افزایش کارایی فرایند ساکاریفیکاسیون تفاله چغندرقند با استفاده از موتانت برتر قارچ Trichoderma reesei برای تولید بیواتانل

نوع مقاله : کامل علمی - پژوهشی

نویسندگان

1 استادیار گروه گیاهپزشکی، پژوهشکده کشاورزی هسته‎ای، پژوهشگاه علوم و فنون هسته‎ای، سازمان انرژی اتمی ایران

2 کارشناس ارشد گروه گیاهپزشکی، پژوهشکده کشاورزی هسته‎ای، پژوهشگاه علوم و فنون هسته‎ای، سازمان انرژی اتمی ایران

3 دانشیار گروه بیوتکتنولوژی، دانشگاه پیام نور،

4 کارشناس ارشد بیوتکنولوژی کشاورزی، دانشگاه پیام نور کرج

چکیده

تفاله چغندرقند یکی از ضایعات جانبی صنایع تولید قند می‎باشد که به علت دارا بودن درصد بالایی از مواد لیگنوسلولزی می‎تواند یکی از گزینه‎های قابل توجه جهت تولید آنزیم سلولاز، ساکاریفیکاسیون آنزیمی و تولید الکل از آن باشد. قارچ Trichoderma spp. یکی از ارگانیسم­های مهم تولید­کننده  دامنه وسیعی از آنزیم‎های تجزیه کننده سلولز در طبیعت است. در این پژوهش از تفاله چغندرقند در محیط تخمیر قارچ تریکودرما استفاده شد و با استفاده از 21 جدایه موتانت پرتو گاما قارچ T. reesei، آنزیم سلولاز در شرایط دمایی °C 28 و سرعت همزدن rpm 180برای مدت 72 ساعت تولید گردید. توانایی تولید آنزیم‎های تجزیه کننده سلولز در کلیه جدایه‎ها مورد ارزیابی قرار گرفت. فعالیت آنزیم‎های اندوگلوکاناز، اگزوگلوکاناز و سلولاز کل در جدایه موتانت T. r M5 بالاترین مقادیر فعالیت آنزیمی را در بین جدایه های موتانت و جدایه والد اولیه نشان داد. هم‎چنین جدایه مذکور دارای فعالیت بتا-گلوکوزیدازی مناسبی بود. پروفایل پروتئینی جدایه موتانت T.r M5 با استفاده از آزمون SDS-PAGE بررسی شد. جدایه فوق دارای باندهای آنزیمی متعددی در وزن‎های مولکولی مختلف بود که مربوط به آنزیم‎های EG IV، Cel 3C، Cel 3D، Cel 3A، Cel 7A، Cel 6A، Cel 5A و Cel 61A بودند. نتایج این تحقیق نشان داد که جدایه موتانت T. r M5 بالاترین کارایی را بین جدایه‎های موتانت برای ساکاریفیکاسیون تفاله چغندرقند داراست. با استفاده از آنزیم های تولیدی از این جدایه، ساکاریفیکاسیون تفاله چغندر به مدت یک ساعت انجام شد و میزان تولید الکل از قندهای آزاد شده در محیط با استفاده از مخمرهای صنعتی Saccharomyces cerevisiae وCluyveromyces marxianus مورد ارزیابی قرار گرفت. میزان تولید الکل در تیمار ساکاریفیکاسیون با T. r M5، حدود 2-5/1 برابر بیشتر از والد اولیه خود (T. reesei) بود.

کلیدواژه‌ها

عنوان مقاله [English]

Increasing the efficiency of sugar beet pulp saccharification by Trichoderma reesei superior mutants for bioethanol production

نویسندگان [English]

  • S. Shahbazi 1
  • H. Askari 2
  • A. Ebrahimi 3
  • M. Safaeie 4
  • M. Karimi 4
1 Assistant professor of Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of IRAN (AEOI), Alborz, Iran
2 Master expert of Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of IRAN (AEOI), Iran
3 Associate Professor, Department of Biotechnology, Payame Noor University, Iran
4 Department of Biotechnology, Payame Noor University, Iran
چکیده [English]

Sugar beet pulp is one of the sugar industries by-products which can be used for cellulase enzyme production, Enzymatic Saccharification, and alcohol production due to its high percentage of lingo-cellulosic content. Trichoderma spp. is an important fungus that produces a wide range of cellulytic enzymes. In this study, cellulase enzyme was produced by placing sugar beet pulp in Trichoderma fermentation media together with 21 gamma irradiated T. reesei mutants and then shaking at 180 rpm at 28 °C for 72 h. All isolates were screened for cellulytic enzyme production. T. r M5 mutanthad the highest level of endo-glucanase, total cellulase, and exo-glucanase enzymeactivity among the all mutants and primary parental isolates. It also had optimum ß-glucosidase activity. The protein profile of T. r M5 mutant was analyzed using SDS-PAGE test. T. r M5  had  different enzymatic bands with variable molecular weight related to EG IV, Cel 3C, Cel 3D, Cel 3A, Cel 7A, Cel 6A, Cel 5A, and Cel 61A enzymes. Results showed that T. r M5 mutant had the highest efficiency for sugar beet pulp saccharification among the all mutants. Sugar beet pulp saccharification was carried out within 1 h using enzymes produced by this mutant. The amount of alcohol production from sugar released by industrial yeast stains Saccharomyces cerevisiae and Kluyveromyces marxianus was evaluated. Alcohol production in T. r M 5 was 1.5-2 times more than its parent, T. reesei .

کلیدواژه‌ها [English]

  • Bio-ethanol
  • cellulase
  • Kluyveromyces marxianus
  • mutation
  • Saccharification
  • Saccharomyces cerevisiae
  • Trichoderma reesei

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