Characterization of a maltodextrin glucosidase from Klebsiella variicola of the GH13_21 subfamily
DOI:
https://doi.org/10.4025/actascitechnol.v48i1.73755Keywords:
Maltodextrin glucosidase; Klebsiella variicola; Recombinant expression; MalZ.Abstract
Klebsiella variicola is a Gram-negative bacterium belonging to the Klebsiella pneumoniae species complex. It is frequently isolated from many plants, where it is involved in nitrogen fixation and growth. However, it has also been described as associated with disease in humans and animals. Escherichia coli and K. pneumoniae are related members of the Enterobacteriaceae family that metabolize maltose and maltodextrins. However, only isolates of the K. pneumoniae species complex metabolize starch. In E. coli, four enzymes metabolize maltodextrins, including a periplasmic ?-amylase (MalS), a cytoplasmatic amylomaltase (MalQ), a maltodextrin phosphorylase (MalP), and a maltodextrin glucosidase (MalZ). Considering the medical and agricultural importance of K. variicola and to better understand its role in maltodextrin degradation, the malZ gene was cloned from an isolate of this species, and the coded protein was recombinantly expressed and characterized. The K. variicola MalZ protein sequence was 79% identical to the E. coli MalZ and is the fifth enzyme and second maltodextrin glucosidase characterized from the GH13_21 CAZy subfamily. Its modeled monomeric structure shows ?-stranded amino- and carboxy-terminal domains and a catalytic domain with a (?/?)8-barrel. The 70 kDa expressed enzyme could promote glucose release from maltoheptaose and other maltodextrins. The enzyme’s optimum temperature was 30°C. The KM and Vmax for maltoheptaose were 32.7 mM and 2.58 ?mol/min of glucose released, respectively. Considering the characteristics of degrading maltodextrins, the release of glucose from this substrate, and the high profit of recombinant expression, the enzyme expressed in this work could be used in the industrial degradation of starch as a saccharifying enzyme.
Downloads
References
Adewale, P., Yancheshmeh, M. S., & Lam, E. (2022). Starch modification for non-food, industrial applications: Market intelligence and critical review. Carbohydrate Polymers, 291, 119590. https://doi.org/10.1016/j.carbpol.2022.119590
Ahn, W. C., An, Y., Song, K. M., Park, K. H., Lee, S., Oh, B. H., Park, J. T., & Woo, E. J. (2022). Dimeric architecture of maltodextrin glucosidase (MalZ) provides insights into the substrate recognition and hydrolysis mechanism. Biochemical and Biophysical Research Communications, 586, 49–54. https://doi.org/10.1016/j.bbrc.2021.11.070
Benassi, V. M., Pasin, T. M., Facchini, F. D. A., Jorge, J. A., & Polizeli, M. de L. T. de M. (2014). A novel glucoamylase activated by manganese and calcium produced in submerged fermentation by Aspergillus phoenicis. Journal of Basic Microbiology, 54(5), 333–339. https://doi.org/10.1002/jobm.201200515
Binder, F., Huber, O., & Böck, A. (1986). Cyclodextrin-glycosyltransferase from Klebsiella pneumoniae M5a1: cloning, nucleotide sequence and expression. Gene, 47(2–3), 269–277. https://doi.org/10.1016/0378-1119(86)90070-3
Bloch, M. A., & Raibaud, O. (1986). Comparison of the malA regions of Escherichia coli and Klebsiella pneumoniae. Journal of Bacteriology, 168(3), 1220–1227. https://doi.org/10.1128/jb.168.3.1220-1227.1986
Boel, E., Brady, L., Brzozowski, A. M., Derewenda, Z., Dodson, G. G., Jensen, V. J., Petersen, S. B., Swift, H., Thim, L., & Woldike, H. F. (1990). Calcium binding in ?-amylases: an X-ray diffraction study at 2.1-Å resolution of two enzymes from Aspergillus. Biochemistry, 29(26), 6244–6249. https://doi.org/10.1021/bi00478a019
Bradford, 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/10.1006/abio.1976.9999
Buisson, G., Duée, E., Haser, R., & Payan, F. (1987). Three-dimensional structure of porcine pancreatic ?-amylase at 2.9 Å resolution. Role of calcium in structure and activity. The EMBO Journal, 6(13), 3909–3916. https://doi.org/10.1002/j.1460-2075.1987.tb02731.x
Canteri, M. G., Althaus, R. A., Virgens Filho, J. S., Giglioti, É. A., & Godoy, C. V. (2001). SASM-AGRI - System for analyses and separation of averages in agriculture experiments using the methods of Scott-Knott, Tukey and Duncan. Brazilian Journal of Agrocomputation, 1(2), 18–24. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/175651/1/SASM-AGRI.pdf
Chapman, J., Ismail, A., & Dinu, C. (2018). Industrial applications of enzymes: Recent advances, techniques, and outlooks. Catalysts, 8(6), 238. https://doi.org/10.3390/catal8060238
Chung, C. T., Niemela, S. L., & Miller, R. H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences, 86(7), 2172–2175. https://doi.org/10.1073/pnas.86.7.2172
Cihan, A. C., Yildiz, E. D., Sahin, E., & Mutlu, O. (2018). Introduction of novel thermostable ?-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related ?-amylases under five individual GH13 subfamilies. World Journal of Microbiology and Biotechnology, 34(7), 95. https://doi.org/10.1007/s11274-018-2478-8
D’Enfert, C., Chapon, C., & Pugsley, A. P. (1987). Export and secretion of the lipoprotein pullulanase by Klebsiella pneumoniae. Molecular Microbiology, 1(3), 107–116. https://doi.org/10.1111/j.1365-2958.1987.tb00534.x
Dippel, R., & Boos, W. (2005). The maltodextrin system of Escherichia coli: metabolism and transport. Journal of Bacteriology, 187(24), 8322–8331. https://doi.org/10.1128/JB.187.24.8322-8331.2005
El-Fallal, A., Abou, M., El-Sayed, A., & Omar, N. (2012). Starch and microbial ?-amylases: From concepts to biotechnological applications. In Carbohydrates - Comprehensive Studies on Glycobiology and Glycotechnology (Vol. 21, pp. 259–288). InTech. https://doi.org/10.5772/51571
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39(4), 783. https://doi.org/10.2307/2408678
Freundlieb, S., & Boos, W. (1986). Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein. Journal of Biological Chemistry, 261(6), 2946–2953. https://doi.org/10.1016/S0021-9258(17)35878-7
Gopinath, S. C. B., Anbu, P., Arshad, M. K. M., Lakshmipriya, T., Voon, C. H., Hashim, U., & Chinni, S. V. (2017). Biotechnological processes in microbial amylase production. BioMed Research International, 2017, Article ID 1272193. https://doi.org/10.1155/2017/1272193
Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., & Chauhan, B. (2003). Microbial ?-amylases: a biotechnological perspective. Process Biochem, 38(11), 1599–1616. https://doi.org/10.1016/S0032-9592(03)00053-0
Gurung, N., Ray, S., Bose, S., & Rai, V. (2013). A broader view: Microbial enzymes and their relevance in industries, medicine, and beyond. BioMed Research International, 2013, Article ID 329121. https://doi.org/10.1155/2013/329121
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98. https://doi.org/10.14601/Phytopathol_Mediterr-14998u1.29
Hudson, C. M., Bent, Z. W., Meagher, R. J., & Williams, K. P. (2014). Resistance determinants and mobile genetic elements of an NDM-1-encoding Klebsiella pneumoniae strain. PLoS ONE, 9(6), e99209. https://doi.org/10.1371/journal.pone.0099209
Jane?ek, Š. (2002). How many conserved sequence regions are there in the ?-amylase family? Biologia, Bratislava, 57(11), 29–41. http://imb.savba.sk/~janecek/Papers/PDF/Biologia_02_Suppl_11.pdf
Jane?ek, Š., & Gabriško, M. (2016). Remarkable evolutionary relatedness among the enzymes and proteins from the ?-amylase family. Cellular and Molecular Life Sciences, 73(14), 2707–2725. https://doi.org/10.1007/s00018-016-2246-6
Jane?ek, Š., Mare?ek, F., MacGregor, E. A., & Svensson, B. (2019). Starch-binding domains as CBM families–history, occurrence, structure, function and evolution. Biotechnology Advances, 37(8), 107451. https://doi.org/10.1016/j.biotechadv.2019.107451
Jane?ek, Š., & Svensson, B. (2022). How many ?-amylase GH families are there in the CAZy database? Amylase, 6(1), 1–10. https://doi.org/10.1515/amylase-2022-0001
Jane?ek, Š., Svensson, B., & MacGregor, E. A. (2014). ?-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cellular and Molecular Life Sciences, 71(7), 1149–1170. https://doi.org/10.1007/s00018-013-1388-z
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. E. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10(6), 845–858. https://doi.org/10.1038/nprot.2015.053
Laemmli. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. https://doi.org/10.1038/227680a0
Läufer, A. (2017). Starch biorefinery enzymes. Advances in Biochemical Engineering/Biotechnology, 166, 137–152. https://doi.org/10.1007/10_2016_60
Lin, L., Wei, C., Chen, M., Wang, H., Li, Y., Li, Y., Yang, L., & An, Q. (2015). Complete genome sequence of endophytic nitrogen-fixing Klebsiella variicola strain DX120E. Standards in Genomic Sciences, 10(1), 22. https://doi.org/10.1186/s40793-015-0004-2
Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., & Henrissat, B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Research, 42(D1), D490–D495. https://doi.org/10.1093/nar/gkt1178
McNicholas, S., Potterton, E., Wilson, K. S., & Noble, M. E. M. (2011). Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallographica Section D Biological Crystallography, 67(4), 386–394. https://doi.org/10.1107/S0907444911007281
Mehta, D., & Satyanarayana, T. (2016). Bacterial and archaeal ?-amylases: Diversity and amelioration of the desirable characteristics for industrial applications. Frontiers in Microbiology, 7, 1129. https://doi.org/10.3389/fmicb.2016.01129
Momma, M. (2000). Cloning and sequencing of the maltohexaose-producing amylase gene of Klebsiella pneumoniae. Bioscience, Biotechnology, and Biochemistry, 64(2), 428–431. https://doi.org/10.1271/bbb.64.428
Movahedpour, A., Asadi, M., Khatami, S. H., Taheri?Anganeh, M., Adelipour, M., Shabaninejad, Z., Ahmadi, N., Irajie, C., & Mousavi, P. (2022). A brief overview on the application and sources of ??amylase and expression hosts properties in order to production of recombinant ??amylase. Biotechnology and Applied Biochemistry, 69(2), 650–659. https://doi.org/10.1002/bab.2140
Nakajima, R., Imanaka, T., & Aiba, S. (1986). Comparison of amino acid sequences of eleven different ?-amylases. Applied Microbiology and Biotechnology, 23(5), 355–360. https://doi.org/10.1007/BF00257032
Ohtaki, A., Iguchi, A., Mizuno, M., Tonozuka, T., Sakano, Y., & Kamitori, S. (2003). Mutual conversion of substrate specificities of Thermoactinomyces vulgaris R-47 ?-amylases TVAI and TVAII by site-directed mutagenesis. Carbohydrate Research, 338(15), 1553–1558. https://doi.org/10.1016/S0008-6215(03)00219-2
Palmer, T. N., Ryman, B. E., & Whelan, W. J. (1976). The action pattern of amylomaltase from Escherichia coli. European Journal of Biochemistry, 69(1), 105–115. https://doi.org/10.1111/j.1432-1033.1976.tb10863.x
Pandey, A., Nigam, P., Soccol, C. R., Soccol, V. T., Singh, D., & Mohan, R. (2000). Advances in microbial amylases. Biotechnology and Applied Biochemistry, 31(2), 135. https://doi.org/10.1042/BA19990073
Paul, J. S., Gupta, N., Beliya, E., Tiwari, S., & Jadhav, S. K. (2021). Aspects and recent trends in microbial ?-amylase: A review. Applied Biochemistry and Biotechnology, 193(8), 2649–2698. https://doi.org/10.1007/s12010-021-03546-4
Puyet, A. (1993). Expression of bacterial genes involved in maltose metabolism. World Journal of Microbiology and Biotechnology, 9(4), 455–460. https://doi.org/10.1007/BF00328033
Rodríguez-Medina, N., Barrios-Camacho, H., Duran-Bedolla, J., & Garza-Ramos, U. (2019). Klebsiella variicola: an emerging pathogen in humans. Emerging Microbes & Infections, 8(1), 973–988. https://doi.org/10.1080/22221751.2019.1634981
Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Sambrook, J., & Russell, D. (2001). Molecular cloning: a laboratory manual, third edition. Spring Harbor Laboratory Press.
Santos, F. C. dos, & Barbosa-Tessmann, I. P. (2019). Recombinant expression, purification, and characterization of a cyclodextrinase from Massilia timonae. Protein Expression and Purification, 154, 74–84. https://doi.org/10.1016/j.pep.2018.08.013
Santos, F. C. dos, de Castro, F. F., Apolonio, T. M., Yoshida, L., Martim, D. B., Tessmann, D. J., & Barbosa-Tessmann, I. P. (2019). Isolation, diversity, and biotechnological potential of maize (Zea mays L.) grains bacteria. Genetics and Molecular Research, 18(3), gmr18320. https://doi.org/10.4238/gmr18320
Schwartz, M. (1987). The maltose regulon. In F. C. Neidhardt (Ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology (Vol. 2, pp. 1482–1502). American Society of Microbiology.
Sindhu, R., Binod, P., Madhavan, A., Beevi, U. S., Mathew, A. K., Abraham, A., Pandey, A., & Kumar, V. (2017). Molecular improvements in microbial ?-amylases for enhanced stability and catalytic efficiency. Bioresource Technology, 245, 1740–1748. https://doi.org/10.1016/j.biortech.2017.04.098
Singh, R., Kumar, M., Mittal, A., & Mehta, P. K. (2016). Microbial enzymes: Industrial progress in 21st century. 3 Biotech, 6(2), 174. https://doi.org/10.1007/s13205-016-0485-8
Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K. M., Soccol, C. R., & Pandey, A. (2006). ?-Amylases from microbial sources – An overview on recent developments. Food Technology and Biotechnology, 44(2), 173–184. https://hrcak.srce.hr/file/161876
Stam, M. R., Danchin, E. G. J., Rancurel, C., Coutinho, P. M., & Henrissat, B. (2006). Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of ?-amylase-related proteins. Protein Engineering Design and Selection, 19(12), 555–562. https://doi.org/10.1093/protein/gzl044
Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38(7), 3022–3027. https://doi.org/10.1093/molbev/msab120
Tapio, S., Yeh, F., Shuman, H. A., & Boos, W. (1991). The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase. Journal of Biological Chemistry, 266(29), 19450–19458. https://doi.org/10.1016/S0021-9258(18)55017-1
Trinder, P. (1969). Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology, 22(2), 158–161. https://doi.org/10.1136/jcp.22.2.158
Van der Maarel, M. J. E. C., Van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H., & Dijkhuizen, L. (2002). Properties and applications of starch-converting enzymes of the ?-amylase family. Journal of Biotechnology, 94(2), 137–155. https://doi.org/10.1016/S0168-1656(01)00407-2
Wang, X., Kan, G., Ren, X., Yu, G., Shi, C., Xie, Q., Wen, H., & Betenbaugh, M. (2018). Molecular cloning and characterization of a novel ?-amylase from Antarctic Sea ice bacterium Pseudoalteromonas sp. M175 and its primary application in detergent. BioMed Research International, 2018, Article ID 3258383. https://doi.org/10.1155/2018/3258383
Zuckerkandl, E., & Pauling, L. (1965). Evolutionary divergence and convergence in proteins. In H. J. Bryson, V., & Vogel (Eds.), Evolving Genes and Proteins (pp. 97–166). Elsevier. https://doi.org/10.1016/B978-1-4832-2734-4.50017-6
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Fabiane Cristina dos Santos, Ione Parra Barbosa-Tessmann (Autor)
This work is licensed under a Creative Commons Attribution 4.0 International License.
DECLARATION OF ORIGINALITY AND COPYRIGHTS
I Declare that current article is original and has not been submitted for publication, in part or in whole, to any other national or international journal.
The copyrights belong exclusively to the authors. Published content is licensed under Creative Commons Attribution 4.0 (CC BY 4.0) guidelines, which allows sharing (copy and distribution of the material in any medium or format) and adaptation (remix, transform, and build upon the material) for any purpose, even commercially, under the terms of attribution.
Read this link for further information on how to use CC BY 4.0 properly.
8.png){kind=logo}
