Skip to main content
SpringerLink
Log in

Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli

  • Original Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg2+, Zn2+, Al3+ and K+, while it was slightly enhanced by 5 and 9 % with Cu2+ and Fe2+ metal ions, respectively.

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
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. ElMekawy A, Hegab HM, El-Baz A, Hudson SM (2013) Kinetic properties and role of bacterial chitin deacetylase in the bioconversion of chitin to chitosan. Recent Pat Biotechnol 7:234–241

    Article  CAS  Google Scholar 

  2. Atanasova N, Kitayska T, Yankov D et al (2009) Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli. Biochem Eng J 46:278–285

    Article  Google Scholar 

  3. Svensson D, Adlercreutz P (2011) Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides. J Mol Catal B Enzym 69:147–153

    Article  CAS  Google Scholar 

  4. Astray G, Gonzalez-Barreiro C, Mejuto JC et al (2009) A review on the use of cyclodextrins in foods. Food Hydrocoll 23:1631–1640

    Article  CAS  Google Scholar 

  5. Moriwaki C, Ferreira LR, Rodella JRT, Matioli G (2009) A novel cyclodextrin glycosyltransferase from Bacillus sphaericus strain 41: production, characterization and catalytic properties. Biochem Eng J 48:124–131

    Article  CAS  Google Scholar 

  6. Del Valle EMM (2004) Cyclodextrins and their uses: a review. Process Biochem 39:1033–1046

    Article  Google Scholar 

  7. Otero-Espinar FJ, Luzardo-Alvarez A, Blanco-Méndez J (2010) Cyclodextrins: more than pharmaceutical excipients. Mini Rev Med Chem 10:715–725

    Article  CAS  Google Scholar 

  8. Rahman K, Illias RM, Hassan O et al (2006) Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme. Enzyme Microb Technol 39:74–84

    Article  CAS  Google Scholar 

  9. Yue Y, Song B, Xie T et al (2014) Enhancement of α-cyclodextrin product specificity by enriching histidines of α-cyclodextrin glucanotransferase at remote subsite– 6. Process Biochem 49:230–236

    Article  CAS  Google Scholar 

  10. Takano T, Fukuda M, Monma M et al (1986) Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene. J Bacteriol 166:1118–1122

    CAS  Google Scholar 

  11. Atanasova N, Kitayska T, Bojadjieva I et al (2011) A novel cyclodextrin glucanotransferase from alkaliphilic Bacillus pseudalcaliphilus 20RF: purification and properties. Process Biochem 46:116–122

    Article  CAS  Google Scholar 

  12. Zain WSWM, Illias RM, Salleh MM et al (2007) Production of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. TS1-1: optimization of carbon and nitrogen concentration in the feed medium using central composite design. Biochem Eng J 33:26–33

    Article  CAS  Google Scholar 

  13. Safarikova M, Atanasova N, Ivanova V et al (2007) Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of magnetic biocatalysts from cells of Bacillus circulans ATCC 21783. Process Biochem 42:1454–1459

    Article  CAS  Google Scholar 

  14. Mathew S, Hedström M, Adlercreutz P (2012) Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase. Process Biochem 47:528–532

    Article  CAS  Google Scholar 

  15. Ibrahim ASS, Al-Salamah AA, El-Toni AM et al (2014) Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double mesoporous core–shell silica nanospheres. Electron J Biotechnol 17: 55–64

  16. Tai A, Iwaoka Y, Ito H (2013) Highly efficient and regioselective production of an erythorbic acid glucoside using cyclodextrin glucanotransferase from Thermoanaerobacter sp. and amyloglucosidase. J Mol Catal B Enzym 92:19–23

    Article  CAS  Google Scholar 

  17. Mathew S, Adlercreutz P (2013) Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp. Biochem Eng J 79:187–193

    Article  CAS  Google Scholar 

  18. Nguyen DHD, Tran PL, Li D et al (2014) Modification of rice grain starch for lump-free cooked rice using thermostable disproportionating enzymes. Food Res, Int

    Google Scholar 

  19. Singh M, Sharma R, Banerjee U (2002) Biotechnological applications of cyclodextrins. Biotechnol Adv 20:341–359

    Article  CAS  Google Scholar 

  20. Biwer A, Antranikian G, Heinzle E (2002) Enzymatic production of cyclodextrins. Appl Microbiol Biotechnol 59:609–617

    Article  CAS  Google Scholar 

  21. Martins RF, Davids W, Abu Al-Soud W et al (2001) Starch-hydrolyzing bacteria from Ethiopian soda lakes. Extremophiles 5:135–144

    Article  CAS  Google Scholar 

  22. Park CS, Park KH, Kim SH (1989) A rapid screening method for alkaline β-cyclodextrin glucanotransferase using phenolphthalein-methyl orange containing solid medium. Agric Biol Chem 53:1167–1169

    Article  CAS  Google Scholar 

  23. Goel A, Nene SN (1995) Modifications in the phenolphthalein method for spectrophotometric estimation of beta cyclodextrin. Starch–Stärke 47:399–400

    Article  CAS  Google Scholar 

  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  25. Kitahata S, Tsuyama N, Okada S (1974) Purification and some properties of cyclodextrin glycosyltransferase from a strain of Bacillus species. Agric Biol Chem 38:387–393

    Article  CAS  Google Scholar 

  26. Barham D, Trinder P (1972) An improved colour reagent for the determination of blood glucose by the oxidase system. Analyst 97:142–145

    Article  CAS  Google Scholar 

  27. Trinder P (1969) Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J Clin Pathol 22:158–161

    Article  CAS  Google Scholar 

  28. Scopes RK (1994) Protein purification: principles and practice, Springer, New York, pp 85–92

  29. Nomoto M, Chen C, Sheu D (1986) Purification and characterization of cyclodextrin glucanotransferase from an alkalophilic bacterium of Taiwan. Agric Biol Chem 50:2701–2707

    Article  CAS  Google Scholar 

  30. Cao X, Jin Z, Wang X, Chen F (2005) A novel cyclodextrin glycosyltransferase from an alkalophilic Bacillus species: purification and characterization. Food Res Int 38:309–314

    Article  CAS  Google Scholar 

  31. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  32. Jemli S, Messaoud EB, Ayadi-Zouari D et al (2007) A β-cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: purification, properties and potential use in bread-making. Biochem Eng J 34:44–50

    Article  CAS  Google Scholar 

  33. Rahman K (2005) Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1. Universiti Teknologi, Malaysia

    Google Scholar 

  34. Sian HK, Said M, Hassan O et al (2005) Purification and characterization of cyclodextrin glucosyltransferase from alkalophilic Bacillus sp. G1. Process Biochem 40:1101–1111

    Article  CAS  Google Scholar 

  35. Yampayont P, Iizuka M, Ito K, Limpaseni T (2006) Isolation of cyclodextrin producing thermotolerant Paenibacillus sp. from waste of starch factory and some properties of the cyclodextrin glycosyltransferase. J Incl Phenom Macrocycl Chem 56:203–207

    Article  CAS  Google Scholar 

  36. Doukyu N, Kuwahara H, Aono R (2003) Isolation of Paenibacillus illinoisensis that produces cyclodextrin glucanotransferase resistant to organic solvents. Biosci Biotechnol Biochem 67:334–340

    Article  CAS  Google Scholar 

  37. Illias RM, Tien SF, Rahman RA et al (2003) Application of factorial design to study the effects of temperature, initial pH and agitation on the production of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1. Sci Asia 29:135–140

    Article  CAS  Google Scholar 

  38. Higuti lma H, Grande SW, Sacco R, do Nascimento AJ (2003) Isolation of alkalophilic CGTase-producing bacteria and characterization of cyclodextrin-glycosyltransferase. Braz Arch Biol Technol 46:183–186

    Article  Google Scholar 

  39. Bonilha PRM, Menocci V, Goulart AJ et al (2006) Cyclodextrin glycosyltransferase from Bacillus licheniformis: optimization of production and its properties. Braz J Microbiol 37:317–323

    Article  CAS  Google Scholar 

  40. Ramli N, Abd-Aziz S, Hassan MA et al (2010) Potential cyclodextrin glycosyltransferase producer from locally isolated bacteria. Afr J Biotechnol 9:7317–7321

    CAS  Google Scholar 

  41. Chen WC, Lin JM, Liu KJ (1994) Production of cyclodextrins glucanotransferase by fed batch fermentation with sugar supply. J Chin Agric Chem Soc 32:565–573

    CAS  Google Scholar 

  42. Kuo C-C, Lin C-A, Chen J-Y et al (2009) Production of cyclodextrin glucanotransferase from an alkalophilic Bacillus sp. by pH-stat fed-batch fermentation. Biotechnol Lett 31:1723–1727

    Article  CAS  Google Scholar 

  43. Park YS, Dohjima T, Okabe M (1996) Enhanced alpha-amylase production in recombinant Bacillus brevis by fed-batch culture with amino acid control. Biotechnol Bioeng 49:36–44

    Article  CAS  Google Scholar 

  44. Letsididi R, Sun T, Mu W et al (2011) Production of a thermoactive β-cyclodextrin glycosyltransferase with a high starch hydrolytic activity from an alkalitolerant Bacillus licheniformis Sk 13.002 strain. Asian J Biotechnol 3:214–225

    Article  CAS  Google Scholar 

  45. Gawande BN, Patkar AY (1999) Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS-22. Biotechnol Bioeng 64:168–173

    Article  CAS  Google Scholar 

  46. Singh J, Vohra R, Sahoo D (2004) Enhanced production of alkaline proteases by Bacillus sphaericus using fed-batch culture. Process Biochem 39:1093–1101

    Article  CAS  Google Scholar 

  47. Kitcha S, Cheirsilp B, Maneerat S (2008) Cyclodextrin glycosyltransferase from a newly isolated alkalophilic Bacillus sp. C26. Songklanakarin J Sci Technol 30:723–728

    Google Scholar 

  48. ElMekawy A, El-Baz A, Soliman EA, Hudson SM (2013) Statistical modeling and optimization of chitosan production from Absidia coerulea using response surface methodology. Curr Biotechnol 2:125–133

    Article  CAS  Google Scholar 

  49. Varavinit S, Sanguanpong V, Shobsngob S (1998) Utilization of brewery yeast waste and thai glutinous rice starch in the production of cyclodextrin glycosyltransferase. J Jpn Foods Ingred 176:112–130

    Google Scholar 

  50. Savergave LS, Dhule SS, Jogdand VV et al (2008) Production and single step purification of cyclodextrin glycosyltransferase from alkalophilic Bacillus firmus by ion exchange chromatography. Biochem Eng J 39:510–515

    Article  CAS  Google Scholar 

  51. Ai-Noi S, Abd-Aziz S, Alitheen N et al (2008) Optimization of cyclodextrin glycosyltransferase production by response surface methodology approach. Biotechnology 7:10–18

  52. Jung S-W, Kim T-K, Lee K-W, Lee Y-H (2007) Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilicBacillus sp. BL-12 and intermolecular transglycosylation of stevioside. Biotechnol Bioprocess Eng 12:207–212

    Article  CAS  Google Scholar 

  53. Pishtiyski I, Popova V, Zhekova B (2008) Characterization of cyclodextrin glucanotransferase produced by Bacillus megaterium. Appl Biochem Biotechnol 144:263–272

    Article  CAS  Google Scholar 

  54. Gawande BN, Patkar AY (2001) Purification and properties of a novel raw starch degrading α-cyclodextrin glucanotransferase from Klebsiella pneumoniae AS-22. Enzym Microb Technol 28:735–743

    Article  CAS  Google Scholar 

  55. Ong RM, Goh KM, Mahadi NM et al (2008) Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli. J Ind Microbiol Biotechnol 35:1705–1714

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Genetic Engineering and Biotechnology Research Institute, University of Sadat City (USC), Sadat City, Egypt

    Ashraf F. Elbaz, Ahmed Sobhi & Ahmed ElMekawy

  2. Chemical and Biomedical Engineering Department, Ohio State University (OSU), Columbus, OH, USA

    Ashraf F. Elbaz

Authors
  1. Ashraf F. Elbaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ahmed Sobhi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ahmed ElMekawy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ahmed ElMekawy.

Additional information

All authors equally contributed to this paper.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Elbaz, A.F., Sobhi, A. & ElMekawy, A. Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli. Bioprocess Biosyst Eng 38, 767–776 (2015). https://doi.org/10.1007/s00449-014-1318-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-014-1318-y

Keywords

Search

Navigation