Evaluation of enzymatic activity and identification of potent proteolytic and chitinolytic bacteria isolated from crab shell waste
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Abstract. Dliyauddin M, Ardyati T, Suharjono. 2020. Evaluation of enzymatic activity and Identification of Potent Proteolytic and Chitinolytic Bacteria Isolated from Crab Shell Waste. Biodiversitas 21: 211-218. Proteolytic and chitinolytic bacteria are needed to degrade protein-chitin fibers in crab shell waste. These bacteria are important for enhancing the degradation rate of crab shell waste in the environment. This study aims to study the enzymatic activity and identify potent proteolytic and chitinolytic bacteria based on 16S rDNA sequences. The bacteria were isolated from crab shell waste using skim milk agar and colloidal chitin agar for proteolytic and chitinolytic bacteria, respectively. The bacterial isolates were screened based on their protease and chitinase activity using casein and chitin as substrates, respectively. The selected isolates were identified based on 16S rDNA sequences similarity. A total of 20 proteolytic and 22 chitinolytic bacteria were successfully isolated from the samples. The isolate of AP9, AP5, and BP14 had a high protease activity. However, only BP14 and CK20 showed a high chitinase activity. The isolate of AP9 was identified as Bacillus subtilis, AP5, and CK20 were identified as Bacillus licheniformis, and BP14 was identified as Bacillus cereus. AP5 dan CK20 were classified as non-pathogenic isolates.
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References
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Degering C, Eggert T, Puls M, Bongaerts J, Evers S, Maurer KH, Jaeger KE. 2010. Optimization of Protease Secretion in Bacillus subtilis and Bacillus licheniformis by Screening of Homologous and Heterologous Signal Peptides. Appl. Environ. Microbiol. 76: 6370–6376.
Elieh-Ali-Komi D, and Hamblin MR. 2016. Chitin and Chitosan: Production and Application of Versatile Biomedical Nanomaterials. IJAR 4: 411–427.
Faramarzi M, Fazeli M, Yazdi MT, Adrangi S, Al Ahmadi KJ, Tasharrofi N, Mohseni FA. 2009. Optimization of Cultural Conditions for Production of Chitinase by a Soil Isolate of Massilia timonae. Biotechnology 8: 93-99.
Fulzele R, DeSa E, Yadav A, Shouche Y, Bhadekar R. 2011. Characterization of Novel Extracellular Protease Produced by Marine Bacterial Isolate From The Indian Ocean. Braz. J. Microbiol. 42: 1364–1373.
Gadgey K, and Bahekar A. 2017. Investigation of Mechanichal Properties of Crab Shell: A Review. IJLTET 1: 268–281.
Gomaa EZ. 2012. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: Their potential in Antifungal Biocontrol. J. Microbiol. 50: 103–111.
Gortari MC, and Hours RA. 2013. Biotechnological Processes for Chitin Recovery Out of Crustacean Waste: A Mini-Review, Electron. J. Biotechnol. 16 :14
Hajji S, Younes I, Ghorbel-Bellaaj O, Hajji R, Rinaudo M., Nasri M, Jellouli K. 2014. Structural Differences Between Chitin and Chitosan Extracted from Three Different Marine Sources. Int. J. Biol. Macromol. 65 : 298–306.
Hammami I, Siala R, Jridi M, Ktari N, Nasri M, Triki MA. 2013. Partial Purification and Characterization of Chiio8, A Novel Antifungal Chitinase Produced by Bacillus cereus IO8. J. Appl. Microbiol. 115: 358–366.
Hsieh YC, Wu YJ, Chiang TY, Kuo CY, Shrestha KL, Chao CF, Chen CJ. 2010. Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis. J. Biol. Chem. 285: 31603–31615.
Jayakumar R, Prabaharan M, Sudheesh KPT, Nair SV, Tamura H. 2011. Biomaterials Based on Chitin and Chitosan in Wound Dressing Applications. Biotechnol. Adv. 29: 322–337.
Jo GH, Jung WJ, Kuk JH, Oh KT, Kim YJ, Park RD. 2008. Screening of Protease-Producing Serratia marcescens FS-3 and Its Application To Deproteinization of Crab Shell Waste for Chitin Extraction. Carbohydrate Polymers 74:504–508
Karadag D, Mäkinen AE, Efimova E, Puhakka JA. 2009. Thermophilic Biohydrogen Production by An Anaerobic Heat Treated-Hot Spring Culture. Bioresour. Technol. 100: 5790–5795.
Kraisangsri J, Nalinanon S, Riebroy S, Yarnpakdee S, Ganesan P. 2018. Pysicochemical Characteristics of Glucosamine From Blue Swimming Crab (Portunus pelagicus) Shell Prepared by Acid Hydrolysis. Walailak J Sci & Tech. 15: 869-877.
Kumari S, and Rath PK. 2012. Extraction and Characterization of Chitin and Chitosan From (Labeo Rohit) Fsh Scales, Proc. Mater. Sci. 6 : 482–489.
Kuzu SB, Güvenmez HK, Denizci AA. 2012. Production of a Thermostable and Alkaline Chitinase by Bacillus thuringiensis subsp. kurstaki Strain HBK-51, Biotechnol. Res. Int. 2012: 135498.
Lamine BM, Lamine BM, Bouziane A. 2012. Optimization of the Chitinase Production by Serratia Marcescens DSM 30121T and Biological Control of Locusts. Journal of Biotechnology and Biomaterials, 2, 13-138. http://dx.doi.org/10.4172/2155-952X.1000133
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Mageswari A, Subramanian P, Chandrasekaran S, Karthikeyan S, Gothandam KM. 2017. Systematic Functional Analysis and Application Of A Cold-Active Serine Protease from a Novel Chryseobacterium sp. Food Chem. 217: 18–27.
Mulyawati AI, Jatmiko YD, Mustafa I, Ardyati T, Suharjono. 2019. Diversity of Lactic Acid Bacteria Isolated from Fermented Mare’s Milk Products Based on PCR-RFLP analysis. EES 230: 012104.
Nelson DL, and Cox MM. 2005. Lehninger: Principles of Biochemistry. WH Freeman and Company, New York.
Nouri M, Khodaiyan F, Razavi SH, Mousavi M. 2016. Improvement of Chitosan Production from Persian Gulf Shrimp Waste by Response Surface Methodology. Food Hydrocolloid. 59: 50–58.
Ordó˜nez-Del Pazo T, Antelo LT, Franco-Uría A, Pérez-Martín RI, Sotelo CG, Alonso AA. 2014. Fish Discards Management in Selected Spanish and Portuguese Métiers: Identi?cation and Potential Valorisation, Trends Food Sci. Technol. 36 : 29–43.
Pant G, Prakash A, Pavani JVP, Bera S, Deviram GVNS, Kumar A, Prasuna RG. 2015. Production, Optimization and Partial Purification of Protease from Bacillus subtilis. JTUSCI 9: 50–55.
Rachanamol RS, Lipton AP, Thankamani V, Sarika AR, Selvin J. 2017. Production of Protease Showing Antibacterial Activity by Bacillus subtilis VCDA Associated with Tropical Marine Sponge Callyspongia diffusa. J. Microbial & Biochemical Technol. 9:6
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. 1998. Molecular and Biotechnological Aspects of Microbial Proteases. Microbiol. Mol. Biol. Rev. 62: 597–635.
Raut S, Sen SK, Kabir NA, Satpathy S, Raut S. 2012. Isolation and Characterization of Protease Producing Bacteria from Upper Respiratory Tract of Wild Chicken. Bioinformation 8: 326–330.
Riniwati H, Harahab N, Carla TY. 2017. Analysis of Indonesia Crab Export Competitiveness In International Market. IRMM 7: 23-27.
Rustad T, Storro I, Slizyte R. 2011. Possibilities for The Utilization Of Marine by-Products. Int. J. Food Sci. Technol. 46: 2001–2014.
Saima, Kuddus M, Roohi, Ahmad IZ. 2013. Isolation of Novel Chitinolytic Bacteria and Production Optimization of Extracellular Chitinase. JGEB. 11: 39–46.
Saleem F, Younas A, Bashir R, Naz S, Munir N, Shakoori AR. 2014. Molecular Cloning and Characterization of Exochitinase a Gene of Indigenous Bacillus thuringiensis Isolates. Pakistan Journal of Zoology. 46 :1491-1501.
Sanmartin E, Arboleya JC, Iloro I, Escuredo K, Elortza F, Moreno FJ. 2012. Proteomic Analysis Of Processing By-Products from Canned And Fresh Tuna: Identi?cation of Potentially Functional Food Proteins. Food Chem. 134: 1211–1219.
Senol M, Nadaroglu H, Dikbas N, Kotan R. 2014. Purification of Chitinase Enzymes From Bacillus Subtilis Bacteria TV-125, Investigation of Kinetic Properties And Antifungal Activity Against Fusarium culmorum. Ann. Clin. Microbiol. Antimicrob. 13: 35.
Senphan T, Benjakul S, Kishimura H. 2014. Characteristics and Antioxidative Activity Of Carotenoproteins from Shells of Paci?c White Shrimp Extracted Using Hepatopancreas Proteases. Food Biosci. 5 : 54–63.
Setia IN, and Suharjono. 2015. Chitinolytic Assay and Identification of Bacteria Isolated from Shrimp Waste Based on 16S rDNA Sequences. Advances in Microbiology. 5: 58153.
Susanto JP, and Sopiah N. 2003. Effect of Metals and Concentration on Growth and Activity of Proteolytic Bacteria in the Deproteination Process of Small Skin. J. Environ Technol. 4: 40-46.
Tennalli G, Udapudi B, Naik P. 2012. Isolation of Proteolytic Bacteria and Characterization of their Proteolytic Activity. IJAEST 2: 185-192.
Tolaimate A, Desbrieres J, Rhazia M, Alagui A. 2003. Contribution to The Preparation of Chitins and Chitosans with Controlled Physico-Chemical Properties. Polymer 44 : 7939–7952.
Zarei M, Aminzadeh S, Zolgharnein H, Safahieh A, Daliri M, Noghabi KA, Motallebi A. 2011. Characterization of a Chitinase with Antifungal Activity from a Native Serratia marcescens B4A. Braz. J. Microbiol. 42: 1017–1029.
Azuma K, Ifuku S, Osaki T, Okamoto Y, Minami S. 2014. Preparation and Biomedical Applications of Chitin and Chitosan Nanofibers. J. Biomed. Nanotechnol. 10: 2891–2920.
Bailly JE, and Ollis DF. 1986. Biochemical Engineering Fundamentals. McGraw-Hill, New York.
Cupp-Enyard C. 2008. Sigma’s Non-specific Protease Activity Assay - Casein as a Substrate. JoVE 19: 899.
Degering C, Eggert T, Puls M, Bongaerts J, Evers S, Maurer KH, Jaeger KE. 2010. Optimization of Protease Secretion in Bacillus subtilis and Bacillus licheniformis by Screening of Homologous and Heterologous Signal Peptides. Appl. Environ. Microbiol. 76: 6370–6376.
Elieh-Ali-Komi D, and Hamblin MR. 2016. Chitin and Chitosan: Production and Application of Versatile Biomedical Nanomaterials. IJAR 4: 411–427.
Faramarzi M, Fazeli M, Yazdi MT, Adrangi S, Al Ahmadi KJ, Tasharrofi N, Mohseni FA. 2009. Optimization of Cultural Conditions for Production of Chitinase by a Soil Isolate of Massilia timonae. Biotechnology 8: 93-99.
Fulzele R, DeSa E, Yadav A, Shouche Y, Bhadekar R. 2011. Characterization of Novel Extracellular Protease Produced by Marine Bacterial Isolate From The Indian Ocean. Braz. J. Microbiol. 42: 1364–1373.
Gadgey K, and Bahekar A. 2017. Investigation of Mechanichal Properties of Crab Shell: A Review. IJLTET 1: 268–281.
Gomaa EZ. 2012. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: Their potential in Antifungal Biocontrol. J. Microbiol. 50: 103–111.
Gortari MC, and Hours RA. 2013. Biotechnological Processes for Chitin Recovery Out of Crustacean Waste: A Mini-Review, Electron. J. Biotechnol. 16 :14
Hajji S, Younes I, Ghorbel-Bellaaj O, Hajji R, Rinaudo M., Nasri M, Jellouli K. 2014. Structural Differences Between Chitin and Chitosan Extracted from Three Different Marine Sources. Int. J. Biol. Macromol. 65 : 298–306.
Hammami I, Siala R, Jridi M, Ktari N, Nasri M, Triki MA. 2013. Partial Purification and Characterization of Chiio8, A Novel Antifungal Chitinase Produced by Bacillus cereus IO8. J. Appl. Microbiol. 115: 358–366.
Hsieh YC, Wu YJ, Chiang TY, Kuo CY, Shrestha KL, Chao CF, Chen CJ. 2010. Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis. J. Biol. Chem. 285: 31603–31615.
Jayakumar R, Prabaharan M, Sudheesh KPT, Nair SV, Tamura H. 2011. Biomaterials Based on Chitin and Chitosan in Wound Dressing Applications. Biotechnol. Adv. 29: 322–337.
Jo GH, Jung WJ, Kuk JH, Oh KT, Kim YJ, Park RD. 2008. Screening of Protease-Producing Serratia marcescens FS-3 and Its Application To Deproteinization of Crab Shell Waste for Chitin Extraction. Carbohydrate Polymers 74:504–508
Karadag D, Mäkinen AE, Efimova E, Puhakka JA. 2009. Thermophilic Biohydrogen Production by An Anaerobic Heat Treated-Hot Spring Culture. Bioresour. Technol. 100: 5790–5795.
Kraisangsri J, Nalinanon S, Riebroy S, Yarnpakdee S, Ganesan P. 2018. Pysicochemical Characteristics of Glucosamine From Blue Swimming Crab (Portunus pelagicus) Shell Prepared by Acid Hydrolysis. Walailak J Sci & Tech. 15: 869-877.
Kumari S, and Rath PK. 2012. Extraction and Characterization of Chitin and Chitosan From (Labeo Rohit) Fsh Scales, Proc. Mater. Sci. 6 : 482–489.
Kuzu SB, Güvenmez HK, Denizci AA. 2012. Production of a Thermostable and Alkaline Chitinase by Bacillus thuringiensis subsp. kurstaki Strain HBK-51, Biotechnol. Res. Int. 2012: 135498.
Lamine BM, Lamine BM, Bouziane A. 2012. Optimization of the Chitinase Production by Serratia Marcescens DSM 30121T and Biological Control of Locusts. Journal of Biotechnology and Biomaterials, 2, 13-138. http://dx.doi.org/10.4172/2155-952X.1000133
Lopes C, Antelo LT, Franco-Uría A, Alonso AA, Pérez-Martín R. 2017. Chitin Production From Crustacean Biomass: Sustainability Assessment of Chemical and Enzymatic Processes, J. Clean. Prod. http://dx.doi.org/10.1016/j.jclepro.2017.01.082
Mageswari A, Subramanian P, Chandrasekaran S, Karthikeyan S, Gothandam KM. 2017. Systematic Functional Analysis and Application Of A Cold-Active Serine Protease from a Novel Chryseobacterium sp. Food Chem. 217: 18–27.
Mulyawati AI, Jatmiko YD, Mustafa I, Ardyati T, Suharjono. 2019. Diversity of Lactic Acid Bacteria Isolated from Fermented Mare’s Milk Products Based on PCR-RFLP analysis. EES 230: 012104.
Nelson DL, and Cox MM. 2005. Lehninger: Principles of Biochemistry. WH Freeman and Company, New York.
Nouri M, Khodaiyan F, Razavi SH, Mousavi M. 2016. Improvement of Chitosan Production from Persian Gulf Shrimp Waste by Response Surface Methodology. Food Hydrocolloid. 59: 50–58.
Ordó˜nez-Del Pazo T, Antelo LT, Franco-Uría A, Pérez-Martín RI, Sotelo CG, Alonso AA. 2014. Fish Discards Management in Selected Spanish and Portuguese Métiers: Identi?cation and Potential Valorisation, Trends Food Sci. Technol. 36 : 29–43.
Pant G, Prakash A, Pavani JVP, Bera S, Deviram GVNS, Kumar A, Prasuna RG. 2015. Production, Optimization and Partial Purification of Protease from Bacillus subtilis. JTUSCI 9: 50–55.
Rachanamol RS, Lipton AP, Thankamani V, Sarika AR, Selvin J. 2017. Production of Protease Showing Antibacterial Activity by Bacillus subtilis VCDA Associated with Tropical Marine Sponge Callyspongia diffusa. J. Microbial & Biochemical Technol. 9:6
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. 1998. Molecular and Biotechnological Aspects of Microbial Proteases. Microbiol. Mol. Biol. Rev. 62: 597–635.
Raut S, Sen SK, Kabir NA, Satpathy S, Raut S. 2012. Isolation and Characterization of Protease Producing Bacteria from Upper Respiratory Tract of Wild Chicken. Bioinformation 8: 326–330.
Riniwati H, Harahab N, Carla TY. 2017. Analysis of Indonesia Crab Export Competitiveness In International Market. IRMM 7: 23-27.
Rustad T, Storro I, Slizyte R. 2011. Possibilities for The Utilization Of Marine by-Products. Int. J. Food Sci. Technol. 46: 2001–2014.
Saima, Kuddus M, Roohi, Ahmad IZ. 2013. Isolation of Novel Chitinolytic Bacteria and Production Optimization of Extracellular Chitinase. JGEB. 11: 39–46.
Saleem F, Younas A, Bashir R, Naz S, Munir N, Shakoori AR. 2014. Molecular Cloning and Characterization of Exochitinase a Gene of Indigenous Bacillus thuringiensis Isolates. Pakistan Journal of Zoology. 46 :1491-1501.
Sanmartin E, Arboleya JC, Iloro I, Escuredo K, Elortza F, Moreno FJ. 2012. Proteomic Analysis Of Processing By-Products from Canned And Fresh Tuna: Identi?cation of Potentially Functional Food Proteins. Food Chem. 134: 1211–1219.
Senol M, Nadaroglu H, Dikbas N, Kotan R. 2014. Purification of Chitinase Enzymes From Bacillus Subtilis Bacteria TV-125, Investigation of Kinetic Properties And Antifungal Activity Against Fusarium culmorum. Ann. Clin. Microbiol. Antimicrob. 13: 35.
Senphan T, Benjakul S, Kishimura H. 2014. Characteristics and Antioxidative Activity Of Carotenoproteins from Shells of Paci?c White Shrimp Extracted Using Hepatopancreas Proteases. Food Biosci. 5 : 54–63.
Setia IN, and Suharjono. 2015. Chitinolytic Assay and Identification of Bacteria Isolated from Shrimp Waste Based on 16S rDNA Sequences. Advances in Microbiology. 5: 58153.
Susanto JP, and Sopiah N. 2003. Effect of Metals and Concentration on Growth and Activity of Proteolytic Bacteria in the Deproteination Process of Small Skin. J. Environ Technol. 4: 40-46.
Tennalli G, Udapudi B, Naik P. 2012. Isolation of Proteolytic Bacteria and Characterization of their Proteolytic Activity. IJAEST 2: 185-192.
Tolaimate A, Desbrieres J, Rhazia M, Alagui A. 2003. Contribution to The Preparation of Chitins and Chitosans with Controlled Physico-Chemical Properties. Polymer 44 : 7939–7952.
Zarei M, Aminzadeh S, Zolgharnein H, Safahieh A, Daliri M, Noghabi KA, Motallebi A. 2011. Characterization of a Chitinase with Antifungal Activity from a Native Serratia marcescens B4A. Braz. J. Microbiol. 42: 1017–1029.
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