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
Ahari Mostafavi H. The use of nuclear technology for weeds and plant disease management, Second National Conference on the application of nuclear technology for agricultural sciences and natural resources. 9-10 June. 2009;pp:331-335. (In Persian)

Adney WS, Mohagheghi A, Thomas SR, Himmel M. Comparison of protein contents of cellulose preparations in a worldwide round-robin assay. In: Saddler, J.N., Penner, M.H. (Eds.), Enzymatic Degradation of Insoluble Carbohydrates, ACS Symposium Series 618. American Chemical Society, Washington. 1995; pp. 256–271.

Barr BK, Hsieh Y-L, Ganem B, Wilson, DB. Identification of two functionally different classes of exocellulases. Biochemistry. 1996; 35: 586-592.

Bhikhabhai R, Johansson G, Pettersson G. Isolation of cellulolytic enzymes from Trichoderma reesei QM 9414. J ApplBiochem. 1984; 6: 336-345.

Cannon RE, Anderson SM. Biogenesis of Bacterial Cellulose, Critical Reviews in Microbiology. 1991; 17, 435-447.

Divne C, Ståhlberg J, Teeri TT, Jones TA. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei. J. Mol. Biol. 1998; 275: 309-325.

Fägerstam L, Håkansson U, Pettersson G, Andersson L. Purification of three different cellulolutic enzymes from Trichoderma viride QM 9414 on a large scale. In Proceedings of Bioconversion Symposium, 1977; pp. 165-178. Indian Institute of Technology, New Delhi.

Fägerstam LG, Pettersson LG. The 1,4-beta-glucan cellobiohydrolases of Trichoderma reesei QM 9414. A new type of cellulolytic synergism.  FEBS Letters. 1980; 119: 97-100.

Foreman PK, Brown D, Dankmeyer L, Dean R,  Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS, Meerman  HJ, Mitchell T, Mitchinson C, Olivares HA, Teunissen PJ, Yao J, Ward M. Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichodermareesei. J Biol Chem. 2003; 278: 31988-31997.

Grishutin SG, Gusakov AV, Markov AV, Ustinov BB, Semenova M, Sinitsyn AP, Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. Biochim. Biophys. Acta. 2004; 1674: 268–281.

Henrissat B, Driguez H, Viet C, Shulein M. Synergism of cellulases from Trichoderma reesei in the degradation of cellulose. Bio/Technol. 1985; 3:722– 726.

Ilmen M, Saloheimo A, Onnela M-L, Penttila ME. Regulation of cellulose gene expression in the filamentous fungus Trichodermareesei. Appl Environ Microbiol. 1997; 63:1298–306.

Jun H, Kieselbach T, Jönsson L J, Enzyme production by filamentous fungi: analysis of the secretome of Trichoderma reesei grown on unconventional carbon source. Microbial Cell Factories. 2011; 10:68.

Karlsson J, Saloheimo M, Siika-aho M, Tenkanen M, Penttilä M, Tjerneld F. Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei. Eur. J. Biochem. 2001; 268: 6498-6507.

Klemm D, Philipp B, Heinze T, Heinze U, Wagenknecht W. Comprehensive cellulose chemistry. I. Fundamentals and analytical methods. Weinheim: Wiley-VCH. 1998.

Klyosov AA. Cellulases of the third generation. In: Aubert J-P, Beguin P, Millet J, editors. Biochemistry and genetics of cellulose degradation. London: Academic Press. 1988; p 87–99.

Klyosov AA. Trends in biochemistry and enzymology of cellulose degradation. Biochemistry. 1990; 29:10577–10585.

Laemmli UK, Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, 1970; 227: 680-685.

Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS. Microbial cellulose utilization: Fundamentals and biotechnology. Microbiology and Molecular Biology Reviews. 2002; 66:506-577.

Maki M, Leung KT, Qin W. The prospects of cellulose-producing bacteria for the bioconversion of lignocellulosic biomass.  Int J Biol Sci. 2009; 5: 500–516.

Medve J, Stahlberg J, Tjerneld F. Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose. Biotechnol Bioeng. 1994; 44:1064– 1073.

Moradi R, Shahbazi S, Ahari Mostafavi H. Determine the appropriate dose of radiation in order to induce the desired mutation effects of morphological investigation of Trichoderma, First National Congress of Agricultural Science and New Technologies. 10-12 Septamber. 2010; pp:29.

Nidetzky B, Claeyssens M. Specific quantification of Trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase-cellulose binding studies. Biotechnol Bioeng. 1994; 44:961–966.

Nidetzky B, Steiner W. A new approach for modeling cellulase-cellulose adsorption and the kinetics of the enzymatic hydrolysis of microcrystalline cellulose. Biotechnol Bioeng. 1993; 42:469– 479.

Reinikainen T. The cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei VTT publications 206 (1994) ISBN 951-38-4644-x.

Shoemaker SP, Brown RD. Enzymatic activities of endo-1, 4-h-D-glucanases purified from Trichodermaviride. Biochim BiophysActa. 1978; 523:133– 146.

Shoemaker S, Schweickaut V, Ladner M, Gelfand D, Kwok S, Myambo K, Innis M, Molecular cloning of exocellobiohydrolase I derived from Trichoderma reesei strain L27. Bio/Technology. 1983; 1:691–696.

Srisodsuk M, Kleman-Leyer K, Keranen S, Kirk TK, Teeri TT. Modes of action on cotton and bacterial cellulose of a homologous endoglucanase-exoglucanase pair from Trichoderma reesei. Eur J Biochem. 1998; 251(3):885–892..

Ståhlberg J. Functional organization of cellulases from Trichoderma reesei. In Doctoral thesis. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science. 1991; 344. 45pp, Uppsala. ISBN 91-554-2800-2. Uppsala University.

Teeri T, Koivula A. 1995. Cellulose degradation by native and engineered fungal cellulases. Carbohydr. Eur. 12, 28–33.

Valjamae P, Sild V, Pettersson G, Johansson G. The initial kinetics of hydrolysis by cellobiohydrolases I and II is consistent with a cellulose surface - erosion model. Eur. J. Biochem. 1998; 253:469 – 475.

Van Tilbeurgh H, Claeyssens M, Bruyne CK. Te use of 4-methylum-belliferyl and other chromophoric glycosides in the study of cellulolytic enzymes. FEBS Lett. 1982; 149: 152–156.

VanTilbeurgh H, Pettersson G, Bhikabhai R, Boeck H, Claeyssens M. Studies of the cellulolytic system of Trichodermareesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-beta-glucan cellobiohydrolase II. Eur. J. Biochem. 1985; 148: 329–334.

Watson DL, Wilson DB, Walker LP. Synergism in binary mixtures of Thermo bifidafusca cellulases Cel6B, Cel9A, and Cel5A on BMCC and Avicel. Appl. Biochem. Biotechnol. 2002; 101:97– 111.

Wen Z, Liao W, Chen Sh. Production of cellulase by Trichoderma reesei from dairy manure. Bioresour. Technol. 2005; 96: 491-499.

Wood TM, Bhat KM. Methods for measuring cellulase activities. Methods Enzymol 1988; 160:87– 117.

Xin Z, Yinbo Q, Peiji G, Acceleration of ethanol production from paper mill waste fiber by supplementation with β-glucosidase. Enzyme Microb. Technol. 1993; 15: 62–65.

Zaia DAM, Zaia CTBV, Lichtig J. Determinatio de proteinastotais via espectrofometria: vantagens e desvantagens dos métodosexistentes. Quim. 1998; 21: 787–793.

Zhang S, Wolfgang DE, Wilson DB. Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis. Biotechnol. Bioeng. 1999; 66:35– 41.

Zhang Y-HP, Lynd LR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: non complexes cellulase systems. Biotechnol. Bioeng, 2004; 88:797–824.

Zhang Y-HP, Lynd LR. A Functionally Based Model for Hydrolysis of Cellulose by Fungal Cellulase. You have free access to this content. Biotechnology and Bioengineering, 2006; 94(5):888-898.

Zhao H, Kwak JH, Zhang ZC, Brown HM, Arey BW, Holladay JE. Studying cellulose fiber structure by SEM, XRD, NMR and acid hydrolysis. Carbohydrate Polymers. 2007; 68: 235-241.