Cloning of glucoamylase gene from Aspergillus niger and its expression in Pichia pastoris

Year 2024, Volume: 33 Issue: 2, 119 - 126

Meryem Damla Ozdemir Alkis Dilek Göktürk Osman Gülnaz , Mehmet İnan

https://doi.org/10.38042/biotechstudies.1601283

Abstract

Glucoamylase (1,4-α-glucosidase) is a crucial commercial enzyme responsible for the conversion of starch, glycogen, and oligosaccharides into D-glucose through the hydrolysis of their non-reducing terminal glycosidic bonds. While many microorganisms, including bacteria, yeasts, and fungi, can produce glucoamylase, fungal glucoamylase is the preferred choice for industrial applications. The goal of this study was to produce the glucoamylase enzyme recombinantly in Pichia pastoris. To achieve this, the glaA gene from Aspergillus niger, responsible for encoding the glucoamylase enzyme, was cloned into a plasmid (pGAPZα-A) under the control of the GAP promoter and subsequently transferred into P. pastoris. The gene was verified through sequence analysis, while the effectiveness of transfection was validated using colony PCR and enzyme activity assays. The results demonstrated that the recombinant P. pastoris strain successfully secreted a substantial amount of glucoamylase (307.05 mg/L). The activity of the recombinant enzyme was measured at 79 U/mL.min. The enzyme exhibited robust activity over a broad range of temperatures (50-80°C) and various pH levels (pH 5-10), retaining 92-60% of its maximum activity. In conclusion, this study highlights the potential for laboratory-scale production of the glucoamylase enzyme, crucial for various industries, from a cost-effective and easily cultivable recombinant yeast strain, P. pastoris.

Keywords

Aspergillus niger, Enzyme activity, Glucoamylase, Industrial enzyme, Pichia pastoris, Recombinant DNA technology

Supporting Institution

Work supported by the Scientific Projects Unit of Adana Science and Technology University under project numbers 16103002 and 16103004.

Project Number

16103002, 16103004

Thanks

The authors thank Dr. Zeynep YÜCE from the Department of Medical Biology, Faculty of Medicine, Dokuz Eylül University for her kind laboratory and material support in colony PCR and protein quantitation experiments.

References

• Adrio, J. L., & Demain, A. L. (2010). Recombinant Organisms For Production of İndustrial Products. Bioengineered Bugs, 1(2), 116–131. https://doi.org/10.4161/bbug.1.2.10484
• Aehle, W. (2007). Enzymes in Industry : Production and Applications , 3rd Edition (W. Aehle, Ed.; third). WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Bagheri, A., Khodarahmi, R., & Mostafaie, A. (2014). Purification and Biochemical Characterisation of Glucoamylase From A Newly İsolated Aspergillus Niger: Relation to Starch Processing. Food Chemistry, 161, 270–278. https://doi.org/10.1016/j.foodchem.2014.03.095
• Bernfeld, P. (1955). Amylases, α and β. In Methods in Enzymology (Vol. 1, pp. 149–158). https://doi.org/10.1016/0076-6879(55)01021-5
• Burgard, J., Grünwald‐Gruber, C., Altmann, F., Zanghellini, J., Valli, M., Mattanovich, D., & Gasser, B. (2020). The secretome of Pichia pastoris in fed‐batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time. Microbial Biotechnology, 13(2), 479–494. https://doi.org/10.1111/1751-7915.13499
• Chen, J., Zhang, Y.-Q., Zhao, C.-Q., Li, A.-N., Zhou, Q.-X., & Li, D.-C. (2007). Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris. Journal of Applied Microbiology, 103(6), 2277–2284. https://doi.org/10.1111/j.1365-672.2007.03475.x
• Christakopoulos, P., & Topakas, E. (2012). Editorial Note: Advances in Enzymology and Enzyme Engineering. Computational and Structural Biotechnology Journal, 2(3), 1. https://doi.org/10.5936/csbj.201209001
• Fierobe, H. P., Mirgorodskaya, E., Frandsen, T. P., Roepstorff, P., & Svensson, B. (1997). Overexpression and characterization of Aspergillus awamori wild-type and mutant glucoamylase secreted by the methylotrophic yeast Pichia pastoris: comparison with wild-type recombinant glucoamylase produced using Saccharomyces cerevisiae and Asperg. Protein Expression and Purification, 9(2), 159–170. https://doi.org/10.1006/prep.1996.0689
• Fleißner, A., & Dersch, P. (2010). Expression and Export: Recombinant Protein Production Systems for Aspergillus. Applied Microbiology and Biotechnology, 87(4), 1255–1270. https://doi.org/10.1007/s00253-010-2672-6
• Hua, H., Luo, H., Bai, Y., Wang, K., Niu, C., Huang, H., Shi, P., Wang, C., Yang, P., & Yao, B. (2014). A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity. PLoS ONE, 9(11), e113581. https://doi.org/10.1371/journal.pone.0113581
• James, J. A., & Lee, B. H. (1997). Glucoamylases: Microbial Sources, Industrial Applications and Molecular Biology — A Review. Journal of Food Biochemistry, 21(6), 1–52. https://doi.org/10.1111/j.1745- 4514.1997.tb00223.x
• Karakaş, B., Inan, M., & Certel, M. (2010). Expression and characterization of Bacillus subtilis PY22 α-amylase in Pichia pastoris. Journal of Molecular Catalysis B: Enzymatic, 64(3–4), 129–134. https://doi.org/10.1016/j.molcatb.2009.07.006
• Karim, K. M. R., Husaini, A., Hossain, M. A., Sing, N. N., Mohd Sinang, F., Hussain, M. H. M., & Roslan, H. A. (2016). Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris. BioMed Research International, 2016. https://doi.org/10.1155/2016/5962028
• Kumar, P., & Satyanarayana, T. (2009). Microbial glucoamylases: characteristics and applications. Critical Reviews in Biotechnology, 29(3), 225–255. https://doi.org/10.1080/07388550903136076
• Kwon, M. J., Jørgensen, T. R., Nitsche, B. M., Arentshorst, M., Park, J., Ram, A. F. J., & Meyer, V. (2012). The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger. BMC Genomics, 13(1). https://doi.org/10.1186/1471-2164-13-701
• Li, Z., Ji, K., Dong, W., Ye, X., Wu, J., Zhou, J., Wang, F., Chen, Q., Fu, L., Li, S., Huang, Y., & Cui, Z. (2017). Cloning, heterologous expression, and enzymatic characterization of a novel glucoamylase GlucaM from Corallococcus sp. strain EGB. Protein Expression and Purification, 129(June), 122–127. https://doi.org/10.1016/j.pep.2015.06.009
• Liu, S. H., Chou, W. I., Sheu, C. C., & Chang, M. D. T. (2005). Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations. Biochemical and Biophysical Research Communications, 326(4), 817–824. https://doi.org/10.1016/J.BBRC.2004.11.112
• Longoni, P., Leelavathi, S., Doria, E., Reddy, V. S., & Cella, R. (2015). Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification. BioMed Research International, 2015. https://doi.org/10.1155/2015/289759
• Lu, X., Sun, J., Nimtz, M., Wissing, J., Zeng, A.-P., & Rinas, U. (2010). The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate. Microbial Cell Factories, 9, 23. https://doi.org/10.1186/1475-2859-9-23
• Macauley‐Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast, 22(4), 249–270. https://doi.org/10.1002/yea.1208
• Norouzian, D., Akbarzadeh, A., Scharer, J. M., & Moo Young, M. (2006). Fungal glucoamylases. Biotechnology Advances, 24(1), 80–85. https://doi.org/10.1016/j.biotechadv.2005.06.003 Pan, Y., Yang, J., Wu, J., Yang, L., & Fang, H. (2022). Current advances of Pichia pastoris as cell factories for production of recombinant proteins. Frontiers in Microbiology, 13. https://doi.org/10.3389/fmicb.2022.1059777
• Pandey, A. (1995). Glucoamylase Research: An Overview. Starch - Stärke, 47(11), 439–445. https://doi.org/10.1002/star.19950471108
• Pasin, T. M., Betini, J. H. A., de Lucas, R. C., & Polizeli, M. de L. T. de M. (2024). Biochemical characterization of an acid‐thermostable glucoamylase from Aspergillus japonicus with potential application in the paper bio‐deinking. Biotechnology Progress, 40(1). https://doi.org/10.1002/btpr.3384
• Paszczvdski, A., & Miedziak, I. (1982). A simple method of affinity chromatography for the purification of glucoamylase obtained from Aspergilhs niger C. 149(1), 63–66.
• Pfister, B., Sánchez-Ferrer, A., Diaz, A., Lu, K., Otto, C., Holler, M., Shaik, F. R., Meier, F., Mezzenga, R., & Zeeman, S. C. (2016). Recreating the synthesis of starch granules in yeast. ELife, 5(NOVEMBER2016), 1–29. https://doi.org/10.7554/eLife.15552
• Satyanarayana, T., Noorwez, S. M., Kumar, S., Rao, J. L. U. M., Ezhilvannan, M., & Kaur, P. (2004). Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles. Biochemical Society Transactions, 32(Pt 2), 276–278.
• Sauer, J., Sigurskjold, B. W., Christensen, U., Frandsen, T. P., Mirgorodskaya, E., Harrison, M., Roepstor, P., & Svensson, B. (2000). Glucoamylase : structure / function relationships , and protein engineering. Science, 1543.
• Simpson, R. J. (2006). Bulk Precipitation of Proteins by Ammonium Sulfate. Cold Spring Harbor Protocols, 2006(1), pdb. prot 4308. https://doi.org/10.1101/pdb.prot4308
• Storms, R., Zheng, Y., Li, H., Sillaots, S., Martinez-Perez, A., & Tsang, A. (2005). Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger. Plasmid, 53(3), 191–204. https://doi.org/10.1016/j.plasmid.2004.10.001
• Thorsen, T. S., Johnsen, A. H., Josefsen, K., & Jensen, B. (2006). Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1764(4), 671–676. https://doi.org/10.1016/J.BBAPAP.2006.01.009
• Tong, L., Huang, H., Zheng, J., Wang, X., Bai, Y., Wang, X., Wang, Y., Tu, T., Yao, B., Qin, X., & Luo, H. (2022). Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris. Microbial Cell Factories, 21(1), 95. https://doi.org/10.1186/s12934-022-01833-1
• Tong, L., Zheng, J., Wang, X., Wang, X., Huang, H., Yang, H., Tu, T., Wang, Y., Bai, Y., Yao, B., Luo, H., & Qin, X. (2021). Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications. Biotechnology for Biofuels, 14(1), 202. https://doi.org/10.1186/s13068-021-02052-3
• Wucherpfennig, T., Hestler, T., & Krull, R. (2011). Morphology engineering - Osmolality and its effect on Aspergillus niger morphology and productivity. Microbial Cell Factories, 10, 1–15. https://doi.org/10.1186/1475-2859-10-58
• Xiao, Z., Storms, R., & Tsang, A. (2006). A quantitative starch – iodine method for measuring alpha-amylase and glucoamylase activities. Analytical Biochemistry, 362(MAY 2006), 146–148. https://doi.org/10.1016/j.ab.2006.01.036
• Xu, Q.-S., Yan, Y.-S., & Feng, J.-X. (2016). Efficient hydrolysis of raw starch and ethanol fermentation: a novel raw starch-digesting glucoamylase from Penicillium oxalicum. Biotechnology for Biofuels, 9, 216. https://doi.org/10.1186/s13068-016-0636-5
• Zong, X., Wen, L., Wang, Y., & Li, L. (2022). Research progress of glucoamylase with industrial potential. Journal of Food Biochemistry, 46(7). https://doi.org/10.1111/jfbc.14099