Fine-Tuning of Protein Extraction From Wall-Deficient Chlamydomonas reinhardtii Using Liquid Nitrogen and Sonication-Assisted Cell Disruption

Year 2022, Volume: 11 Issue: 1, 32 - 40, 28.03.2022

Arzu Yıldırım

https://doi.org/10.33714/masteb.1057346

Cited By: 1

Abstract

Disruption methods used to extract proteins from the cell often require optimization in terms of yield increase and molecular integrity according to the cell type. Most cell lysis methods primarily target the cell wall. However, even for the wall-deficient strains, efficient extraction of molecules in or attached to membranous structures is a delicate process. In this study, we optimized the protein extraction technique for a cell wall deficient strain of Chlamydomonas reinhardtii, which is also a preferred material for most of the recombinant protein production studies. Liquid nitrogen (LN) was evaluated for efficient protein extraction from wall-less strain. The results were compared with sonic treatments, which were optimized in terms of applied power and duration. The results showed that sonication at 25% power for 20 seconds of three rounds provided optimum results for the protein integrity and extraction yield (74.13±2 µg/mL and 185.32±5 mg/g). Although LN has provided similar results in terms of protein content compared to sonication, (70.15±4.43 µg/mL and 175.37±11.09 mg/g maximum), it revealed low efficiency in extracting intact proteins from sub-compartments of the cell.

Keywords

Chlamydomonas, Cell disruption, Protein, Wall-deficiency

Thanks

The author thanks Elif İşel and Gülseren Özduman for their technical assistance during SDS PAGE analysis and fluorescence microscopy.

References

• Ahmad, N., Mehmood, M. A., & Malik S. (2020). Recombinant protein production in microalgae: emerging trends. Protein & Peptide Letters, 27(2), 105–110. http://doi.org/10.2174/0929866526666191014124855
• Alhattab, M., Kermanshahi-Pour, A., & Brooks, M. S. -L. (2019) Microalgae disruption techniques for product recovery: Influence of cell wall composition. Journal of Applied Phycology, 31, 61–88. https://doi.org/10.1007/s10811-018-1560-9
• Avhad, D. N., Niphadkar, S. S., & Rathod, V. K. (2014). Ultrasound assisted three phase partitioning of a fibrinolytic enzyme. Ultrasonics Sonochemistry, 21(2), 628–633. http://doi.org/10.1016/j.ultsonch.2013.10.002
• Bensalem, S., Pareau, D., Cinquin, B., Francaiz, O., Le Pioufle, B., & Lopes, F. (2020). Impact of pulsed electric fields and mechanical compressions on the permeability and structure of Chlamydomonas reinhardtii cells. Scientific Reports, 10, 2668. https://doi.org/10.1038/s41598-020-59404-6
• Bleakley, S., & Hayes, M. (2017). Algal proteins: Extraction, application and challenges concerning production. Foods, 6(5), 33. https://doi.org/10.3390/foods6050033
• Borkhsenious, O. N., Mason, C. B., & Moroney, J. V. (1998). The intracellular localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. Plant Physiology, 116(4), 1585–1591. https://doi.org/10.1104/pp.116.4.1585
• Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
• Chiong, T., Acquah, C., Lau, S. Y., Khor, E. H., & Danquah, M. K. (2016). Microalgal-based protein by-products: Extraction, purification, and applications. In Dhillon, G. S. (Ed.), Protein Byproducts; Transformation from Environmental Burden into Value-Added Products (pp. 213-234). Academic Press. http://doi.org/10.1016/B978-0-12-802391-4.00012-4
• D’Hondt, E., Martin-Juarez, J., Bolado, S., Kasperoviciene, J., Koreiviene, J., Sulcius, S., Elst, K., Bastiaens, L. (2017), Cell disruption technologies. In Gonzalez-Fernandez, C., & Muñoz, R. (Eds.), Microalgae-Based Biofuels and Bioproducts: From Feedstock Cultivation to End Products (pp. 133-154). Woodhead Publishing. https://doi.org/10.1016/B978-0-08-101023-5.00006-6
• Dixon, C., & Wilken, L. R. (2018). Green microalgae biomolecule separations and recovery. Bioresources and Bioprocessing, 5, 14. https://doi.org/10.1186/s40643-018-0199-3
• Doron, L., Segal, N., & Shapira, M. (2016). Transgene expression in microalgae-from tools to applications. Frontiers in Plant Science, 7, 505. https://doi.org/10.3389/fpls.2016.00505
• Dyo, Y. M., & Purton S. (2018). The algal chloroplast as a synthetic biology platform for production of therapeutic proteins. Microbiology, 164(2), 113–121. https://doi.org/10.1099/mic.0.000599
• Gerde, J. A., Montalbo-Lomboy, M., Yao L., Grewell, D., & Wang, T. (2012). Evaluation of microalgae cell disruption by ultrasonic treatment. Bioresource Technology, 125, 175–181. https://doi.org/10.1016/j.biortech.2012.08.110
• Gong, Y., Hu, H., Gao, Y., Xu, X., & Gao, H. (2011). Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects. Journal of Industrial Microbiology and Biotechnology, 38(12), 1879–1890. https://doi.org/10.1007/s10295-011-1032-6
• Goodenough, U. W., & Heuser, J. E. (1985). The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique. Journal of Cell Biology, 101(4), 1550–1568. https://doi.org/10.1083/jcb.101.4.1550
• Grewe, S., Ballottari, M., Alcocer, M., D’Andrea, C., Blifernez-Klassen, O., Hankamer, B., Mussgnug, J. H., Bassi, R., & Krusea, O. (2014). Light-harvesting complex protein LHCBM9 is critical for photosystem II activity and hydrogen production in Chlamydomonas reinhardtii. The Plant Cell, 26(4), 1598–1611. https://doi.org/10.1105/tpc.114.124198
• Harris, E. H. (1989). The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press. 780p. https://doi.org/10.1126/science.246.4936.1503-a
• Hummel, E., Guttmann, P., Werner, S., Tarek, B., Schneider, G., Kunz, M., Frangakis, A. S., & Westermann, B. (2012). 3D ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft X-ray tomography. Plos One, 7(12), e53293. https://doi.org/10.1371/journal.pone.0053293
• Jaenicke, L., Kuhne, W., Spessert, R., Wahle, U., & Waffenschmidt, S. (1987). Cell-wall lytic enzymes (autolysins) of Chlamydomonas reinhardtii are (hydroxy)proline-specific proteases. European Journal of Biochemistry, 170(1-2), 485-491. https://doi.org/10.1111/j.1432-1033.1987.tb13725.x
• Kay, R. A. (1991). Microalgae as food and supplement. Critical Reviews in Food Science and Nutrition, 30(6), 555-573. http://doi.org/10.1080/10408399109527556
• Kuhavichanan, A., Kusolkumbot, P., Sirisattha, S., & Areeprasert, C. (2018). Mechanical extraction of protein solution from microalgae by ultrasonication. IOP Conference Series: Earth and Environmental Science, 159, 012009. https://doi.org/10.1088/1755-1315/159/1/012009
• Lai, Y. C., Chang, C. H., Chen, C. Y., Chang, J. S., & Ng, I. S. (2019). Towards protein production and application by using Chlorella species as circular economy. Bioresource Technology, 289, 121625. https://doi.org/10.1016/j.biortech.2019.121625
• Lam, P. G., Kolk, J. A., Chordia, A., Vermue, H. M., Olivieri, G., Eppink, M. H. M., & Wijffels, R. H. (2017). Mild and selective protein release of cell wall deficient microalgae with pulsed electric field. ACS Sustainable Chemistry Engineering, 5(7), 6046-6053. https://doi.org/10.1021/acssuschemeng.7b00892
• Maxwell, K., & Johnson, G. N. (2000). Chlorophyll fluorescence-a practical guide. Journal of Experimental Botany, 51(345), 659-668. https://doi.org/10.1093/jexbot/51.345.659
• Newman, S. M., Gillham, N. W., Harris, E. H., Johnson, A. M., & Boynton, J. E. (1991). Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii. Molecular and General Genetics MGG, 230, 65–74. https://doi.org/10.1007/BF00290652
• Patnaik, R., Singh, N. K., Bagchi, S. K., Rao, P. S., & Mallick, N. (2019). Utilization of Scenedesmus obliquus protein as a replacement of the commercially available fish meal under an algal refinery approach. Frontiers in Microbiology, 10, 2114. https://doi.org/10.3389/fmicb.2019.02114
• Rasala, B. A., & Mayfield, S. P. (2015). Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynthesis Research, 123(3), 227-39.https://doi.org/10.1007/s11120-014-9994-7
• Rosales-Mendoza, S., Paz-Maldonado, L. M. T., & Soria-Guerra, R. E. (2012). Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: Current status and perspectives. Plant Cell Reports, 31, 479-494 http://doi.org/10.1007/s00299-011-1186-8
• Sager, R., & Granick, S. (1953). Nutritional studies with Chlamydomonas reinhardtii. Annals of the New York Academy of Sciences, 56(5), 831-838. https://doi.org/10.1111/j.1749-6632.1953.tb30261.x
• Saloméa, P. A., & Merchant, S. S. (2019). A series of fortunate events: Introducing Chlamydomonas as a reference organism. Plant Cell, 31, 1682–1707. https://doi.org/10.1105/tpc.18.00952
• Sotto-Sierra, L., Dixon, C. K., & Wilken, L. R. (2017). Enzymatic cell disruption of the microalgae Chlamydomonas reinhardtii for lipid and protein extraction. Algal Research, 25, 149–159. https://doi.org/10.1016/j.algal.2017.04.004
• Sotto-Sierra, L., Stoykovab, P., & Nikolova, Z. L. (2018). Extraction and fractionation of microalgae-based protein products. Algal Research, 36, 175–192. https://doi.org/10.1016/j.algal.2018.10.023
• Stirk, W. A., Bálint, P., Vambe, M., Lovász, C., Molnár, Z., van Staden, J., & Ördög, V. (2020). Effect of cell disruption methods on the extraction of bioactive metabolites from microalgal biomass. Journal of Biotechnology, 307, 35-43. https://doi.org/10.1016/j.jbiotec.2019.10.012
• Sudhani, H. P., García-Murria, M. J., Marín-Navarro, J., García-Ferris, C., Peñarrubia, L., & Moreno, J. (2015). Purification of Rubisco from Chlamydomonas reinhardtii. Bio-Protocol, 5(23), e1673. https://doi.org/10.21769/BioProtoc.1673
• Torres-Tiji, Y., Fields, F. J., & Mayfield, S. P. (2020). Microalgae as a future food source. Biotechnology Advances, 41, 107536. https://doi.org/10.1016/j.biotechadv.2020.107536
• Wang, J., & Yin, Y., (2018). Fermentative hydrogen production using pretreated microalgal biomass as feedstock. Microbial Cell Factories, 17, 22. https://doi.org/10.1186/s12934-018-0871-5
• Wells, M. L., Potin, P., Craigie, J. S., Raven, J. A., Merchant, S. S., Helliwell, K. E., Smith, A. G., Camire, M. E., & Brawley, S. H. (2017). Algae as nutritional and functional food sources: revisiting our understanding. Journal of Applied Phycology, 29(2), 949–982. https://doi.org/10.1007/s10811-016-0974-5
• White, A. L., & Melis, A. (2006). Biochemistry of hydrogen metabolism in Chlamydomonas reinhardtii wild type and a Rubisco-less mutant. International Journal of Hydrogen Energy, 31(4), 455–464. https://doi.org/10.1016/j.ijhydene.2005.04.028
• Zheng, S., Zhang, G., Wang, H., Long, Z., Wei, T., & Li, Q. (2021). Progress in ultrasound-assisted extraction of the value-added products from microorganisms. World Journal of Microbiology and Biotechnology, 37, 71. https://doi.org/10.1007/s11274-021-03037-y