The development and use of an ELISA-based method to follow the distribution of cellulase monocomponents during the hydrolysis of pretreated corn stover

Biotechnology for Biofuels

Jack N Saddler¹  Valdeir Arantes¹  Jinguang Hu¹  Amadeus Y Pribowo¹ 

[1]Forest Products Biotechnology/Bioenergy Group, University of British Columbia, 2424 Main Mall, Vancouver, British Columbia, V6T1Z4, Canada

关键词: ELISA;    Adsorption;    Enzyme;    Cellulase;    Cellulose;   

Others  :  798028
DOI  :  10.1186/1754-6834-6-80

 received in 2013-02-06, accepted in 2013-05-10,  发布年份 2013

【 摘 要 】

Background

It is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requires the synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to the complexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack of specific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrate interaction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in a typical “cellulase mixture” need to work cooperatively to achieve effective hydrolysis, the action of one enzyme is likely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature of the lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us to follow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commercially realistic biomass substrates.

Results

A high throughput immunoassay that could quantitatively and specifically follow individual cellulase enzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb, respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to specifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II (Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosic supernatants could be minimized by dilution.

Conclusion

A double-antibody sandwich ELISA was able to detect and quantify individual enzymes when present in cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 – 1 μg/ml), provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay was employed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, that were present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS). All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profiles observed with the ELISA method were in agreement with earlier work where more labour intensive enzyme assay techniques were used.

【 授权许可】

   
2013 Pribowo et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Pribowo A, Arantes V, Saddler JN: The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover. Enzyme Microb Technol 2012, 50(3):195-203.
• [2]Banerjee G, Scott-Craig JS, Walton JD: Improving enzymes for biomass conversion: a basic research perspective. Bioen Res 2010, 3(1):82-92.
• [3]Humbird D, Davis R, Tao L, Kinchin C, Hsu D, Aden A, Schoen P, Lukas J, Olthof B, Worley M, Sexton D, Dudgeon D: Process design and economics for biochemical conversion of lignocellulosic biomass to ethanol - dilute-acid pretreatment and enzymatic hydrolysis of corn stover. NREL/TP 2011, 5100-47764.
• [4]Kumar L, Chandra R, Chung PA, Saddler J: Can the same steam pretreatment conditions be used for most softwoods to achieve good, enzymatic hydrolysis and sugar yields? Bioresour Technol 2010, 101(20):7827-7833.
• [5]Del Rio LF, Chandra RP, Saddler JN: The effect of varying organosolv pretreatment chemicals on the physicochemical properties and cellulolytic hydrolysis of mountain pine beetle-killed lodgepole pine. Appl Biochem Biotechnol 2010, 161(1–8):1-21.
• [6]Darias R, Villalonga R: Functional stabilization of cellulase by covalent modification with chitosan. J Chem Technol Biotechnol 2001, 76(5):489-493.
• [7]Boer H, Koivula A: The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A. Eur J Biochem 2003, 270(5):841-848.
• [8]Tu MB, Zhang X, Kurabi A, Gilkes N, Mabee W, Saddler J: Immobilization of beta-glucosidase on Eupergit C for lignocellulose hydrolysis. Biotechnol Lett 2006, 28(3):151-156.
• [9]Tu M, Chandra RP, Saddler JN: Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated lodgepole pine. Biotechnol Prog 2007, 23(5):1130-1137.
• [10]Sharrock KR: Cellulase assay-methods - a Review. J Biochem Biophys Methods 1988, 17(2):81-105.
• [11]Medve J, Lee D, Tjerneld F: Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography. J Chromatogr 1998, 808(1–2):153-165.
• [12]Medve J, Karlsson J, Lee D, Tjerneld F: Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes. Biotechnol Bioeng 1998, 59(5):621-634.
• [13]Yu AHC, Lee D, Saddler JN: A quantitative approach to the study of the adsorption-desorption of cellulase components in a crude cellulase mixture. Biotechnol Tech 1993, 7(10):713-718.
• [14]Gao D, Chundawat SPS, Uppugundla N, Balan V, Dale BE: Binding characteristics of trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass. Biotechnol Bioeng 2011, 108(8):1788-1800.
• [15]Varnai A, Viikari L, Marjamaa K, Siika-aho M: Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates. Bioresour Technol 2011, 102(2):1220-1227.
• [16]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(2):469-475.
• [17]Igarashi K, Uchihashi T, Koivula A, Wada M, Kimura S, Okamoto T, Penttila M, Ando T, Samejima M: Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface. Science 2011, 333(6047):1279-1282.
• [18]Ramos LP, Zandona A, Deschamps FC, Saddler JN: The effect of Trichoderma cellulases on the fine structure of a bleached softwood kraft pulp. Enzyme Microb Technol 1999, 24(7):371-380.
• [19]KOLBE J, KUBICEK C: Quantification and identification of the main components of the trichoderma cellulase complex with monoclonal-antibodies using an Enzyme-Linked-Immunosorbent-Assay (Elisa). Appl Microbiol Biotechnol 1990, 34(1):26-30.
• [20]Conde S, Suyama K, Itoh K, Yamamoto H: Application of commercially available fenitrothion-ELISA kit for soil residue analysis. J Pestic Sci 2008, 33(1):51-57.
• [21]DROW D, MANNING D: Indirect sandwich enzyme-linked Immunosorbent-assay for rapid detection of streptococcus-pneumoniae type-3 antigen. J Clin Microbiol 1980, 11(6):641-645.
• [22]VOLLER A, BARTLETT A, BIDWELL D: Enzyme immunoassays with special reference to elisa techniques. J Clin Pathol 1978, 31(6):507-520.
• [23]Riske FJ, Eveleigh DE, Macmillan JD: Double-antibody sandwich enzyme-linked-immunosorbent-assay for cellobiohydrolase-i. Appl Environ Microbiol 1990, 56(11):3261-3265.
• [24]BUHLER R: Double-antibody sandwich enzyme-linked-immunosorbent-assay for quantitation of endoglucanase-i of trichoderma-reesei. Appl Environ Microbiol 1991, 57(11):3317-3321.
• [25]Crowther JR: The ELISA Guidebook: Totowa. NJ, USA: Humana Press; 2000.
• [26]Lynd L, Zhang Y: Quantitative determination of cellulase concentration as distinct from cell concentration in studies of microbial cellulose utilization: Analytical framework and methodological approach. Biotechnol Bioeng 2002, 77(4):467-475.
• [27]Toscano I, Gascon J, Marco M, Rocha J, Barcelo D: Atrazine interaction with tropical humic substances by Enzyme Linked Immunosorbent Assay. Analusis 1998, 26(3):130-134.
• [28]Krotzky A, Zeeh B: Pesticides report .33. Immunoassays for residue analysis of agrochemicals: Proposed guidelines for precision, standardization and quality control. Pure Appl Chem 1995, 67(12):2065-2088.
• [29]Palonen H, Tenkanen M, Linder M: Dynamic interaction of Trichoderma reesei cellobiohydrolases Ce16A and Ce17A and cellulose at equilibrium and during hydrolysis. Appl Environ Microbiol 1999, 65(12):5229-5233.
• [30]Gama F, Vilanova M, Mota M: Exo- and endo-glucanolytic activity of cellulases purified from Trichoderma reesei. Biotechnol Tech 1998, 12(9):677-681.
• [31]Rosgaard L, Pedersen S, Langston J, Akerhielm D, Cherry JR, Meyer AS: Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated barley straw substrates. Biotechnol Prog 2007, 23(6):1270-1276.
• [32]Zhou J, Wang Y, Chu J, Zhuang Y, Zhang S, Yin P: Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100–14. Bioresour Technol 2008, 99(15):6826-6833.
• [33]Starcher B: A ninhydrin-based assay to quantitate the total protein content of tissue samples. Anal Biochem 2001, 292(1):125-129.
• [34]Ohgren K, Galbe M, Zacchi G: Optimization of steam pretreatment of SO2-impregnated corn stover for fuel ethanol production. Appl Biochem Biotechnol 2005, 121:1055-1067.
• [35]Wu M, Chang K, Gregg D, Boussaid A, Beatson R, Saddler J: Optimization of steam explosion to enhance hemicellulose recovery and enzymatic hydrolysis of cellulose in softwoods. Appl Biochem Biotechnol 1999, 77–9:47-54.
• [36]Bura R, Chandra R, Saddler J: Influence of xylan on the enzymatic hydrolysis of steam-pretreated corn stover and hybrid poplar. Biotechnol Prog 2009, 25(2):315-322.
• [37]Arantes V, Saddler JN: Cellulose accessibility limits the effectiveness of minimal cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates. Biotechnol Biofuels 2011, 4:3. BioMed Central Full Text
• [38]Berlin A, Maximenko V, Bura R, Kang KY, Gilkes N, Saddler J: A rapid microassay to evaluate enzymatic hydrolysis of lignocellulosic substrates. Biotechnol Bioeng 2006, 93(5):880-886.