Impact of Metal Ion Substitution on the Activity and Stability of Saccharifying Raw Starch Digesting Amylase from Aspergillus carbonarius
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Published
Jan 7, 2020
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1-11
Main Article Content
Tochukwu Nwamaka Nwagu
Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka Campus, Nigeria.
Hideki Aoyagi
Department of Life Sciences and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan.
Bartholomew Okolo
Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka Campus, Nigeria.
Abstract
Though raw starch digesting amylases can be utilized in numerous bioprocesses, poor activity and stability remain a limiting factor. In this study, the effect of metal ion substitution on the activity and stability of the RSDA from Aspergillus carbonarius was investigated. The amylase was inactivated using the chelating agent ethylene di aminotetraacetic acid (EDTA) and reactivated using different metal ions. The effect of different metal ions on the reactivation of the amylase activity was investigated. Impact of the metal ions on the stability of the amylase was also studied. Kinetic constants of the native enzyme were compared to the metal reactivated holoenzyme. Most efficient was 5 mM concentration of Co2+ with 94.6% activity recovery. Others included 5 mM Zn2+ (77.7%) and 5 mM Ca2+ (68.7%). Incubating the Co2+ activated amylase in 10 mM Mn2+ further stimulated the activity of the amylase to 136.7%. Compared to the metal ions tested, Mn2+ had the most stabilizing effect on the amylase; the amylase exhibited 148.2% and 136.5% activity at 70ºC and 80ºC respectively in the presence of 5 mM Mn2+. Ca2+ inhibited the amylase activity and inhibition rate increased with increasing concentration of Ca2+ concentrations. Km of the reactivated amylase was 0.18 mg/ml.
Keywords:
Metal ion substitution, raw starch hydrolysis, activity, stability, re-activation.
Article Details
How to Cite
Nwamaka Nwagu, T., Aoyagi, H., & Okolo, B. (2020). Impact of Metal Ion Substitution on the Activity and Stability of Saccharifying Raw Starch Digesting Amylase from Aspergillus carbonarius. Journal of Advances in Biology & Biotechnology, 22(4), 1-11. https://doi.org/10.9734/jabb/2019/v22i430127
Section
Original Research Article
References
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Available:https://www.ncbi.nlm.nih.gov/pubmed/12719434
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digesting amylase from Aspergillus carbonarius. Journal of Science of Food and Agriculture. 2001;81:329–336.
Available:https://onlinelibrary.wiley.com/doi/abs/10.1002/1097-0010%28200102% 2981%3A3%3C329%3A%3AAID-JSFA 815%3E3.0.CO%3B2-3
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options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius. International Journal
of Biological Macro-molecule. 2017;99:641-647.
Available:https://doi.org/10.1016/j.ijbiomac.2017.03.022
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Available:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107500/
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Available:https://www.ajol.info/index.php/ajb/article/view/15097/91003
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DOI: 10.3923/ajb.2011
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Nwagu TN, Okolo BN, Aoyagi H. Stabilization of a raw-starch-digesting amylase by multipoint covalent attachment on glutaraldehyde-activated amberlite beads. Journal of Microbiology Bio-technology. 2012;22:628-636.
Available:http://www.academicjournals.org/app/webroot/article/article1387208446_Nwagu%20et%20al.pdf
Belmonte L, Mansy SS. Metal catalysts and the origin of life. Elements. 2016; 12(6):413-418. Available:https://doi.org/10.2113/gselements.12.6.413
Zhou W, Liu J. Multi-metal-dependent nucleic acid enzymes. Critical Rev. Metallomics. 2018;10:30-48.
Available:https://www.ncbi.nlm.nih.gov/pubmed/29094140
Nonaka T, Fujihashi M, Kita A, Hagihara H, Ozaki K, Ito S, Miki K. Crystal structure of calcium-free α-amylase from Bacillus sp. strain KSM-K38 (AmyK38) and its sodium ion binding sites. Journal of Biology and Chemistry. 2003;278:24818-24824.
Available:https://www.ncbi.nlm.nih.gov/pubmed/12719434
Saboury AA, Karbassi F. Thermodynamic studies on the interaction of calcium ions with alpha-amylase. Thermochimica Acta. 2000;362:121-129.
Available:https://www.sciencedirect.com/science/article/abs/pii/S0040603100005797
Vielle C, Zeikus GJ. Hyperthermophilic enzymes: Sources, use, and molecular mechanisms for thermostability.
Microbiology and Molecular Biology Review. 2001;6:1-43.
Available:https://mmbr.asm.org/content/65/1/1.long
Machius M, Declerck N, Huber R, Weigand G. Activation of Bacillus licheniformis alpha-amylase through a disorder→order transition of the substrate-binding site mediated by a calcium–sodium–calcium metal triad. Structure. 1998;6:281–292.
Available:https://www.cell.com/structure/fulltext/S0969-2126(98)00032-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS096921269800032X%3Fshowall%3Dtrue
Buisson G, Duee E, Haser R, Payan F. Three dimensional structure of porcine pancreatic α-amylase at 2.9 Å
resolution. Role of calcium in structure and activity. The Embo Journal. 1987;6:3909-3916.
Available:https://www.ncbi.nlm.nih.gov/pubmed/3502087
McCall KA, Huang C, Fierke CA. Function and mechanism of zinc metalloenzymes. The Journal of Nutrition. 2000;130:1437S-1446S. Available:https://academic.oup.com/jn/article/130/5/1437S/4686409
Okwuenu PC, Agbo KU, Ezugwu AL, Eze SO, Chilaka FC. Effect of divalent metal ions on glucoamylase activity of glucoamylase isolated from Aspergillus niger. Fermentation Technology. 2017;6:1.
Available:https://www.longdom.org/abstract/effect-of-divalent-metal-ions-on-glucoamylase-activity-of-glucoamylase-isolated-from-aspergillus-niger-24232.html
D' Amico S, Sohier JS, Feller G. Kinetics and energetics of ligand binding determined by microcalorimetry: Insights
into active site mobility in a psychrophilic α-amylase. Journal of Molecular Bioliogy. 2006;358(5):1296-1304.
Available:https://www.ncbi.nlm.nih.gov/pubmed/16580683
Radfar R, Leaphart A, Brewer JM, Minor W, Odom JD, Dunlap RB. Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica. Bio-chemistry. 2000;39:14481–14486.
Available:https://www.ncbi.nlm.nih.gov/pubmed/11087401
Morokuma K, Musaev DG, Vreven T, Basch TH, Torrent M, Khoroshun DV. Model studies of the structures, reactivities and reaction mechanisms of meta-lloenzymes. IBM Journal of Research and Development. 2001;45: 367–395.
Available:https://ieeexplore.ieee.org/document/5389058
Mulrooney SB, Hausinger RP. Metal ion dependence of recombinant Escherichia coli allantoinase. Journal of Bacteriology. 2003;185:126-134.
Available:https://jb.asm.org/content/185/1/126.long
Wojciechowski CL, Cardia JP, Kantrowitz ER. Alkaline phosphatase from the hyper thermophilic bacterium Thermotoga
maritime requires cobalt for activity. Protein Science. 2002;11:903-911. Available:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373536/
Adamczak M, Krishna SH. Strategies for improving enzyme for efficient biocatalysis. Food Technology and Biotechnology. 2004;42:251–264.
Available:https://www.researchgate.net/publication/229052820_Strategies_for_Improving_Enzymes_for_Efficient_Biocatalysis
Nwagu TN, Okolo B, Aoyagi H, Yoshida S. Improved yield and stability of amylase by multipoint covalent binding on glutaral-dehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions. Process Biochemistry. 2013;48:1031-1038. Available:https://www.sciencedirect.com/science/article/abs/pii/S1359511313002110
Bano S, Qader SA, Aman A, Azhar A. Partial purification and some properties of α-amylase from Bacillus subtilis KIBGE-HAS. Indian Journal of Biochemistry and Biophysics. 2009;46:401-404.
Available:https://www.ncbi.nlm.nih.gov/pubmed/20027871
Zhang Z, Zhang R, Chen L, McClements DJ. Encapsulation of lactase (β-galactosidase) into k-carrageenan-based hydrogel beads: Impact of environmental conditions on enzyme activity. Food Chemistry. 2016;200:69-75.
Available:https://www.ncbi.nlm.nih.gov/pubmed/26830562
Najafi MF, Kembhavi A. One step purification and characterization of an extracellular α-amylase from marine Vibrio sp. Enzyme and Microbial Technology. 2005;36:535-539.
Available:https://doi.org/10.1016/j.enzmictec.2004.11.014
Miller GL. Use of dinitro-salicylic acid reagent for determination of reducing sugars. Analytical Chemistry. 1959;31: 426-428.
Available:https://pubs.acs.org/doi/pdf/10.1021/ac60147a030
Chen Y, Naik SG, Krzstek J, Shin S, Nelson WH, Xue S, Yang JJ, Davidson VL, Liu A. Role of calcium in metalloenzymes:
Effects of calcium removal on the axial ligation geometry and magnetic properties of the catalytic diheme center in MauG. Biochemistry. 2012;51:1586−1597.
Available:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294375/
Okolo BN, Ire F, Ezeogu L, Anyanwu C, Odibo FJC. Purification and some properties of a novel raw starch-
digesting amylase from Aspergillus carbonarius. Journal of Science of Food and Agriculture. 2001;81:329–336.
Available:https://onlinelibrary.wiley.com/doi/abs/10.1002/1097-0010%28200102% 2981%3A3%3C329%3A%3AAID-JSFA 815%3E3.0.CO%3B2-3
Marchal LM, Jonkers J, Franke GT, De Gooijer CD, Tramper J. The effect of process conditions on the α-amylolytic
hydrolysis of amylopectin potato starch: An experimental design approach. Bio-technology and Bioengineering. 1999; 62:348–357.
Available:https://onlinelibrary.wiley.com/doi/abs/10.1002/%28SICI%291097-0290%2819990205%2962%3A3%3C348%3A%3AAID-BIT11%3E3.0.CO%3B2-F
Dahot MU, Saboury AA, Ghobadi S, Moosavi-Movahedi AA. Properties of alpha amylase from Moringa oleifera
seeds. Journal of Biological Sciences. 2001;1(8): 747-749.
Available:https://scialert.net/abstract/?doi=jbs.2001.747.749
Saxena R, Singh R. Amylase production by solid-state fermentation of agro-industrial wastes using Bacillus sp.
Brazilian Journal of Microbiology. 2011;42: 1334-1342.
Available:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768732/
Hashida Y, Inouye K. Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the
suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin. Journal of Biochemistry.
2007;141:879-888.
Available:https://www.ncbi.nlm.nih.gov/pubmed/17405797
Alabi G, Sanni D, Bamidele F, Adeleke. Purification and characterization of α-amylase from Bacillus subtilis
isolated from cassava processing sites. Journal of Bioremediation and Biodegradation. 2017; 8:6.
Available:https://www.omicsonline.org/open-access/purification-and-characterization-of-945amylase-
from-bacillus-subtilis-isolated-from-cassava-processing-sites-2155-6199-1000417-96162.html
Nwagu TN, Okolo B, Aoyagi H, Yoshida. Chemical modification with phthalic anhydride and chitosan: Viable
options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius. International Journal
of Biological Macro-molecule. 2017;99:641-647.
Available:https://doi.org/10.1016/j.ijbiomac.2017.03.022
Yin H, Yang Z, Nie X, Li S, Xuyang S, Gao C. et al. Functional and cooperative stabilization of a two-metal
(Ca, Zn) center in α-amylase derived from Flavo-bacteriaceae species. Scientific Reports. 2017;7:17933.
Available:https://www.nature.com/articles/s41598-017-18085-4
Ghosh M, Grunden AM, Dunn DM, Weiss W, Adams MW. Characterization of native and recombinant
forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyper thermophilic
archaeon Pyrococcus furiosus. Journal of Bacteriology. 1998;180:4781-4789.
Available:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107500/
Babu KR, Satyanarayana T. Extracellular calcium-inhibited alpha-amylase of Bacillus coagulans B 49.
Enzyme and Microbial Technology. 1993;15:1066–1069.
Available:https://doi.org/10.1016/0141-0229(93)90056-8
Rao MS, Reddy NS, Venkateswara G, Sambasiva Rao KRS. Studies on the extraction and characterization
of thermostable α-amylase from pericarp of Borassus indica. African Journal of Biotechnology. 2005;4:289-291.
Available:https://www.ajol.info/index.php/ajb/article/view/15097/91003
Prakash O, Jaiswal N, Pandey RK. Effect of metal ions, EDTA and sulfhydrl reagents on soybean
amylase activity. Asian Journal of Biochemistry; 2011.
DOI: 10.3923/ajb.2011
Available:https://scialert.net/fulltextmobile/?doi=ajb.2011.282.290
Bibi Z, Nawaz M, Salam I, Waqs M, Aman A, Ul Qader S. Significance of metal ions, solvents
and surfactants to improve the xylan degrading behaviour of β-1,4-D-xylanohydrolase from
Geobacillus stearothermophilus KIBGE-IB29. Bio-catalysis & Agricultural Biotechnology. 2018;17:242-246. Available:https://doi.org/10.1016/j.bcab.2018.11.028
Suvd D, Fujimoto Z, Takase K, Matsumura M, Mizuno H. Crystal structure of Bacillus stearothermophilus
α-amylase: Possible factors determining the thermostability. Journal of Biochemistry. 2001;129:461-468.
Available:https://www.jstage.jst.go.jp/article/biochemistry1922/129/3/129_3_461/_pdf/-char/en
Boel E, Brady L, Brzozowski AM, Derewenda Z, Dodson GG, Jensen VJ, Peterson SB, Swift H,
Thim L, Woldike HF. Calcium binding in α-amylases: An X-ray diffraction study at 2.1-Å resolution
of two enzymes from Aspergillus. Biochemistry. 1990;29:6244-6249. Available:https://www.ncbi.nlm.nih.gov/pubmed/2207069
Ceci LN, Lozano JE. Amylase for apple juice processing: Effects of pH, heat and Ca2+ ions.
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Available:https://www.ftb.com.hr/images/pdfarticles/2002/January-March/40-33.pdf
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