ﺷﻨﺎﺳﺎﯾﯽ، ﮐﻠﻮن، ﺑﯿﺎن و ﺧﺎﻟﺺﺳﺎزي آﻧﺰﯾﻢ ﻧﻮﺗﺮﮐﯿﺐ ﺟﺪﯾﺪ زاﯾﻼﻧﺎز ﺑﺎ ﻣﻘﺎوﻣﺖ ﺳﺎﺧﺘﺎري ﺑﺎﻻ از ﺟﻤﻌﯿﺖ ﻣﯿﮑﺮوﺑﯽ ﺷﮑﻤﺒﻪ ﮔﻮﺳﻔﻨﺪ

Identification, cloning, and expression of the novel recombinant xylanase with stable structure from the sheep rumen microbiota

ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ﻧﻘﺶ ﻣﻬﻢ آﻧﺰﯾﻢﻫﺎ در ﺗﺴﻬﯿﻞ ﻋﻤﻠﮑﺮد ﻓﺮاﯾﻨﺪﻫﺎي ﻣﺨﺘﻠـﻒ، اﺳـﺘﻔﺎده از آﻧﻬـﺎ در اﻏﻠـﺐ ﺻﻨﺎﯾﻊ ﺑﺴﯿﺎر ﻣﻮرد ﺗﻮﺟﻪ اﺳﺖ. درﺻﺪ ﺑﺴﯿﺎر ﭘﺎﯾﯿﻨﯽ از ﻣﯿﮑﺮوارﮔﺎﻧﯿﺴﻢﻫﺎي ﺗﻮﻟﯿﺪ ﮐﻨﻨﺪه آﻧﺰﯾﻢﻫـﺎي ﺟﺪﯾﺪ در ﻣﺤﯿﻂ آزﻣﺎﯾﺸﮕﺎﻫﯽ ﻗﺎﺑﻞ ﮐﺸﺖ ﻣﯽﺑﺎﺷﻨﺪ، اﯾﻦ درﺣﺎﻟﯽ اﺳﺖ ﮐﻪ ﻣﺘﺎژﻧﻮم ﻣﻨﺒﻊ ﻋﻈﯿﻤـﯽ از اﻃﻼﻋﺎت ژﻧﺘﯿﮑﯽ آﻧﺰﯾﻢﻫﺎي ﻧﺎﺷﻨﺎﺧﺘﻪ را ﻣﯽﺗﻮاﻧﺪ در اﺧﺘﯿﺎر ﻣﺎ ﻗﺮار دﻫﺪ. ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻫﻤﯿﺖ اﺳﺘﻔﺎده از آﻧﺰﯾﻢﻫﺎ در ﺻﻨﺎﯾﻊ ﮔﻮﻧﺎﮔﻮن، آﻧﺰﯾﻢﻫﺎﯾﯽ ﺑﺎ ﺳﺎﺧﺘﺎر ﭘﺎﯾﺪار و ﻣﻘﺎوم در ﺑﺮاﺑﺮ ﺣـﺮارت، ﮐـﺎرﺑﺮد و ﻋﻤﻠﮑﺮد ﺑﻬﺘﺮي را ﻧﺸﺎن ﻣﯽدﻫﻨﺪ و ﺗﺤﻘﯿﻘﺎت زﯾﺎدي در زﻣﯿﻨﻪ ﺷﻨﺎﺳﺎﯾﯽ و ﺗﻮﻟﯿـﺪ آﻧﻬـﺎ ﺑـﻪ ﺻـﻮرت ﭘﯿﻮﺳﺘﻪ ﺻﻮرت ﮔﺮﻓﺘﻪ اﺳﺖ. در اﯾﻦ ﭘﮋوﻫﺶ ﺑﺎ اﺳﺘﻔﺎده از روشﻫﺎي ﻣﺤﺎﺳﺒﺎﺗﯽ، ﭘﯿﺸﮕﻮﯾﯽ ﺗﻌﯿﯿﻦ ﺳﺎﺧﺘﺎر و اﺳﺘﻔﺎده از ﺗﻮاﻟﯽ آﻧﺰﯾﻢﻫﺎي زاﯾﻼﻧﺎز ﻣﺴﺘﺨﺮج از دادهﻫﺎي ﻣﺘﺎژﻧﻮم ﺷﮑﻤﺒﻪ ﮔﻮﺳﻔﻨﺪ، آﻧﺰﯾﻢ زاﯾﻼﻧﺎزي ﺑﺎ ﺳﺎﺧﺘﺎر ﺑﺴﯿﺎر ﻣﻘﺎوم ﺷﻨﺎﺳﺎﯾﯽ و ﺑﻪﺻﻮرت ﻧﻮﺗﺮﮐﯿﺐ ﺗﻮﻟﯿﺪ ﺷﺪ. آﻧﺰﯾﻢ زاﯾﻼﻧﺎز ﻧﻮﺗﺮﮐﯿﺐ ﻣﻘﺎوم ﺑﻪ ﺣﺮارت، PersiXyn5 ﻧﺎمﮔﺬاري ﺷﺪه و از DNA اﺳﺘﺨﺮاج ﺷﺪه از ﻣﺤﺘﻮﯾﺎت ﺷﮑﻤﺒﻪ ﮔﻮﺳﻔﻨﺪ ﮐﻠﻮن و در ﺑـﺎﮐﺘﺮيE.coli ﺑﯿـﺎن و ﺧـﺎﻟﺺ ﺷـﺪ ﻓﻌﺎﻟﯿﺖ وﯾﮋه و ﭘﺎراﻣﺘﺮﻫﺎي ﮐﯿﻨﺘﯿﮑﯽ Km و Vmax ﺑﺮاي اﯾﻦ آﻧﺰﯾﻢ ﻣﺤﺎﺳﺒﻪ ﺷﺪه و ﻓﻌﺎﻟﯿﺖ ﺑﻬﯿﻨﻪ اﯾﻦ آﻧـﺰﯾﻢ در دﻣﺎي 80 درﺟﻪ ﺳﺎﻧﺘﯽﮔﺮاد وpH 8 ﻣﺸﺎﻫﺪه ﺷﺪ. آﻧﺰﯾﻢ زاﯾﻼﻧﺎز ﻧﻮﺗﺮﮐﯿـﺐ ﺟﺪﯾـﺪ ﭘـﺲ از2 ﺳـﺎﻋﺖ ﺗﯿﻤﺎر در دﻣﺎي 90 درﺟﻪ ﺳﺎﻧﺘﯽﮔﺮاد 58 درﺻﺪ ﻓﻌﺎﻟﯿﺖ ﺧﻮد را ﺣﻔﻆ ﮐﺮد. ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﯾﻨﮑﻪ آﻧﺰﯾﻢ زاﯾﻼﻧﺎز ﻣﻮرد ﻣﻄﺎﻟﻌﻪ ﻣﻘﺎوم در دﻣﺎي ﺑﺎﻻ و ﻓﻌﺎل در ﻣﺤﯿﻂ ﻗﻠﯿﺎﯾﯽ اﺳﺖ ﺑﺮاي اﺳﺘﻔﺎده در ﺻﻨﺎﯾﻊ ﮐﺎﻏﺬﺳﺎزي، ﺗﻬﯿﻪ ﺧﻮراك ﻃﯿﻮر و ﺗﻮﻟﯿﺪ ﺳﻮﺧﺖ زﯾﺴﺘﯽ ﻣﻨﺎﺳﺐ ﻣﯽﺑﺎﺷﺪ.

Enzymes play an essential role in catalyzing the reactions for multiple industrial applications. One of these critical industries with a worldwide application is paper and pulp, which is cost-effective in increasing attention. Xylanases are potential enzymes that proved their abilities in a broad range of applications, specifically in the paper and pulp industry as a biobleaching agent and dye removal biocatalyst. In these decades, the production of novel enzymes from natural sources is conceivable, especially with applying the culture-independent method of metagenome. This practical approach provides the opportunity to identify the novel enzymes from uncultivable microbial diversities. Concerning the importance of the thermostable enzymes for industrial applications and their better action in harsh conditions, this study aimed to identify novel thermostable xylanase from metagenomic data of sheep rumen by applying the in-silico screening. The thermostable xylanase was extracted from the ruminal DNA and after cloning and expression named PersiXyn5. The enzymes' kinetic parameters, including Km, Vmax, and its specific activity, were examined. The enzyme was optimally active at 80 ℃ and pH 8 and could retain 58% of its maximum activity after 2h of incubation at 90 ℃. The thermostable, alkali PersiXyn5 was an efficient enzyme in the paper industry and poultry feed and fuel applications