Production, purification and characterization of chitinase from Micromonospora sp. AR17

https://doi.org/10.22146/ijbiotech.77137

Yohanes Harvinda(1), Ustadi Ustadi(2), Masagus Muhammad Prima Putra(3*)

(1) Fisheries Science Postgraduate Program, Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, Jalan Flora A4, Bulaksumur, Yogyakarta 55281, Indonesia
(2) Fisheries Science Postgraduate Program, Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, Jalan Flora A4, Bulaksumur, Yogyakarta 55281, Indonesia
(3) Fisheries Science Postgraduate Program, Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, Jalan Flora A4, Bulaksumur, Yogyakarta 55281, Indonesia
(*) Corresponding Author

Abstract


N‐acetylglucosamine (NAG) is the monomer product of chitin, which has been widely used as a bioactive com‐ pound in applications such as anti‐tumor, anti‐microbial, and antioxidant activities. In production, biological processes using enzymes are preferable to chemicals due to environmental issues. This study aims to determine the activity, purity level, and molecular weight of purified chitinase from Micromonospora sp. AR17 determines the concentration of NAG produced by purified chitinase that has been characterized. Chitinase was produced by fermentation in colloidal chitin broth at 40 °C, pH 7, for 7 days, while chitinase activity was checked every 24 h. The optimal fermentation time was used to produce chitinase for a further purification step. Enzyme purification was carried out by ultrafiltration, ammonium sulfate precipitation, ion exchange chromatography (Q Sepharose Fast Flow), and gel filtration (Sephacryl S‐300). The purified enzyme was then char‐ acterized for optimum time, pH, and temperature to produce NAG. The results suggested that the fourth day was the optimal time for chitinase production, with chitinase activity of 0.0040 U/mL and a NAG concentration of 7.62 µg/mL. The purifica‐ tion step successfully increased the purity by 6.82 times with chitinase‐specific activity at 1.4648 U/mg. Production of NAG with purified chitinase produced a NAG concentration of 32.472 µg/mL with an incubation time of 30 min at 40 °C and pH 7.

Keywords


Chitinase; Micromonospora sp. AR17; N‐acetylglucosamine; Purification

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References

Agheshlouie M, Mohammadzadeh R, Azam G. 2015. Comparative study fungal chitinase of beauveria bassiana strain HFW­05 and bacterial chitinase of Micromonospora sp. L5 using bioinformatics analysis. International Conference on Research in Science and Technology URL https://www.sid.ir/paper/92985 0/en.

Ahmed EA, Hassan EA, Tobgy KM, Ramadan EM. 2014. Evaluation of rhizobacteria of some medicinal plants for plant growth promotion and biological control. Ann. Agric. Sci. 59(2):273–280. doi:10.1016/j.aoas.2014.11.016.

Akeed Y, Atrash F, Naffaa W. 2020. Partial purification and characterization of chitinase produced by Bacillus licheniformis B307. Heliyon 6(5):e03858. doi:10.1016/j.heliyon.2020.e03858.

Amin D, Abolmaaty A, Tolba S, Abdallah N, Wellington E. 2018. Phylogenic Characteristics of a Unique Antagonistic Micromonospora sp. Rc5 to S. aureus Isolated from Sinai Desert of Egypt. Annu. Res. Rev. Biol. 22(2):1–15. doi:10.9734/arrb/2018/38318.

Ansari WA, Krishna R, Zeyad MT, Singh S, Yadav AK. 2020. Endophytic actinomycetes­mediated modulation of defense and systemic resistance confers host plant fitness under biotic stress conditions. Singapore: Springer. doi:10.1007/978­981­15­3028­9_10.

Arnold LD, Solomon NA. 1986. Manual of industrial microbiology and biotechnology. Washington: American Society for Microbiology. Azam MS, Kim EJ, Yang HS, Kim JK. 2014. High antioxidant and DNA protection activities of Nacetylglucosamine (GlcNAc) and chitobiose produced by exolytic chitinase from Bacillus cereus EW5. Springerplus 3(1):1–11. doi:10.1186/2193­ 1801­3­354.

Bhagwat P, Amobonye A, Singh S, Pillai S. 2021. A comparative analysis of GH18 chitinases and their isoforms from Beauveria bassiana: An insilico approach. Process Biochem. 100:207–216. doi:10.1016/j.procbio.2020.10.012.

Bisswanger H. 2014. Enzyme assays. Perspect. Sci. 1(1­ 6):41–55. doi:10.1016/j.pisc.2014.02.005. Bradford M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of ProteinDye Binding. Anal. Biochem. 72(1­2):248–254. doi:10.1006/abio.1976.9999.

Brzezinska MS, Jankiewicz U, Burkowska A, Walczak M. 2014. Chitinolytic microorganisms and their possible application in environmental protection. Curr. Microbiol. 68(1):71–81. doi:10.1007/s00284­013­0440­4.

Cardozo FA, Facchinatto WM, Colnago LA, Campana-Filho SP, Pessoa A. 2019. Bioproduction of Nacetyl­glucosamine from colloidal α­chitin using an enzyme cocktail produced by Aeromonas caviae CHZ306. World J. Microbiol. Biotechnol. 35(8):1– 13. doi:10.1007/s11274­019­2694­x.

Gallagher SR. 2012. SDS­polyacrylamide gel electrophoresis (SDS­PAGE). Curr. Protoc. Essent. Lab. Tech. 2012(SUPPL.6):7.3.1–7.3.28. doi:10.1002/9780470089941.et0703s06.

Haliza W, Suhartono MT. 2012. Karakteristik Kitinase Dari Mikrobia. Bul. Teknol. Pasca Panen 8(1):1–14. Hamid R, Khan MA, Ahmad M, Ahmad MM, Abdin MZ, Musarrat J, Javed S. 2013. Chitinases: An update. J. Pharm. Bioallied Sci. 5(1):21–29. doi:10.4103/0975­ 7406.106559.

Herdyastuti N, Cahyaningrum SE. 2017. Analysis of N­acetylglucosamine from enzymatic degradation of amorphous chitin. Rasayan J. Chem. 10(1):226–233. doi:10.7324/RJC.2017.1011582.

Herdyastuti N, Fauziah RW, Prabowo YY, Apriliana IA. 2021. Diversity of chitinolytic bacteria from shrimp farms and their antifungal activity. J. Nat. Sci. Biol. Med. 12(3):317–324.

Horak I, Engelbrecht G, van Rensburg PJ, Claassens S. 2019. Microbial metabolomics: essential definitions and the importance of cultivation conditions for utilizing Bacillus species as bionematicides. J. Appl. Microbiol. 127(2):326–343. doi:10.1111/jam.14218.

Hsu SC, Lockwood JL. 1975. Powdered Chitin Agar as a Selective Medium for Enumeration of Actinomycetes in Water and Soil. Appl. Microbiol. 29(3):422–426. doi:10.1128/am.29.3.422­426.1975.

Karthik N, Binod P, Pandey A. 2015. Purification and characterisation of an acidic and antifungal chitinase produced by a Streptomyces sp. Bioresour. Technol. 188:195–201. doi:10.1016/j.biortech.2015.03.006.

Keffeler EC, Parthasarathy S, Abdullahi ZH, Hancock LE. 2021. Metabolism of poly­b1,4­N­acetylglucosamine substrates and importation of n­acetylglucosamine and glucosamine by enterococcus faecalis. J. Bacteriol. 203(21):e00371. doi:10.1128/JB.00371­21.

Kim TI, Lim DH, Baek KS, Jang SS, Park BY, Mayakrishnan V. 2018. Production of chitinase from Escherichia fergusonii, chitosanase from Chryseobacterium indologenes, Comamonas koreensis and its application in N­acetylglucosamine production. Int. J. Biol. Macromol. 112:1115–1121. doi:10.1016/j.ijbiomac.2018.02.056.

Li J, Zheng J, Liang Y, Yan R, Xu X, Lin J. 2020. Expression and characterization of a chitinase from Serratia marcescens. Protein Expr. Purif. 171:105613. doi:10.1016/j.pep.2020.105613.

Liaqat F, Eltem R. 2018. Chitooligosaccharides and their biological activities: A comprehensive review. Carbohydr. Polym. 184:243–259. doi:10.1016/j.carbpol.2017.12.067.

Liu S, Li Z, Yu B, Wang S, Shen Y, Cong H. 2020. Recent advances on protein separation and purification methods. Adv. Colloid Interface Sci. 284:1–23. doi:10.1016/j.cis.2020.102254.

Lu Y, Wang N, He J, Li Y, Gao X, Huang L, Yan X. 2018. Expression and characterization of a novel chitinase with antifungal activity from a rare actinomycete, Saccharothrix yanglingensis Hhs.015. Protein Expr. Purif. 143:45–51. doi:10.1016/j.pep.2017.10.013.

Luo L, Meng H, Gu JD. 2017. Microbial extracellular enzymes in biogeochemical cycling of ecosystems. J. Environ. Manage. 197:539–549. doi:10.1016/j.jenvman.2017.04.023.

Nagpure A, Gupta RK. 2013. Purification and characterization of an extracellular chitinase from antagonistic Streptomyces violaceusniger. J. Basic Microbiol. 53(5):429–439. doi:10.1002/jobm.201100648.

Nawani NN, Kapadnis BP, Das AD, Rao AS, Mahajan SK. 2002. Purification and characterization of a thermophilic and acidophilic chitinase from Microbispora sp. V2. J. Appl. Microbiol. 93(6):965–975. doi:10.1046/j.1365­2672.2002.01766.x.

O’Riordan A, McHale ML, Gallagher J, McHale AP. 1989. Chitinase production following coimmobilization of Micromonospora chalcae with chitin in calcium alginate. Biotechnol. Lett. 11(10):735–738. doi:10.1007/BF01044107.

Patantis G, Zilda DS, Fawzya YN, Chasanah E. 2019. Purification of chitosanase from Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 for chitooligosacharide production. IOP Conf. Ser. Earth Environ. Sci. 404(1):1–8. doi:10.1088/1755­ 1315/404/1/012078.

Qin Z, Zhao L. 2019. The history of chito/chitin oligosaccharides and its monomer. Singapore: Springer. doi:10.1007/978­981­13­9402­7_1.

Reissig JL, Storminger JL, Leloir LF. 1955. A modified colorimetric method for the estimation of Nacetylamino sugars. J. Biol. Chem. 217(2):959–966. doi:10.1016/s0021­9258(18)65959­9.

Revathi M, Saravanan R, Shanmugam A. 2012. Production and characterization of chitinase from Vibrio species, a head waste of shrimp Metapenaeus dobsonii (Miers, 1878) and chitin of Sepiella inermis Orbigny, 1848. Adv. Biosci. Biotechnol. 03(04):392– 397. doi:10.4236/abb.2012.34056.

Roy JC, Salaün F, Giraud S, Ferri A. 2017. Solubility of Chitin: Solvents, Solution Behaviors and Their Related Mechanisms. Rijeka: IntechOpen. doi:10.5772/intechopen.71385.

Secundo F. 2013. Conformational changes of enzymes upon immobilisation. Chem. Soc. Rev. 42(15):6250– 6261. doi:10.1039/c3cs35495d.

Stoykov YM, Pavlov AI, Krastanov AI. 2015. Chitinase biotechnology: Production, purification, and application. Eng. Life Sci. 15(1):30–38. doi:10.1002/elsc.201400173.

Suganthi M, Senthilkumar P, Arvinth S, Chandrashekara KN. 2017. Chitinase from Pseudomonas fluorescens and its insecticidal activity against Helopeltis theivora. J. Gen. Appl. Microbiol. 63(4):222–227. doi:10.2323/jgam.2016.11.001.

Su’i M, Suprihana S. 2013. Fraksinasi enzim lipase dari endospora kelapa dengan metode salting out. Agritech 33(4):377–383.

Suryadi Y, Priyatno TP, Samudra IM, Susilowati DN, Lawati N, Kustaman E. 2016. Pemurnian Parsial dan Karakterisasi Kitinase Asal Jamur Entomopatogen Beauveria bassiana Isolat BB200109. J. AgroBiogen 9(2):77–84. doi:10.21082/jbio.v9n2.2013.p77­84.

Swiontek Brzezinska M, Jankiewicz U, Lisiecki K. 2013. Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis. Appl. Biochem. Microbiol. 49(2):154–159. doi:10.1134/S0003683813020014.

Therien JP, Hammerer F, Friščić T, Auclair K. 2019. Mechanoenzymatic Breakdown of Chitinous Material to N­Acetylglucosamine: The Benefits of a Solventless Environment. ChemSusChem 12(15):3481– 3490. doi:10.1002/cssc.201901310.

Tran TN, Doan CT, Nguyen VB, Nguyen AD, Wang SL. 2019. The isolation of chitinase from Streptomyces thermocarboxydus and its application in the preparation of chitin oligomers. Res. Chem. Intermed. 45(2):727–742. doi:10.1007/s11164­018­3639­y.

Wang SL, Liang TW, Yen YH. 2011. Bioconversion of chitin­containing wastes for the production of enzymes and bioactive materials. Carbohydr. Polym. 84(2):732–742. doi:10.1016/j.carbpol.2010.06.022.

Wyllie JA, McKay MV, Barrow AS, Soares da Costa TP. 2022. Biosynthesis of uridine diphosphate N­Acetylglucosamine: An underexploited pathway in the search for novel antibiotics? IUBMB Life 74(12):1232–1252. doi:10.1002/iub.2664.

Yang S, Jiang Z, Liu Y, Ma S. 2019. Preparation of Chitin Oligosaccharides and Its Monomer. Singapore: Springer. doi:10.1007/978­981­13­9402­7_4.

Zeng YB, Hsiao HM, Chan SH, Wang YH, Lin YY, Kuo YH, Guh JH, Liang PH. 2014. Synthesis and anti­cancer activity of a glycosyl library of N ­acetylglucosamine­bearing oleanolic acid. Mol. Divers. 18(1):13–23. doi:10.1007/s11030­013­9480­ 8.

Agheshlouie M, Mohammadzadeh R, Azam G. 2015.
Comparative study fungal chitinase of beauveria
bassiana strain HFW­05 and bacterial chitinase of
Micromonospora sp. L5 using bioinformatics analysis. International Conference on Research in Science
and Technology URL https://www.sid.ir/paper/92985
0/en.
Ahmed EA, Hassan EA, Tobgy KM, Ramadan EM.
2014. Evaluation of rhizobacteria of some medicinal plants for plant growth promotion and biological control. Ann. Agric. Sci. 59(2):273–280.
doi:10.1016/j.aoas.2014.11.016.
Akeed Y, Atrash F, Naffaa W. 2020. Partial purification and characterization of chitinase produced by
Bacillus licheniformis B307. Heliyon 6(5):e03858.
doi:10.1016/j.heliyon.2020.e03858.
Amin D, Abolmaaty A, Tolba S, Abdallah N, Wellington
E. 2018. Phylogenic Characteristics of a Unique Antagonistic Micromonospora sp. Rc5 to S. aureus Isolated from Sinai Desert of Egypt. Annu. Res. Rev.
Biol. 22(2):1–15. doi:10.9734/arrb/2018/38318.
Ansari WA, Krishna R, Zeyad MT, Singh S, Yadav AK.
2020. Endophytic actinomycetes­mediated modulation of defense and systemic resistance confers host
plant fitness under biotic stress conditions. Singapore:
Springer. doi:10.1007/978­981­15­3028­9_10.
Arnold LD, Solomon NA. 1986. Manual of industrial microbiology and biotechnology. Washington: American Society for Microbiology.
Azam MS, Kim EJ, Yang HS, Kim JK. 2014. High
antioxidant and DNA protection activities of Nacetylglucosamine (GlcNAc) and chitobiose produced by exolytic chitinase from Bacillus cereus
EW5. Springerplus 3(1):1–11. doi:10.1186/2193­
1801­3­354.
Bhagwat P, Amobonye A, Singh S, Pillai S. 2021.
A comparative analysis of GH18 chitinases and
their isoforms from Beauveria bassiana: An insilico approach. Process Biochem. 100:207–216.
doi:10.1016/j.procbio.2020.10.012.
Bisswanger H. 2014. Enzyme assays. Perspect. Sci. 1(1­
6):41–55. doi:10.1016/j.pisc.2014.02.005.
Bradford M. 1976. A Rapid and Sensitive Method
for the Quantitation of Microgram Quantities
of Protein Utilizing the Principle of ProteinDye Binding. Anal. Biochem. 72(1­2):248–254.
doi:10.1006/abio.1976.9999.
Brzezinska MS, Jankiewicz U, Burkowska A, Walczak M.
2014. Chitinolytic microorganisms and their possible
application in environmental protection. Curr. Microbiol. 68(1):71–81. doi:10.1007/s00284­013­0440­4.
Cardozo FA, Facchinatto WM, Colnago LA, Campana



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