Abstract
A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli. 16S rRNA sequence of the strain shared 99.46% similarities with closely related type species. Also, the genome sequence shared ANI values below 92% and dDDH values below 52% with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The AmyF gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (< 24%) with functionally characterized recombinant halophilic alpha-amylases. The recombinant alpha-amylase was successfully purified from Ni–NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25–75 °C) and pH (4–9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0–4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca2+ ion had no activating effect, but high concentrations of the ion (40–200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg2+, Zn2+, Hg2+ and Fe3+. However, it was inhibited only at very high concentrations of these metal ions. Cu2+ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.
Similar content being viewed by others
References
Altschul SF, Gish W, Miller W, Ew M, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Amoozegar MA, Samareh-Abolhasani B, Shafiei M, Didari M, Hamedi J (2013) Production of halothermotolerant α-amylase from a moderately halophilic bacterium, Nesterenkonia Strain F. Prog Biol Sci 2:85–97
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISSMODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201
Auch AF, Jan M, Klenk HP, Göker M (2010) Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genom Sci 2:117
Auiewiriyanukul W, Saburi W, Kato K, Yao M, Mori H (2018) Function and structure of GH13_31 α-glucosidase with high α-(1→4)-glucosidic linkage specificity and transglucosylation activity. FEBS Lett 592:2268–2281
Bolhuis A, Kwan D, Thomas JR (2008) Halophilic adaptations of proteins. In: Siddiqui KS, Thomas T, Uversky V (eds) Protein adaptation in extremophiles: design, selection and applications. Nova Science Publishers, New York, pp 71–104
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinform 10:421
Coronado MA, Vargas C, Mellado E, Tegos G, Drainas C, Nieto JNJ, Ventosa A (2000) The alpha-amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization. Microbiology 146:861–868
Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461
Feller G, Lonhienne T, Deroanne C, Libioulle C, Van Beeumen J, Gerday C (1992) Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the antarctic psychrotroph Alteromonas haloplanctis A23. J Biol Chem 267:5217–5221
Gomes J, Steiner W (2004) The biocatalytic potential of extremophiles and extremozymes: review. Food Tech Biotech 42:223–235
Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P et al (2007) DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91
Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial α-amylases: a biotechnological perspective. Process Biochem 38:1599–1616
Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316
Hoffman BJ, Broadwater JA, Johnson P, Harper J, Fox BG, Kenealy WR (1995) Lactose fed-batch overexpression of recombinant metalloproteins in Escherichia coli BL21 (DE3): process control yielding high levels of metal-incorporated, soluble protein. Protein Expr Purif 6:646–654
Hough DW, Danson MJ (1999) Extremozymes. Curr Opin Chem Biol 3:39–46
Hutcheon GW, Vasisht N, Bolhuis A (2005) Characterization of a highly stable alpha-amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles 9:487–495
Jabbour D, Sorger A, Sahm K, Antranikian G (2012) A highly thermoactive and salt-tolerant a-amylase isolated from a pilotplant biogas reactor. Appl Microbiol Biotechnol 97:2971–2978
Janeček Š, Zámocká B (2020) A new GH13 subfamily represented by the α-amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles 24:207–217
Kelley L, Mezulis S, Yates C et al (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858
Kobayashi T, Kanai H, Aono R, Horikoshi K, Kudo T (1994) Cloning, expression, and nucleotide sequence of the alpha-amylase gene from the haloalkaliphilic archaeon Natronococcus sp. strain Ah-36. J Bacteriol 176:5131–5134
Kumar S, Grewal J, Sadaf A, Hemamalini R, Khare SK (2016) Halophiles as a source of polyextremophilic alpha-amylase for industrial applications. AIMS Microbiol 2:1–26
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lee I, Kim YO, Park SC, Chun J (2016) OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100–1103
MacGregor EA, Janecek S, Svensson B (2001) Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes. Biochim Biophys Acta 1546:1–20
Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4:91–98
Meier-Koltho JP, Auch AF, Klenk HP, Göker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform 14:60
Mijts BN, Patel BK (2002) Cloning, sequencing and expression of an α-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzymea. Microbiology 148:2343–2349
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Mobini-Dehkordi M, Javan FA (2012) Application of alpha-amylase in biotechnology. J Biol Today’s World 1:39–50
Mojsov K (2012) Microbial alpha-amylases and their industrial applications: a review. IntJ Manag IT Eng 2:583–609
Nielsen JE, Borchert TV (2000) Protein engineering of bacterial alpha-amylases. Biochim Biophys Acta 1543:253–274
Nigam PS (2013) Microbial enzymes with special characteristics for biotechnological applications. Biomolecules 3:597–611
Onodera M, Yatsunami R, Tsukimura W, Fukui T, Nakasone K, Takashina T, Nakamura S (2013) Gene analysis, expression, and characterization of an intracellular α-amylase from the extremely halophilic archaeon Haloarcula japonica. Biosci Biotechnol Biochem 77:281–288
Oren A (2010) Industrial and environmental applications of halophilic microorganisms. Environ Technol 31:825–834
Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R (2000) Advances in microbial amylases. Biotechnol Appl Biochem 31:135–152
Qin Y, Huang Z, Liu Z (2014) A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning, heterologous expression and biochemical characterization. Extremophiles 18:271–281
Richter M, Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106:19126–19131
Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J (2015) JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32:929–931
Santorelli M, Maurelli L, Pocsfalvi G, Fiume I, Squillaci G, La Cara F, Del Monaco G, Morana A (2016) Isolation and characterisation of a novel alpha-amylase from the extreme haloarchaeon Haloterrigena turkmenica. Int J Biol Macromol 92:174–184
Sarikhan S, Azarbaijani R, Yeganeh LP, Fazeli AS, Amoozegar MA, Salekdeh GH (2011) Draft genome sequence of Nesterenkonia sp. strain F, isolated from Aran-Bidgol Salt Lake in Iran. J Bacteriol 193:5580–5580
Shafiei M, Ziaee AA, Amoozegar MA (2010) Purification and biochemical characterization of a novel SDS and surfactant stable, raw starch digesting, and halophilic α-amylase from a moderately halophilic bacterium, Nesterenkonia sp. strain F. Process Biochem 45:694–699
Shafiei M, Ziaee AA, Amoozegar MA (2011) Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F. J Ind Microbiol Biotechnol 38:275–281
Shafiei M, Ziaee AA, Amoozegar MA (2012) Purification and characterization of a halophilic alpha-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F. Extremophiles 16:627–635
Shen X, Saburi W et al (2015) Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism. Acta Crystallogr D Biol Crystallogr 71:1382–1391
Van Den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6:213–218
Van der Maarel MJ, van der Veen B, Uitdehaag JC, Leemhuis H, Dijkhuizen L (2002) Properties and applications of starch-converting enzymes of the α-amylase family. J Biotechnol 94:137–155
Ventosa A, Nieto JJ (1995) Biotechnological applications and potentialities of halophilic microorganisms. World J Microbiol Biotechnol 11:85–94
Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62:504–544
Ventosa A, Sanches-Porro C, Martin S, Mellado E (2005) Halophilic archaea and bacteria as a source of extracellular hydrolytic enzymes. In: Gunde-Cimerman N, Oren A, Plemenitaš A (eds) Adaptation to life at high salt concentrations in Archaea, Bacteria, and Eukarya. Springer, Dordrecht, pp 339–354
Wang X, Kan G, Ren X, Yu G, Shi C, Xie Q, Wen H, Betenbaugh M (2018) 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 1:1–15. https://doi.org/10.1155/2018/3258383
Wang G, Luo M, Lin J, Lin Y, Yan R, Streit WR, Ye X (2019) A new extremely halophilic, calcium-independent and surfactant-resistant alpha-amylase from Alkalibacterium sp. SL3. J Microbiol Biotechnol 29:765–775
Wei Y, Wang X, Liang J, Li X, Du L, Huang R (2013) Identification of a halophilic α-amylase gene from Escherichia coli JM109 and characterization of the recombinant enzyme. Biotechnol Lett 35:1061–1065
Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35:W407–W410
Yamaguchi R, Tokunaga H, Ishibashi M, Arakawa T, Tokunaga M (2011) Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians. Appl Microbiol Biotechnol 89:673–684
Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER Suite: protein structure and function prediction. Nat Methods 12:7–8
Yoon SH, Ha SM, Lim JM, Kwon SJ, Chun J (2017) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281–1286
Acknowledgements
Financial support for this work was provided by the Research grant from Shahid Chamran University of Ahvaz.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Erko Stackebrandt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Solat, N., Shafiei, M. A novel pH and thermo-tolerant halophilic alpha-amylase from moderate halophile Nesterenkonia sp. strain F: gene analysis, molecular cloning, heterologous expression and biochemical characterization. Arch Microbiol 203, 3641–3655 (2021). https://doi.org/10.1007/s00203-021-02359-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00203-021-02359-7