Skip to main content
SpringerLink
Log in

Calcium pectinate-agar beads as improved carriers for β-d-galactosidase and their thermodynamics investigation

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

Abstract

Polyethyleneimine (PEI) glutaraldehyde-refined calcium pectinate (CaP)-agar beads were presented as improved covalent immobilization matrices. The CaP-agar beads exhibited incremented mechanical stability which facilitated their handling. The beads’ concoction and activation processes were honed using the Box–Behnken design which recommended utilizing 5.4% agar, and a 2.95% PEI solution of pH 8.67. The honed CaP-agar beads established a more efficient ionic interaction with PEI which enabled the immobilization of more enzyme while utilizing less PEI than that required to activate the neat CaP beads. Furthermore, the activated CaP-agar beads granted superior operational stability to the immobilized enzyme, β-d-galactosidase (βgal), where it preserved 86.84 ± 0.37% of its precursive activity during its thirteenth reusability round. The CaP-agar immobilized βgal (iβgal) also showed incremented storage stability where it preserved 85.05 ± 3.32% of its precursive activity after 38 days of storage. The thermal stability of the iβgal was shown to be superior to that of the free enzyme as the iβgal exhibited incremented thermodynamic parameters, such as the t1/2 values, the D values, the thermal denaturation activation energy, the enthalpies, and the Gibb’s free energies. The βgal’s immobilization onto the activated CaP-agar beads also shifted the enzyme’s optimal pH from 4.6–5.1 to 3.3–4.9, whereas its optimal temperature was retained at 55 °C. The procured biocatalyst was exploited to efficiently hydrolyze the lactose in whey permeate.

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
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Abdel-Wahab WA, Karam EA, Hassan ME, Kansoh AL, Esawy MA, Awad GEA (2018) Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of operational covalent immobilization. Int J Biol Macromol 113(2018):159–170

    Article  CAS  Google Scholar 

  • Ansari SA, Husain Q (2011) Immobilization of Aspergillus oryzae β-galactosidase on concanavalin A-layered calcium alginate-cellulose beads and its application in lactose hydrolysis in continuous spiral bed reactors. Pol J Chem Technol 13(4):15–20

    Article  Google Scholar 

  • Awad GEA, Ghanem AF, Abdel-Wahab WA, Wahba MI (2020) Functionalized κ-carrageenan/hyperbranched poly(amidoamine) for protease immobilization: thermodynamics and stability studies. Int J Biol Macromol 148(2020):1140–1155

    Article  CAS  Google Scholar 

  • Barkovec M, Koper GJM (1997) Proton binding characteristics of branched polyelectrolytes. Macromolecules 30(7):2151–2158

    Article  Google Scholar 

  • Bayramoglu G, Tunali Y, Arica MY (2007) Immobilization of β-galactosidase onto magnetic poly(GMA–MMA) beads for hydrolysis of lactose in bed reactor. Catal Commun 8(7):1094–1101

    Article  CAS  Google Scholar 

  • Contesini FJ, Ibarguren C, Grosso CRF, Carvalho PDO, Sato HH (2012) Immobilization of glucosyltransferase from Erwinia sp. using two different techniques. J Biotechnol 158(3):137–143

    Article  CAS  Google Scholar 

  • Da Silva OS, de Oliveira RL, Silva JDC, Converti A, Porto TS (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

    Article  Google Scholar 

  • Delattre C, Fenoradosoa TA, Michaud P (2011) Galactans: an overview of their most important sourcing and applications as natural polysaccharides. Braz Arch Biol Technol 54(6):1075–1092

    Article  CAS  Google Scholar 

  • Dutra Rosolen M, Gennari A, Volpato G, de Souza CFV (2015) Lactose hydrolysis in milk and dairy whey using microbial β-galactosidases. Enzyme Res 2015:806240

    Article  Google Scholar 

  • Fatarella E, Spinelli D, Ruzzante M, Pogni R (2014) Nylon 6 film and nanofiber carriers: preparation and laccase immobilization performance. J Mol Catal B: Enzym 102:41–47

    Article  CAS  Google Scholar 

  • Ferreira MM, Santiago FLB, da Silva NAG, Luiz JHH, Fernandéz-Lafuente R, Mendes AA, Hirata DB (2018) Different strategies to immobilize lipase from Geotrichum candidum: Kinetic and thermodynamic studies. Process Biochem 67:55–63

    Article  CAS  Google Scholar 

  • Freile-Pelegrín Y, Robledo D, Armisén R, García-Reina G (1996) Seasonal changes in agar characteristics of two populations of Pterocladia capillacea in Gran Canaria. Spain J Appl Phycol 8(3):239–246

    Article  Google Scholar 

  • Geiger B, Nguyen HM, Wenig S, Nguyen HA, Lorenz C, Kittl R, Mathiesen G, Eijsink VGH, Haltrich D, Nguyen TH (2016) From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus. Biochem Eng J 116:45–53

    Article  CAS  Google Scholar 

  • Guidini CZ, Fischer J, de Resende MM, Cardoso VL, Ribeiro EJ (2011) β-Galactosidase of Aspergillus oryzae immobilized in an ion exchange resin combining the ionic-binding and crosslinking methods: Kinetics and stability during the hydrolysis of lactose. J Mol Catal B: Enzym 71(3–4):139–145

    Article  CAS  Google Scholar 

  • Granjeiro PA, Cavagis ADM, Leite LDC, Ferreira CV, Granjeiro JM, Aoyama H (2004) The thermal stability of a castor bean seed acid phosphatase. Mol Cell Biochem 266(1–2):11–15

    Article  CAS  Google Scholar 

  • Güleç HA, Gürda S, Albayrak N, Mutlu M (2010) Immobilization of Aspergillus oryzae β-galactosidase on low pressure plasma-modified cellulose acetate membrane using polyethyleneimine for production of galactooligosaccharide. Biotechnol Bioprocess Eng 15:1006–1015

    Article  Google Scholar 

  • Hatzinikolaou DG, Katsifas E, Mamma D, Karagouni AD, Christakopoulos P, Kekos D (2005) Modeling of the simultaneous hydrolysis–ultrafiltration of whey permeate by a thermostable β-galactosidase from Aspergillus niger. Biochem Eng J 24(2):161–172

    Article  CAS  Google Scholar 

  • Kishore D, Kayastha AM (2012) Optimisation of immobilization conditions for chick pea β-galactosidase (CpGAL) to alkylamine glass using response surface methodology and its applications in lactose hydrolysis. Food Chem 134(3):1650–1657

    Article  CAS  Google Scholar 

  • Klein MP, Hackenhaar CR, Lorenzoni ASG, Rodrigues RC, Costa TMH, Ninow JL, Hertz PF (2016) Chitosan crosslinked with genipin as support matrix for application in food process: support characterization and β-d-galactosidase immobilization. Carbohydr Polym 137:184–190

    Article  CAS  Google Scholar 

  • Maksimainen MM, Lampio A, Mertanen M, Turunen O, Rouvinen J (2013) The crystal structure of acidic β-galactosidase from Aspergillus oryzae. Int J Biol Macromol 60:109–115

    Article  CAS  Google Scholar 

  • Makwana D, Castaňo J, Somani RS, Bajaj HC (2020) Characterization of Agar-CMC/Ag-MMT nanocomposite and evaluation of antibacterial and mechanical properties for packaging applications. Arab J Chem 13(1):3092–3099

    Article  CAS  Google Scholar 

  • Martínez YN, Cavello I, Hours R, Cavalitto S, Castro GR (2013) Immobilized keratinase and enrofloxacin loaded on pectin PVA cryogel patches for antimicrobial treatment. Bioresour Technol 145:280–284

    Article  Google Scholar 

  • Mollaei M, Abdollahpour S, Atashgahi S, Abbasi H, Masoomi F, Rad I, Lotfi AS, Zahiri HS, Vali H, Noghabi KA (2010) Enhanced phenol degradation by Pseudomonas sp SA01: gaining insight into the novel single and hybrid immobilizations. J Hazard Mater 175(1–3):284–292

    Article  CAS  Google Scholar 

  • Mitchell AJ, Wimpenny JWT (1997) The ffects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria. J Appl Microbiol 83(1):76–84

    Article  CAS  Google Scholar 

  • Munarin F, Petrini P, Barcellona G, Roversi T, Piazza L, Visai L, Tanzi MC (2014) Reactive hydroxyapatite fillers for pectin biocomposites. Mater Sci Eng C 45:154–161

    Article  CAS  Google Scholar 

  • Panesar PS, Kumari S, Panesar R (2010) Potential applications of immobilized β-galactosidase in food processing industries. Enzyme Res 2010:473137

    Article  Google Scholar 

  • Pessela BCC, Fernández-Lafuente R, Fuentes M, Vián A, García JL, Carrascosa AV, Mateo C, Guisánb JM (2003) Reversible immobilization of a thermophilic β-galactosidase via ionic adsorption on PEI-coated Sepabeads. Enzyme Microb Technol 32(3–4):369–374

    Article  CAS  Google Scholar 

  • Rai AK, Prakash O, Singh J, Singh PM (2013) Immobilization of cauliflower myrosinase on agar agar matrix and its application with various effectors. Adv Biochem 1(3):51–56

    Article  CAS  Google Scholar 

  • Rodrigues RC, Ortiz C, Berenguer-Murcia A, Torres R, Fernández-Lafuente R (2013) Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev 42(15):6290–6307

    Article  CAS  Google Scholar 

  • S. D. Fine-Chem Limited (2020) Price list 2019–2020. https://www.sdfine.com/pricelistpdf.pdf. Accessed 22 July 2020

  • Sigma-Aldrich (2020) Poly(ethyleneimine) solution. https://www.sigmaaldrich.com/catalog/product/sial/p3143?lang=en&region=EG. Accessed 22 July 2020

  • Soares LN, Falleiros S, Cabral BV, Fischer J, Guidini CZ, Cardoso VL, De Resende MM, Ribeiro EJ (2017) Improvement of recovered activity and stability of the Aspergillus oryzae β-galactosidase immobilized on Duolite®A568 by combination of immobilization methods. Chem Ind Chem Eng Q 23:495–506

    Article  CAS  Google Scholar 

  • Sriamornsak P, Thirawong N, Puttipipatkhachorn S (2005) Emulsion gel beads of calcium pectinate capable of floating on the gastric fluid: effect of some additives hardening agent or coating on release behavior of metronidazole. Eur J Pharm Sci 24(4):363–373

    Article  CAS  Google Scholar 

  • Torres R, Pessela BCC, Fuentes M, Mateo C, Munilla R, Fernández- Lafuente R, Guisán J (2006) Supports coated with PEI as a new tool in chromatography. Enzyme Microb Technol 39(4):711–716

    Article  CAS  Google Scholar 

  • Toyama Y, Sahara R, Lino Y, Kubota K (2011) pH dependence of rheological properties of gelatin gel mixed with agar or agarose. Trans Mater Res Soc Jpn 36(3):383–386

    Article  CAS  Google Scholar 

  • Wahba MI (2016) Treated calcium pectinate beads for the covalent immobilization of β-d-galactosidase. Int J Biol Macromol 91:877–886

    Article  CAS  Google Scholar 

  • Wahba MI (2017) Porous chitosan beads of superior mechanical properties for the covalent immobilization of enzymes. Int J Biol Macromol 105(1):894–904

    Article  CAS  Google Scholar 

  • Wahba MI, Hassan ME (2015) Novel grafted agar disks for the covalent immobilization of β-d-galactosidase. Biopolymers 103(12):675–684

    Article  CAS  Google Scholar 

  • Wahba MI, Hassan ME (2017) Agar-carrageenan hydrogel blend as a carrier for the covalent immobilization of β-d-galactosidase. Macromol Res 25(9):913–923

    Article  CAS  Google Scholar 

  • Wu J, Yu HQ (2007) Biosorption of 2,4-dichlorophenol by immobilized white-rot fungus Phanerochaete chrysosporium from aqueous solutions. Bioresour Technol 98:253–259

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by the National Research Centre, Egypt.

Author information

Authors and Affiliations

  1. Department of Chemistry of Natural and Microbial Products, National Research Centre, El-Behooth St., Dokki, Giza, Egypt

    Marwa I. Wahba

  2. Centre of Scientific Excellence-Group of Advanced Materials and Nanotechnology, National Research Centre, El-Behooth St., Dokki, Giza, Egypt

    Marwa I. Wahba

Authors
  1. Marwa I. Wahba
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marwa I. Wahba.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wahba, M.I. Calcium pectinate-agar beads as improved carriers for β-d-galactosidase and their thermodynamics investigation. 3 Biotech 10, 356 (2020). https://doi.org/10.1007/s13205-020-02341-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-020-02341-y

Keywords

Search

Navigation