Isolation, identification, and characterization of neopullulanase from Thermomonas hydrothermalis GKE 08

Year 2024, Volume: 5 Issue: 2, 130 - 139, 30.08.2024

Songul Yildiz

https://doi.org/10.51753/flsrt.1447335

Abstract

The production of neopullulanase from thermophiles, such as Thermomonas hydrothermalis GKE 08, has great importance due to the enzyme’s unique thermophilic nature. This characteristic results in enhanced stability and functionality at elevated temperatures. It is known that this is a very important issue for industrial processes that require efficient catalysis under extreme conditions. The investigation of pullulanase from T. hydrothermalis GKE 08 showed significant results. Optimal conditions for enzyme production were determined, with peak activity observed in the presence of 1.5% soluble pullulan and 0.5% peptone. The study delved into the pH and temperature dynamics, identifying an optimal pH of 7.0 and a temperature of 55°C. Notably, the neopullulanase exhibited time-dependent stability, retaining 72% activity after 1 hour but declining to 50% after 2 hours. Purified pullulanase from T. hydrothermalis GKE 08 displayed optimal activity at pH 7.0, with a subsequent time-dependent decline observed during incubation at this pH: retaining 72% activity after 1 hour, approximately 50% after 2 hours, and a significant 77% loss after one day. Furthermore, the enzyme displayed remarkable thermostability at 60°C, with 88% activity after 30 minutes. Metal ion studies indicated susceptibility to inhibition by Cu2+, Mg2+, and Zn2+, while Ca2+ stimulated activity up to 138% at higher concentrations. The enzyme’s response to specific reagents revealed sensitivity to SDS and EDTA, while urea surprisingly enhanced activity to 85%. The study enhances understanding of pullulanase behavior, offering valuable insights for biotechnological and industrial applications.

Keywords

Neopullulanase, Thermomonas hydrothermalis, thermophiles, thermozyme

References

• Andersen, J. M., Barrangou, R., Hachem, M. A., Lahtinen, S. J., Goh, Y. J., Svensson, B., & Klaenhammer, T. R. (2012). Transcriptional analysis of prebiotic uptake and catabolism by Lactobacillus acidophilus NCFM. PLoS ONE, 7(9), e44409.
• Araujo, R., Casal, M., & Cavaco-Paulo, A. (2008). Application of enzymes for textile fibres processing. Biocatalysis and Biotransformation, 26(5), 332-349.
• Asha, R., Niyonzima, F. N., & Sunil, S. M. (2013). Purification and properties of pullulanase from Bacillus halodurans. International Research Journal of Biological Sciences, 2(3), 35-43.
• Bajpai, P. (2023). Developments and applications of enzymes from thermophilic microorganisms. Elsevier, 1-314.
• Balolong, M. P., Chae, J. P., & Kang, D. K. (2016). Expression and characterisation of neopullulanase from Lactobacillus mucosae. Biotechnology Letters, 38, 1753-1760.
• Boersma, B. (2024). Biochemical characterisation of an alpha-amylase with pullulan hydrolase type III characteristics derived from a hot spring metagenomics library, Doctoral Dissertation, (pp. 1-123). University of the Western Cape.
• Bruins, M. E., Janssen, A. E., & Boom, R. M. (2001). Thermozymes and their applications: a review of recent literature and patents. Applied Biochemistry and Biotechnology, 90, 155-186.
• Brunswick, J. M., Kelly, C. T., & Fogarty, W. M. (1999). The amylopullulanase of Bacillus sp. DSM 405. Applied Microbiology and Biotechnology, 51, 170-175.
• Bukhari, D. A., Bibi, Z., Ullah, A., & Rehman, A. (2024). Isolation, characterization, and cloning of thermostable pullulanase from Geobacillus stearothermophilus ADM-11. Saudi Journal of Biological Sciences, 31(2), 103901.
• Castro, G. R., Baigorí, M. D., Méndez, B. S., & Siñeriz, F. (1993). Effects of pH and temperature on the continuous production of amylolytic enzymes by bacillus amyloliquefaciens MIR‐41. Journal of Chemical Technology & Biotechnology, 58(3), 277-280.
• Choi, J. S., Kim, J. W., Cho, H. R., Kim, K. Y., Lee, J. K., Sohn, J. H., & Ku, S. K. (2014). Laxative effects of fermented rice extract in rats with loperamide induced constipation. Experimental and Therapeutic Medicine, 8(6), 1847-1854.
• Das, R., & Kayastha, A. M. (2023). An Overview on Starch Processing and Key Enzymes. Industrial Starch Debranching Enzymes, 1-20.
• Davaeifar, S., Shariati, P., Tabandeh, F., & Yakhchali, B. (2015). Isolation and identification of a new Bacillus cereus strain and characterization of its neopullulanase. Applied Food Biotechnology, 2(2), 39-45.
• de Souza, C. K., Ghosh, T., Lukhmana, N., Tahiliani, S., Priyadarshi, R., Hoffmann, T. G., ... & Han, S. S. (2023). Pullulan as a sustainable biopolymer for versatile applications: A review. Materials Today Communications, 36, 106477.
• de Souza, P. M. (2010). Application of microbial α-amylase in industry-A review. Brazilian Journal of Microbiology, 41, 850-861.
• Duffner, F., Bertoldo, C., Andersen, J. T., Wagner, K., & Antranikian, G. (2000). A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis. Journal of Bacteriology, 182(22), 6331-6338.
• Ece, S., Evran, S., Janda, J. O., Merkl, R., & Sterner, R. (2015). Improving thermal and detergent stability of Bacillus stearothermophilus neopullulanase by rational enzyme design. Protein Engineering, Design and Selection, 28(6), 147-151.
• Egharevba, H. O. (2019). Chemical properties of starch and its application in the food industry. In: Emeje M. (ed) Chemical Properties of Starch (pp. 63-88). IntechOpen.
• Giordano, D. (2020). Bioactive molecules from extreme environments. Marine Drugs, 18(12), 640.
• Gomes, I., Gomes, J., & Steiner, W. (2003). Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization. Bioresource Technology, 90(2), 207-214.
• Hii, S. L., Tan, J. S., Ling, T. C., & Ariff, A. B. (2012). Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Research, 2012(1), 921362.
• Hondoh, H., Kuriki, T., & Matsuura, Y. (2003). Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase. Journal of Molecular Biology, 326(1), 177-188.
• Hussian, C. H. A. C., & Leong, W. Y. (2023). Thermostable enzyme research advances: a bibliometric analysis. Journal of Genetic Engineering and Biotechnology, 21(1), 37.
• Imanaka, T., & Kuriki, T. (1989). Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan. Journal of Bacteriology, 171(1), 369-374.
• Jafari, F., Kiani-Ghaleh, F., Eftekhari, S., Razzaghshoar Razlighi, M., Nazari, N., Hajirajabi, M., ... & Sharafieh, G. (2022). Cloning, overexpression, and structural characterization of a novel archaeal thermostable neopullulanase from Desulfurococcus mucosus DSM 2162. Preparative Biochemistry & Biotechnology, 52(10), 1190-1201.
• Kamasaka, H., Sugimoto, K., Takata, H., Nishimura, T., & Kuriki, T. (2002). Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin. Applied and Environmental Microbiology, 68(4), 1658-1664.
• Kanno, M., & Tomimura, E. (1985). A plate culture method for the simultaneous for the simultaneous detection of bacteria producing pullulan-and/or starch-hydrolyzing enzymes. Agricultural and Biological Chemistry, 49(5), 1529-1530.
• Kim, C. H., Nashiru, O., & Ko, J. H. (1996). Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24. FEMS Microbiology Letters, 138(2-3), 147-152.
• Kim, E. J., Kim, Y. J., Yang, S. K., Seo, Y. J., Seo, D. H., Lim, S., ... & Park, C. S. (2024). Effects of carbohydrate binding module of pullulanase type I on the raw starch rearrangement by enhancing the hydrolysis activity. Food Bioscience, 61, 104657.
• Kim, J. H., Sunako, M., Ono, H., Murooka, Y., Fukusaki, E., & Yamashita, M. (2008). Characterization of gene encoding amylopullulanase from plant-originated lactic acid bacterium, Lactobacillus plantarum L137. Journal of Bioscience and Bioengineering, 106(5), 449-459.
• Kłosowski, G., Mikulski, D., Czupryński, B., & Kotarska, K. (2010). Characterisation of fermentation of high-gravity maize mashes with the application of pullulanase, proteolytic enzymes and enzymes degrading non-starch polysaccharides. Journal of Bioscience and Bioengineering, 109(5), 466-471.
• Kumar, S., Dangi, A. K., Shukla, P., Baishya, D., & Khare, S. K. (2019). Thermozymes: adaptive strategies and tools for their biotechnological applications. Bioresource Technology, 278, 372-382.
• Kuriki, T., Okada, S., & Imanaka, T. (1988). New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis. Journal of Bacteriology, 170(4), 1554-1559.
• Kuriki, T., Tsuda, M., & Imanaka, T. (1992). Continuous production of panose by immobilized neopullulanase. Journal of Fermentation and Bioengineering, 73(3), 198-202.
• Labes, A., Karlsson, E. N., Fridjonsson, O. H., Turner, P., Hreggvidson, G. O., Kristjansson, J. K., ... & Schönheit, P. (2008). Novel members of glycoside hydrolase family 13 derived from environmental DNA. Applied and Environmental Microbiology, 74(6), 1914-1921.
• Lee, J. H., Kim, J. H., Zhu, I. H., Zhan, X. B., Lee, J. W., Shin, D. H., & Kim, S. K. (2001). Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans. Biotechnology Letters, 23, 817-820.
• Ling HiiSiew, L. H., Chuan LingTau, C. L., Rosfarizan Mohamad, R. M., & Ariff, A. B. (2009). Characterization of pullulanase type II from Bacillus cereus H1. American Journal of Biochemistry and Biotechnology, 5(4), 170-179.
• Madigan, M. T., Martinko, J. M., & Parker, J. (1997). Brock biology of microorganisms (Vol. 11). Upper Saddle River, NJ: Prentice Hall.
• Mäkeläinen, H., Hasselwander, O., Rautonen, N., & Ouwehand, A. C. (2009). Panose, a new prebiotic candidate. Letters in Applied Microbiology, 49(6), 666-672.
• Miao, M., & BeMiller, J. N. (2023). Enzymatic approaches for structuring starch to improve functionality. Annual Review of Food Science and Technology, 14(1), 271-295.
• Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426-428.
• Møller, M. S., Henriksen, A., & Svensson, B. (2016). Structure and function of α-glucan debranching enzymes. Cellular and Molecular Life Sciences, 73, 2619-2641.
• Morgan, F. J., Adams, K. R., & Priest, F. G. (1979). A cultural method for the detection of pullulan—degrading enzymes in bacteria and its application to the genus Bacillus. Journal of Applied Microbiology, 46(2), 291-294.
• Naik, B., Kumar, V., Goyal, S. K., Dutt Tripathi, A., Mishra, S., Joakim Saris, P. E., ... & Rustagi, S. (2023). Pullulanase: unleashing the power of enzyme with a promising future in the food industry. Frontiers in Bioengineering and Biotechnology, 11, 1139611.
• Nair, S. U., Singhal, R. S., & Kamat, M. Y. (2007). Induction of pullulanase production in Bacillus cereus FDTA-13. Bioresource Technology, 98(4), 856-859.
• Nair, S. U., Singhal, R. S., & Kamat, M. Y. (2006). Enhanced production of thermostable pullulanase type 1 using Bacillus cereus FDTA 13 and its mutant. Food Technology and Biotechnology, 44(2), 275-282.
• Obi, S. K. C., & Odibo, F. J. C. (1984). Partial purification and characterization of a thermostable actinomycete β-amylase. Applied and Environmental Microbiology, 47(3), 571-575.
• Park, B. R., MubarakAli, D., & Kim, J. W. (2023). Identification of a novel cyclomaltodextrinase annotated as a neopullulanase in the genome of Bacillus cereus. Archives of Microbiology, 205(3), 86.
• Raha, M. A. N. I. D. I. P. A., Kawagishi, I., Müller, V., Kihara, M., & Macnab, R. M. (1992). Escherichia coli produces a cytoplasmic alpha-amylase, AmyA. Journal of Bacteriology, 174(20), 6644-6652.
• Roy, A., Messaoud, E. B., & Bejar, S. (2003). Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp. US149. Enzyme and Microbial Technology, 33(5), 720-724.
• Schäfers, C., Blank, S., Wiebusch, S., Elleuche, S., & Antranikian, G. (2017). Complete genome sequence of Thermus brockianus GE-1 reveals key enzymes of xylan/xylose metabolism. Standards in Genomic Sciences, 12, 1-9.
• Sharma, S., Vaid, S., Bhat, B., Singh, S., & Bajaj, B. K. (2019). Thermostable enzymes for industrial biotechnology. In: Singh R. S., Singhania R. R., Pandey A., Larroche C. (eds) Advances in Enzyme Technology (pp. 469-495). Elsevier.
• Swamy, M. V., & Seenayya, G. (1996). Thermostable pullulanase and α-amylase activity from Clostridium thermosulfurogenes SV9-Optmization of culture conditions for enzyme production. Process Biochemistry, 31(2), 157-162.
• Tang, K., Kobayashi, R. S., Champreda, V., Eurwilaichitr, L., & Tanapongpipat, S. (2008). Isolation and characterization of a novel thermostable neopullulanase-like enzyme from a hot spring in Thailand. Bioscience, Biotechnology, and Biochemistry, 72(6), 1448-1456.
• Tsunehiro, J., Matsukubo, T., Shiota, M., & Takaesu, Y. (1997). Effects of a hydrogenated isomaltooligosaccharide mixture on glucan synthesis and on caries development in rats. Bioscience, Biotechnology, and Biochemistry, 61(12), 2015-2018.
• Wu, X., Dou, B., Wang, B., Liu, M., Shao, R., Lu, J., ... & Wang, S. (2023). Improved stability and hydrolysates of hyperthermophilic GH57 type II pullulanase from the deep-sea archaeon Thermococcus siculi HJ21 by truncation. Catalysts, 13(3), 453.
• Yasar Yildiz, S. (2024). Exploring the hot springs of golan: a source of thermophilic bacteria and enzymes with industrial promise. Current Microbiology, 81(4), 101.
• Zhao, Y., Liu, Y., Fu, Q., Zhou, Y., Qin, R., Xiong, H., & Wang, Y. (2023). Domain analysis and site-directed mutagenesis of a thermophilic pullulanase from Thermotoga maritima MSB8. Research Square, 1-11.