APPLICATION OF PROTEINASE K TO FACILITATE FUNGAL CELL LYSIS IN COMMERCIAL SILICA COLUMN-BASED DNA ISOLATION KIT
Abstract
Efficient isolation of high-quality genomic DNA is essential for molecular analyses such as PCR and sequencing in Fungal studies. However, the rigid cell walls of mold, composed predominantly of chitin and β-glucans pose significant barrier to standard lysis protocols. This study investigates the integration of Proteinase K into a commercial silica column-based DNA isolation kit to enhance lysis efficiency and DNA recovery from mold cells. Multiple isolates of fungi were subjected to DNA isolation using modified protocol incorporating Proteinase K treatment during the lysis step. The resulting DNA was quantified spectrophotometrically and evaluated for amplifiability via PCR using fungi specific barcoding of Internal Transcribed Spacer (ITS) region, consisting of ITS 5 and ITS 4 primer. The findings demonstrate that the inclusion of Proteinase K along with incubation time significantly improves cell disruption, leading to increased DNA yield and successful PCR amplification of ITS region across all tested isolates. These results underscore the utility of Proteinase K as a robust, cost-effective enzymatic supplement to conventional fungi DNA isolation workflows, especially when working with structurally complex cell wall mold taxa.
References
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Duong, V.A., Lee, H., 2023. Bottom-up proteomics: advancements in sample preparation. International Journal of Molecular Sciences, 24(6), p.5350.
Fathurahman, F.R., Suman, A., Mahmoud, H.H.A., & Nuryanto, A., 2025. Comparison of Two Isolation Methods for Naturally Preserved DNA in Ambergris. Biosaintifika: Journal of Biology & Biology Education, 17(1).
Galliano, I., Dapra, V., Zaniol, E., Alliaudi, C., Graziano, E., Montanari, P., Calvi, C., & Bergallo, M., 2021. Comparison of methods for isolating fungal DNA. Practical Laboratory Medicine, 25, p.e00221.
Ghassemi, N., Poulhazan, A., Deligey, F., Mentink-Vigier, F., Marcotte, I., & Wang, T., 2021. Solid-state NMR investigations of extracellular matrixes and cell walls of algae, bacteria, fungi, and plants. Chemical Reviews, 122(10), pp.10036-10086.
Goyani, K.H., Mukhopadhyaya, P.N., 2023. Evolution of nucleic acid extraction methods and its role in development of complex human diagnostic solutions with special reference to tuberculosis: A review. Magna Scientia Advanced Research and Reviews, 9(02), pp.115-146.
Jillwin, J., Rudramurthy, S.M., Singh, S., Bal, A., Das, A., Radotra, B., Prakash, H., Dhaliwal, M., Kaur, H., Ghosh, A.K., & Chakrabarti, A., 2021. Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues. Journal of medical microbiology, 70(2), p.001282.
Kalendar, R., Boronnikova, S., & Seppänen, M., 2021. Isolation and purification of DNA from complicated biological samples. Molecular plant taxonomy: methods and protocols, pp.57-67.
Khan, N.A., Asaf, S., Ahmad, W., Jan, R., Bilal, S., Khan, I., Khan, A.L., Kim, K.M., & Al-Harrasi, A., 2023. Diversity, lifestyle, genomics, and their functional role of cochliobolus, bipolaris, and curvularia species in environmental remediation and plant growth promotion under biotic and abiotic stressors. Journal of Fungi, 9(2), p.254.
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Lucena-Aguilar G., Sánchez-López A.M., Barberán-Aceituno C., Carrillo-Ávila J.A., López-Guerrero JA, Aguilar-Quesada R., 2016. DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis. Biopreserv Biobank 14(4):264-70.
Luna, N., Páez-Triana, L., Ramírez, A.L., Muñoz, M., Goméz, M., Medina, J.E., Urbano, P., Barragán, K., Ariza, C., Martínez, D., & Hernández, C., 2024. Microbial community dynamics in blood, faeces and oral secretions of neotropical bats in Casanare, Colombia. Scientific Reports, 14(1), p.25808.
Matlock, B, 2015. Assessment of Nucleic Acid Purity. www.thermoscientific.com
Manzo, M., Serra, A., Pedone, E., Pirone, L., Scognamiglio, V., De Felice, M., & De Falco, M., 2024. DNA polymerase I large fragment from Deinococcus radiodurans, a candidate for a cutting-edge room-temperature LAMP. International Journal of Molecular Sciences, 25(3), p.1392.
Minkner, R., Boonyakida, J., Park, E.Y., & Wätzig, H., 2022. Oligonucleotide separation techniques for purification and analysis: What can we learn for today's tasks?. Electrophoresis, 43(23-24), pp.2402-2427.
Oyeleye, A., & Normi, Y.M., 2018. Chitinase: diversity, limitations, and trends in engineering for suitable applications. Bioscience reports, 38(4), p.BSR2018032300.
Panlilio, H. & Rice, C.V., 2021. The role of extracellular DNA in the formation, architecture, stability, and treatment of bacterial biofilms. Biotechnology and bioengineering, 118(6), pp.2129-2141.
Ruggieri, J., Kemp, R., Forman, S., & Van Eden, M.E., 2016. Techniques for nucleic acid purification from plant, animal, and microbial samples. Sample Preparation Techniques for Soil, Plant, and Animal Samples, pp.41-52.
Russell, D.W., Sambrook, J., 2001. Molecular cloning: a laboratory manual (Vol. 1, p. 112). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory
Sankar, S.A., Girijan, S.K., Shambhugowda, Y.B., Nosad, S., & Narayanane, S., 2025. Comparative analysis of DNA extraction methods from seamount sediments: evaluation of yield, purity, and fragment integrity. Ecological Genetics and Genomics, p.100364.
Saputra F., Indra A. I. N., Djuminar A., Merdekawati F., & Nurhayati B., 2024. Concentration and purity of DNA extraction with sonication and spin column methods from the sputum sample of tuberculosis patient. Current Biomedicine, 2(2), 84-92.
Saravanan, R., Choong, Y.K., Lim, C.H., Lim, L.M., Petrlova, J., & Schmidtchen, A., 2021. Cell-free DNA promotes thrombin autolysis and generation of thrombin-derived C-terminal fragments. Frontiers in Immunology, 12, p.593020.
Sidstedt, M., Romsos, E.L., Hedell, R., Ansell, R., Steffen, C.R., Vallone, P.M., Rådström, P., & Hedman, J., 2017. Accurate digital polymerase chain reaction quantification of challenging samples applying inhibitor-tolerant DNA polymerases. Analytical chemistry, 89(3), pp.1642-1649.
Vajpayee, K., Dash, H.R., Parekh, P.B. and Shukla, R.K., 2023. PCR inhibitors and facilitators–Their role in forensic DNA analysis. Forensic Science International, 349, p.111773.
Wang, C., You, Z., Fu, J., Chen, S., Bai, D., Zhao, H., Song, P., Jia, X., Yuan, X., Xu, W., & Zhao, Q., 2022. Application of metagenomic next-generation sequencing in the diagnosis of pulmonary invasive fungal disease. Frontiers in cellular and infection microbiology, 12, p.949505.
White, T.J., Bruns, T., Lee, S.J.W.T., & Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18(1), pp.315-322.
Yang, Q., Downey, R., Stark, J.S., Johnstone, G.J., & Mitchell, J.G., 2025. The Microbial Ecology of Antarctic Sponges. Microbial Ecology, 88(1), pp.1-18.
Al-Madboly, L.A., Aboulmagd, A., El-Salam, M.A., Kushkevych, I., & El-Morsi, R.M., 2024. Microbial enzymes as powerful natural anti-biofilm candidates. Microbial Cell Factories, 23(1), p.343.
Barcenilla, C., Cobo-Díaz, J.F., De Filippis, F., Valentino, V., Cabrera Rubio, R., O’Neil, D., Mahler de Sanchez, L., Armanini, F., Carlino, N., Blanco-Míguez, A., & Pinto, F., 2024. Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments. Nature Protocols, 19(5), pp.1291-1310.
Corbu, V.M., Gheorghe-Barbu, I., Dumbravă, A.Ș., Vrâncianu, C.O., & Șesan, T.E., 2023. Current insights in fungal importance—a comprehensive review. Microorganisms, 11(6), p.1384.
Duong, V.A., Lee, H., 2023. Bottom-up proteomics: advancements in sample preparation. International Journal of Molecular Sciences, 24(6), p.5350.
Fathurahman, F.R., Suman, A., Mahmoud, H.H.A., & Nuryanto, A., 2025. Comparison of Two Isolation Methods for Naturally Preserved DNA in Ambergris. Biosaintifika: Journal of Biology & Biology Education, 17(1).
Galliano, I., Dapra, V., Zaniol, E., Alliaudi, C., Graziano, E., Montanari, P., Calvi, C., & Bergallo, M., 2021. Comparison of methods for isolating fungal DNA. Practical Laboratory Medicine, 25, p.e00221.
Ghassemi, N., Poulhazan, A., Deligey, F., Mentink-Vigier, F., Marcotte, I., & Wang, T., 2021. Solid-state NMR investigations of extracellular matrixes and cell walls of algae, bacteria, fungi, and plants. Chemical Reviews, 122(10), pp.10036-10086.
Goyani, K.H., Mukhopadhyaya, P.N., 2023. Evolution of nucleic acid extraction methods and its role in development of complex human diagnostic solutions with special reference to tuberculosis: A review. Magna Scientia Advanced Research and Reviews, 9(02), pp.115-146.
Jillwin, J., Rudramurthy, S.M., Singh, S., Bal, A., Das, A., Radotra, B., Prakash, H., Dhaliwal, M., Kaur, H., Ghosh, A.K., & Chakrabarti, A., 2021. Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues. Journal of medical microbiology, 70(2), p.001282.
Kalendar, R., Boronnikova, S., & Seppänen, M., 2021. Isolation and purification of DNA from complicated biological samples. Molecular plant taxonomy: methods and protocols, pp.57-67.
Khan, N.A., Asaf, S., Ahmad, W., Jan, R., Bilal, S., Khan, I., Khan, A.L., Kim, K.M., & Al-Harrasi, A., 2023. Diversity, lifestyle, genomics, and their functional role of cochliobolus, bipolaris, and curvularia species in environmental remediation and plant growth promotion under biotic and abiotic stressors. Journal of Fungi, 9(2), p.254.
Khan, A.M., Bhadauria, S., 2019. Molecular characterization of keratin degrading fungi isolated from semi-arid soil by PCR using ITS4 and ITS5 primers. Journal of King Saud University-Science, 31(4), pp.1418-1423. Khan, A.M. and Bhadauria, S., 2019. Molecular characterization of keratin degrading fungi isolated from semi-arid soil by PCR using ITS4 and ITS5 primers. Journal of King Saud University-Science, 31(4), pp.1418-1423.
Kauserud, H., 2023. ITS alchemy: On the use of ITS as a DNA marker in fungal ecology. Fungal Ecology, 65(2023) 101274
Kovács-Simon, A., Fones, H.N., 2024. Use of chitin: DNA ratio to assess growth form in fungal cells. BMC biology, 22(1), p.10.
Kumari, A., Tripathi, A.H., Anand, R., Tewari, L.M., Joshi, Y., Bajpai, R., Upreti, D.K., Joshi, P., & Upadhyay, S.K., 2023. Streamlined and Cost-Effective Genomic DNA Extraction Method for Lichens, Mushrooms, and Endolichenic Fungi: Enabling DNA Barcoding and Molecular Research. Journal of Advanced Zoology, 44, pp.674-685. Kumari, A., Tripathi, A.H., Anand, R., Tewari, L.M., Joshi, Y., Bajpai, R., Upreti, D.K., Joshi, P. and Upadhyay, S.K., 2023. Streamlined and Cost-Effective Genomic DNA Extraction Method for Lichens, Mushrooms, and Endolichenic Fungi: Enabling DNA Barcoding and Molecular Research. Journal of Advanced Zoology, 44, pp.674-685.
Langsiri, N., Meyer, W., Irinyi, L., Worasilchai, N., Pombubpa, N., Wongsurawat, T., Jenjaroenpun, P., Luangsa-Ard, J.J., & Chindamporn, A., 2025. Optimizing fungal DNA extraction and purification for Oxford Nanopore untargeted shotgun metagenomic sequencing from simulated hemoculture specimens. mSystems, pp.e01166-24.
Lucena-Aguilar G., Sánchez-López A.M., Barberán-Aceituno C., Carrillo-Ávila J.A., López-Guerrero JA, Aguilar-Quesada R., 2016. DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis. Biopreserv Biobank 14(4):264-70.
Luna, N., Páez-Triana, L., Ramírez, A.L., Muñoz, M., Goméz, M., Medina, J.E., Urbano, P., Barragán, K., Ariza, C., Martínez, D., & Hernández, C., 2024. Microbial community dynamics in blood, faeces and oral secretions of neotropical bats in Casanare, Colombia. Scientific Reports, 14(1), p.25808.
Matlock, B, 2015. Assessment of Nucleic Acid Purity. www.thermoscientific.com
Manzo, M., Serra, A., Pedone, E., Pirone, L., Scognamiglio, V., De Felice, M., & De Falco, M., 2024. DNA polymerase I large fragment from Deinococcus radiodurans, a candidate for a cutting-edge room-temperature LAMP. International Journal of Molecular Sciences, 25(3), p.1392.
Minkner, R., Boonyakida, J., Park, E.Y., & Wätzig, H., 2022. Oligonucleotide separation techniques for purification and analysis: What can we learn for today's tasks?. Electrophoresis, 43(23-24), pp.2402-2427.
Oyeleye, A., & Normi, Y.M., 2018. Chitinase: diversity, limitations, and trends in engineering for suitable applications. Bioscience reports, 38(4), p.BSR2018032300.
Panlilio, H. & Rice, C.V., 2021. The role of extracellular DNA in the formation, architecture, stability, and treatment of bacterial biofilms. Biotechnology and bioengineering, 118(6), pp.2129-2141.
Ruggieri, J., Kemp, R., Forman, S., & Van Eden, M.E., 2016. Techniques for nucleic acid purification from plant, animal, and microbial samples. Sample Preparation Techniques for Soil, Plant, and Animal Samples, pp.41-52.
Russell, D.W., Sambrook, J., 2001. Molecular cloning: a laboratory manual (Vol. 1, p. 112). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory
Sankar, S.A., Girijan, S.K., Shambhugowda, Y.B., Nosad, S., & Narayanane, S., 2025. Comparative analysis of DNA extraction methods from seamount sediments: evaluation of yield, purity, and fragment integrity. Ecological Genetics and Genomics, p.100364.
Saputra F., Indra A. I. N., Djuminar A., Merdekawati F., & Nurhayati B., 2024. Concentration and purity of DNA extraction with sonication and spin column methods from the sputum sample of tuberculosis patient. Current Biomedicine, 2(2), 84-92.
Saravanan, R., Choong, Y.K., Lim, C.H., Lim, L.M., Petrlova, J., & Schmidtchen, A., 2021. Cell-free DNA promotes thrombin autolysis and generation of thrombin-derived C-terminal fragments. Frontiers in Immunology, 12, p.593020.
Sidstedt, M., Romsos, E.L., Hedell, R., Ansell, R., Steffen, C.R., Vallone, P.M., Rådström, P., & Hedman, J., 2017. Accurate digital polymerase chain reaction quantification of challenging samples applying inhibitor-tolerant DNA polymerases. Analytical chemistry, 89(3), pp.1642-1649.
Vajpayee, K., Dash, H.R., Parekh, P.B. and Shukla, R.K., 2023. PCR inhibitors and facilitators–Their role in forensic DNA analysis. Forensic Science International, 349, p.111773.
Wang, C., You, Z., Fu, J., Chen, S., Bai, D., Zhao, H., Song, P., Jia, X., Yuan, X., Xu, W., & Zhao, Q., 2022. Application of metagenomic next-generation sequencing in the diagnosis of pulmonary invasive fungal disease. Frontiers in cellular and infection microbiology, 12, p.949505.
White, T.J., Bruns, T., Lee, S.J.W.T., & Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18(1), pp.315-322.
Yang, Q., Downey, R., Stark, J.S., Johnstone, G.J., & Mitchell, J.G., 2025. The Microbial Ecology of Antarctic Sponges. Microbial Ecology, 88(1), pp.1-18.
Published
2025-06-24
How to Cite
ESHANANDA, Yuriza.
APPLICATION OF PROTEINASE K TO FACILITATE FUNGAL CELL LYSIS IN COMMERCIAL SILICA COLUMN-BASED DNA ISOLATION KIT.
Journal of Biodiversity and Maritime Study, [S.l.], v. 2, n. 1, p. 1-8, june 2025.
Available at: <https://jos.unsoed.ac.id/index.php/mandara/article/view/16142>. Date accessed: 19 sep. 2025.
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