Abstract
The gene encoding N-acetylmuramoyl-l-alanine amidase in Latilactobacillus sakei isolated from a fermented meat product was cloned in two forms: its complete sequence (AmiC) and a truncated sequence without one of its anchoring LysM domains (AmiLysM4). The objective of this work was to evaluate the effect of LysM domain deletion on antibacterial activity as well the biochemical characterization of each recombinant protein. AmiC and AmiLysM4 were expressed in Escherichia coli BL21. Using a zymography method, two bands with lytic activity were observed, which were confirmed by LC–MS/MS analysis, with molecular masses of 71 kDa (AmiC) and 66 kDa (AmiLysM4). The recombinant proteins were active against Listeria innocua and Staphylococcus aureus strains. The inhibitory spectrum of AmiLysM4 was broader than AmiC as it showed inhibition of Leuconostoc mesenteroides and Weissella viridescens, both microorganisms associated with food decomposition. Optimal temperature and pH values were determined for both proteins using l-alanine-p-nitroanilide hydrochloride as a substrate for N-acetylmuramoyl-l-alanine amidase activity. Both proteins showed similar maximum activity values for pH (8) and temperature (50 °C). Furthermore, structural predictions did not show differences for the catalytic region, but differences were found for the region called 2dom-AmiLysM4, which includes 4 of the 5 LysM domains. Therefore, modification of the LysM domain offers new tools for the development of novel food biopreservatives.
Graphic abstract
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Afshar D, Rafiee F, Kheirandish M, Moghadam SO, Azarsa M (2020) Autolysin (lytA) recombinant protein: a potential target for developing vaccines against pneumococcal infections. Clin Exp Vaccine Res 9(2):76. https://doi.org/10.7774/cevr.2020.9.2.76
Akcapinar GB, Kappel L, Sezerman OU, Seidl-Seiboth V (2015) Molecular diversity of LysM carbohydrate-binding motifs in fungi. Curr Genet 61:103–113. https://doi.org/10.1007/s00294-014-0471-9
Anba-Mondoloni J, Chaillou S, Zagorec M, Champomier-Verges M (2013) Catabolism of N-acetylneuraminic acid, a fitness function of the food-borne lactic acid bacterium Lactobacillus sakei, involves two newly characterized proteins. Appl Environ Microbiol 79:2012–2018. https://doi.org/10.1128/AEM.03301-12
Buist G, Steen A, Kok J, Kuipers OP (2008) LysM, a widely distributed protein motif for binding to (peptido) glycans. Mol Microbiol 68:838–847. https://doi.org/10.1111/j.1365-2958.2008.06211.x
Chang Y (2020) Bacteriophage-derived endolysins applied as potent biocontrol agents to enhance food safety. Microorganisms 8(5):724. https://doi.org/10.3390/microorganisms8050724
Comi G, Andyanto D, Manzano M, Iacumin L (2016) Lactococcus lactis and Lactobacillus sakei as bio-protective culture to eliminate Leuconostoc mesenteroides spoilage and improve the shelf life and sensorial characteristics of commercial cooked bacon. Food Microbiol 58:16–22. https://doi.org/10.1016/j.fm.2016.03.001
Do T, Page JE, Walker S (2020) Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes. J Biol Chem 295(10):3347–3361. https://doi.org/10.1074/jbc.REV119.010155
Eckert C, Lecerf M, Dubost L, Arthur M, Mesnage S (2006) Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis. J Bacteriol 188(24):8513–8519. https://doi.org/10.1128/JB.01145-06
Etobayeva I, Linden SB, Alem F, Harb L, Rizkalla L, Mosier PD, Johnson AA, de Louise T, Hakami RM, Nelson DC (2018) Discovery and biochemical characterization of PlyP56, PlyN74, and PlyTB40—Bacillus specific endolysins. Viruses 10(5):276. https://doi.org/10.3390/v10050276
Fessard A, Remize F (2017) Why are weissella spp. Not used as commercial starter cultures for food fermentation? Fermentation 3(3):38. https://doi.org/10.3390/fermentation3030038
García-Cano I, Ponce-Alquicira E (2015) Lactobacillus sakei isolated from fermented meat: external antibacterial activity against lactic acid bacteria and pathogenic strains. 6th congress of European microbiologists, FEMS Maastricht Nederland
García-Cano I, Campos-Gómez M, Contreras-Cruz M, Serrano-Maldonado C, González-Canto A, Peña-Montes C, Rodríguez-Sanoja R, Sánchez S, Farrés A (2015) Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042. Appl Microbiol Biotechnol 99:8563–8573. https://doi.org/10.1007/s00253-015-6593-2
García-Cano I, Rocha-Mendoza D, Kosmerl E, Zhang L, Jiménez-Flores R (2020) Technically relevant enzymes and proteins produced by LAB suitable for industrial and biological activity. Appl Microbiol Biotechnol 104(4):1401–1422. https://doi.org/10.1007/s00253-019-10322-2
Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: The proteomics protocols handbook, Humana Press, Totowa, pp 571–607. https://doi.org/10.1385/1-59259-890-0:571
Gonzalez-Delgado LS, Walters-Morgan H, Salamaga B, Robertson AJ, Hounslow AM, Jagielska E, Sabala I, Williamson MP, Lovering AL, Mesnage S (2020) Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b. Nat Chem Biol 16(1):24–30. https://doi.org/10.1038/s41589-019-0393-4
Harney SJ, Simopoulos ND, Ikawa M (1967) Cell wall constituents of Leuconostoc citrovorum and Leuconostoc mesenteroides. J Bacteriol 93(1):273–277. https://doi.org/10.1128/jb.93.1.273-277.1967
Hemamalini N, Ezhilmathi S, Mercy AA (2020) Recombinant protein expression optimization in Escherichia coli: a review. Indian J Anim Res 54(6):653–660. https://doi.org/10.18805/ijar.B-3808
Kamboj K, Vasquez A, Balada-Llasat JM (2015) Identification and significance of Weissella species infections. Front Microbiol 6:1204. https://doi.org/10.3389/fmicb.2015.01204
Khoury GA, Smadbeck J, Kieslich CA, Floudas CA (2014) Protein folding and de novo protein design for biotechnological applications. Trends Biotechnol 32(2):99–109. https://doi.org/10.1016/j.tibtech.2013.10.008
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Leclerc D, Asselin A (1989) Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can J Microbiol 35(8):749–753. https://doi.org/10.1139/m89-125
Liburdi K, Benucci I, Esti M (2014) Lysozyme in wine: an overview of current and future applications. Compr Rev Food Sci Food Saf 13(5):1062–1073. https://doi.org/10.1111/1541-4337.12102
Lopez-Arvizu A, Rocha-Mendoza D, Ponce-Alquicira E, García-Cano I (2021) Characterization of antibacterial activity of a N-acetylmuramoyl-L-alanine amidase produced by Latilactobacillus sakei isolated from salami. World J Microbiol Biotechnol 37:65. https://doi.org/10.1007/s11274-021-03033-2
Lorenzo JM, Munekata PE, Dominguez R, Pateiro M, Saraiva JA, Franco D (2018) Main groups of microorganisms of relevance for food safety and stability: general aspects and overall description. In: Innovative technologies for food preservation. Academic Press, New York, pp 53–107. https://doi.org/10.1016/B978-0-12-811031-7.00003-0
Low LY, Yang C, Perego M, Osterman A, Liddington R (2011) Role of net charge on catalytic domain and influence of cell wall binding domain on bactericidal activity, specificity, and host range of phage lysins. J Biol Chem 286(39):34391–34403. https://doi.org/10.1074/jbc.M111.244160
Mapelli C, Barbiroli AG, De Benedetti S, Musatti A, Rollini MS (2018) Antilisterial bacteriocins for food security: the case of sakacin A. Encycl Food Secur Sustain 2:385–392. https://doi.org/10.1016/B978-0-08-100596-5.22150-1
Mesnage S, Dellarole M, Baxter N, Rouget J, Dimitrov J, Wang N, Fujimoto Y, Hounslow A, Lacroix-Desmazes S, Fukase K, Foster S, Williamson M (2014) Molecular basis for bacterial peptidoglycan recognition by LysM domains. Nat Commun 5:4269. https://doi.org/10.1038/ncomms5269
Najjari A, Amairi H, Chaillou S, Mora D, Boudabous A, Zagorec M, Ouzari H (2016) Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei. J Adv Res 7(1):155–163. https://doi.org/10.1016/j.jare.2015.04.004
Park JH, Ahn HJ, Kim SG, Chung CH (2013) Dextran-like exopolysaccharide-producing Leuconostoc and Weissella from kimchi and its ingredients. Food Sci Biotechnol 22(4):1047–1053. https://doi.org/10.1007/s10068-013-0182-x
Sabala I, Jonsson I-M, Tarkowski A, Bochtler M (2012) Anti-staphylococcal activities of lysostaphin and LytM catalytic domain. BMC Microbiol 12:97. https://doi.org/10.1186/1471-2180-12-97
Sánchez-Vallet A, Saleem-Batcha R, Kombrink A, Hansen G, Valkenburg DJ, Thomma BP, Mesters JR (2013) Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. Elife 2:e00790. https://doi.org/10.7554/eLife.00790.001
Schmelcher M, Loessner MJ (2016) Bacteriophage endolysins: applications for food safety. Curr Opin Biotechnol 37:76–87. https://doi.org/10.1016/j.copbio.2015.10.005
Serrano-Maldonado CE, García-Cano I, González-Canto A, Ruiz-May E, Elizalde-Contreras JM, Quirasco M (2018) Cloning and characterization of a novel N-acetylglucoaminidase (AtlD) from Enterococcus faecalis. J Mol Microbiol Biotechnol 28:14–27. https://doi.org/10.1159/000486757
Steen A, Buist G, Hoursburgh G, Venema G, Kuipers O, Foster S, Kok J (2005) AcmA of Lactococcus lactis an N-acetylglucoaminidase with an optimal number of LysM domains for proper functioning. FEBS J 11:2854–2868. https://doi.org/10.1111/j.1742-4658.2005.04706.x
Sun L, Zhang Y, Guo X, Zhang L, Zhang W, Man C, Jiang Y (2020) Characterization and transcriptomic basis of biofilm formation by Lactobacillus plantarum J26 isolated from traditional fermented dairy products. LWT. https://doi.org/10.1016/j.lwt.2020.109333
Visweswaran GRR, Leenhouts K, van Roosmalen M, Kok J, Buist G (2014) Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications. Appl Microbiol Biotechnol 98(10):4331–4345. https://doi.org/10.1007/s00253-014-5633-7
Vollmer W, Blanot D, de Pedro MA (2008) Peptidoglycan structure and architecture. FEMS Microbiol Rev 32:149–167. https://doi.org/10.1111/j.1574-6976.2007.00094.x
Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296–W303. https://doi.org/10.1093/nar/gky427
Wong JE, Blaise M (2013) Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the putative NlpC/P60 endopeptidase, TTHA0266, from Thermus thermophilus HB8. Acta Crystallogr F-Struct Biol Cryst Commun 69(11):1291–1294. https://doi.org/10.1107/S1744309113027164
Wong JE, Blaise M (2020) Report of false positives when using zymography to assess peptidoglycan hydrolytic activity of an endopeptidase with multiple LysM domains. Biochimie. https://doi.org/10.1016/j.biochi.2020.07.014
Wong JE, Midtgaard SR, Gysel K, Thygesen MB, Sorensen KK, Jensen KJ, Stougaard J, Thirup S, Blaise M (2015) An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase. Acta Crystallogr Sect-D Biol Crystallogr 71:592–605. https://doi.org/10.1107/S139900471402793X
Xu Y, Wang T, Kong J, Wang HL (2015) Identification and functional characterization of AclB, a novel cell-separating enzyme from Lactobacillus casei. Int J Food Microbiol 203:93–100. https://doi.org/10.1016/j.ijfoodmicro.2015.03.011
Zagorec M, Champomier V, Marie C (2017) Lactobacillus sakei: A starter for sausage fermentation, a protective culture for Meat products. Microorganisms 5:56. https://doi.org/10.3390/microorganisms5030056
Acknowledgements
The authors wish to thank the Consejo Nacional de Ciencia y Tecnología (CONACYT) in Mexico for the student scholarship, as well as the Graduate program Posgrado en Biotecnología, UAM.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors state no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
López-Arvizu, A., Rocha-Mendoza, D., Farrés, A. et al. Improved antimicrobial spectrum of the N-acetylmuramoyl-l-alanine amidase from Latilactobacillus sakei upon LysM domain deletion. World J Microbiol Biotechnol 37, 196 (2021). https://doi.org/10.1007/s11274-021-03169-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-021-03169-1