شناسايي يك پروتئين مهاركننده فعاليت آميلازي در بذر گندم

Identification of a protein that inhibit amylase activity in seed of wheat

ﭘﺮوﺗﺌﻴﻦﻫﺎي ذﺧﻴﺮهاي ﻣﻮﺟﻮد در داﻧﻪ ﺑﺴﻴﺎري از ﮔﻴﺎﻫﺎن در زﻣﻴﻨﻪﻫﺎي ﻣﺘﻨﻮﻋﻲ ﻣﺎﻧﻨﺪ ﺗﻐﺬﻳﻪ اﻧﺴﺎن، ﺗﻐﺬﻳﻪ دام، ﺷﻴﻤﻲ ﭘﺮوﺗﺌﻴﻦ،داروﺳﺎزي، ﺑﻴﻮﺷﻴﻤﻲ ﮔﻴﺎﻫﻲ و ﭘﺰﺷﻜﻲ ﻣﻮرد ﻣﻄﺎﻟﻌﻪ ﻗﺮار ﻣﻲ ﮔﻴﺮﻧﺪ. ﺗﻮاﻧﺎﻳﻲ اﻳﻦ ﺗﺮﻛﻴﺒﺎت ﺑﺮاي اﻳﺠﺎد ﻣﺸﻜﻼت ﺗﻐﺬﻳﻪاي، اﺛﺮات ﺳﻤﻲ و داروﻳﻲ ﻫﻨﮕﺎﻣﻲ ﻛﻪ ﺑﻪ ﻋﻨﻮان ﻏﺬاﻫﺎي ﮔﻴﺎﻫﻲ ﻣﺼﺮف ﻣﻲ ﺷﻮﻧﺪ ﺑﺎﻋﺚ ﮔﺮدﻳﺪه ﻛﻪ در زﻣﻴﻨﻪٔ ◌ ﭘﺮاﻛﻨﺪﮔﻲ آﻧﻬﺎ در ﮔﻴﺎﻫﺎن ﻣﻄﺎﻟﻌﺎت ﻣﺘﻌﺪدي اﻧﺠﺎم ﺷﻮد. دراﻳﻦ ﭘﮋوﻫﺶ ﭘﺮوﺗﺌﻴﻦﻫﺎي ﺑﺬر ﮔﻨﺪم وارﻳﺘﻪ زرﻳﻦ اﺳﺘﺨﺮاج و ﺧﺎﻟﺺ ﺳﺎزي ﭘﺮوﺗﺌﻴﻦ ﻣﻮردﻧﻈﺮ ﺑﺎ روش رﺳﻮب ﺟﺰءﺑﻪ ﺟﺰء ﺑﺎ آﻣﻮﻧﻴﻮم ﺳﻮﻟﻔﺎت، دﻳﺎﻟﻴﺰ و ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌﻮﻳﺾ ﻳﻮﻧﻲ اﻧﺠﺎم ﮔﺮدﻳﺪ. ﺑﻌﺪ از ﺧﺎﻟﺺﺳﺎزي ﺑﺎ روش ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺳﺮﻳﻊ ﭘﺮوﺗﺌﻴﻦ ﺑﺎ ﻓﺎز ﻣﺎﻳﻊ، وﻳﮋﮔﻲﻫﺎي اﻟﻜﺘﺮوﻓﻮزي اﻳﻦ ﭘﺮوﺗﺌﻴﻦ ﺑﺎ روش اﻟﻜﺘﺮوﻓﻮرز ژل ﺳﺪﻳﻢ دو دﺳﻴﻞ ﺳﻮﻟﻔﺎت ﭘﻠﻲ آﻛﺮﻳﻞ آﻣﻴﺪ ﺑﺮرﺳﻲ ﺷﺪ. ﻣﻬﺎرﻛﻨﻨﺪﮔﻲ ﭘﺮوﺗﺌﻴﻦ ﻣﻮرد ﻧﻈﺮ در ﺑﺮاﺑﺮ ﻓﻌﺎﻟﻴﺖ آﻟﻔﺎآﻣﻴﻼز اﺳﺘﺎﻧﺪارد ﺑﺎ ﻣﻨﺸﺄ ﺑﺎﻛﺘﺮﻳﺎﻳﻲ( و ﺑﺰاق اﻧﺴﺎﻧﻲ ﺑﺎ اﺳﺘﻔﺎده از روش ﺑﺮﻧﻔﻠﺪ ﻣﻮرد ﺳﻨﺠﺶ ﻗﺮار ﮔﺮﻓﺖ. دﻳﺎﮔﺮام ﺧﺎﻟﺺ ﺳﺎزي و ﺟﻤﻊ آوري ﭘﺮوﺗﺌﻴﻦ ﻣﻮرد ﻧﻈﺮ را ﻧﺸﺎن داد. اﻟﮕﻮي اﻟﻜﺘﺮوﻓﻮرزي اﻳﻦ ﭘﺮوﺗﺌﻴﻦ ﺑﺎ ﺣﺮﻛﺖ ﻧﺴﺒﻲ 0/59 و 0/60 ﺗﺄﻳﻴﺪي ﺑﺮ ﻓﺮاﻳﻨﺪ ﺧﺎﻟﺺ ﺳﺎزي ﭘﺮوﺗﺌﻴﻦ ﻣﻲﺑﺎﺷﺪ. ﻓﻌﺎﻟﻴﺖ آﻟﻔﺎآﻣﻴﻼز اﺳﺘﺎﻧﺪارد ﺑﺎ ﻣﻨﺸﺄ ﺑﺎﻛﺘﺮﻳﺎﻳﻲ( ﺑﻪ ﻣﻴﺰان 89/97 درﺻﺪ و ﻫﻤﭽﻨﻴﻦ ﻓﻌﺎﻟﻴﺖ ﻫﻴﺪروﻟﻴﺘﻴﻚ ﺑﺰاق اﻧﺴﺎﻧﻲ ﺗﻮﺳﻂ اﻳﻦ ﭘﺮوﺗﺌﻴﻦ ﺑﻪ ﻣﻴﺰان97/07 درﺻﺪ ﻛﺎﻫﺶ ﻧﺸﺎن داد. ﺑﻪ ﻃﻮرﻛﻠﻲ ﺟﺪاﺳﺎزي، ﺧﺎﻟﺺ ﺳﺎزي و ﻣﻬﺎرﻛﻨﻨﺪﮔﻲ آﻟﻔﺎ آﻣﻴﻼزي ﭘﺮوﺗﺌﻴﻦ اﺳﺘﺨﺮاج ﺷﺪه از ﺑﺬر ﮔﻨﺪم زرﻳﻦ دراﻳﻦ ﭘﮋوﻫﺶ ﺗﺄﻳﻴﺪ ﮔﺮدﻳﺪ. ﻣﻬﺎر آﻟﻔﺎآﻣﻴﻼز ﺑﺎﻛﺘﺮﻳﺎﻳﻲ ﺗﻮﺳﻂ اﻳﻦ ﭘﺮوﺗﺌﻴﻦ ﺑﺎ اﺳﺘﻔﺎده از روﺷﻬﺎي ﻧﻮﻳﻦ و اﺑﺰارﻫﺎي ﺑﻴﻮﺗﻜﻨﻮﻟﻮژي و ﻣﻬﻨﺪﺳﻲ ژﻧﺘﻴﻚ در ﻛﻨﺘﺮل ﻃﺒﻴﻌﻲ ﺑﻴﻤﺎرﻳﻬﺎ و آﻓﺎت ﮔﻴﺎﻫﻲ ﻣﻲﺗﻮاﻧﺪ ﻣﻮردﺗﻮﺟﻪ ﻣﺤﻘﻘﻴﻦ ﻛﺸﺎورزي ﻗﺮارﮔﻴﺮد. اﻳﻦ ﻣﻬﺎرﻛﻨﻨﺪه آﻟﻔﺎآﻣﻴﻼز ﻫﻤﭽﻨﻴﻦ ﻣﻲﺗﻮاﻧﺪ در درﻣﺎن دﻳﺎﺑﺖ و ﻧﺎرﺳﺎﻳﻲ ﻫﺎي ﮔﻮارﺷﻲ و اﺻﻼح رژﻳﻢ ﻫﺎي ﻏﺬاﻳﻲ ﺑﻪ ﻣﻨﻈﻮر ﻛﺎﻫﺶ وزن ﻛﺎرﺑﺮد داﺷﺘﻪ ﺑﺎﺷﺪ.

Storage proteins which found in seeds of many plants are studied in various fields such as human and animal nutrition, chemistry of protein, pharmacology, plant biochemistry and medicinal plants. The ability of these compounds to cause nutritional problems and toxic effects when used as foods has led to several studies on their dispersion in plants. In this research, seed proteins of wheat (Zarrin variety) have been extracted. The protein of interest was purified by ammonium sulfate precipitation method; dialysis and ion exchange chromatography. After purification by Fast protein liquid chromatography (FPLC), electrophoretic properties of this protein were investigated by Sodium dodecyl-polyacrylamide gel electrophoresis )SDS-PAGE( method. Inhibitory activity of this Protein against Bacterial alpha-amylase and human saliva were measured using the Bernfeld method. FPLC diagram illustrates the purification and collection of the desired protein. The electrophoretic pattern of this protein with relative mobility of 0.60 and 0.59 confirmed the purification process. Hydrolytic activity of bacterial and human saliva Alpha- amylase decreased by this protein 89.97% and 97.07% respectively. In general the isolation, purification and alpha-amylase inhibition property of this protein which extracted from wheat seed were confirmed in this study. Inhibition of bacterial alpha-amylase by this protein can be considered by agricultural researchers in biological control of parasites and pests. This alpha-amylase inhibitor can also be used to treat diabetes, digestive deficiencies and modify dietary regimens to reduce weight.