Ferdowsi University of Mashhad

Document Type : Research Articles

Authors

1 Agricultural Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

2 Biotechnology and Plant Breeding Department, Ferdowsi University of Mashhad, Mashhad, Iran

3 Ornamental Biotechnology Department, ACECR, Mashhad Branch, Mashhad, Iran

Abstract

Cellulose which is extremely produced by plants, can be used for biofuel production but this function needs chemical or enzymatic digestion. Cellulose hydrolysis of plant wastes for ethanol production requires a mixture of three enzyme groups, including endoglucanases, exoglucanases, and beta-glucosidases. The cellobiohydrolase enzyme (Cel6B) from Thermobifidia fusca has been used for cellulase activity extensively. This research aimed to express recombinant Cel6B enzyme in Pichia pastoris. For this purpose, cel6B gene in control of AOX1 promoter (methanol inducible) was introduced into Pichia pastoris. Amplification of cel6B gene was performed by PCR technique and then introduced into the Phil-S1 yeast vector. The recombinant construct contained the cel6B gene sequence and PHO1 signal sequence as secretion signal was transferred into Pichia pastoris GS115 strain. The transformed yeast cells expressed the recombinant Cel6B to yield 2.104 U (µmol/min)/ml of culture medium. Purified recombinant enzyme showed the best activity at 60 °C and pH 4.5 and this was agreed with optimum conditions for recombinant Cel6B enzymes which were produced in other systems. The purity of the enzyme was examined by SDS–PAGE technique, and a single band with a molecular weight about 59.6 kDa was observed. As cel6B gene sequence was not optimized for expression in the Pichia pastoris yeast, this could be one of the reasons for low level activity of recombinant Cel6B enzyme. This thermostable enzyme can be used for cellulolytic digestion of biomaterials in biofuel production research and other uses.

Keywords

Abd Rahman R. N. Z., Geok L. P., Basri M. and Saleh, A. B. (2004) Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K. Bioresource Technology 96:429-43
Alami N. H., Kuswytasari N. D., Zulaika E. and Shovitri M. (2017) Optimization of cellulase production by Candida G3.2 from the rhizosphere of gunung anyar mangrove surabaya. Proceeding of International Conference on Green Technology 8 (1):399-406.
Fang H. and Xia L. (2015) Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw. Biomass and Bioenergy 78:99-109.
Fang H., Zhao R., Li C. and Zhao C. (2019) Simultaneous enhancement of the beta–exo synergism and exo–exo synergism in Trichoderma reesei cellulase to increase the cellulose degrading capability. Microbial Cell Factories 18(1):1-14.
Gomez del Pulgar E.M. and Saadeddin A. (2014) The cellulolytic system of Thermobifida fusca. Critical Reviews in Microbiology 40(3):236-247.
Harshvardhan K., Mishr A. and Jha B. (2013). Purification and characterization of cellulase from a marine Bacillus sp. H1666: a potential agent for single step saccharification of seaweed biomass. Journal of Molecular Catalysis B: Enzymatic 93:51-56.
Kazzaz A. E. and Fatehi P. (2020) Technical lignin and its potential modification routes: a mini-review. Industrial Crops and Products 154:112732.
Laemmli U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680-685.
Lindenmuth B. E. and McDonald K. A. (2011) Production and characterization of Acidothermus cellulolyticus endoglucanase in Pichia pastoris. Protein Expression and Purification 77(2):153-158.
Murao S., Sakamoto R. and Arai M. (1988) Cellulase of Aspergillus aculeatus. In methods in Enzymology. Academic Press. London, 274-299 pp.
Lu W., Li D. and Wu Y. (2003) Influence of water activity and temperature on xylanase biosynthesis in pilot-scale solid-state fermentation by Aspergillus sulphurous. Enzyme and Microbial Technology 32:305-311.
Rai P., Tiwari S. and dan Gaur R. (2012). Optimization of process parameters for cellulase production by novel thermotolerant yeast. BioResources 7:5401-5414.
Srivastava N., Srivastava M., Mishra P. K., Gupta V. K., Molina G., Rodriguez-Couto S., et al. (2018) Applications of fungal cellulases in biofuel production: advances and limitations. Renewable and Sustainable Energy Reviews 82:2379-2386.
Sun F. F., Bai R., Yang H., Wang F., He J., Wang C., et al. (2016). Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris. Enzyme and Microbial Technology 92:107-116.
Sun Y. and Cheng J. (2002) Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresource Technology 83(1):1-11.
Taha, A. S. J., Taha, A. J., & Faisal, Z. G. (2015). Purification and kinetic study on cellulase produced by local Trichoderma viride. Nature and Science, 13(1), 87-90.
Taha, A. S. J., Taha, A. J. and Faisal, Z. G. (2015) Purification and kinetic study on cellulase produced by local Trichoderma viride. Nature and Science 13:87-90.
Tenkanen M., Vršanská M., Siika-aho M., Wong D. W., Puchart V., Penttilä M., et al. (2012) Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity. FEBS Journal 280(1):285-301.
Volossiouk T., Robb E. J. and Nazar R. N. (1995) Direct DNA extraction for PCR-mediated assays of soil organisms. Applied and Environmental Microbiology 61(11):3972-3976.
Vuong T. V. and Wilson D. B. (2009). Processivity, synergism, and substrate specificity of Thermobifida fusca Cel6B. Applied and Environmental Microbiology 75(21):6655-6661.
Vuong T. V. and Wilson D. B. (2009). The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B. The FEBS Journal 276(14):.3837-3845.
Wu M., Bu L., Vuong T.V., Wilson D.B., Crowley M.F., Sandgren M., et al. (2013). Loop motions important to product expulsion in the Thermobifida fusca glycoside hydrolase family 6 cellobiohydrolase from structural and computational studies. Journal of Biological Chemistry 288(46):33107-33117.
Xiao W., Li H., Xia W., Yang Y., Hu P., Zhou S., et al.(2019) Co-expression of cellulase and xylanase genes in Sacchromyces cerevisiae toward enhanced bioethanol production from corn stover. Bioengineered 10(1):513-521.
Zhang N., Wright T., Wang X., Savary B. J. and Xu J. (2020) Production of thermostable endo-1, 5-α-L-arabinanase in Pichia pastoris for enzymatically releasing functional oligosaccharides from sugar beet pulp. Applied Microbiology and Biotechnology 104(4):1595-1607.
Zhang P., Yuan X., Du Y. and Li J. J. (2018). Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris. BMC Biotechnology, 18(1):1-9.
Zhang Y.P., Hong J. and Ye X. (2009). Cellulase assays. In Biofuels. Humana Press, Totowa, NJ, 213-231 pp.
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