بيان ترشحي ژن بتا-گزايلوزيداز باكتريايي حاوي برچسب هيستيدين در پيكيا پاستوريس

Secretive expression of bacterial β-xylosidase gene including hexahistidin-tag in Pichia pastoris

ﺳﺎﺑﻘﻪ و ﻫﺪف: ﺑﺘﺎ-ﮔﺰاﻳﻠﻮزﻳﺪازﻫﺎ (EC 3.2.1.37)، از ﻣﻬﻢ ﺗﺮﻳﻦ آﻧﺰﻳﻢ ﻫﺎ در ﻣﺠﻤﻮﻋﻪ آﻧﺰﻳﻢ ﻫـﺎي ﻫﻤـﻲ ﺳـﻠﻮﻻزي ﺑـﺎ ﭘﺘﺎﻧﺴﻴﻞ ﻛﺎرﺑﺮد ﺑﺎﻻ در ﺻﻨﻌﺖ از ﺟﻤﻠﻪ ﺗﻮﻟﻴﺪ ﺑﻴﻮاﺗﺎﻧﻮل، از ﺧﺎﻧﻮاده ﮔﻠﻴﻜﻮزﻳﺪازﻫﺎ ﺑﻮده ﻛﻪ ﺣـﺬف ﻣﺘـﻮاﻟﻲ ﺑﻘﺎﻳـﺎي ﺑﺘـﺎ ﮔﺰاﻳﻠﻮزﻳﻞ را از اﻧﺘﻬﺎي ﻏﻴﺮ اﺣﻴﺎ ﻛﻨﻨﺪه ي ﮔﺰاﻳﻠﻮﺑﻴﻮز و ﮔﺰاﻳﻠﻮاﻟﻴﮕﻮﺳﺎﻛﺎرﻳﺪﻫﺎي ﺧﻄﻲ ﺑﺎ ﭘﻴﻮﻧﺪﻫﺎي β-1,4 اﻧﺠﺎم داده و ﺑﻪ ﻫﻤﺮاه ﺑﺘﺎ-ﮔﺰاﻳﻼﻧﺎزﻫﺎ، ﺑﺮاي دﭘﻠﻴﻤﺮﻳﺰه ﻛﺮدن ﻛﺎﻣﻞ ﮔﺰاﻳﻼن ﺿﺮوري ﻣﻲ ﺑﺎﺷﻨﺪ. ﻫﺪف از اﻳﻦ ﺗﺤﻘﻴـﻖ ﺑﻴـﺎن ﺗﺮﺷـﺤﻲ ژن ﺑﺘﺎ-ﮔﺰاﻳﻠﻮزﻳﺪاز ﺑﺎﻛﺘﺮﻳﺎﻳﻲ ﺣﺎوي ﺑﺮﭼﺴﺐ ﻫﻴﺴﺘﻴﺪﻳﻦ در ﻣﻴﺰﺑﺎن ﺑﻴﺎﻧﻲ ﭘﻴﻜﻴﺎﭘﺎﺳﺘﻮرﻳﺲ، ﺟﻬﺖ رﺳﻴﺪن ﺑﻪ اﻓﺰاﻳﺶ ﺳﻄﺢ ﺑﻴﺎن و ﺳﭙﺲ ﺧﺎﻟﺺ ﺳﺎزي ﻧﺴﺒﻲ آﻧﺰﻳﻢ ﺑﻮد. ﻣﻮاد و روش ﻫﺎ: در اﻳﻦ ﺗﺤﻘﻴﻖ ﺗﻮاﻟﻲ ﭘﺮوﺗﺌﻴﻨﻲ آﻧﺰﻳﻢ ﺑﺘﺎ-ﮔﺰاﻳﻠﻮزﻳﺪاز ﺑﺎﻛﺘﺮﻳﺎﻳﻲ ﭘﺲ از ﺑﻬﻴﻨـﻪ ﺳـﺎزي ﻛـﺪون ﺑـﺮاي ﻣﻴﺰﺑﺎن ﺑﻴﺎﻧﻲ Pichia pastoris، و اﻓﺰودن ﺗﻮاﻟﻲ 6xHis-tag ﺑﺎ ﻃﺮاﺣﻲ ﭘﺮاﻳﻤﺮﻫﺎي ﻣﻨﺎﺳﺐ، ﺑﻪ ﻣﻴﺰﺑـﺎن ﻣﺨﻤـﺮ ي اﻧﺘﻘـﺎل ﻳﺎﻓﺖ. ﻳﺎﻓﺘﻪ ﻫﺎ: ﻧﺘﺎﻳﺞ ﺑﻪ دﺳﺖ آﻣﺪه در ﻛﺸﺖ ﻓﻼﺳﻚ، ﺗﻮﻟﻴﺪ U/L 40 آﻧﺰﻳﻢ ﺑﺘﺎ-ﮔﺰاﻳﻠﻮزﻳﺪاز ﻧﻮﺗﺮﻛﻴﺐ ﺑـﺎ ﻓﻌﺎﻟﻴـﺖ وﻳـﮋه ي U/mg 0/6 را در ﻣﺤﻴﻂ ﺑﻴﺎﻧﻲ BMMY ﻧﺸﺎن داد. ﻧﺘﻴﺠﻪ ﮔﻴﺮي: ﻛﻠﻮﻧﻴﻨﮓ و ﺑﻴﺎن ﻣﻮﻓﻖ ژن ﺑﺘﺎ-ﮔﺰاﻳﻠﻮزﻳﺪاز ﺑﺎﻛﺘﺮﻳﺎﻳﻲ ﺟﻬﺖ ﺑﻪ دﺳـﺖ آوردن آﻧـﺰﻳﻢ ﻓﻌـﺎل و ﭘﺎ ﻳـﺪار از ﺳﻴﺴﺘﻢ ﺑﻴﺎﻧﻲ ﻣﺨﻤﺮ، ﺑﺎ ﻣﻘﺪار ﺑﺎﻻي ﺑﻴﺎن آﻧﺰﻳﻢ اﻧﺠﺎم ﺷﺪ.

Introduction: β-D-Xylosidases (EC 3.2.1.37), one of the most important hemicellulases with high potential industrial application such as bioethanol production, are exo-type glycosidases that catalyze the successive removal of β-xylosyl residues from the non-reducing termini of xylobiose and higher linear β- 1,4-xylooligosaccharides and in conjunction with β-xylanases are essential in completely depolymerizing xylan. The aim of this study was secretive expression of bacterial β-xylosidase gene including hexahistidintag in Pichia pastoris in order to achieve high level expression and enzyme purification subsequently. Materials and Methods: In this study, after optimizing the sequence of protein encoding bacterial β- xylosidase based on the expression codon usage of Pichia pastoris and addition of 6xHis-tag sequence through suitable designed primers to gene, transformation of recombinant plasmids was performed through electroporation method into the expression yeast. Results: The results showed that the recombinant β-xylosidase production was 40 U/L with 0/6 U/mg specific activity in flask culture. Conclusion: Cloning and successful expression of bacterial β-xylosidase gene was carried out in order to obtain an active and stable enzyme with high level expression from yeast expression system.