بهبود فعاليت آنزيم‌هاي تجزيه‌كننده سلولز با جهش‌زايي در قارچ Trichoderma reesei

Improvement of Cellulose Degrading Enzymes Activity by Mutagenesis in Trichoderma reesei Fungi

ﭼﮑﯿﺪه ﻗﺎرچ Trichoderma reesei ﯾﮑﯽ از ﮔﻮﻧﻪﻫﺎي ﻣﻬﻢ ﺗﻮﻟﯿﺪﮐﻨﻨﺪه آﻧﺰﯾﻢﻫﺎي ﺗﺠﺰﯾﻪﮐﻨﻨﺪه ﺳﻠﻮﻟﺰ در ﻃﺒﯿﻌﺖ اﺳﺖ. در اﯾﻦ ﭘﮋوﻫﺶ 21 ﺟﺪاﯾﻪ ﺟﻬﺶﯾﺎﻓﺘﻪ ﭘﺮﺗﻮ ﮔﺎﻣﺎ از ﻗﺎرچ T. reesei ﺑﺮاي ﺗﻮﻟﯿﺪ آﻧﺰﯾﻢ ﺳﻠﻮﻻز روي ﻣﺤﯿﻂ ﻣﺤﺘﻮي ﺳﻠﻮﻟﺰ ﮐﻠﻮﺋﯿﺪي ﻏﺮﺑﺎلﮔﺮي ﺷﺪ. ﻏﻠﻈﺖ ﭘﺮوﺗﺌﯿﻦﻫﺎي ﺧﺎرج ﺳﻠﻮﻟﯽ ﺟﺪاﯾﻪﻫﺎي وﺣﺸﯽ و ﺟﻬﺶﯾﺎﻓﺘﻪ ﺑﺎ اﺳﺘﻔﺎده از روش ﺑﺮدﻓﻮرد اﻧﺪازهﮔﯿﺮي ﺷﺪ. آوﯾﺴﻞ، ﮐﺮﺑﻮﮐﺴﯽ ﻣﺘﯿﻞ ﺳﻠﻮﻟﺰ و ﮐﺎﻏﺬ ﺻﺎﻓﯽ ﺑﺮاي اﻧﺪازهﮔﯿﺮي ﻓﻌﺎﻟﯿﺖ ﺳﻠﻮﻻزي اﺳﺘﻔﺎده ﺷﺪ. ﻫﻢﭼﻨﯿﻦ ﺧﻠﻮص و ﺗﺮﮐﯿﺐ ﭘﺮوﺗﺌﯿﻦﻫﺎي ﻏﻨﯽ از آﻧﺰﯾﻢ ﺑﺎ اﺳﺘﻔﺎده از آزﻣﻮن اﻟﮑﺘﺮوﻓﻮرز ژل ﭘﻠﯽآﮐﺮﯾﻞآﻣﯿﺪ ﻣﻮرد ارزﯾﺎﺑﯽ ﻗﺮار ﮔﺮﻓﺖ. ﺑﺎﻻﺗﺮﯾﻦ ﻣﯿﺰان ﺗﻮﻟﯿﺪ ﭘﺮوﺗﺌﯿﻦ ﺧﺎرج ﺳﻠﻮﻟﯽ در ﺟﺪاﯾﻪﻫﺎي T. r M7 و T. r M17 ﻣﺸﺎﻫﺪه ﺷﺪ. ﻓﻌﺎﻟﯿﺖ آﻧﺰﯾﻢﻫﺎي اﻧﺪوﮔﻠﻮﮐﺎﻧﺎز، اﮔﺰوﮔﻠﻮﮐﺎﻧﺎز و ﺳﻠﻮﻻز ﮐﻞ در ﺟﺪاﯾﻪ ﺟﻬﺶﯾﺎﻓﺘﻪ T. r M8 ﺑﺎﻻﺗﺮﯾﻦ ﻣﻘﺎدﯾﺮ ﻓﻌﺎﻟﯿﺖ آﻧﺰﯾﻤﯽ را در ﺑﯿﻦ ﺟﺪاﯾﻪﻫﺎي ﺟﻬﺶﯾﺎﻓﺘﻪ و وﺣﺸﯽ ﻧﺸﺎن داد. اﺧﺘﻼف وزن ﻣﻮﻟﮑﻮﻟﯽ ﺑﺎﻧﺪﻫﺎي آﻧﺰﯾﻤﯽ ﻧﺸﺎن داد ﮐﻪ آﻧﺰﯾﻢﻫﺎي Cel 12A ،Cel 1A ،EG IV، Cel 5A ،Cel 6A ،Cel 7A ،Cel 3A ،Cel 45A و Cel 61A ﺳﻠﻮﻟﺰ ﮐﻠﻮﺋﯿﺪي را ﺑﻪﺻﻮرت ﻫﻢاﻓﺰاﯾﯽ ﻫﯿﺪروﻟﯿﺰ ﻣﯽﮐﻨﻨﺪ. اﯾﻦ ﻧﺘﺎﯾﺞ ﻧﺸﺎن ﻣﯽدﻫﺪ ﮐﻪ ﺟﻬﺶزاﯾﯽ ﺑﺎ ﭘﺮﺗﻮ ﮔﺎﻣﺎ ﺑﺮاي دﺳﺘﯿﺎﺑﯽ ﺑﻪ ﺟﻬﺶﯾﺎﻓﺘﻪﻫﺎي ﺗﺮﯾﮑﻮدرﻣﺎ ﺑﺎ ﺗﻮﻟﯿﺪ ﺑﺎﻻي آﻧﺰﯾﻢ ﺳﻠﻮﻻز اﻣﮑﺎنﭘﺬﯾﺮ اﺳﺖ.

Trichoderma reesei is one of the most important fungi producing cellulose degrading enzymes in nature. In this study, 21 mutated isolate via gamma irradiation of T. reesei were screened for cellulase enzyme production on a colloidal cellulose medium. The concentration of extracellular proteins of wild and mutant isolates were measured using Bradford's method. Avicel, carboxymethyl cellulose and filter paper were used to measure cellulase activity. The purity and composition of enzyme-rich proteins were also evaluated using polyacrylamide gel electrophoresis (SDS-PAGE) test. The highest amount of extracellular protein production was observed in T. r M7 and T. r M17 isolates. Activity of endoglucanase, exoglucanase and total cellulase enzymes in mutated isolate T. r M8 showed the highest levels of enzymatic activity among mutant and wild isolates. The difference in the molecular weight of the enzyme bands showed that the EG 4, Cel 1A, Cel 12A, Cel 45A, Cel 3A, Cel 7A, Cel 6A, Cel 5A and Cel 61A enzymes hydrolyzed the colloidal cellulose, synergictly. These results indicate that, mutation induced by gamma radiation is possible to obtain high producing Trichoderma mutants of cellulase enzymes.