Oleosins (24 and 18 kDa) Are Hydrolyzed Not Only in Extracted Soybean Oil Bodies but Also in Soybean Germination

Abstract

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

Label-freein situimaging of oil body dynamics and chemistry in germination

“…High (23–24 kDa) and low (15–16 and 17–18 kDa) molecular weight soya bean oleosins were visible starting from day 1 but decreased until they were undetectable at day 5 (figure 4 c ). Similar degradation of oleosins was previously reported [16,60] in soya beans. This verifies that oil bodies start as oleosin-coated oil bodies to become non-oleosin-coated oil bodies during the early days of germination [34].…”

“…Twenty-five per cent sucrose (w/w) was added to the raw soya milk and the pH was adjusted to 11.0 with 1 mol l −1 sodium hydroxide to remove weakly bound proteins, such as conglycinin, glycinin, allergenic proteins and oleosin proteases. This made subsequent Bradford measurements more specific for only strongly bound proteins such as oleosins, caloleosins and steroleosins [16,36,38]. We note that using high alkaline pH reduces the total amount of oleosins compared with neutral extraction [39], which could cause a systematic offset for subsequent oil body size measurements from laser diffraction and Bradford measurements of protein quantity.…”

“…The mixture was centrifuged again (15 000 g , 4°C, 3 h). This sequence of steps was repeated once more to remove any residual storage proteins in the oil bodies [37] and to denature the proteases Bd 30 K and P34 [16]. Finally, the washed oil bodies were collected with a spoon, dispersed in 10 ml phosphate-buffered saline (PBS), and stored at 4°C for protein and lipid analysis.…”

“…Although previous studies have shown that oleosin disappears from oil bodies during germination [16,17] and that oil bodies fuse when oleosin is genetically suppressed [5,6], it is unknown if oil bodies grow or shrink during unperturbed, native germination and how this correlates to oleosin levels. Recent work has shown that oleosins are degraded prior to lipid mobilization from oil bodies via a ubiquitination–proteasome pathway.…”

Macronutrients and Micronutrients of Soybean Oil Bodies Extracted at Different pH

“…The 1st step to examine the components of soybean OBs is the aqueous extraction, but it is known that many OB extrinsic proteins are bound to OBs in the extraction (Chen and Ono ; Zhao and others ; Chen and others ). In order to obtain uncontaminated OBs, detergent washing, ionic elution, and treatment with a chaotropic agent were used by researchers in the field of plant physiology (Tzen and others ), which was improper in the processing of food ingredients.…”