In Silico Analysis of Bioactive Peptides in Invasive Sea Grass Halophila stipulacea

Halophila stipulacea is a well-known invasive marine sea grass in the Mediterranean Sea. Having been introduced into the Mediterranean Sea via the Suez Channel, it is considered a Lessepsian migrant. Although, unlike other invasive marine seaweeds, it has not demonstrated serious negative impacts on indigenous species, it does have remarkable invasive properties. The present in-silico study reveals the biotechnological features of H. stipulacea by showing bioactive peptides from its rubisc/o protein. These are features such as antioxidant and hypolipideamic activities, dipeptidyl peptidase-IV and angiotensin converting enzyme inhibitions. The reported data open up new applications for such bioactive peptides in the field of pharmacy, medicine and also the food industry.


Introduction
Invasive species are becoming an important problem in the Mediterranean Sea where many vectors exist, including inflowing water from the Suez Channel and ships' ballast waters. One of the best-known invasive sea grasses is Halophila stipulacea (Forsskål) Ascherson [1], which was first reported in the Mediterranean Sea by Friscth [2]. This sea grass is abundant in the eastern Mediterranean Basin and very common along the Turkish coastline [3]. No validated eradication method for this invasive species is described in the scientific literature. A few reports mention the negative impacts of H.stipulacea on indigenous species [4], although Willette and Ambrose [5] mention that little is known of H. stipulacea's effects in its recently discovered Caribbean locations, while Van Tussenbroek et al. [6] report that H.stipulacea may be harmful for native species. Although most efforts so far have been devoted to finding alternative ways of evaluating non-indigenous species [7,8], invasive species secrete interesting secondary metabolites that can be exploited economically under the title of blue biotechnology. Indeed, recent years have seen growing interest in blue biotechnology-based products [9]. For example, bioactive peptides are one of the candidate targets that can be isolated from invasive species. However, to the best of our knowledge, the bioactive peptides from invasive H. stipulacea have not been assessed, and the aim of the present contribution is to fill in this gap in the literature.
Bioactive peptides (hereafter BPs) comprise 3-20 free amino acid food protein fragments [10] composed of covalently bonded (amide/peptide bonds) amino acids [11]. According to the BP database The frequency with which fragments with given activity were released by enzymes (A E ) and the relative frequency of release of fragments with given activity by enzymes (W) were calculated based on Equations (2) and (3), respectively.
where d is the number of fragments with given activity in the protein sequence that could be released by enzymes, and N is the number of amino acid residues in the protein chain. The relative frequency of release of fragments with given activity by selected enzymes (W) is given by: The values of A E and A are defined according to Equations (1) and (2), respectively. Potential biological activity of protein (B) [µM −1 ]: In Equation (4), a i is the number of repetitions of the i-th bioactive fragment in protein sequence, EC 50i is the concentration of the i-th bioactive peptide corresponding to its half-maximal activity [µM], k is the number of different fragments with given activity and N is the number of amino acid residues [35].
The theoretical degree of hydrolysis (DH t ) was also calculated using the following Equation (5): In Equation (5), d is number of hydrolyzed peptide bonds and D is total number of peptide bonds in a protein chain.
The relative activity of fragments with given activity released by selected enzymes (V) is: In Equation (6), B E is the activity of fragments potentially released by proteolytic enzyme (enzymes) and B is the potential biological activity of the protein.
The amino acid composition of protein was determined based on protein sequences, using the ProtParam program [37] available at [38].

Results
H. stipulacea large chain RubisCO was retrieved from expasy.org. After in silico proteolytic fragmentation of the RubisCO by BIOPEP tools, bioactive peptides were obtained. The raw data can be found in Appendix A (Tables A1-A56). BIOPEP parameters (A, A E , W, BH t and V) were extracted from the raw data and the results are presented in the Tables 1-10.
The angiotensin-converting enzyme (ACE) inhibitor properties of bioactive peptides from RubisCO of H. stipulacea were given in Table 1. The values were not calculated for prolyl endopeptidase, clostripain, thrombin, glutamyl endopeptidase II, Xaa-dipeptidase, chymosin, ginger protease (zingipain). From this table, maximum and minimum A values were found to be 0.5833 and 0.5808, respectively. The highest values were observed in V-protease, endopeptidase, V-8 protease (Glutamyl endopeptidase), and the minimum values were found in trypsin, plasmin and oligopeptidase B. The maximum A E value was found to be 0.0874 (calpain 2) and the minimum A E values were found to be 0.0097 (plasmin, oligopeptidase B, tripsin). The maximum and minimum relative frequency of release of fragments (W) were found if RubisCO is cleaved by calpain 2 (0.1500) and V-protease, glycyl endopeptidase, V8-protease (glutamyl endopeptidase) (0.0084).       The highest B value was found to be 0.0055 (subtilisin) and the lowest B values were found to be 0 for trypsin, pepsin, plasmin, pancreatic elastase II, oligopeptidase B, glycyl endopeptidase, oligopeptidase F and V-8 protease (Glutamyl endopeptidase). The maximum and minimum V values were found to be 0.2057 and 0.0002, respectively. The maximum value was observed in subtilisin and the minimum value was found in glycyl endopeptidase. ACE is known as dipeptidyl carboxypeptidase and one of its major roles is controlling blood pressure [39]. In the literature, Agirbasli and Cavas [13] evaluated the frequency of occurrence (A) values of ACE inhibitor peptides in Caulerpa RubisCO and found that C. racemosa var. lamourouxii, C. taxifolia and C. racemosa f. occidentalis had the highest A values (0.4330, 0.4330 and 0.3993, respectively) and C. racemosa var. turbinata exhibited the lowest (0.3822). Another study revealed that C. microphysa had potential ACE inhibitory activity as a result of pepsin cleavage [40]. However, the number of bioactive peptides in the BIOPEP database has increased, and so the frequency values might have been altered.
The antioxidative properties of BPs from RubisCO of H. stipulacea are listed in Table 2. Results reveal that maximum and minimum A values were 0.07282 and 0.07280, respectively. The minimum A value was obtained when proteinase K was used as protease. Minimum A values were found in calpain 2 and proteinase P1 (lactocepin). The maximum and minimum A E values were obtained as 0.0243 (proteinase K) and 0.0049 (chmytripsin, cathepsin, chymase, papain, ficin, leukocyte elastase, metridin, bromelain and pepsin), respectively. Proteinase K had the maximum W value (0.3338) and chymase, papain, ficin, leukocyte elastase, metridin, bromelain and pepsin had the lowest W values (0.0673). When thrombin, endopeptidase II, Xaa-Pro dipeptidase, chymosin and ginger protease (zingipain) were used for cleaving, no antioxidative fragment from RubisCO of H. stipulacea was found. The inhibition of lipid peroxidation, scavenging of radicals and metal chelation are among the antioxidative properties of BPs [41]. In the literature, the antioxidative activity of BPs have been evaluated in-silico. According to a recent study, the highest A value for the antioxidative properties of RubisCO was found in Caulerpa taxifolia (0.0785) samples and C. cylindracea (0.0759) species [13]. Also, Udenigwe et al. [19,30] found the maximum and minimum A value of antioxidative properties of RubisCO of cereal crops to be 0.0568 and 0.0464, respectively [19].
The inhibition effects of bioactive peptides from RubisCO of H. stipulacea on dipeptidyl peptidase IV (DPP-IV) (E.C. 3.4.14.5) are given in Table 3. According to the results, prolyl oligopeptidase, V-protease, clostripain and glycyl endopeptidase have the maximum (0.6533) and cathepsin, chymase and metridin have the minimum (0.6497) A value. The maximum A E value was 0.1214 when calpain 2 was used as a protease in DPP-IV inhibitor activity. The minimum A E value (0.0049) was found in prolyl oligopeptidase, V-protease, clostripain and glycyl endopeptidase. Calpain 2 had the maximum (0.1866) and prolyl oligopeptidase, V-protease and clostripain had the minimum (0.0075) W value. We found very low B values for all the enzymes studied. The highest V value was found in pepsin (pH > 2) (0.4569), while tripsin, prolyl oligopeptidase, V8-protease, plasmin, clostripain, oligopeptidase B, glycyl endopeptidase and proteinase P1 had the lowest V value (0.0000). DPP-IV is crucial in glucose metabolism and it degrades the incretins [42]. Thus, DPP-IV inhibitors play a major role in type-2 diabetes mellitus in which insulin secretion and blood glucose level stability are of great importance. [42]. Agirbasli and Cavas found the A value of DPP-IV to be between 0.0550 and 0.0714 in all of Caulerpa species [13]. They also mentioned that caulerpenyne is found in Caulerpa species and its alpha-amylase inhibition activity may play an important role in reducing starch degradation. In another in silico study carried out by Udenigwe et al. [19,30], rice and oat showed the highest A value (0.0758). Table 4 shows the ubiquitin-mediated proteolysis (UbMP) activating properties of bioactive peptides from RubisCO of H. stipulacea. The A value was 0.0146 in pancreatic elastase, leukocyte elastase, proteinase P1 (lactocepin) and pepsin (pH > 2). The maximum A E value was 0.0097 (pancreatic elestase) and the minimum A E values were 0.0049 for leukocyte elastase and proteinase P1. Also, when thrombin, endopeptidase II and Xaa-Pro dipeptidase, chymosin and ginger protease were used, a ubiquitin-mediated proteolysis fragment from H. stipulacea was not found. Pancreatic elastase and pepsin have the highest W value (0.6644) and leukocyte elastase and proteinase P1 have the lowest W value (0.3356). No B or W value was found for UbMP properties of bioactive peptides from RubisCO of H. stipulacea. UbMP is crucial for brain development [43] and its absence causes neurodegenerative diseases such as Parkinson's and Alzheimer's [44]. In the literature, Minkiewicz et al. [36] carried out an in-silico evaluation of bovine meat proteins and found that the highest A value of activating UbMP was 0.028 in tropomyosin α-1 chain [36].
Results for bioactive regulating fragments from H. stipulacea by proteases are shown in Table 5. The results reveal that the only A value (0.0146) was found in chymotrypsin, ficin, calpain 2 and pepsin. The maximum A E value (0.0049) was obtained in chymotrypsin, ficin, calpain 2 and pepsin. Chymotrypsin, ficin, calpain 2 and pepsin had the same W values (0.3356). B and V values were not found for the bioactive regulating activity of H. stipulacea by proteases. Table 6 shows the antithrombotic activity of BPs from H. stipulacea. According to the results, maximum A, A E and W values (0.0097, 0.0049 and 0.5052, respectively) were obtained for chymotrypsin, calpain 2 and pepsin. The B and V values of the antithrombotic properties of H. stipulacea by proteases were not found. Antithrombic activity is essential for the reduction of thrombin. In the study of Agirbasli and Cavas [13], the A values of antithrombic activity of BPs from Caulerpa genus were found within the range of 0.0010 to 0.0100 [13].
The antiamnestic activity values of the bioactive peptides are given in Table 7. The only A value found (0.0097) was found in chymotrypsin, calpain 2 and pepsin. Also, the maximum A E and W values (0.0049 and 0.5052, respectively) were obtained by chymotrypsin, calpain 2 and pepsin. Agirbasli and Cavas [13] found the A values of antiamnestic activity of BPs from Caulerpa genus to be within the range of 0.0010 to 0.0100, the same as for antihrombic activity, perhaps because they act in the similar pathway way [13].
The results of stimulating fragments of H. stipulacea are given in Table 8. The highest A, A E and W values (0.0340, 0.0049 and 0.1441) were obtained by papain and pepsin (pH > 2). Table 9 provides the immunomodulating activity results of BPs from H. stipulacea. The results reveal that calpain 2 has the highest A, A E and W values (0.0097, 0.0049 and 0.5052, respectively). It is interesting to note A, A E , W, B or V values could not be calculated for other enzymes in this study.
In-silico analysis is regarded as an important tool by food scientists since in-silico results may reflect in-vitro and in-vivo results [10,[45][46][47][48]. Lafarga et al. [48] defined new bioactive peptides that show ACE and DPP IV inhibition. They confirmed their biological activity by synthetic tripeptides. Sayd et al. [49] also used a similar strategy, grouping the bioactive meat proteins into three categories based on their digestion dynamic. In recent years, there has been a growth in meat consumption as a result of an increasing population. This demand may increase the use of growth hormones, which, in some countries, are banned, but in others allowed [50]. Therefore, an alternative protein source to meat would be of great interest. In this respect, marine seaweeds and seagrasses can be exploited on an industrial scale since there is no hormone ingredient.

Discussion
Blue biotechnology and blue growth have become two of the hottest topics in recent years. The evaluation of invasive species may open up a new door in the search for novel agents such as vaccines, secondary metabolites and medicines. The present paper reveals that H.stipulacea contains bioactive peptides. These peptides can be harvested and evaluated in the countries affected. However, any possible industrial collection of H.stipulacea would have to be approved by local authorities. Since H.stipulacea forms a mixed vegetation with local Mediterranean macrophytes and seaweeds, its uncontrolled collection might damage the local species. In conclusion, invasive species in the Mediterranean Sea contain very important secondary metabolites and bioactive peptides. Instead of applying blunt eradication methods, biotechnological evaluation is needed.