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

Abstract

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.

Mechanistic Insight Into the Production of Collagen Oligopeptides by the S8 Family Protease A4095

“…This result is in agreement with previous studies, which showed that Gly, Pro, and Hyp are abundant in collagen hydrolysates. 37,38 In the hydrolysate, the molecular weights of 62.48% of peptides were less than 1000 Da, and 30.36% less than 500 Da (Table 4 and Figure S3). For comparison, the oligopeptide contents reported in the collagen hydrolysates prepared with other proteases are shown in Table 5.…”

Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities

“…With the majority of active serine hydrolases identified from our screen being annotated as subtilases (peptidase family S8) or penicillin acylases (peptidase family S15; Tab.1–2), we indeed detected thermostable representatives of the functionally highly diverse family of serine hydrolases that already find application for industrial purposes. In particular, proteases from the subtilisin family are utilized in the laundry industry 60 or for the production of valuable chemicals from proteinaceous substrates 61,62 , whereas penicillin acylases are important enzymes for the production of 6-aminopenicillanic acid (6-APA), the β-lactam nucleus used in the manufacturing process of semi-synthetic antibiotics, through hydrolysis of natural penicillins 63 .…”

Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities

“…Altogether, these results show that our eABPP approach enabled the identification of functionally diverse serine hydrolases, ranging from various proteases to esterases/lipases up to serine hydrolases with uncharacterized functions (e.g., DUFs or UPFs), thus being superior over a simple bioinformatic metagenome annotation procedure. Of note, the majority of the identified active serine hydrolases are annotated as subtilases or penicillin acylases, which demonstrates that the applied eABPP screen indeed allows the detection of thermostable representatives of the functionally highly diverse family of serine hydrolases that already find application for industrial purposes [66][67][68][69].…”