Login Register Cart 0
Generic placeholder image

Current Enzyme Inhibition

Editor-in-Chief

ISSN (Print): 1573-4080
ISSN (Online): 1875-6662

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Kinetic Behaviour of Amylase According to pH: A New Perspective for Starch Hydrolysis Process

Author(s): Ravneet K. Grewal*, Baldeep Kaur and Gagandeep Kaur

Volume 16, Issue 2, 2020

Page: [135 - 144] Pages: 10

DOI: 10.2174/1573408016666200316114808

Price: $65

Abstract

Background: Amylases are the most widely used biocatalysts in starch saccharification and detergent industries. However, commercially available amylases have few limitations viz. limited activity at low or high pH and Ca2+ dependency.

Objective: The quest for exploiting amylase for diverse applications to improve the industrial processes in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence of metal ions as a function of pH.

Methods: The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of ions, as a function of substrate concentration in Lineweaver-Burk Plot.

Results: Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and 7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5.

Conclusion: These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore, a time-saving and cost-effective solution is proposed to overcome the challenges of the existing methodology of starch hydrolysis in starch and detergent industries.

Keywords: Activator, amylase detergent, inhibitor, saccharification, starch liquefaction.

« Previous Next »
Graphical Abstract

[1]
de Sauza, P.M.; Magalhaes, P.O Application of microbial α amylase in industry - A review. Braz. J. Microbiol., 2010, 41, 850-41861.
[2]
Gopinath, S.C.B.; Anbu, P.; Arshad, M.K.; Lakshmipriya, T.; Voon, C.H.; Hashim, U.; Chinni, S.V. Biotechnological processes in microbial amylase production. BioMed Res. Int., 2017, 20171272193
[http://dx.doi.org/10.1155/2017/1272193] [PMID: 28280725]
[3]
Sharma, A.; Satyanrayana, T. Microbial acid-stable α- amylases: Characteristics, genetic engineering and applications. Process Biochem., 2013, 48, 201-211.
[http://dx.doi.org/10.1016/j.procbio.2012.12.018]
[4]
Wang, X.; Kan, G.; Ren, X.; Yu, G.; Shi, C.; Xie, Q.; Wen, H.; Betenbaugh, M. Molecular cloning and characterization of a novel α-amylase from antarctic sea ice Bacterium Pseudoalteromonas sp. M175 and its primary application in detergent. BioMed Res. Int., 2018, 20183258383
[http://dx.doi.org/10.1155/2018/3258383] [PMID: 30050926]
[5]
Karaki, N.; Aljawish, A.; Humeau, C.; Muniglia, L.; Jasniewski, J. Enzymatic modification of polysaccharides: Mechanisms, properties, and potential applications: A review. Enzyme Microb. Technol., 2016, 90, 1-18.
[http://dx.doi.org/10.1016/j.enzmictec.2016.04.004] [PMID: 27241287]
[6]
Mehta, D.; Satyanarayana, T. Bacterial and archaeal α-amylases: diversity and amelioration of the desirable characteristics for industrial applications. Front. Microbiol., 2016, 7, 1129-1150.
[http://dx.doi.org/10.3389/fmicb.2016.01129] [PMID: 27516755]
[7]
Simair, A.A.; Qureshi, A.S.; Khushk, I.; Ali, C.H.; Lashari, S.; Bhutto, M.A.; Mangrio, G.S.; Lu, C. Production and partial characterization of α-amylase enzyme from bacillus sp. BCC 01-50 and potential applications. BioMed Res. Int., 2017, 2017, 9173040-9173049.
[http://dx.doi.org/10.1155/2017/9173040] [PMID: 28168200]
[8]
Ali, E.H.; El-Nagdy, M.A.; Al-Garni, S.M.; Ahmed, M.S.; Rawaa, A.M. Enhancement of alpha amylase production by Aspergillus flavus 11685 on mandarin (Citrus reticulate) peel using submerged fermentation. Eur. J. Biol. Res., 2017, 7(3), 154-164.
[9]
Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P.M.; Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res., 2014, 42(Database issue), D490-D495.
[http://dx.doi.org/10.1093/nar/gkt1178] [PMID: 24270786]
[10]
Prabha, T.R. A simple method for total genomic DNA extraction from water moulds. Curr. Sci., 2013, 104(3), 345-347.
[11]
White, T.J.; Bruns, T.; Lee, S.; Taylor, J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications; Inn, M.A.; Gelfand, D.H.; Sninsky, J.J; White, T.J., Ed.; Academic Press Inc.: New York, 1990, pp. 315-322.
[12]
Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol., 1990, 215(3), 403-410.
[http://dx.doi.org/10.1016/S0022-2836(05)80360-2] [PMID: 2231712]
[13]
Kim, O.S.; Cho, Y.J.; Lee, K.; Yoon, S.H.; Kim, M.; Na, H.; Park, S.C.; Jeon, Y.S.; Lee, J.H.; Yi, H.; Won, S.; Chun, J. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol., 2012, 62(Pt 3), 716-721.
[http://dx.doi.org/10.1099/ijs.0.038075-0] [PMID: 22140171]
[14]
Gertz, G.M. BLAST Scoring Parameters, 2005 March; 16..
[15]
States, D.J.; Gish, W.; Altschul, S.F. Improved sensitivity of nucleic acid database searches using application-specific scoring matrices. Methods, 1991, 3, 6-70.
[http://dx.doi.org/10.1016/S1046-2023(05)80165-3]
[16]
Karlin, S.; Altschul, S.F. Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc. Natl. Acad. Sci. USA, 1990, 87(6), 2264-68.
[http://dx.doi.org/10.1073/pnas.87.6.2264]
[17]
Myers, E.W.; Miller, W. Optimal alignments in linear space. Comput. Appl. Biosci., 1988, 4(1), 11-17.
[PMID: 3382986]
[18]
Saitou, N.; Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 1987, 4(4), 406-425.
[PMID: 3447015]
[19]
Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol., 2013, 30(12), 2725-2729.
[http://dx.doi.org/10.1093/molbev/mst197] [PMID: 24132122]
[20]
Annamalai, N.; Thavasi, R.; Vijayalakshmi, S.; Balasubramanian, T. Extraction, Purification and characterization of thermostable, alkaline tolerant α- amylase from Bacillus cereus. Indian J. Microbiol., 2011, 51(4), 424-429.
[http://dx.doi.org/10.1007/s12088-011-0160-z] [PMID: 23024403]
[21]
Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951, 193(1), 265-275.
[PMID: 14907713]
[22]
Miller, G.L. Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Anal. Chem., 1959, 31, 426-429.
[http://dx.doi.org/10.1021/ac60147a030]
[23]
Ali, S.; Hossain, Z. Characteristics for glucoamylase from Aspergillus terreus. J. Appl. Bacteriol., 1991, 71, 44-146.
[http://dx.doi.org/10.1111/j.1365-2672.1991.tb02970.x]
[24]
Carlson, M.; Sphor, A.B.; Nielson, J.; Villadesh, J. Morphology and physiology of an α-amylase producing strain of Aspergillus oryzae during batch cultivation. Biotechnol. Bioeng., 1996, 4, 266-276.
[http://dx.doi.org/10.1002/(SICI)1097-0290(19960205)49:3<266:AID-BIT4>3.0.CO;2-I]
[25]
Mamo, G.; Gashe, A.B.; Gessesse, A. A highly thermostable amylase from a newly isolated thermophilic Bacillus sp. J. Appl. Microbiol., 1999, 86, 557-560.
[http://dx.doi.org/10.1046/j.1365-2672.1999.00685.x]
[26]
Wang, J.; Li, Yu.; Lu, F. Molecular cloning and biochemical characterization of an α-amylase family from Aspergillus niger. Electron. J. Biotechnol., 2018, 32, 55-62.
[http://dx.doi.org/10.1016/j.ejbt.2018.01.004]
[27]
Bozic, N.; Ruiz, J.; Santin, J.L.; Vujcic, Z. Production and properties of the highly efficient raw starch digesting α-amylase from a Bacillus licheniformis ATCC 9945a. Biochem. Eng. J., 2011, 53(2), 203-209.
[http://dx.doi.org/10.1016/j.bej.2010.10.014]
[28]
Abdel-Fattah, Y.R.; Soliman, A.N.; El-Toukhy, N.M.; El-Gendi, H.; Ahmed, R.S. Production, purification, and characterization, of thermotable α-amylase produced by Bacillus licheniformis isolate A120. J. Chem., 2013, 673173-673184.
[29]
da Silva, T.M.; Maller, A. Evidence of high production levels of thermostable dextrinizing and saccharogenic amylase by A. niger. Afr. J. Biotechnol., 2013, 12(15), 1874-1881.
[http://dx.doi.org/10.5897/AJB12.2830]
[30]
Agüloğlu Fincan, S.; Enez, B.; Özdemir, S.; Matpan Bekler, F. Purification and characterization of thermostable α-amylase from thermophilic Anoxybacillus flavithermus. Carbohydr. Polym., 2014, 102, 144-150.
[http://dx.doi.org/10.1016/j.carbpol.2013.10.048] [PMID: 24507266]
[31]
Adegoke, S.A.; Odibo, F.J.C. Production, purification and characterization of α-amylase of Aspergillus sydowii IMI 502692 Plant cell biotechnology and molecular biology., 2019, 20(23-24), 1050-1058.
[32]
Sethi, B.K.; Nanda, P.K.; Sahoo, S.; Sena, S. Characterization of purified α-amylase produced by Aspergillus terreus NCFT 4269.10 using pearl millet as substrate. Cogent Food Agric., 2016, 2, 1-11.
[http://dx.doi.org/10.1080/23311932.2016.1158902]
[33]
Femi-Ola, T.O.; Olowe, B.M. Characterization of alpha amylase from Bacillus subtilis BS5 from Amitermes evuncifer Silvestri, Research. J. Microbiol., 2011, 6(2), 140-146.
[34]
Vyas, G.; Sharma, N.; Sharma, N. Purification and characterization of α-amylase from a novel thermoalkalophilic strain of Bacillus sonorensis GV2 isolated from mushroom compost. Int. Res. J. Pure Appl. Chem., 2019, 19(3), 1-14.
[http://dx.doi.org/10.9734/irjpac/2019/v19i330111]
[35]
Abdulaal, W.H. Purification and characterization of α-amylase from Trichoderma pseudokoningii. BMC Biochem., 2018, 19(1), 4.
[http://dx.doi.org/10.1186/s12858-018-0094-8] [PMID: 29902965]
[36]
Okwuenu, P.C.; Agbo, K.U.; Ezugwu, A.L.; Eze, S.O.; Chilaka, F.C. Effects of divalent metal ions on glucomylase activity of glucoamylase isolated from A. niger. Ferment. Technol., 2017, 6(1), 1-6.
[37]
Boel, E.; Brady, L.; Brzozowski, A.M.; Derewenda, Z.; Dodson, G.G.; Jensen, V.J.; Petersen, S.B.; Swift, H.; Thim, L.; Woldike, H.F. Calcium binding in α-amylases: an X-ray diffraction study at 2.1-A resolution of two enzymes from Aspergillus. Biochemistry, 1990, 29(26), 6244-6249.
[http://dx.doi.org/10.1021/bi00478a019] [PMID: 2207069]
[38]
Machius, M.; Declerck, N.; Huber, R.; Wiegand, 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(3), 281-292.
[http://dx.doi.org/10.1016/S0969-2126(98)00032-X] [PMID: 9551551]
[39]
Larson, S.B.; Greenwood, A.; Cascio, D.; Day, J.; McPherson, A. Refined molecular structure of pig pancreatic α-amylase at 2.1 A resolution. J. Mol. Biol., 1994, 235(5), 1560-1584.
[http://dx.doi.org/10.1006/jmbi.1994.1107] [PMID: 8107092]
[40]
Brzozowski, A.M.; Lawson, D.M.; Turkenburg, J.P.; Bisgaard Frantzen, H.; Svendsen, A.; Borchert, T.V.; Dauter, Z.; Wilson, K.S.; Davies, G.J. Structural analysis of native and ligand complexes. Biochemistry, 2000, 39, 9099-9107.
[http://dx.doi.org/10.1021/bi0000317] [PMID: 10924103]
[41]
Suvd, D.; Fujimoto, Z.; Takase, K.; Matsumura, M.; Mizuno, H. Crystal structure of Bacillus stearothermophilus α-amylase: possible factors determining the thermostability. J. Biochem., 2001, 129(3), 461-468.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a002878] [PMID: 11226887]
[42]
Ghollasi, M.; Khajeh, K.; Naderi-Manesh, H.; Ghasemi, A. Engineering of a Bacillus α-amylase with improved thermostability and calcium independency. Appl. Biochem. Biotechnol., 2010, 162(2), 444-459.
[http://dx.doi.org/10.1007/s12010-009-8879-2] [PMID: 20177823]
[43]
Gupta, R.; Gigra, P.; Mohapatra, H.; Kuma, V.G.; Chauhan, B. Microbial α-amylases: A biotechnological perspective. Process Biochem., 2003, 38, 1599-1616.
[http://dx.doi.org/10.1016/S0032-9592(03)00053-0]
[44]
Bedade, D.; Deska, J.; Bankar, S.; Bejar, S.; Singhal, R.; Shamekh, S. Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization. Prep. Biochem. Biotechnol., 2018, 48(6), 549-555.
[http://dx.doi.org/10.1080/10826068.2018.1476876] [PMID: 29889602]
[45]
Machovic, M.; Stefan, J. Amlyotic enzymes: Types, Structure and specificities.. In: J. Polaina, MacCabe AP, Eds., Industrial Enzymes: Structure, Function and Applications,; Springer: Netherlands, 2007, pp. 3- 18.
[46]
Declerck, N.; Machius, M.; Wiegand, G.; Huber, R.; Gaillardin, C. Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J. Mol. Biol., 2000, 301(4), 1041-1057.
[http://dx.doi.org/10.1006/jmbi.2000.4025] [PMID: 10966804]
[47]
Priyadharshini, R.; Gunasekaran, P. Site-directed mutagenesis of the calcium-binding site of α-amylase of Bacillus licheniformis. Biotechnol. Lett., 2007, 29(10), 1493-1499.
[http://dx.doi.org/10.1007/s10529-007-9428-0] [PMID: 17598074]
[48]
Antranikian, G. Microbial degradation of starch.Microbial degradation of natural products; Winkelmann, G., Ed.; VCH: Germany, 1992, pp. 27-56.
[49]
Satyanaryana, T.; Rao, J.L.U.M.; Ezhilvannan, M. α- amylase. In:Enzyme Technology; Pandey, A.; Webb, C.; Soccol, CA.; Larroche, C., Eds.; Asiatech Publishers:: New Delhi,, 2005; pp. 89- 220.

Rights & Permissions Print Cite
Article Metrics
18
© 2024 Bentham Science Publishers | Privacy Policy