Bioproduction of Chitin Hydrolysate Containing N-Acetylglucosamine by Serratia marcescens PT6 Crude Chitinase and Its Effects on Bacterial Growth Inhibition in Various Temperature

Indun Dewi Puspita(1*), Susana Endah Ratnawati(2), Hendri Setiawan(3), Murwantoko Murwantoko(4), Ustadi Ustadi(5), David Ratkowsky(6), Mark Tamplin(7)

(1) Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, 55281 Yogyakarta, Indonesia
(2) Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, 55281 Yogyakarta, Indonesia
(3) Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, 55281 Yogyakarta, Indonesia
(4) Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, 55281 Yogyakarta, Indonesia
(5) Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, 55281 Yogyakarta, Indonesia
(6) Tasmanian Institute of Agriculture, 7001 Tasmania, Australia
(7) Tasmanian Institute of Agriculture, 7001 Tasmania, Australia
(*) Corresponding Author


N-acetylglucosamine (GlcNAc), a chitin monomer, can be used as a natural preservative to ensure food quality and safety. Combining natural preservatives with low storage temperature offers physical hurdles to bacterial growth in food. This study aimed to produce chitin hydrolysate containing GlcNAc using Serratia marcescens PT6 crude chitinase and investigate its effect on bacterial growth rate as a function of temperature. Crude chitinase from partial purification was used to hydrolyze 1.3% colloidal chitin. The optimal enzymatic conditions were pH 6 and 45˚C for 120 min, at an enzyme:substrate ratio of 1:1, yielding a 65.6 µg/mL GlcNAc. Inhibitory activity of hydrolysate containing 2.5-7.5 ppm GlcNAc on Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus was measured at 4, 15, and 30oC in nutrient broth. Bacterial growth was measured using of optical density for each combination of GlcNAc concentration and temperature. Growth curves fitted by the Baranyi and Roberts model were developed using DMFit software. The growth rate was converted to the square root and then modeled as a function of temperature using the Ratkowsky square root model. Incubation temperature exerted a pronounced effect on the inhibition of all bacterial species (P<0.0001), with the greatest effect observed for E. coli at 30°C (P<0.0001), and the least effect for V. parahaemolyticus (P=0.0878). The inhibitory effect of GlcNAc in chitin hydrolysate was only significant for E. coli (P<0.0001) and S. aureus (P=0.0041). This study revealed that the effect of temperature in growth inhibition was more significant than GlcNAc addition. However, a reduction in bacterial growth with the addition of GlcNAc at 30°C was observed, which may be effective for food encountered thermal abuse conditions. Further investigation of the effect of GlcNAc on bacteria structure and metabolism is required to elucidate the mechanism of GlcNAc as a food preservative.


Antibacterial; Chitinase; Growth rate; N-acetylglucosamine; Serratia marcescens

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Abidin, M.Z., Kourmentza, C., Karatzas, A.K., and Niranjan, K. (2019). “Enzymatic hydrolysis of thermally pre-treated chitin and antimicrobial activity of N, N’-diacetylchitobiose,” J. Chem. Technol. Biotechnol., 94(8), 2529–2536.

Ahuja, V., Bhatt, A.K., Sharma, V., Rathour, R.K., Rana, N., Bhatia, R.K., Varjani, S., Kumar, M., Magdouli, S., Yang, Y.H., and Bhatia, S.K. (2021). “Advances in glucosamine production from waste biomass and microbial fermentation technology and its applications,” Biomass Convers. Biorefin., s13399-021-01968-y.

Aktuganov, G.E., Galimzianova, N.F., Gilvanova, E.A., Kuzmina, L.Y., Boyko, T.F., Safina, V.R., and Melentiev, A.I. (2018). “Characterization of chitinase produced by the alkaliphilic Bacillus mannanilyticus IB-OR17 B1 strain,” Appl. Biochem. Microbiol., 54, 505–511.

Baranyi, J., and Roberts, T.A. (1994). “A dynamic approach to predicting bacterial growth in food.” Int. J. Food Microbiol., 23, 277–294.

Benhabiles, M.S., Salah, R., Lounici, H., Drouiche, N., Goosen, M.F.A., and Mameri N. (2012). “Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste,” Food Hydrocoll., 29, 48–56.

Byun, S.M., No, H.K., Hong, J.H., Lee, S.I., and Prinyawiwatkul, W. (2013). “Comparison of physicochemical, binding, antioxidant and antibacterial properties of chitosans prepared from ground and entire crab leg shells,” Int. J. Food Sci. Technol., 48(1), 136–142.

Cao, S., Liu, Y., Shi, L., Zhu, W., and Wang, H. (2022). “N-Acetylglucosamine as a platform chemical produced from renewable resources: Opportunity, challenge, and future prospects,” Green Chem., 24(2), 493–509.

Chen, M.J., Chen, K.N., and Lin, C.W. (2004). “Optimization of the viability of probiotics in a fermented milk drink by the response surface method,” Asian-Australas. J. Anim. Sci., 17(5), 705–711.

Costa, M.D.A.A., Owen, R.A., Tammsalu, T., Buchanan, G., Palmer, T., and Sargent, F. (2019). “Controlling and co-ordinating chitinase secretion in a Serratia marcescens population,” Microbiology, 165(11), 1233–1244.

Doi, S., Higashino, H., Karatsu, A., Masuda, A., and Masuda, T. (2022). “Identification of polyphenol and reductone antioxidants in the caramelization product of N-acetylglucosamine,” ACS Food Sci. Technol., 2(7), 1135–1140.

Dotto, G.L., Vieira, M.L., and Pinto, L.A. (2015). “Use of chitosan solutions for the microbiological shelflife extension of papaya fruits during storage at room temperature,” LWT- Food Sci. Technol., 64(1), 126–130.

Emruzi, Z., Keshavarz, M., Gholami, D., Aminzadeh, S., and Noori, A.R. (2020). “Kinetic and thermo-inactivation thermodynamic parameters of a novel isolated Serratia marcescens B4A chitinase,” Biomacromol. J., 6(1), 46–55.

Garcia, L.G.S., de Melo Guedes, G.M., da Silva, M.L.Q., Castelo-Branco, D.S.C.M., Sidrim, J.J.C., de Aguiar Cordeiro, R., Rocha, M.F.G., Vieira, R.S., and Brilhante, R.S.N. (2018). “Effect of the molecular weight of chitosan on its antifungal activity against Candida spp. in planktonic cells and biofilm,” Carbohydr. Polym., 195, 662–669.

Gottesman, S. (2019). “Trouble is coming: Signaling pathways that regulate general stress responses in bacteria,” J. Biol. Chem., 294(31), 11685–11700.

Gutiérrez-Román, M.I., Dunn, M.F., Tinoco-Valencia, R., Holguín-Meléndez, F., Huerta-Palacios, G., and Guillén-Navarro, K. (2014). “Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP),” World J. Microbiol. Biotechnol., 30, 33–42.

Kanatt, S.R., Rao, M.S., Chawla, S.P., and Sharma, A. (2013). “Effects of chitosan coating on shelf-life of ready-to-cook meat products during chilled storage,” LWT-Food Sci. Technol., 53(1), 321–326.

Kuk, J.H., Jung, W.J., Jo, G.H., Ahn, J.S., Kim K.Y., and Park, R.D. (2005). “Selective preparation of N-acetyl-D-Glucosamine and N.N’-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp.,” Biotechnol. Lett., 27(1), 7–11.

Kulikov, S.N., Tikhonov, V.E., Bezrodnykh, E.A., Lopatin, S.A., and Varlamov, V.P. (2015). “Comparative evaluation of antimicrobial activity of oligochitosans against Klebsiella pneumoniae,” Russ. J. Bioorganic Chem., 41, 57–62.

Lee, Y.H., Park, S.Y., Hwang, Y.J., and Park, J.K. (2022). “Molecular weight determination of chitosan with antibacterial activity using matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis,” Macromol. Res., 30(2), 90–98.

Li, J., Zheng, J., Liang, Y., Yan, R., Xu, X., and Lin, J. (2020). “Expression and characterization of a chitinase from Serratia marcescens,” Protein Expr. Purif., 171, 105613.

Li, Y., Zhou, D., Hu, S., Xiao, X., Yu, Y., and Li, X. (2018). “Transcriptomic analysis by RNA-seq of Escherichia coli O157: H7 response to prolonged cold stress,” LWT-Food Sci. Technol., 97, 17–24.

Liu, J., Xu, Q., Wu, Y., Sun, D., Zhu, J., Liu, C., and Liu, W. (2023). “Carbohydrate-binding modules of ChiB and ChiC promote the chitinolytic system of Serratia marcescens BWL1001. Enzyme Microb. Technol., 162, 110118.

Liu, Z., Ge, X., Lu, Y., Dong, S., Zhao, Y., and Zeng, M. (2012). “Effects of chitosan molecular weight and degree of deacetylation on the properties of gelatine-based films,” Food Hydrocoll., 26(1), 311–317.

Patil, N.S. and Jadhav, J.P. (2014). “Enzymatic production of N-acetyl-D-glucosamine by solid state fermentation of chitinase by Penicillium ochrochloron MTCC 517 using agricultural residues,” Int. Biodeter. Biodegr., 91, 9–17.

Phillips, J. (2019). Fundamentals of Enzymology. ED-Tech Press, United Kingdom.

Purushotham, P., Sarma, P.V.S.R.N., and Podile, A.R. (2012). “Multiple chitinases of an endophytic Serratia proteamaculans 568 generate chitin oligomers,” Bioresource Technol., 112, 261–269.

Rahman, M.H., Hjeljord, L.G., Aam, B.B., Sørlie, M., and Tronsmo, A. (2015). “Antifungal effect of chito-oligosaccharides with different degrees of polymerization,” Eur. J. Plant Pathol., 141, 147–158.

Ratkowsky, D.A., Olley, J., McMeekin, T.A., and Ball, A. (1982). “Relationship between temperature and growth rate of bacterial cultures,” J. Bacteriol., 149, 1–5.

Raut, A.V., Satvekar, R.K., Rohiwal, S.S., Tiwari, A.P., Gnanamani, A., Pushpavanam, S., Nanaware, S.G., and Pawar, S.H. (2016). “In vitro biocompatibility and antimicrobial activity of chitin monomer obtain from hollow fiber membrane,” Des. Monomers Polym., 19(5), 445–455.

Reesha, K.V., Panda, S.K., Bindu, J., and Varghese, T.O. (2015). “Development and characterization of an LDPE/chitosan composite antimicrobial film for chilled fish storage,” Int. J. Biol. Macromol., 79, 934–942.

Reissig, J.L., Strominger, J.L., and Leloir, L.F. (1955). “A modified colorimetric method for the estimation of N-acetylamino sugars,” J. Biol. Chem., 217(2), 959–966.

Tao, A., Wang, T., Pang, F., Zheng, X., Ayra-Pardo, C., Huang, S., Xu, R., Liu, F., Li, J., Wei, Y., Wang, Z., Niu, Q., and Li, D. (2022). “Characterization of a novel chitinolytic Serratia marcescens strain TC-1 with broad insecticidal spectrum,” AMB Express, 12(1), 1–13.

Taokaew, S. and Kriangkrai, W. (2023). “Chitinase-assisted bioconversion of chitinous waste for development of value-added chito-oligosaccharides products,” Biology, 12(1), 87.

Triwijayani, A.U., Puspita, I.D., Murwantoko, and Ustadi. (2018). “Identification of chitinolytic bacteria isolated from shrimp pond sediment and characterization of their chitinase encoding gene,” IOP Conf. Series: Earth and Environmental Science, 139(1), 012051.

Tsai, G.J. and Su, W.H. (1999). “Antibacterial Activity of Shrimp Chitosan against Escherischia coli,” J. Food Protect., 62, 239–243.

Tsironi, T., Houhoula, D., and Taoukis, P. (2020). “Hurdle technology for fish preservation,” Aquaculture and Fisheries, 5(2), 65–71.

Tuveng, T.R., Hagen, L.H., Mekasha, S., Frank, J., Arntzen, M.Ø., Vaaje-Kolstad, G., and Eijsink, V.G.H. (2017). “Genomic, proteomic and biochemical analysis of the chitinolytic machinery of Serratia marcescens BJL200,” Biochim. Biophys. Acta, 1865, 4, 414–421.

Wassenaar, W. (2004). U.S. Patent Application 2004/0071855.

Yuan, G., Lv, H., Tang, W., Zhang, X., and Sun, H. (2016). “Effect of chitosan coating combined with pomegranate peel extract on the quality of Pacific white shrimp during iced storage,” Food Control, 59, 818–823.

Zhang, Y., Burkhardt, D.H., Rouskin, S., Li, G.W., Weissman, J.S., and Gross, C.A. (2018). “A stress response that monitors and regulates mRNA structure is central to cold shock adaptation,” Mol. cell, 70(2), 274–286.


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ASEAN Journal of Chemical Engineering  (print ISSN 1655-4418; online ISSN 2655-5409) is published by Chemical Engineering Department, Faculty of Engineering, Universitas Gadjah Mada.