Functional Properties Of Yam Storage Protein And Mechanism Of Trypsin Inhibitor Activity

Yam, the tuber of Dioscorea spp., is a perennial twining herb. Yams are rich in resources, extensive acreage, high yield and a long history of medicinal. General compositions (fresh-weight basis) 1% to 3% crude protein had been reported. Yam storage protein (YSP) accounts for approximately 80-85% of the total soluble proteins. Many studies showed that YSP exhibited biological activities both in vitro and in vivo, including the enzymatic, antioxidant, antihypertensive, immunomodulatory, lectin and airway epithelial cell protecting activities.The extraction of YSP was optimized using combination of one-factor-at-a-time method, Box-Behnken design (BBD) and response surface methodology (RSM). Three factors influencing the extraction yield of YSPs were investigated. The results showed that all these three factors had a notable effect on the extraction yield of YSPs and their significance could be ranked in a decreasing order of pH> liquid/material ratio> extraction time. The optimal extraction conditions were determined as 8.5,3:1 (mL/g) and 20 min for pH, liquid/material ratio and extraction time, respectively. The predicted extraction yield of YSPs under the optimized conditions was 57.88%, agreeing with the experimental value (57.74%).The proximate composition, SDS-PAGE, amino acid composition, intrinsic fluorescence intensity, FT-IR and antioxidant activity were determined. The YSP was composed of 65.31% protein,2.30% fat,1.62% crude fiber,3.28% ash and 18.52% total carbohydrate. Without 2-ME,there was 2 protein bands, while in the presence of 2-ME, there was only one single protein band with molecular weight of 29 kDa, which is suggesting that YSP contain intramolecular disulfide bond. Asp and Glu were the most abundant amino acids found in YSP. The percentage of some essential amino acids or amino acid pairs of the YSP in the present study fulfilled or exceeded their respective percentages stated in the’ideal protein’. With the increase of pH, the fluorescence Fmax appear first decreased and then increased, which is may be closed by the protein structure into an open structure, some internal hydrophobic amino acids are exposed to the fluorescence signal. FT-IR determination showed that YSP have some important functional groups. YSP have DPPH radical scavenging activity, hydroxyl radical scavenging activity and superoxide radical scavenging activity, also DPPH and hydroxyl radical scavenging activity in a concentration-dependent manner.To evaluate under the influence of pH and ionic strength, YSP’s solubility, water and oil binding capacity, emulsifying, foaming and gelation properties were investigated. In distilled water, as pH shifted away from the pI (3.5), with the increase of pH, solubility, emulsifying activity index (EAI), foam capacity (FC) and foam stability (FS) of the YSP increased drastically. The solubility and emulsion stability index (ESI) of the YSP in 0.5 M and 1.0 M were lower than those in water and 0.1 M NaCl. The oil-holding capacity of YSP was found to be 3.20 mL oil/g of protein. The water holding capacity of YSP have not been detected. As pH>4, an increment in NaCl concentration reduced the EAI and ESI. In the very acid region (pH 1-3), increasing the NaCl concentration from 0.1 M to 1.0 M increased the EAI of the YSP, then the EAI values increased again until pH 7 after which the values fell again. The minimal gelation concentration of YSP was 8%(w/v) in distilled water. The composite gel in the presence of 0.1 M NaCl has smaller pores and dense gel compared to the control which also provided higher gel firmness. The high concentration of salt (0.5 M and 1 M NaCl) in the uneven, the gap increases the large pore structure which also reduced the gel strength.It has been reported that dioscorin has trypsin inhibitor (TI) activity. The Cluspro 2.0 server was used for docking of dioscorin into the porcine trypsin (pdb:1S5S). Based on the similarity of the structure characteristics between dioscorin and knottin structural family proteins (disulfide, β-strands and loops), C28S mutation was introduced to investigate the impact of structure stability obtained by the disulfide linkage (Cys28-Cys187) on the inhibitory activity of dioscorin. Moreover, R43A and R43W mutations were introduced to investigate the inhibitory mechanism in detail. We found that C28S dioscorin showed big decreases in inhibitory activity, R43A dioscorin only showed small decreases in inhibitory activity, almost all of the inhibitory activity was lost for R43W dioscorin. So we can know that C28S mutants may change the complete structure of Dioscorin, which will make other non reactive sites become flexible, and change its inhibitory activity. Arg43 is the key amino acid of the trypsin inhibitor activity. Trp43 larger side chains have steric hindrance, so that it is almost impossible to connect with the active pocket of trypsin, so TI activity almost disappeared.