Skip to main content
SpringerLink
Log in

Structure-based rational design and introduction of arginines on the surface of an alkaline α-amylase from Alkalimonas amylolytica for improved thermostability

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

In this study, the thermostability of an alkaline α-amylase from Alkalimonas amylolytica was significantly improved through structure-based rational and the introduction of multiple arginines (Arg) on the protein surface. Based on an analysis of the tertiary structure, seven residues (glutamine (Gln) 166, Gln 169, serine (Ser) 270, lysine (Lys) 315, Gln 327, asparagine (Asn) 346, and Asn 423) were selected as engineering targets and individually replaced with arginine. Five of the seven single-mutated enzymes—S270R, K315R, Q327R, N346R, and N423R—showed enhanced thermostability. Multiple arginines were subsequently introduced on the protein surface, and the quintuple-mutated enzyme S270R/K315R/Q327R/N346R/N423R showed a 6.4-fold improvement in half-life at 60 and a 5.4 °C increase in melting temperature (T m) compared with those of wild-type enzyme. Concomitantly, the optimal temperature, optimal pH, and catalytic efficiency of this mutated enzyme also improved. The mutated enzyme displayed a large shift in optimal pH from 9.5 to 11.0. In addition, the optimum temperature increased from 50 to 55 °C, and the catalytic efficiency (k cat/K m) increased from 1.8 × 104 to 3.6 × 104 L/(g · min). The intramolecular interactions of mutated enzymes that contributed to increased thermostability were examined through comparative analysis of the model structures of wild-type and mutated enzymes. The thermostable mutated enzymes generated in this study have potential applications in the textile industry.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Ali MB, Khemakhem B, Robert X, Haser R, Bejar S (2006) Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain. Biochem J 394:51–56

    Article  PubMed  PubMed Central  Google Scholar 

  • Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA (2012) Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microbiol 78:3458–3464

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27:343–350

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Borders C, Broadwater JA, Bekeny PA, Salmon JE, Lee AS, Eldridge AM, Pett VB (1994) A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens. Protein Sci 3:541–548

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Cesarini S, Bofill C, Pastor F, Reetz MT, Diaz P (2012) A thermostable variant of P. aeruginosa cold-adapted LipC obtained by rational design and saturation mutagenesis. Process Biochem 47:2064–2071

    Article  CAS  Google Scholar 

  • Danson MJ, Hough DW, Russell RJ, Taylor GL, Pearl L (1996) Enzyme thermostability and thermoactivity. Protein Eng 9:629–630

    Article  PubMed  CAS  Google Scholar 

  • Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C (2000) Probing structural determinants specifying high thermostability in Bacillus licheniformis α-amylase. J Mol Biol 301:1041–1057

    Article  PubMed  CAS  Google Scholar 

  • Donald JE, Kulp DW, DeGrado WF (2011) Salt bridges: geometrically specific, designable interactions. Proteins 79:898–915

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Dong SW, Lu DN (2008) Kinetics of the thermal inactivation of Bacillus subtilis α-amylase and its application on the desizing of cotton fabrics. J Appl Polym Sci 109:3733–3738

    Article  CAS  Google Scholar 

  • Fuhrmann GF, Völker B (1993) Misuse of graphical analysis in nonlinear sugar transport kinetics by Eadie-Hofstee plots. Biochim Biophys Acta 1145:180–182

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–191

    Article  PubMed  CAS  Google Scholar 

  • Mabrouk SB, Aghajari N, Ali MB, Messaoud EB, Juy M, Haser R, Bejar S (2011) Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions. Bioresour Technol 102:1740–1746

    Article  PubMed  Google Scholar 

  • Masui A, Fujiwara N, Imanaka T (1994) Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions. Appl Environ Microbiol 60:3579–3584

    PubMed  CAS  PubMed Central  Google Scholar 

  • Mossavarali S, Hosseinkhani S, Ranjbar B, Miroliaei M (2006) Stepwise modification of lysine residues of glucose oxidase with citraconic anhydride. Int J Biol Macromol 39:192–196

    Article  PubMed  CAS  Google Scholar 

  • Murakami S, Nagasaki K, Nishimoto H, Shigematu R, Umesaki J, Takenaka S, Kaulpiboon J, Prousoontorn M, Limpaseni T, Pongsawasdi P (2008) Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli. Enzym Microb Technol 43:321–328

    Article  CAS  Google Scholar 

  • Ó'Fágáin C (2003) Enzyme stabilization-recent experimental progress. Enzym Microb Technol 33:137–149

    Article  Google Scholar 

  • Ordu EB, Sessions RB, Clarke AR, Karagüler NG (2013) Effect of surface electrostatic interactions on the stability and folding of formate dehydrogenase from Candida methylica. J Mol Catal B Enzym 95:23–28

    Article  CAS  Google Scholar 

  • Prakash O, Jaiswal N (2010) α-Amylase: an ideal representative of thermostable enzymes. Appl Biochem Biotechnol 160:2401–2414

    Article  PubMed  Google Scholar 

  • Rahimzadeh M, Khajeh K, Mirshahi M, Khayatian M, Schwarzenbacher R (2012) Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase. Int J Biol Macromol 50:1175–1182

    Article  PubMed  CAS  Google Scholar 

  • Roy JK, Mukherjee AK (2013) Applications of a high maltose forming, thermo-stable α-amylase from an extremely alkalophilic Bacillus licheniformis strain AS08E in food and laundry detergent industries. Biochem Eng J 77:220–230

    Article  CAS  Google Scholar 

  • Strickler SS, Gribenko AV, Gribenko AV, Keiffer TR, Tomlinson J, Reihle T, Loladze VV, Makhatadze GI (2006) Protein stability and surface electrostatics: a charged relationship. Biochemistry 45:2761–2766

    Article  PubMed  CAS  Google Scholar 

  • Svensson B (1994) Protein engineering in the α-amylase family: catalytic mechanism, substrate specificity, and stability. Plant Mol Biol 25:141–157

    Article  PubMed  CAS  Google Scholar 

  • Tan TC, Mijts BN, Swaminathan K, Patel BKC, Divne C (2008) Crystal structure of the polyextremophilic α-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch. J Mol Biol 378:852–870

    Article  PubMed  Google Scholar 

  • Tomschy A, Brugger R, Lehmann M, Svendsen A, Vogel K, Kostrewa D, Lassen SF, Burger D, Kronenberger A, van Loon AP (2002) Engineering of phytase for improved activity at low pH. Appl Environ Microbiol 68:1907–1913

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Uitdehaag JC, Mosi R, Kalk KH, van der Veen BA, Dijkhuizen L, Withers SG, Dijkstra BW (1999) X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family. Nat Struct Mol Biol 6:432–436

    Article  CAS  Google Scholar 

  • Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Vogt G, Woell S, Argos P (1997) Protein thermal stability, hydrogen bonds, and ion pairs. J Mol Biol 269:631–643

    Article  PubMed  CAS  Google Scholar 

  • Yang ZR, Thomson R, McNeil P, Esnouf RM (2005) RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 21:3369–3376

    Article  PubMed  CAS  Google Scholar 

  • Yang HQ, Liu L, Shin HD, Chen RR, Li JH, Du GC, Chen J (2013a) Comparative analysis of heterologous expression, biochemical characterization optimal production of an alkaline α-amylase from alkaliphilic Alkalimonas amylolytica in Escherichia coli and Pichia pastoris. Biotechnol Prog 29:39–47

    Article  PubMed  CAS  Google Scholar 

  • Yang HQ, Lu XY, Liu L, Li J, Shin HD, Chen RR, Du G, Chen J (2013b) Fusion of an oligopeptide to the N terminus of an alkaline α-amylase from Alkalimonas amylolytica simultaneously improves the enzyme's catalytic efficiency, thermal stability, and resistance to oxidation. Appl Environ Microbiol 79:3049–3058

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Yang HQ, Liu L, Shin HD, Li JH, Du GC, Chen J (2013c) Structure-guided systems-level engineering of oxidation-prone methionine residues in catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica for significant improvement of both oxidative stability and catalytic efficiency. PLoS ONE 8:e57403

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Yokota K, Satou K, Ohki SY (2006) Comparative analysis of protein thermostability: differences in amino acid content and substitution at the surfaces and in the core regions of thermophilic and mesophilic proteins. Sci Technol Adv Mater 7:255–262

    Article  CAS  Google Scholar 

  • Zhou CY, Zhang MC, Wang YT, Guo WY, Liu ZH, Wang Y, Wang W (2013) Enhancement of the thermo-alkali-stability of xylanase II from Aspergillus usamii E001 by site-directed mutagenesis. Afr J Microbiol Res 7:1535–1542

    CAS  Google Scholar 

Download references

Acknowledgments

This project was supported by 863 Program (2012AA022202), 973 Program (2012CB720806), and 111 Project (111-2-06).

Author information

Authors and Affiliations

  1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Zhuangmei Deng, Haiquan Yang, Jianghua Li & Long Liu

  2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Zhuangmei Deng, Haiquan Yang, Jianghua Li & Long Liu

  3. School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, 30332, USA

    Hyun-dong Shin

Authors
  1. Zhuangmei Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Haiquan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hyun-dong Shin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jianghua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Long Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Long Liu.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 209 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Deng, Z., Yang, H., Shin, Hd. et al. Structure-based rational design and introduction of arginines on the surface of an alkaline α-amylase from Alkalimonas amylolytica for improved thermostability. Appl Microbiol Biotechnol 98, 8937–8945 (2014). https://doi.org/10.1007/s00253-014-5790-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-5790-8

Keywords

Search

Navigation