanaWO24 DAP IV) complexed with an inhibitor at 2.2 Å resolution. The subunit structure is composed of two domains, the N-terminal eight-bladed β-propeller domain and the C-terminal alpha/beta/alpha sandwich catalytic domain. These structural features are conserved with clan SC S9 family. However, the N-terminal domain contains a unique helix region that extends over the active site acting as a lid, gating substrate or product access. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique helix region is conserved in some kind of bacterial DPP4s except for mammalian DPP4s and some bacterial DPP4s. Some asaccharolytic and anaerobic bacteria can be used protein or peptides as an energy source. Therefore, these bacteria secrete many types of proteases and peptidases. Especially, the elucidation of degradation mechanisms of collagen, including proline and hydroxyproline, are very important from the point of view of host tissue breakdown in pathogens. Our findings suggest that different ligand recognition mechanisms from the bacterial DPP IV to mammalian DPP4 raise the possibility of an antimicrobial development targeting DPP IV from bacteria.