A malto-oligosaccharide forming thermostable acidic α-amylase from Bacillus stercoris YSP18 isolated from sediment of Sikkim hot spring: heterologous expression and characterisation

Abstract

The current study deals with cloning and expressing a maltogenic α-amylase gene from thermophilic Bacillus stercoris YSP18 (AmyYSP) in Escherichia coli BL21 (DE3). AmyYSP belongs to the GH13_20 subfamily of Glycoside Hydrolases and entails five conserved regions found in maltogenic α-amylases. As a monomer of 67 kDa, AmyYSP exhibits maximal activity at 80°C, pH 5.0 and retains>75% residual activity at 70–100°C and pH 5.0–8.0. The kinetic and thermodynamic studies displayed that it has a high affinity for soluble starch (K_m = 1.54±0.236 mgmL⁻¹), exhibits a longer half-life (38.5 h at 80°C and 8.88 h at 100°C), and a higher E_aD value of 3824±1.03 kJ mol⁻¹. It was characterised as a Ca²⁺-independent α-amylase, resistant to various denaturing additives. It hydrolyses soluble starch and raw corn starch efficiently, liberating glucose, maltose, maltotriose and maltotetraose as the main products. The thermostable and acid-stable, maltooligosaccharide forming AmyYSP is a versatile enzyme with prerequisites for successful application in starch-saccharification industries.

Keywords

Bacillus stercoris / α-amylase / Thermozymes / Maltooligosaccharides / Glycosyl hydrolases

References

[1] Mumcu H, Kayrav A, İsleyen ND, Karaguler NG. Cloning and characterization of thermostable amylopullulanase TbbApu and its C-terminal truncated variants with enhanced activity in organic solvents. Enzyme Microb Technol. 2023, 164: 110176

[2] Sharma P, Mondal K, Mondal KC, Thakur N. Hunt for α-amylase from metagenome and strategies to improve its thermostability: a systematic review. World J Microbiol Biotechnol. 2022;38. https://doi.org/10.1007/s11274-022-03396-0.

[3] Bláhová M, Štefuca V, Hronská H, Rosenberg M. Maltooligosaccharides: Properties, Production and Applications. Molecules. 2023;28. https://doi.org/10.3390/molecules28073281.

[4] Pan S, Ding N, Ren J, Gu Z, Li C, Hong Y, et al. . Maltooligosaccharide-forming amylase: Characteristics, preparation, and application. Biotechnol Adv. 2017, 35: 619-32

[5] Yip YS, Manas NHA, Jaafar NR, Rahman RA, Puspaningsih NNT, Illias RM. Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase from Bacillus lehensis G1 for maltooligosaccharides synthesis. Int J Biol Macromol. 2023;242. https://doi.org/10.1016/j.ijbiomac.2023.124675.

[6] Mondal S, Mondal K, Halder SK, Thakur N, Mondal KC. Microbial Amylase: Old but still at the forefront of all major industrial enzymes. Biocatal Agric Biotechnol. 2022, 45: 102509

[7] Parashar D, Satyanarayana T. An Insight Into Ameliorating Production, Catalytic Efficiency, Thermostability and Starch Saccharification of Acid-Stable α-Amylases From Acidophiles. Front Bioeng Biotechnol. 2018;6. https://doi.org/10.3389/fbioe.2018.00125.

[8] Najar IN, Sharma P, Das S, Sherpa MT, Kumar S, Thakur N. Bacterial diversity, physicochemical and geothermometry of South Asian hot springs. Curr Res Microb Sci. 2022, 3: 100125

[9] Najar IN, Das S, Kumar S, Sharma P, Mondal K, Sherpa MT, et al. Coexistence of Heavy Metal Tolerance and Antibiotic Resistance in Thermophilic Bacteria Belonging to Genus Geobacillus. Front Microbiol. 2022;13. https://doi.org/10.3389/fmicb.2022.914037.

[10] Mondal K, Sharma P, Najar IN, Mondal S, Kumar S, Tamang S, et al. . Cloning, expression, and characterization of thermostable α-amylase of a thermophilic bacterium isolated from hot-spring of Sikkim and sustainable immobilization on coconut coir. Syst Microbiol Biomanufacturing. 2024

[11] Najar IN, Sherpa MT, Das S, Thakur N. Bacterial diversity and functional metagenomics expounding the diversity of xenobiotics, stress, defense and CRISPR gene ontology providing eco-efficiency to Himalayan Hot Springs. Funct Integr Genomics. 2020, 20: 479-96

[12] Tamura K, Stecher G, Kumar S. Mol Biol Evol. 2021;38:3022–7. https://doi.org/10.1093/molbev/msab120. MEGA11: Molecular Evolutionary Genetics Analysis Version 11.

[13] Erickson K. The Jukes-Cantor Model of Molecular Evolution. PRIMUS. 2010, 20: 438-45

[14] Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1991, 280: 309-16

[15] LOWRY OH, ROSEBROUGH NJ FARRAL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951, 193: 265-75

[16] LAEMMLI UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970, 227: 680-5

[17] FUWA HA NEW METHOD FOR MICRODETERMINATION OF AMYLASE ACTIVITY BYTHE USE OF AMYLOSE AS THE SUBSTRATE. J Biochem. 1954, 41: 583-603

[18] Mehta D, Satyanarayana T. Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans. J Mol Catal B Enzym. 2013, 85–86: 229-38

[19] Park B-R, MubarakAli D, Kim J-W. Identification of a novel cyclomaltodextrinase annotated as a neopullulanase in the genome of Bacillus cereus. Arch Microbiol. 2023, 205: 86

[20] Ranjani V, Janeček Š, Chai KP, Shahir S, Rahman RNZRA, Chan K-G, et al. . Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase. Sci Rep. 2014, 4: 5850

[21] Plaza-Vinuesa L, Hernandez-Hernandez O, Moreno FJ, De Las Rivas B, Munõz R. Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1. Microb. Cell Fact. 2019;18. https://doi.org/10.1186/s12934-019-1237-3.

[22] Cha HJ, Yoon HG, Kim YW, Lee HS, Kim JW, Kweon KS, et al. . Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose. Eur J Biochem. 1998, 253: 251-62

[23] Nasrollahi S, Golalizadeh L, Sajedi RH, Taghdir M, Asghari SM, Rassa M. Substrate preference of a Geobacillus maltogenic amylase: A kinetic and thermodynamic analysis. Int J Biol Macromol. 2013, 60: 1-9

[24] Kim IC, Cha JH, Kim JR, Jang SY, Seo BC, Cheong TK, et al. . Catalytic properties of the cloned amylase from Bacillus licheniformis. J Biol Chem. 1992, 267: 22108-14

[25] Fincan SA, Özdemir S, Karakaya A, Enez B, Mustafov SD, Ulutaş MS, et al. . Purification and characterization of thermostable α-amylase produced from Bacillus licheniformis So-B3 and its potential in hydrolyzing raw starch. Life Sci. 2021, 264: 118639

[26] Ozdemir S, Fincan SA, Karakaya A, Enez B. A novel raw starch hydrolyzing thermostable α-amylase produced by newly isolated Bacillus mojavensis SO-10: Purification, characterization and usage in starch industries. Braz Arch Biol Technol. 2018;61. https://doi.org/10.1590/1678-4324-2018160399.

[27] Wang Y, Pan S, Jiang Z, Liu S, Feng Y, Gu Z, et al. . A novel maltooligosaccharide-forming amylase from Bacillus stearothermophilus. Food Biosci. 2019, 30: 100415

[28] Zhang X, Li C, Chen X, Chio C, Shrestha S, Qin W. Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase. Fermentation 2021;7. https://doi.org/10.3390/fermentation7040227

[29] Shofiyah SS, Yuliani D, Widya N, Sarian FD, Puspasari F, Radjasa OK et al. Isolation, expression, and characterization of raw starch degrading α-amylase from a marine lake Bacillus megaterium NL3. Heliyon. 2020;6. https://doi.org/10.1016/j.heliyon.2020.e05796

[30] Xie F, Quan S, Liu D, Ma H, Li F, Zhou F, et al. . Purification and characterization of a novel α-amylase from a newly isolated Bacillus methylotrophicus strain P11-2. Process Biochem. 2014, 49: 47-53

[31] Shukla RJ, Singh SP. Purification and Characterization of an Amylase from a Newly Isolated Geobacillus thermoleovorans TTIO4 from the Hot Spring of Tuwa-Timba. Starch - Stärke. 2023, 75: 2200254

[32] Wu X, Wang Y, Tong B, Chen X, Chen J. Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4-423. Int J Biol Macromol. 2018, 109: 329-37

[33] Ariaeenejad S, Zolfaghari B, Sadeghian Motahar SF, Kavousi K, Maleki M, Roy S, et al. Highly Efficient Computationally Derived Novel Metagenome α-Amylase With Robust Stability Under Extreme Denaturing Conditions. Front Microbiol. 2021;12. https://doi.org/10.3389/fmicb.2021.713125.

[34] Kohli I, Joshi NC, Varma A. Production, purification and applications of raw starch degrading and calcium-independent α-amylase from soil rich in extremophile. Int J Biol Macromol. 2020, 162: 873-81

[35] Salem K, Elgharbi F, Ben Hlima H, Perduca M, Sayari A, Hmida-Sayari A. Biochemical characterization and structural insights into the high substrate affinity of a dimeric and Ca²⁺ independent Bacillus subtilis α-amylase. Biotechnol Prog. 2020;36. https://doi.org/10.1002/btpr.2964.

[36] Silva-Salinas A, Rodríguez-Delgado M, Gómez-Treviño J, López-Chuken U, Olvera-Carranza C, Blanco-Gámez EA. Novel Thermotolerant Amylase from Bacillus licheniformis Strain LB04: Purification, Characterization and Agar-Agarose. Microorganisms 2021;9. https://doi.org/10.3390/microorganisms9091857

[37] Muniasamy R, Rathnasaamy S. Sustainable production and preparative purification of thermostable alkaline α-amylase by Bacillus simplex (ON754233) employing natural deep eutectic solvent-based extractive fermentation. Sci Rep. 2024, 14: 481

[38] El-Fallal A, Dobara MA, El-Sayed A, Omar N. Chang C-F. Starch and Microbial α-Amylases: From Concepts to Biotechnological Applications. Carbohydrates. 2012, Rijeka, IntechOpen

[39] Kikani BA, Kourien S, Rathod U. Stability and Thermodynamic Attributes of Starch Hydrolyzing α-Amylase of Anoxybacillus rupiensis TS-4. Starch - Stärke. 2020, 72: 1900105

[40] Sudan SK, Kumar N, Kaur I, Sahni G. Production, purification and characterization of raw starch hydrolyzing thermostable acidic α-amylase from hot springs, India. Int J Biol Macromol. 2018, 117: 831-9

[41] Klibanov AM. Improving enzymes by using them in organic solvents. Nature. 2001, 409: 241-6

[42] Slavić MŠ, Kojić M, Margetić A, Stanisavljević N, Gardijan L, Božić N, et al. . Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications. Int J Biol Macromol. 2023, 249: 126055

[43] Hmidet N, Maalej H, Haddar A, Nasri M. A Novel α-Amylase from Bacillus mojavensis A21: Purification and Biochemical Characterization. Appl Biochem Biotechnol. 2010, 162: 1018-30

[44] Al-Quadan F, Akel H, Natshi R. Characteristics of a novel, highly acid- and thermo-stable amylase from thermophilic Bacillus strain HUTBS62 under different environmental conditions. Ann Microbiol. 2011, 61: 887-92

[45] Burhanoğlu T, Sürmeli Y, Şanlı-Mohamed G. Identification and characterization of novel thermostable α-amylase from Geobacillus sp. GS33. Int J Biol Macromol. 2020, 164: 578-85

[46] Shivlata L, Satyanarayana T. Characteristics of Raw Starch-Digesting α-Amylase of Streptomyces badius DB-1 with Transglycosylation Activity and Its Applications. Appl Biochem Biotechnol. 2017, 181: 1283-303

[47] Kikani BA, Singh SP. Single step purification and characterization of a thermostable and calcium independent α-amylase from Bacillus amyloliquifaciens TSWK1-1 isolated from Tulsi Shyam hot spring reservoir, Gujarat (India). Int J Biol Macromol. 2011, 48: 676-81

[48] Ahmed SA, Abdella MAA, El-Sherbiny GM, Ibrahim AM, El-Shamy AR, Atalla SMM, et al. . Catalytic, kinetic and thermal properties of free andimmobilized Bacillus subtilis -MK1 α-amylase on Chitosan-magnetic nanoparticles. Biotechnol Rep. 2020, 26: e00443

[49] Bharwad K, Shekh S, Singh NK, Patel A, Joshi C. Heterologous expression and biochemical characterization of novel multifunctional thermostable α-amylase from hot-spring metagenome. Int J Biol Macromol. 2023;242. https://doi.org/10.1016/j.ijbiomac.2023.124810.

[50] Sen SK, Jana A, Bandyopadhyay P, Das Mohapatra PK, Raut S. Thermostable amylase production from hot spring isolate Exiguobacterium sp: A promising agent for natural detergents. Sustain Chem Pharm. 2016, 3: 59-68

[51] Asoodeh A, Chamani J, Lagzian M. A novel thermostable, acidophilic alpha-amylase from a new thermophilic Bacillus sp. Ferdowsicous isolated from Ferdows hot mineral spring in Iran: Purification and biochemical characterization. Int J Biol Macromol. 2010, 46: 289-97

[52] Matpan Bekler F, Güven K. Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Aǧri. Turk Biol (Poland). 2014, 69: 419-27

[53] Sharif S, Shah AH, Fariq A, Jannat S, Rasheed S, Yasmin A. Optimization of amylase production using response surface methodology from newly isolated thermophilic bacteria. Heliyon. 2023, 9: e12901

[54] Fan S, Lü X, Wei X, Lü R, Feng C, Jin Y, et al. . Computational design of α-amylase from Bacillus licheniformis to increase its activity and stability at high temperatures. Comput Struct Biotechnol J. 2024, 23: 982-9

[55] Yao D, Su L, Li N, Wu J. Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection. Microb Cell Fact. 2019, 18: 69

[56] Mantiri FR, Rumende RRH, Sudewi S. Identification of α-amylase gene by PCR and activity of thermostable α-amylase from thermophilic Anoxybacillus thermarum isolated from Remboken hot spring in Minahasa, Indonesia. IOP Conf. Ser. Earth Environ. Sci. vol. 217, Institute of Physics Publishing; 2019. https://doi.org/10.1088/1755-1315/217/1/012045

[57] Wang S, Jeyaseelan J, Liu Y, Qin W. Characterization and Optimization of Amylase Production in WangLB, a High Amylase-Producing Strain of Bacillus. Appl Biochem Biotechnol. 2016, 180: 136-51

[58] Wang S, Lu Z, Lu M, Qin S, Liu H, Deng X, et al. . Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase. Appl Microbiol Biotechnol. 2008, 80: 605-14

[59] Li J, Gu X, Pan A. Multifunctional α-amylase Amy19 possesses agarase, carrageenase, and cellulase activities. Int J Biol Macromol. 2019, 126: 585-94

[60] Yang H, Liu L, Li J, Du G, Chen J. Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis. Microb Cell Fact. 2011, 10: 77

[61] Yuan S, Yan R, Lin B, Li R, Ye X. Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations. Biochem Biophys Res Commun. 2023, 653: 69-75

[62] Soy S, Nigam VK, Sharma SR. Enhanced production and biochemical characterization of a thermostable amylase from thermophilic bacterium Geobacillus icigianus BITSNS038. J Taibah Univ Sci. 2021, 15: 730-45

[63] Jiang T, Cai M, Huang M, He H, Lu J, Zhou X, et al. . Characterization of a thermostable raw-starch hydrolyzing α-amylase from deep-sea thermophile Geobacillus sp. Protein Expr Purif. 2015, 114: 15-22

[64] Fincan SA, Enez B. Production, purification, and characterization of thermostable a-amylase from thermophilic Geobacillus stearothermophilus. Starch/Staerke. 2014, 66: 182-9

Funding

Department of Biotechnology, Ministry of Science and Technology, India(BT/PR25092/NER/95/1009/2017)

RIGHTS & PERMISSIONS

Jiangnan University