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Isolation, Identification And

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Isolation, Identification And ﻣﺠﻠﻪ زﻳﺴﺖ ﺷﻨﺎﺳﻲ اﻳﺮان ﺟﻠﺪ 22، ﺷﻤﺎره 1، ﺑﻬﺎر 1388 ﺷﻨﺎﺳﺎﻳﻲ و ﺗﻌﻴﻴﻦ ﺧﺼﻮﺻﻴﺎت ﺑﺎﻛﺘﺮﻳﻬﺎي ﻧﻤﻚ دوﺳﺖ ﺗﻮﻟﻴﺪ ﻛﻨﻨﺪه آﻧﺰﻳﻤﻬﺎي ﻫﻴﺪروﻟﺘﻴﻚ ﺟﺪا ﺷﺪه از درﻳﺎﭼﻪ ﻧﻤﻚ آران و ﺑﻴﺪﮔﻞ 3 2 1 ﺣﻤﻴﺪ ﺑﺎﺑﺎوﻟﻴﺎن ، ﻣﺤﻤﺪ ﻋﻠﻲ آﻣﻮزﮔﺎر ،* و اﺣﻤﺪ ﻋﻠﻲ ﭘﻮرﺑﺎﺑﺎﺋﻲ 1 ﺗﻬﺮان، داﻧﺸﮕﺎه ﻋﻠﻮم ﭘﺰﺷﻜﻲ ﺑﻘﻴﻪ اﷲ (ﻋﺞ)، ﻣﺮﻛﺰ ﺗﺤﻘﻴﻘﺎت ﺑﻴﻮﺗﻜﻨﻮﻟﻮژي ﻛﺎرﺑﺮدي و ﻣﺤﻴﻂ زﻳﺴﺖ 2 ﺗﻬﺮان، داﻧﺸﮕﺎه ﺗﻬﺮان ، ﭘﺮدﻳﺲ ﻋﻠﻮم ، داﻧﺸﻜﺪه زﻳﺴﺖ ﺷﻨﺎﺳﻲ ، ﮔﺮوه ﻣﻴﻜﺮوﺑﻴﻮﻟﻮژي، آزﻣﺎﻳﺸﮕﺎه اﻛﺴﺘﺮﻣﻮﻓﻴﻞ 3 ﻗﻢ، داﻧﺸﮕﺎه آزاد اﺳﻼﻣﻲ، داﻧﺸﻜﺪه ﻋﻠﻮم ﮔﺮوه ﻣﻴﻜﺮوﺑﻴﻮﻟﻮژي ﺗﺎرﻳﺦ درﻳﺎﻓﺖ: 20/12/86 ﺗﺎرﻳﺦ ﭘﺬﻳﺮش: 87/9/6 ﭼﻜﻴﺪه درﻳﺎﭼﻪ آران و ﺑﻴﺪﮔﻞ ﻳﻜﻲ از درﻳﺎﭼﻪ ﻫﺎي ﺷﻮر در ﻣﻨﻄﻘﻪ ﻛﻮﻳﺮ ﻣﺮﻛﺰي اﻳﺮان ﻣﻲ ﺑﺎﺷﺪ. اﻳﻦ درﻳﺎﭼﻪ ﺷﻜﻠﻲ ﺷﺒﻴﻪ ﺑﻪ ﻳﻚ ﻣﺜﻠﺚ دارد ﻛﻪ رأس آن ﺑﻪ ﺳﻤﺖ ﺷﻤﺎل ﻣﻲ ﺑﺎﺷﺪ. ﻃﻮل ﻗﺎﻋﺪه اﻳﻦ ﻣﺜﻠﺚ 35 ﻛﻴﻠﻮﻣﺘﺮ و ارﺗﻔﺎع آن 38 ﻛﻴﻠﻮﻣﺘﺮ ﻣﻲ ﺑﺎﺷﺪ. ﻏﺮﺑﺎل ﮔﺮي ﺑﺎﻛﺘﺮﻳﻬﺎ از ﻧﻘﺎط ﻣﺨﺘﻠﻒ درﻳﺎﭼﻪ ﻣﻨﺠﺮ ﺑﻪ ﺟﺪاﺳﺎزي61 ﺑﺎﻛﺘﺮي ﮔﺮم ﻣﺜﺒﺖ و 22 ﺑﺎﻛﺘﺮي ﮔﺮم ﻣﻨﻔﻲ ﻧﻤﻚ دوﺳﺖ ﻧﺴﺒﻲ ﺗﻮاﻧﺎ ﺑﻪ ﺗﻮﻟﻴﺪ ﻫﻴﺪروﻻزﻫﺎي ﻣﺨﺘﻠﻒ ﺷﺪ اﻳﻦ ﺑﺎﻛﺘﺮﻳﻬﺎ ﺑﻪ ﻃﻮر اﭘﺘﻴﻤﻢ در ﻣﺤﻴﻂ ﺑﺎ 15- 5 درﺻﺪ ﻧﻤﻚ ، دﻣﺎي 37-34 درﺟﻪ ﺳﺎﻧﺘﻲ ﮔﺮاد و pH 2/7 رﺷﺪ ﻣﻲ ﻛﻨﻨﺪ ﺗﻌﺪاد ﺳﻮﻳﻪ ﻫﺎي ﺗﻮﻟﻴﺪ ﻛﻨﻨﺪه آﻧﺰﻳﻤﻬﺎي آﻣﻴﻼز، ﭘﺮوﺗﺌﺎز، ﻟﻴﭙﺎز، DNAase، اﻳﻨﻮﻟﻴﻨﺎز، ﮔﺰﻳﻼﻧﺎز، ﻛﺮﺑﻮﻛﺴﻲ ﻣﺘﻴﻞ ﺳﻠﻮﻻز، ﭘﻜﺘﻴﻨﺎز، ﭘﻮﻟﻮﻻﻧﺎز و ﻛﻴﺘﻴﻨﺎز ﺑﻪ ﺗﺮﺗﻴﺐ 32، 20، 27، 40، 40، 9، 11، 24، 16 و 20 ﺑﻮد ﻛﻪ در ﻣﻴﺎن اﻳﻦ ﺳﻮﻳﻪ ﻫﺎ ﺗﻌﺪادي ﻧﻴﺰﻗﺎدر ﺑﻪ ﺗﻮﻟﻴﺪ ﻣﺨﻠﻮﻃﻲ از اﻳﻦ آﻧﺰﻳﻤﻬﺎ ﺑﻮدﻧﺪ. ﺑﻴﺸﺘﺮﻳﻦ آﻧﺰﻳﻤﻬﺎي ﺗﻮﻟﻴﺪ ﺷﺪه در ﺑﺎﺳﻴﻠﻬﺎي ﮔﺮم ﻣﺜﺒﺖ آﻧﺰﻳﻤﻬﺎي DNase و اﻳﻨﻮﻟﻴﻨﺎز و در ﺑﺎﺳﻴﻠﻬﺎي ﮔﺮم ﻣﻨﻔﻲ آﻧﺰﻳﻢ ﻟﻴﭙﺎز و در ﻛﻮﻛﻮﺳﻬﺎي ﮔﺮم ﻣﺜﺒﺖ آﻧﺰﻳﻤﻬﺎي ﭘﻮﻟﻮﻻﻧﺎزو ﻛﺮﺑﻮﻛﺴﻲ ﻣﺘﻴﻞ ﺳﻠﻮﻻز ﺑﻮدﻧﺪ . ﻛﻤﺘﺮﻳﻦ ﻣﻴﺰان آﻧﺰﻳﻢ ﺗﻮﻟﻴﺪ ﺷﺪه ﻣﺮﺑﻮط ﺑﻪ آﻧﺰﻳﻢ ﮔﺰﻳﻼﻧﺎز ﺑﻮد . از اﻳﻦ ﺳﻮﻳﻪ ﻫﺎ، ﭼﻨﺪﻳﻦ ﺳﻮﻳﻪ ﺑﺎ ﺗﻮاﻧﺎﻳﻲ ﺗﻮﻟﻴﺪ ﺗﻨﻮﻋﻲ از آﻧﺰﻳﻤﻬﺎي ارزﺷﻤﻨﺪ اﻧﺘﺨﺎب ﺷﺪﻧﺪ ﻛﻪ ﺷﺎﻣﻞ AMB1 (8 آﻧﺰﻳﻢ) وAMB7, AMB10, AMB1(7 آﻧﺰﻳﻢ) و ,AMB5, AMB6, AMB11 AMB13 (6 آﻧﺰﻳﻢ) وAMB2, AMB3, AMB8, AMB9 (5 آﻧﺰﻳﻢ) و AMB4, AMG1 (4 آﻧﺰﻳﻢ) و AMC1, AMC2 (3 آﻧﺰﻳﻢ) و AMG2 ،AMC17 ،AMC8 ،AMC3 (2 آﻧﺰﻳﻢ) و AMG3 (1 آﻧﺰﻳﻢ) ﺑﻮدﻧﺪ. ﻧﻬﺎﻳﺘﺎً ﺳﻮﻳﻪ ﻫﺎي ﻣﻨﺘﺨﺐ ﻋﻼوه ﺑﺮ ﺻﻔﺎت ﻓﻨﻮﺗﻴﭙﻲ از ﻧﻈﺮ وﻳﮋﮔﻴﻬﺎي ﻓﻴﻠﻮژﻧﻲ و ﻣﻮﻟﻜﻮﻟﻲ ﺑﺎ ﺗﻜﻨﻴﻚ 16S rDNA ﺷﻨﺎﺳﺎﻳﻲ و ﺗﻌﻴﻴﻦ ﺗﻮاﻟﻲ ﺷﺪﻧﺪ، ﻛﻪ ﺑﺎﻛﺘﺮﻳﻬﺎي ﮔﺮم ﻣﺜﺒﺖ ﻣﺘﻌﻠﻖ ﺑﻪ ﺟﻨﺴﻬﺎي Nesterenkonia ، Halobacillus, Thalassobacillus, Bacillus, Salinicoccus، Staphylococcus ،Marinococcus و ﺑﺎﻛﺘﺮﻳﻬﺎي ﮔﺮم ﻣﻨﻔﻲ ﻣﺘﻌﻠﻖ ﺑﻪ ﺟﻨﺴﻬﺎي Idiomarina, Salicola و Halomonas ﺑﻮدﻧﺪ. ﺑﺎﻛﺘﺮي ﺟﻨﺲ Halobacillus ﻓﺮاوان ﺗﺮﻳﻦ ﺑﺎﻛﺘﺮي ﺟﺪا ﺷﺪه از درﻳﺎﭼﻪ و ﺑﻴﺸﺘﺮﻳﻦ ﺗﻨﻮع آﻧﺰﻳﻢ را ﻧﺸﺎن داد . واژه ﻫﺎي ﻛﻠﻴﺪي: اﻛﺴﺘﺮﻣﻮﻓﻴﻞ، ﻧﻤﻚ دوﺳﺖ، آﻧﺰﻳﻤﻬﺎي ﻫﻴﺪروﻟﻴﺘﻴﻚ، ﻏﺮﺑﺎل ﮔﺮي *ﻧﻮﻳﺴﻨﺪه ﻣﺴﺌﻮل، ﺗﻠﻔﻦ ﺗﻤﺎس 02161112723، ﭘﺴﺖ اﻟﻜﺘﺮوﻧﻴﻚ: [email protected] ﻣﻘﺪﻣﻪ درﻳﺎﭼﻪ آران و ﺑﻴﺪﮔﻞ ﻳﻜﻲ از درﻳﺎﭼﻪ ﻫﺎ در ﻣﻨﻄﻘﻪ ﻛﻮﻳﺮ ﺳﻤﺖ ﺷﻤﺎل ﻣﻲ ﺑﺎﺷﺪ . ﻃﻮل ﻗﺎﻋﺪه اﻳﻦ ﻣﺜﻠﺚ 35 ﻛﻴﻠﻮﻣﺘﺮ ﻣﺮﻛﺰي اﻳﺮان و ﺑﺰرﮔﺘﺮﻳﻦ ﭘﻼﻳﺎي ﺷﻮر در اﻳﺮان اﺳﺖ. اﻳﻦ و ارﺗﻔﺎع آن 38 ﻛﻴﻠﻮﻣﺘﺮ ﻣﻲ ﺑﺎﺷﺪ . ﻋﻤﻖ ﻧﻤﻚ آن ﺑﻴﻦ 54 - درﻳﺎﭼﻪ ﺷﻜﻠﻲ ﺷﺒﻴﻪ ﺑﻪ ﻳﻚ ﻣﺜﻠﺚ دارد، ﻛﻪ رأس آن ﺑﻪ 5 ﻣﺘﺮ ﻣﺘﻐﻴﻴﺮ ﻣﻲ ﺑﺎﺷﺪ ﻛﻪ ﺗﻮﺳﻂ ﻻﻳﻪ ﻫﺎي ﺧﺎك رس از 24 ﻣﺠﻠﻪ زﻳﺴﺖ ﺷﻨﺎﺳﻲ اﻳﺮان ﺟﻠﺪ 22، ﺷﻤﺎره 1، ﺑﻬﺎر 1388 ﻳﻜﺪﻳﮕﺮ ﺟﺪا ﺷﺪه اﻧﺪ. ﺗﺮﻛﻴﺒﺎت ﺗﺸﻜﻴﻞ دﻫﻨﺪه ﻧﻤﻚ اﻧﺪ(23)، در اﻳﻦ ﻣﻄﺎﻟﻌﻪ ﺗﻨﻮع زﻳﺴﺘﻲ ﺑﺎﻛﺘﺮﻳﻬﺎي ﻧﻤﻚ درﻳﺎﭼﻪ، ﻣﻮادي از ﻗﺒﻴﻞ ﺳﺪﻳﻢ ﻛﻠﺮاﻳﺪ، ﺳﺪﻳﻢ ﺳﻮﻟﻔﺎت، دوﺳﺖ ﺟﺪا ﺷﺪه از ﺑﺨﺸﻬﺎي ﻣﺨﺘﻠﻒ در درﻳﺎﭼﻪ ﻧﻤﻚ آران ﻣﻨﻴﺰﻳﻢ ﻛﻠﺮاﻳﺪ، ﻣﻨﻴﺰﻳﻢ ﺳﻮﻟﻔﺎت و .... ﺑﻮده و ﺑﻴﺸﺘﺮ ﻓﺼﻮل و ﺑﻴﺪﮔﻞ ﺗﻮﺻﻴﻒ ﺷﺪه اﺳﺖ. ﺳﺎل ﺧﺸﻚ و اﺷﺒﺎع از ﻧﻤﻚ اﺳﺖ. وﻳﮋﮔﻴﻬﺎي ﺷﺎﺧﺺ اﻳﻦ ﻣﻮاد و روﺷﻬﺎ درﻳﺎﭼﻪ از ﺟﻨﺒﻪ ﺷﻴﻤﻴﺎﻳﻲ، ﺑﺎﻻ ﺑﻮدن ﻋﻴﺎر ﻣﻨﻴﺰﻳﻢ آن ﻧﺴﺒﺖ ﺑﻪ ﺳﺎﻳﺮ درﻳﺎﭼﻪ ﻫﺎ و ﺷﻮراﺑﻪ ﻫﺎي ﻣﻄﺎﻟﻌﻪ ﺷﺪه در اﻳﺮان، از ﻧﻤﻮﻧﻪ ﮔﻴﺮي از ﻧﻘﺎط ﻣﺨﺘﻠﻒ درﻳﺎﭼﻪ: ﺑﻪ ﻣﻨﻈﻮر ﺟﺪاﺳﺎزي ﺟﻨﺒﻪ ﺑﻴﻮﻟﻮژﻳﻚ، ﻣﻴﺰان ﺷﻮري اﺷﺒﺎع ﻧﻤﻚ و ﻻﻳﻪ ﻫﺎي رﻧﮕﻲ ﺳﻮﻳﻪ ﻫﺎي ﻧﻤﻚ دوﺳﺖ ﺑﺎ ﺗﻮاﻧﺎﻳﻲ ﺗﻮﻟﻴﺪ آﻧﺰﻳﻤﻬﺎي ﻧﻤﻚ (ﻗﺮﻣﺰ، ﻧﺎرﻧﺠﻲ، ﺳﺒﺰ، ﺳﻴﺎه) و از ﺟﻨﺒﻪ اﻗﺘﺼﺎدي، ﻫﻴﺪروﻟﻴﺘﻴﻚ ﺧﺎرج ﺳﻠﻮﻟﻲ، از ﻧﻘﺎط ﻣﺨﺘﻠﻒ درﻳﺎﭼﻪ ﻧﻤﻚ ﺣﺼﻮل ﻣﻘﺪار زﻳﺎد ﻧﻤﻚ را ﻣﻲ ﺗﻮان ﻧﺎم ﺑﺮد. اﻳﻦ درﻳﺎﭼﻪ ﺑﻪ آران و ﺑﻴﺪﮔﻞ(از 5 ﻧﻘﻄﻪ درﻳﺎﭼﻪ ﻃﺒﻖ ﻋﻼﻣﺖ ﮔﺬاري روي ﻋﻠﺖ ﺑﺎﻻ ﺑﻮدن ﻣﻴﺰان ﺷﻮري ﻳﻚ زﻳﺴﺘﮕﺎه ﻣﻨﺎﺳﺐ ﺑﺮاي ﻧﻘﺸﻪ ﺑﺎ ﻧﺎﻣﻬﺎي ﻣﺴﻴﺮ ﻣﻨﺘﻬﻲ ﺑﻪ درﻳﺎﭼﻪ ، ﻣﻨﻄﻘﻪ ﺟﺰﻳﺮه دﺳﺘﻪ اي از اﻛﺴﺘﺮﻣﻮﻓﻴﻞ ﻫﺎ ﺑﻪ ﻧﺎم ﻧﻤﻚ دوﺳﺘﻬﺎ ﻣﻲ ﺑﺎﺷﺪ. ﺳﺮﮔﺮداﻧﻲ، ﻣﻨﻄﻘﻪ ﻧﻤﻚ ﭘﺎرﺳﻴﺎن، ﻣﻨﻄﻘﻪ ﻧﻤﻚ ﻛﺎﻧﺴﺎر و ﺑﺎﻛﺘﺮﻳﻬﺎي ﻧﻤﻚ دوﺳﺖ ﻣﻴﻜﺮوارﮔﺎﻧﻴﺴﻢ ﻫﺎﻳﻲ ﻫﺴﺘﻨﺪ ﻛﻪ ﻣﻲ ﻣﻨﻄﻘﻪ 18 ﻛﻴﻠﻮﻣﺘﺮي) در دو ﻣﺎه آﺑﺎن و اردﻳﺒﻬﺸﺖ ﻧﻤﻮﻧﻪ ﺗﻮاﻧﻨﺪ ﺑﺼﻮرت ﺑﻬﻴﻨﻪ در ﻣﺤﻴﻂ ﻣﺤﺘﻮي 15 -5 درﺻﺪ ﻧﻤﻚ ﮔﻴﺮي اﻧﺠﺎم ﺷﺪ. ﺷﻜﻞ 1 ﻧﻘﺎط ﻧﻤﻮﻧﻪ ﺑﺮداري را ﺑﺮ روي رﺷﺪ ﻛﻨﻨﺪ (8 و21). اﻟﺒﺘﻪ اﻳﻦ ﻣﺤﺪوده ﻧﻤﻚ ﺑﺴﺘﮕﻲ ﺑﻪ ﻧﻘﺸﻪ و در ﺟﺪول 1 ﻣﺸﺨﺼﺎت ﻧﻘﺎط ﻧﻤﻮﻧﻪ ﺑﺮداري ﻧﺸﺎن ﺗﺮﻛﻴﺒﺎت ﻣﺤﻴﻂ ﻛﺸﺖ و دﻣﺎ دارد. آﻧﻬﺎ ﺑﻪ ﻃﻮر وﺳﻴﻊ در داده ﺷﺪه اﺳﺖ. ﻓﺮاواﻧﻲ ﺳﻮﻳﻪ ﻫﺎي ﺟﺪا ﺷﺪه از ﻣﺤﻠﻬﺎي زﻳﺴﺘﮕﺎﻫﻬﺎي ﺷﻮر ﻣﺎﻧﻨﺪ درﻳﺎﭼﻪ ﻫﺎي ﺷﻮر، ﺧﺎﻛﻬﺎ و ﻧﻤﻮﻧﻪ ﺑﺮداري در ﺟﺪول 2 ﻧﺸﺎن داده ﺷﺪه اﺳﺖ . ﺑﻴﺎﺑﺎﻧﻬﺎي ﻧﻤﻜﺰار، ﺣﻮﺿﭽﻪ ﻫﺎي ﺑﺮداﺷﺖ ﻧﻤﻚ، ﻣﻌﺎدن ﺳﻮﻳﻪ ﻫﺎي ﺑﺎﻛﺘﺮﻳﺎﻳﻲ و ﺷﺮاﻳﻂ رﺷﺪ: ﺟﺪاﺳﺎزي ﺑﺎﻛﺘﺮﻳﻬﺎي 9 ﻧﻤﻚ، ﻏﺬاﻫﺎي ﺷﻮر و دﻳﮕﺮ ﻣﻨﺎﻃﻖ ﭘﺮاﻛﻨﺪه ﻫﺴﺘﻨﺪ ( ﻧﻤﻚ دوﺳﺖ ﺑﺮ روي ﻣﺤﻴﻂ ﻣﻐﺬي آﮔﺎر داري ﺑﻮد ﻛﻪ ﺑﺪان 22 و ). ﺟﻨﺒﻪ ﻫﺎﻳﻲ ﻛﻪ اﺧﻴﺮاً ﺗﻮﺟﻪ ﻣﺤﻘﻘﻴﻦ را ﺑﻪ ﺧﻮد ﺟﻠﺐ ﻧﻤﻜﻬﺎي زﻳﺮ اﻓﺰوده ﺷﺪه اﺳﺖ . ﻛﺮده اﺳﺖ ﺑﻴﺸﺘﺮ در ارﺗﺒﺎط ﺑﺎ ﻣﻜﺎﻧﻴﺴﻢ ﻓﻴﺰﻳﻮﻟﻮژي آداﭘﺘﻪ NaCl,81 g/l ; MgSO4.7H2O, 9.7 g/l ; MgCl2.6H2O, ﺷﺪن اﻳﻦ ﻣﻴﻜﺮوارﮔﺎﻧﻴﺴﻢ ﻫﺎ ﺑﻪ ﺷﻮري ﺑﺎﻻ، اﻛﻮﻟﻮژي و g/l ; CaCl2, 3.6 g/l ; KCl, 2.0 g/l ; NaHCO3, 0.06 7.0 g/l ; NaBr, 0.026 g/l ; Distilled water, 1000 ml ﻛﺎرﺑﺮدﻫﺎي ﺑﻴﻮﺗﻜﻨﻮﻟﻮژي آﻧﻬﺎ ﻣﻲ ﺑﺎﺷﺪ (22). ﺑﺎﻛﺘﺮﻳﻬﺎي pH ﻣﺤﻴﻂ ﺟﺪاﺳﺎزي ﺑﺎ اﺳﺘﻔﺎده از ﭘﺘﺎس 1 ﻣﻮﻻر ﻗﺒﻞ از ﻧﻤﻚ دوﺳﺖ ﻧﺴﺒﻲ ﺑﻪ ﻋﻠﺖ داﺷﺘﻦ ﺧﺼﻮﺻﻴﺎﺗﻲ ﻣﺎﻧﻨﺪ رﺷﺪ اﺗﻮﻛﻼو ﻛﺮدن در 2/7 ﺗﺎ 4/7 ﺗﻨﻈﻴﻢ ﮔﺸﺖ (7). ﺳﺮﻳﻊ، ﻧﻴﺎز ﻏﺬاﻳﻲ ﻛﻢ، اﺳﺘﻔﺎده از ﺗﺮﻛﻴﺒﺎت ﻣﺨﺘﻠﻒ ﺑﻌﻨﻮان ﺗﻨﻬﺎ ﻣﻨﺒﻊ ﻛﺮﺑﻦ و اﻧﺮژي و ﻛﺎﻫﺶ ﺧﻄﺮ آﻟﻮدﮔﻲ در ﻣﺤﻴﻂ و ﺑﺮاي اﻓﺘﺮاق ﺑﺎﻛﺘﺮﻳﻬﺎي ﺗﺤﻤﻞ ﻛﻨﻨﺪه ﻧﻤﻚ از ﺑﺎﻛﺘﺮﻳﻬﺎي ﻧﻤﻚ اﻣﻜﺎن دﺳﺘﻜﺎري ژﻧﺘﻴﻜﻲ ﺑﺮاي اﻫﺪاف ﺗﺠﺎري ﺑﺴﻴﺎر ﻣﻬﻢ دوﺳﺖ ﻧﺴﺒﻲ و ﻣﻄﻠﻖ ﻧﻴﺰ از ﻣﺤﻴﻂ ﻛﺸﺖ ﻧﻮﺗﺮﻳﻨﺖ آﮔﺎر ﻫﺴﺘﻨﺪ. ﻳﻜﻲ از اﺳﺘﻔﺎده ﻫﺎي ﺗﺠﺎري اﻳﻦ ﻣﻴﻜﺮوارﮔﺎﻧﻴﺴﻢ ﻫﺎ ﺳﻨﺘﺘﻴﻚ ﻛﻪ ﺑﺪون ﻧﻤﻚ اﺳﺘﻔﺎده ﺷﺪ. ﺳﻮﻳﻪ ﻫﺎي رﺷﺪ ﻛﻨﻨﺪه اﺳﺘﻔﺎده از آﻧﺰﻳﻤﻬﺎي ﻫﻴﺪروﻻزي ﺧﺎرج ﺳﻠﻮﻟﻲ ﻣﻲ ﺑﺎﺷﺪ، ﺑﺮ روي اﻳﻦ ﻣﺤﻴﻂ، اﺣﺘﻤﺎﻻ ً◌ ﻫﺎﻟﻮﺗﻮﻟﺮاﻧﺖ ﺑﻮدﻧﺪ اﻣﺎ ﻛﻠﻨﻲ زﻳﺮا اﻳﻦ آﻧﺰﻳﻤﻬﺎ ﻧﻪ ﺗﻨﻬﺎ ﺗﺤﻤﻞ ﻛﻨﻨﺪه ﻧﻤﻚ ﻫﺴﺘﻨﺪ، ﺑﻠﻜﻪ ﻫﺎﻳﻲ ﻛﻪ روي اﻳﻦ ﻣﺤﻴﻂ ﺑﺪون ﻧﻤﻚ ﻧﺘﻮاﻧﻨﺪ رﺷﺪ ﻛﻨﻨﺪ ﺗﺤﻤﻞ ﻛﻨﻨﺪه ﺣﺮارت ﻧﻴﺰ ﻣﻲ ﺑﺎﺷﻨﺪ (13). اﮔﺮﭼﻪ اﻳﻦ ﻧﻤﻚ دوﺳﺖ ﻫﺴﺘﻨﺪ. در ﻣﻮرد ﺳﻮﻳﻪ ﻫﺎﻳﻲ ﻛﻪ رﺷﺪ ﺑﺮ روي ﺑﺎﻛﺘﺮﻳﻬﺎ آﻧﺰﻳﻤﻬﺎي ﺧﺎرج ﺳﻠﻮﻟﻲ ﺗﺤﻤﻞ ﻛﻨﻨﺪه ﻧﻤﻚ ﺑﺎ ﻣﺤﻴﻂ ﻓﺎﻗﺪ ﻧﻤﻚ ﻣﺸﺎﻫﺪه ﺷﺪ، رﺷﺪ ﺑﻬﻴﻨﻪ ﺑﺎﻛﺘﺮﻳﻬﺎ در ﭘﺘﺎﻧﺴﻴﻞ ﺑﺎﻻ را ﺑﺮاي اﺳﺘﻔﺎده در ﻓﺮآﻳﻨﺪ ﻫﺎي ﺻﻨﻌﺘﻲ ﺗﻮﻟﻴﺪ ﺣﻀﻮر ﻧﻤﻜﻬﺎي 0 ، 5/2 ، 5 درﺻﺪ ﺗﻌﻴﻴﻦ ﮔﺸﺖ. ﭼﻨﺎﻧﭽﻪ ﻣﻲ ﻛﻨﻨﺪ، ﺗﻨﻬﺎ ﻳﻚ ﺗﻌﺪاد ﻛﻤﻲ از اﻳﻦ آﻧﺰﻳﻤﻬﺎ ﺷﻨﺎﺳﺎﻳﻲ ﺷﺪه 25 ﻣﺠﻠﻪ زﻳﺴﺖ ﺷﻨﺎﺳﻲ اﻳﺮان ﺟﻠﺪ 22، ﺷﻤﺎره 1، ﺑﻬﺎر 1388 ﺳﻮﻳﻪ اﻳﻲ زﻳﺮ 3 درﺻﺪ ﻧﻤﻚ رﺷﺪ ﺑﻬﻴﻨﻪ داﺷﺘﻪ ﺑﺎﺷﺪ ﻫﺎﻟﻮﺗﻮﻟﺮاﻧﺖ، در ﻏﻴﺮ اﻳﻦ ﺻﻮرت ﻧﻤﻚ دوﺳﺖ ﻣﻲ ﺑﺎﺷﺪ . ! 18 1 ﺷﻜﻞ - ﻧﻘﺎط ﻧﻤﻮﻧﻪ ﺑﺮداري ﺑﺮ روي ﻧﻘﺸﻪ ﺟﺪول1- ﻣﺸﺨﺼﺎت ﻧﻘﺎط ﻧﻤﻮﻧﻪ ﺑﺮداري ﺷﺪه ﻧﻘﺎط ﻧﻤﻮﻧﻪ ﺑﺮداري ﻣﺸﺨﺼﺎت ﺟﻐﺮاﻓﻴﺎﻳﻲ دﻣﺎ pH ﻧﻮع ﻧﻤﻮﻧﻪ اﺑﺘﺪاي درﻳﺎﭼﻪ C h: 808 m° 32 7 ﺧﺎك ﺷﻮر و ﻟﺠﻦ ﻧﻤﻜﻲ N: 34.31265 E: 51.7667 ﺟﺰﻳﺮه ﺳﺮﮔﺮداﻧﻲ C h: 809 m° 34 6.8 ﺧﺎك ﺷﻮر، ﻟﺠﻦ ﻧﻤﻜﻲ ، ﻧﻤﻚ ﺑﺎ درﺻﺪ N: 34.58166 E: 51.76385 ﻣﻨﻴﺰﻳﻢ ﺑﺎﻻ ﻣﻨﻄﻘﻪ ﻧﻤﻚ ﭘﺎرﺳﻴﺎن C h: 805 m° 29 7 ﻧﻤﻚ ﺳﻴﺎه، ﻧﻤﻚ ﺳﻔﻴﺪ، ﻧﻤﻚ ﺳﺒﺰو ﻗﺮﻣﺰ N: 34.50729 E: 51.72439 ﻣﻨﻄﻘﻪ ﻧﻤﻚ ﻛﺎﻧﺴﺎر C h: 804 m° 30 7 ﻧﻤﻚ ﺳﻴﺎه، ﻧﻤﻚ ﺳﻔﻴﺪ، ﻧﻤﻚ ﺳﺒﺰو ﻗﺮﻣﺰ N: 34.50106 E: 51.76385 ﻣﻨﻄﻘﻪ 18 ﻛﻴﻠﻮﻣﺘﺮي C h:799 m° 30 7 ﻧﻤﻚ ﻧﺎرﻧﺠﻲ ، ﻗﺮﻣﺰ و ﺳﺒﺰ N: 34.64378 E: 51.83838 وﻳﮋه ﻣﺤﺘﻮي ﺳﻮﺑﺴﺘﺮاي ﻣﻮرد ﻧﻈﺮ و10 درﺻﺪ ﻧﻤﻚ ﻛﺸﺖ داده ﺗﺎ ﺑﺘﻮان ﺗﻮﻟﻴﺪ آﻧﺰﻳﻢ را در آﻧﻬﺎ ﻣﺸﺎﻫﺪه ﻛﺮد . ﻏﺮﺑﺎل ﮔﺮي ﻣﻴﻜﺮوارﮔﺎﻧﻴﺴﻢ ﻫﺎي ﺗﻮﻟﻴﺪ ﻛﻨﻨﺪه آﻧﺰﻳﻤﻬﺎي ﻫﻴﺪروﻟﻴﺘﻴﻚ: در اﻳﻦ ﻣﺮﺣﻠﻪ ﺳﻨﺠﺶ ﺑﻪ روش ﭘﻠﻴﺖ اﻧﺠﺎم ﺗﻮﻟﻴﺪ ﺧﺎرج ﺳﻠﻮﻟﻲ آﻣﻴﻼز: ﺑﺮاي ﺑﺮرﺳﻲ ﺗﻮﻟﻴﺪ آﻧﺰﻳﻢ ﮔﺮدﻳﺪ .ﻫﺮ ﻳﻚ از ﺳﻮﻳﻪ ﻫﺎي ﺟﺪا ﺷﺪه را ﺑﺮ روي ﻣﺤﻴﻂ آﻣﻴﻼز از ﻣﺤﻴﻂ ﺑﺎ ﺗﺮﻛﻴﺐ زﻳﺮ اﺳﺘﻔﺎده ﺷﺪ : : 26 ﻣﺠﻠﻪ زﻳﺴﺖ ﺷﻨﺎﺳﻲ اﻳﺮان ﺟﻠﺪ 22، ﺷﻤﺎره 1، ﺑﻬﺎر 1388 ; درﺻﺪStarch agar, 30 g/l ; NaCl (solar salt), 10 ﭘﻮﺷﻴﺪه ﺷﺪ، ﻇﻬﻮر ﻳﻚ ﻧﺎﺣﻴﻪ ﺷﻔﺎف اﻃﺮاف ﻛﻠﻨﻲ ﻫﺎ ﻧﺸﺎﻧﻪ Distilled water, 1000ml ;pH, 7.2 ﻫﻴﺪروﻟﻴﺰ ﻧﺸﺎﺳﺘﻪ ﻣﻲ ﺑﺎﺷﺪ (5 ). ). ﺑﻌﺪ از اﻧﻜﻮﺑﺎﺳﻴﻮن در دﻣﺎي 37 درﺟﻪ ﺳﺎﻧﺘﻲ ﮔﺮاد ﺑﻪ ﻣﺪت 7 روز، ﭘﻠﻴﺖ ﻫﺎ ﺑﺎ ﻣﺤﻠﻮل (0.3) درﺻﺪ I2, 0.6 درﺻﺪ KI 27 ﻣﺠﻠﻪ زﻳﺴﺖ ﺷﻨﺎﺳﻲ اﻳﺮان ﺟﻠﺪ 22، ﺷﻤﺎره 1، ﺑﻬﺎر 1388 ﺗﻮﻟﻴﺪ ﺧﺎرج ﺳﻠﻮﻟﻲ ﭘﺮوﺗﺌﺎز: Skim milk, 10 درﺻﺪ ; رﺷﺪ در ﺳﻄﺢ ﭘﻠﻴﺖ ﻧﺸﺎﻧﻪ ﺗﻮﻟﻴﺪ آﻧﺰﻳﻢ و اﺳﺘﻔﺎده از اﻳﻨﻮﻟﻴﻦ N.A , 28 g/l ; NaCl (solar salt), 10 درﺻﺪ Agar ,15 ; ﺑﻪ ﻋﻨﻮان ﺗﻨﻬﺎ ﻣﻨﺒﻊ ﻛﺮﺑﻦ ﻣﻲ ﺑﺎﺷﺪ. (1 ) ) g/l ; Distilled water, 1000ml ; pH 7.2 ﺗﻮﻟﻴﺪ ﺧﺎرج ﺳﻠﻮﻟﻲ ﭘﻜﺘﻴﻨﺎز: ﻣﺤﻴﻂ ﻛﺸﺖ ﺗﻮﻟﻴﺪ آﻧﺰﻳﻢ ﺗﺮﻛﻴﺒﺎت ﻣﺤﻴﻂ ﺑﻪ ﺟﺰ Skim milk در 121 درﺟﻪ ﺳﺎﻧﺘﻲ ﭘﻜﺘﻴﻨﺎز ﮔﺮاد 15 دﻗﻴﻘﻪ اﺗﻮﻛﻼو ﮔﺸﺖ و skim milk از ﻃﺮﻳﻖ Pectin, 1 ; (NH ) SO , 0.14 ;K HPO , ﺻﺎف ﻛﺮدن اﺳﺘﺮﻳﻞ ﮔﺸﺖ . 4 2 درﺻﺪ 4 2 4 درﺻﺪ Nutrient ; درﺻﺪMgSO4.7H2O, 0.02 ; درﺻﺪ 0.2 اﻳﺠﺎد ﻫﺎﻟﻪ ﺷﻔﺎف اﻃﺮاف ﻛﻠﻨﻲ ﻫﺎ ﻧﺸﺎﻧﻪ ﺗﻮﻟﻴﺪ آﻧﺰﻳﻢ ﻣﻲ ; FeSO4.7H2O, 5 mg/l ) درﺻﺪ solution, 0.1 20 ﺑﺎﺷﺪ. ( ) ; MnSO4.H2O, 1.6 mg/L ; ZnSO4.7H2O, 1.4 mg/l ,Agar ; درﺻﺪ CaCL2, 2 mg/l ) ;NaCl(solar salt), 10 ﺗﻮﻟﻴﺪ ﺧﺎرج ﺳﻠﻮﻟﻲ ﻟﻴﭙﺎز: .Peptone , 10 g/l ; CaCl2 2 ; Distilled water, 1000ml; pH 7. 2 ,(H2O, 0.1 g/l ; Tween 80, 10 g/l ; NaCl (solar salt درﺻﺪ 10 درﺻﺪDistilled water, 1000ml; pH, 7.2 ; ﭘﻜﺘﻴﻦ از ﻃﺮﻳﻖ ﺻﺎف ﻛﺮدن اﺳﺘﺮﻳﻞ و ﺑﻘﻴﻪ ﻣﺤﻴﻂ در دﻣﺎي ﺑﻌﺪ از ﺗﻬﻴﻪ ﻣﺤﻴﻂ ﻓﻮق در دﻣﺎي 115 درﺟﻪ ﺳﺎﻧﺘﻲ ﮔﺮاد ﺑﻪ 121 درﺟﻪ ﺑﻪ ﻣﺪت 15 دﻗﻴﻘﻪ اﺳﺘﺮﻳﻞ ﺷﺪ .
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