Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

Armaya Badiatul Fitri, Elvi Restiawaty, Maelita Ramdani Moeis

Abstract


One of the important enzymes in cellulase complex is β-glucosidase. In this research, adding signal peptide of inulinase gene from Kluyveromyces marxianus, cloning, and expressing of bglp15.2 gene in S. cerevisiae BY4741 had been done. Gene of bglp15.2 encoding β-glucosidase has 90% identity to nucleotide sequence of Shewanella frigidimarina NCIMB 400 bacteria. Adding nucleotide sequence of signal peptide was aimed to secrete β-glucosidase and had been done with PCR (Polymerase Chain Reaction) method. The addition of nucleotide sequence of signal peptide in bglp15.2 gene had been done succesfully that indicated from nucleotide sequencing result and the increment of amplicon band size in electroferogram of the last addition PCR step. The bglp15.2 and bglp15.2INU gene (the bglp15.2 gene that has signal peptide nucleotide sequence) were cloned in Escherichia coli DH5α using pGEM-T-Easy vector and pBEVY-GL shuttle vector. The pBEVY-GL shuttle vector was used for transforming S. cerevisiae BY4741 with bglp15.2 and bglp15.2INU. The recombinant S. cerevisiae BY4741 carrying bglp15.2INU gene and growing in 48 hours had extracellularly β-glucosidase enzyme activity of 0,0178 U/ml and the intracellularly activity was 0,0181 U/ml. The  β-glucosidase enzyme without signal peptide was not secreted. With K. marxianus inulinase signal peptide, about 50% Bglp15.2INU protein could be secreted. The protein molecular weight of secreted Bglp15.2INU was 44 kDa in SDS-PAGE result.


Keywords


cloning; expression; peptide signal; β-glucosidase; extracellular; enzyme activity

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DOI: https://doi.org/10.15575/biodjati.v2i2.1619

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References


Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., & Struhl, K. (2003) Current Protocols in Molecular Biology, John Wiley and Sons, Inc, 2030 – 2038.

Bergkessel, M., & Guthrie, C. (2013) Colony PCR. Methods in Enzymology 529.

Buchholz, K., Kasche, V., & Bornscheuer, U., T. (2012). Biocatalysts and Enzyme Technology. Germany: Wiley-Blackwell 232 – 236.

Chung, B. H., Nam, S. W., Kim, B. M., & Park, Y. H. (1995). Highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides. Biotechnology and Bioengineering 49: 473-479.

Erawijantari, P. P. (2014). Mutasi dan ekspresi gen β-glukosidase yang diisolasi dari metagenom laut dalam Kepulauan Kawio, Sulawesi Utara, Skripsi, Bandung: Institut Teknologi Bandung.

Fitzgerald, I., & Glick, B. S. (2014). Secretion of a foreign protein from budding yeasts in enhanced by cotranslational translocation and by suppression of vacuolar targetting. Microbial Cell Factories 13(125).

Gao, L., Gao, F., Jiang, X., Zhanga, C., Zhanga, D., Wang, L., Wua, G., & Chena, S. (2014). Biochemical characterization of a new glucosidase (Cel3e) from Penicillium piceum and its application in boosting lignocelluloses bioconversion and forming disaccharide inducers: new insights into the role of glucosidase. Process Biochemistry 49: 768–774.

Gietz, R. D., & Woods, R. A. (2002). Transformation of yeast by lithium acetate / single-stranded carrier DNA / polyethylene glycol method. Methods in Enzymology 350: 87-96.

Gupta, R. K., Patterson, S. S., Ripp, S., Simpson, M. L., & Sayler, G. S. (2003). Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research 4: 305-313.

Hanahan, D., Jessee, J., & Bloom, F., R. (1991). Plasmid transformation of Escherichia coli and other bacteria. Methods in Enzymology 204.

Hasunuma, T., & Kondo, A. (2012). Consolidated Bioprocessing and Simultaneous Saccharification and Fermentation of Lignocellulose to Ethanol with Thermotolerant Yeast Strains. Process Biochemistry 47: 1287–1294.

Hedge, R. S., & Kang, S. W. (2008). The concept of translocational regulation. Journal of Cell Biology 182(2): 225 – 232.

Hong, S. J., Kim, H. J., Kim, J. W., Lee, D. H. & Seo, J. H. (2014). Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyvermoyces marxianus inulinase. Bioprocess and Biosystem Engineering 38: 263 – 272.

Invitrogen. (2008) : User Manual pYES2. Cat. no. V825–20.

Kang, H. A., Nam, S. W., Kwon, K. S., Chung, B. H., & Yu, M. H. (1996). High level secretion of human α1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence. Journal of Biotechnology 48: 15-24.

Lee, C. R., Sung, B. H., Lim, K. M., Kim, M. J., Sohn, M. J., Bae, J. H., & Sohn, J. H. (2017). Co-fermentation using recombinant Saccharomyces cerevisiae yeast strains hyper-secreting different cellulases for the production of cellulosic bioethanol. Scientific Reports 7: 4428.

Lee, W. H., Nan, H., Kim H. J., & Jin, Y. S. (2013). Simultaneous saccharification and fermentation by engineered Saccharomyces cerevisiae without supplementing extracellular β-glucosidase. Journal of Biotechnology 167 : 316– 322.

Menon, V., & Rao, M. (2012). Trends in Bioconversion of Lignocellulose: Biofuels, Platform Chemicals and Biorefinery Concept. Progress in Energy and Combustion Science 38: 522 – 550.

Rakestraw, J. A., Sazinsky, S. L., Piatesi, A., Antipov, E., & Wittrup, K., D. (2009). Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in S. cerevisiae. Biotechnology and Bioengineering 103(6): 1192 – 1201.

Sambrook, J., & Russell, W. (2001). Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor.

Schekman, R., & Novick, P. (1982). Dalam: Strathen, J. N., Jones, E. W., Broach, J. R., eds., Molecular biology of yeast Saccharomyces cerevisae, metabolism, and gene expression. 361-393. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor.

Sigma. (1997): Sigma quality control test procedure: enzymatic assay of α-galactosidase, Sigma-Aldrich, Inc.

Singhania, R., R., Patel, A., K., Sukumaran, R., K., Larroche, C., & Pandey, A. (2012). Role and significance of beta-glucosidases in hydrolysis of cellulose for bioethanol production. Bioresource Technology 127: 500 – 507.

Stahl, G., Salem, S., N., B., Chen, L., Zhao, B., Farabaugh, P, J. (2004). Translational accuracy during exponential, postdiauxic, and stationary growth phases in Saccharomyces cerevisiae. Eukaryotic Cell 3(2): 331 – 338.

Stanbury, P., F., Whitaker, A., & Hall, S., J. (1995). Principles of fermentation technology, Oxford: Butterworth Heinemann, 13 – 16.

Tang, H., Hou, J., Shen, Y., Xu, L., Yang, H., Fang, X., & Bao, X. (2013). High β-glucosidase secretion in Saccharomyces cerevisiae improves the efficiency of cellulase hydrolysis and ethanol production in simultaneous saccharification and fermentation. Journal of Microbiology and Biotechnology 23(11): 1577-1585.

Treebupachatsakul, T., Nakazawa, H., Shinbo, H., Fujikawa, H., Nagaiwa, A., Ochiai, N., Kawaguchi, T., Nikaido, M., Totani, K., Shioya, K., Shida, Y., Morikawa, Y., Ogasawara, W., & Okada, H. (2015). Heterologously expressed Aspergillus aculeatus β-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using Trichoderma reesei cellulases. Journal of Bioscience and Bioengineering 121(1): 27 - 35.

Washburne, M., W., Braun, E., Johnston, G., C., & Singer, R., A. (1993). Stationary phase in the yeast Saccharomyces cerevisiae. Microbiological Reviews 57(2): 383 – 401.

Yarimizu, T., Nakamura, M., Hoshida, H., & Akada, R. (2015). Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus. Microbial Cell Factories 14(20).

Yu, C. H., Dang, Y., Zhou, Z., Wu, C., Zhao, F., Sachs, M. S. & Liu, Y. (2015) Codon usage influences the local rate of translation elongation to regulate co-translational protein folding. Molecular Cell 59: 744 – 754.


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