Abstract
An extracellular acid stable α-amylase from Paecilomyces variotii ATHUM 8891 (PV8891 α-amylase) was purified to homogeneity applying ammonium sulfate fractionation, ion exchange and gel filtration chromatography and exhibited a reduced molecular weight of 75 kDa. The purified enzyme was optimally active at pH 5.0 and 60 °C and stable in acidic pH (3.0–6.0). Km, vmax and kcat for starch hydrolysis were found 1.1 g L−1, 58.5 μmole min−1 (mg protein)−1, and 73.1 s−1, respectively. Amylase activity was marginally enhanced by Ca2+ and Fe2+ ions while Cu2+ ions strongly inhibited it. Thermodynamic parameters determined for starch hydrolysis (Εα, ΔH*, ΔG*, ΔS*, \({\Delta G}_{\mathrm{E}-\mathrm{S}}^{\mathrm{*}}\) and \({\Delta G}_{\mathrm{E}-\mathrm{T}}^{\mathrm{*}}\)) suggests an effective capacity of PV8891 α-amylase towards starch hydrolysis. Thermal stability of PV8891 α-amylase was assessed at different temperatures (30–80 οC). Thermodynamic parameters (\({E}_{\left(a\right)d}\), ΔH*, ΔG*, ΔS*) as well as the integral activity of a continuous system for starch hydrolysis by the PV8891 α-amylase revealed satisfactory thermostability up to 60 °C. The acidic nature and its satisfactory performance at temperatures lower than the industrially used amylases may represent potential applications of PV8891 α-amylase in starch processing industry.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abdulaal WH (2018) Purification and characterization of α-amylase from Trichoderma pseudokoningii. BMC Biochem 19:4. https://doi.org/10.1186/s12858-018-0094-8
Ademakinwa AN, Agunbiade MO, Ayinla ZA, Agboola FK (2019) Optimization of aqueous two-phase partitioning of Aureobasidium pullulans α-amylase via response surface methodology and investigation of its thermodynamic and kinetic properties. Int J Biol Macromol 140:833–841. https://doi.org/10.1016/j.ijbiomac.2019.08.159
Allala F, Bouacema K, Boucherba N, Azzouz Z, Mechri S, Sahnounc M, Benallaoua S, Hacene H, Jaouadi B, Bouanane-Darenfed A (2019) Purification, biochemical, and molecular characterization of a novel extracellular thermostable and alkaline α-amylase from Tepidimonas fonticaldi strain HB23. Int J Biol Macromol 132:558–574. https://doi.org/10.1016/j.ijbiomac.2019.03.201
Anindyawati T, Melliawati R, Ito K, Iizuka M, Minamiura N (1998) Three different types of α-amylases from Aspergillus awamori KT-11: their purifications, properties, and specificities. Biosci Biotech Bioch 62:1351–1357. https://doi.org/10.1271/bbb.62.1351
Asoodeh A, Alemi A, Heydari A, Akbari J (2013) Purification and biochemical characterization of an acidophilic amylase from a newly isolated Bacillus sp. DR90. Extremophiles 17:339–348. https://doi.org/10.1007/s00792-013-0520-1
Baks T, Bruins ME, Matser AM, Janssen AEM, Boom RM (2008) Effect of gelatinization and hydrolysis conditions on the selectivity of starch hydrolysis with α-amylase from Bacillus licheniformis. J Agric Food Chem 56(2):488–495. https://doi.org/10.1021/jf072217j
Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilising principle of protein dye binding. Analyt Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
da Silva OS, de Oliveira RL, de Carvalho Silva J, Converti A, Souza Porto T (2018) Thermodynamic investigation of an alkaline protease from Aspergillus tamarii URM4634: a comparative approach between crude extract and purified enzyme. Int J Biol Macromol 109:1039–1044. https://doi.org/10.1016/j.ijbiomac.2017.11.081
de Oliveira RL, da Silva OS, Converti A, Souza PT (2018) Thermodynamic and kinetic studies on pectinase extracted from Aspergillus aculeatus: free and immobilized enzyme entrapped in alginate beads. Int J Biol Macromol 115:1088–1093. https://doi.org/10.1016/j.ijbiomac.2018.04.154
Dey TB, Banerjee R (2015) Purification, biochemical characterization and application of α-amylase produced by Aspergillus oryzae IFO-30103. Biocatal Agric Biotechnol 4(1):83–90. https://doi.org/10.1016/j.bcab.2014.10.002
Dixon M, Webb EC (1979) Enzyme kinetics. In: Dixon M, Webb EC (eds) Enzymes. Academic Press, New York, pp 47–206
Eyring H, Stearn AE (1939) The application of the theory of absolute reaction rates to protein. Chem Rev 24:253–270. https://doi.org/10.1021/cr60078a005
Gangadharan D, Nampoothiri KM, Sivaramakrishnan S, Pandey A (2009) Biochemical characterization of raw-starch-digesting alpha amylase purified from Bacillus amyloliquefaciens. Appl Biochem Biotechnol 158:653–662. https://doi.org/10.1007/s12010-008-8347-4
Gouzi H, Depagne C, Thibaud C (2012) Kinetics and thermodynamics of the thermal inactivation of polyphenol oxidase in an aqueous extract from Agaricus bisporus. J Agric Food Chem 60:500–506. https://doi.org/10.1021/jf204104g
Goyal Ν, Gupta JK, Soni SK (2005) A novel raw starch digesting thermostable a-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme Microb. Technol 37(7):723–734. https://doi.org/10.1016/j.enzmictec.2005.04.017
Grootegoed JA, Lauwers AM, Heinen W (1973) Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus. Archiv Mikrobiol 90:223–232. https://doi.org/10.1007/BF00424974
Gummadi SN (2003) What is the role of thermodynamics on protein stability? Biotechnol Bioproc E 8:9–18. https://doi.org/10.1007/BF02932892
Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial a-amylases: a biotechnological perspective. Process Biochem 38(11):1599–1616. https://doi.org/10.1016/S0032-9592(03)00053-0
Hasmann FA, Gurpilhares DB, Roberto IC, Converti A, Pessoa A Jr (2007) New combined kinetic and thermodynamic approach to model glucose-6-phosphate dehydrogenase activity and stability. Enzyme Microb Technol 40:849–858. https://doi.org/10.1016/j.enzmictec.2006.06.017
Karam EA, Wahab WAA, Saleh SAA, Hassan ME, Kansoh AL, Esawy MA (2017) Production, immobilization and thermodynamic studies of free and immobilized Aspergillus awamori amylase. Int J Biol Macromol 102:694–703. https://doi.org/10.1016/j.ijbiomac.2017.04.033
Kikani BA, Singh SP (2012) The stability and thermodynamic parameters of a very thermostable and calcium-independent -amylase from a newly isolated bacterium, Anoxybacillus beppuensis TSSC-1. Process Biochem 47(12):1791–1798. https://doi.org/10.1016/j.procbio.2012.06.005
Kikani BA, Singh SP (2015) Enzyme stability, thermodynamics and secondary structures of a-amylase as probed by the CD spectroscopy. Int J Biol Macromol 81:450–460. https://doi.org/10.1016/j.ijbiomac.2015.08.032
Michelin M, Silva TM, Benassi VM, Peixoto-Nogueira SC, Moraes LAB, Leão JM, Jorge JA, Terenzi HF, Polizeli MLTM (2010) Purification and characterization of a thermostable a-amylase produced by the fungus Paecilomyces variotii. Carbohyd Res 345:2348–2353. https://doi.org/10.1016/j.carres.2010.08.013
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. https://doi.org/10.1021/ac60147a030
Nguyen QD, Rezessy-Szabó JM, Claeyssens M, Stals I, Hoschke Á (2002) Purification and characterisation of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus strain ATCC 34626. Enzyme Microb Technol 31:345–352. https://doi.org/10.1016/S0141-0229(02)00128-X
Pace CN (1992) Contribution of the hydrophobic effect to globular protein stability. J Mol Biol 226:29–35. https://doi.org/10.1016/0022-2836(92)90121-Y
Porto TS, Porto CS, Cavalcanti MTH, Filho JLL, Perego P, Porto ALF, Converti A, Pessoa A Jr (2006) Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima. Extract Biotechnol Prog 22:1637–1642. https://doi.org/10.1021/bp0602350
Rana N, Walia A, Gaur A (2013) a-Amylases from microbial sources and its potential applications in various industries. Natl Acad Sci Lett 36(1):9–17. https://doi.org/10.1007/s40009-012-0104-0
Ratanakhanokchai K, Kaneko J, Kamio Y, Izaki K (1992) Purification and properties of a maltotetraose and maltotriose producing amylase from Chloroflexus aurantiacus. Appl Environ Microbiol 58:2490–2494
Riaz M, Perveen R, Javed ΜΡ, Nadeem Η, Rashid ΜΗ (2007) Kinetic and thermodynamic properties of novel glucoamylase from Humicola sp. Enzyme Microb Technol 41:558–564. https://doi.org/10.1016/j.enzmictec.2007.05.010
Robyt J (2008) Starch: Structure, Properties, Chemistry and Enzymology. In: Fraser-Reid BO, Tatsuta K, Thiem J (eds) Glycoscience. Springer, Berlin, pp 1437–1472. https://doi.org/10.1007/978-3-540-30429-6_35
Samanta S, Das A, Halder SK, Jana A, Kar S, Mohapatra PKD, Pati BR, Mondal KC (2014) Thermodynamic and kinetic characteristics of an α-amylase from Bacillus licheniformis SKB4. Acta Biologica Szegediensis 58(2):147–156
Sharma A, Satyanarayana T (2013) Microbial acid-stable α-amylases: characteristics, genetic engineering and applications. Process Biochem 48(2):201–211. https://doi.org/10.1016/j.procbio.2012.12.018
Shukla RJ, Singh SP (2015) Characteristics and thermodynamics of a-amylase from thermophilic actinobacterium, Laceyella sacchari TSI-2. Process Biochem 50(12):2128–2136. https://doi.org/10.1016/j.procbio.2015.10.013
Singh PL, Singh AK, Kumar Y (2018) Kinetics and thermodynamic studies of partially purified alpha amylase produced from Bacillus altitunidis. World J Pharm Pharm Sci 7(2):1362–1376. https://doi.org/10.20959/wjpps20182-11013
Sugumaran Kr, Ponnusami V, Srivastava SN (2012) Partial purification and thermodynamic analysis of thermostable α-amylase from Bacillus cereus MTCC 1305. Int J Pharm Bio Sci 3:407–413
Wu X, Wang Y, Tong B, Chen X, Chen J (2018) Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4–423. Int J Biol Macromol 109:329–337. https://doi.org/10.1016/j.ijbiomac.2017.12.004
Xian L, Wang F, Luo X, Feng Y-L, Feng J-X (2015) Purification and characterization of a highly efficient calcium-independent α-amylase from Talaromyces pinophilus 1–95. PLoS ONE 10(3):e0121531. https://doi.org/10.1371/journal.pone.0121531
Zenin CT, Park YK (1983) Purification and characterization of acid a-amylase from Paecilomyces sp. J Ferment Technol 61:109–112
Zerva A, Savvides AL, Katsifas EA, Karagouni AD, Hatzinikolaou DG (2014) Evaluation of Paecilomyces variotii potential in bioethanol production from lignocellulose through consolidated bioprocessing. Bioresour Technol 162:294–299. https://doi.org/10.1016/j.biortech.2014.03.137
Author information
Authors and Affiliations
Contributions
Conceptualization: DM, DH and DK; Methodology: DM, DH; Formal analysis and investigation: MEA, SK; Writing—original draft preparation: DM; Writing—review and editing: all authors.
Corresponding author
Ethics declarations
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Rights and permissions
About this article
Cite this article
Apostolidi, M.E., Kalantzi, S., Hatzinikolaou, D.G. et al. Catalytic and thermodynamic properties of an acidic α-amylase produced by the fungus Paecilomyces variotii ATHUM 8891. 3 Biotech 10, 311 (2020). https://doi.org/10.1007/s13205-020-02305-2
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-020-02305-2