Skip to content
2000
Volume 21, Issue 5
  • ISSN: 1570-1646
  • E-ISSN: 1875-6247

Abstract

Background

Entrapment is supposed to be the most effective and simple method among various strategies of enzyme immobilization as it preserves the original conformation and biological activity of the enzyme with greater immobilization yield. A suitable and cost-effective protocol for the entrapment of 1,4-α-D-glucan glucanohydrolase obtained from halotolerant ., K11 has been developed.

Objective

The major objective of the present study was to explore halotolerant bacteria as potential producer of 1, 4-α-D-glucan glucanohydrolase from salt mines.

Methods

A total of 11 bacterial strains were isolated and purified using the halophilic medium. Strain K11 was selected on the basis of a large zone of starch hydrolysis. The crude enzyme extract was utilized to entrap in agar-agar scaffolds. Kinetic studies of agar-agar entrapped 1,4-α-D-glucan glucanohydrolase were assessed and compared with the properties of soluble enzyme.

Results

It was observed that optimum immobilization of 1,4-α-D-glucan glucanohydrolase was attained at 4% concentration of agar-agar. Maximum entrapped enzyme activity was noticed after 15 minutes, highlighting the 5-minute increase as compared to the free enzyme. Moreover, temperature maxima for optimal enzyme substrate reaction were recorded to be 30°C for both immobilized and soluble 1,4-α-D-glucan glucanohydrolase, whereas pH maxima of 1,4-α-D-glucan glucanohydrolase were shifted from 6.5 to 7.0 after entrapment. The need for optimum substrate concentration for entrapped amylase activity was recorded to be 3% (gm), and for soluble 1,4-α-D-glucan glucanohydrolase, 2% (gm) starch was required for improved enzymatic efficacy. The reusability studies showed that agar-agar immobilized 1,4-α-D-glucan glucanohydrolase could be consumed up to 6 repeated cycles.

Conclusion

It is concluded that exploited features of immobilized 1,4-α-D-glucan glucanohydrolase enhance its applicability in several industrial processes.

Loading

Article metrics loading...

/content/journals/cp/10.2174/0115701646326690241127100453
2025-01-03
2025-09-26
Loading full text...

Full text loading...

References

  1. SahuP.K. SinghR. ShrivastavaM. DarjeeS. MageshwaranV. PhurailtpamL. RohatgiB. Microbial production of α-amylase from agro-waste: An approach towards biorefinery and bio-economy.Energy Nexus202414100293
    [Google Scholar]
  2. RiazA. AhmadS. SiddiquiA. JabeenF. TariqF. Ul QaderS.A. Fabrication of 1,4-alpha-D-glucan glucanohydrolase holding Gel-Scaffolds using Agar-Agar, a natural polysaccharide and Polyacrylamide, a synthetic organic polymer for continuous liquefaction of starch.Biosci. J.202339e3900910.14393/BJ‑v39n0a2023‑62426
    [Google Scholar]
  3. PutriA.Z. NakagawaT. Microbial α-Amylases in the Industrial Extremozymes.Rev. Agric. Sci2020
    [Google Scholar]
  4. AhmadA. Rahamtullah; Mishra R. Structural and functional adaptation in extremophilic microbial α-amylases.Biophys. Rev.202214
    [Google Scholar]
  5. FarooqM.A. AliS. HassanA. TahirH.M. MumtazS. MumtazS. Biosynthesis and industrial applications of α-amylase: a review.Arch Microbiol.2021203412811292
    [Google Scholar]
  6. UllahI. KhanM.S. KhanS.S. AhmadW. ZhengL. ShahS.U.A. UllahM. IqbalA. Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil.Saudi J. Biol. Sci.202128197097910.1016/j.sjbs.2020.11.01733424389
    [Google Scholar]
  7. HussainaM. JabbaraB. Applications of Alpha (α)-Amylase in Biotechnology: A Review.GU. J. Phytosci.202198105
    [Google Scholar]
  8. RohbanR. AmoozegarM.A. VentosaA. Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. Iran. J. Ind. Microbiol. Biotechnol.20083619037673
    [Google Scholar]
  9. SaxenaR.K. DuttK. AgarwalL. NayyarP. A highly thermostable and alkaline amylase from a Bacillus sp. PN5.Bioresour. Technol.200798226026510.1016/j.biortech.2006.01.01616524725
    [Google Scholar]
  10. MohapatraB.R. BanerjeeU.C. BapujiM. Characterization of a fungal amylase from Mucor sp. associated with the marine sponge Spirastrella sp.J. Biotechnol.1998601-211311710.1016/S0168‑1656(97)00197‑1
    [Google Scholar]
  11. MehtaD. SatyanarayanaT. Bacterial and Archaeal α-Amylases: Diversity and Amelioration of the Desirable Characteristics for Industrial Applications.Front. Micrbiol2016
    [Google Scholar]
  12. ZhuD. QariaM.A. ZhuB. SunJ. YangB. Extremophiles and extremozymes in lignin bioprocessing.Renew. Sustain. Energy Rev.2022157
    [Google Scholar]
  13. SarmientoF. PeraltaR. BlameyJ.M. Cold and hot extremozymes: industrial relevance and current trends.Front. Bioeng. Biotechnol.2015314810.3389/fbioe.2015.0014826539430
    [Google Scholar]
  14. RaiR. SamantaD. GohK.M. ChadhaB.S. SaniR.K. Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1.Int. J. Biol. Macromol.2024257Pt 212867910.1016/j.ijbiomac.2023.12867938072346
    [Google Scholar]
  15. GhattaviS. HomaeiA. Marine enzymes: Classification and application in various industries.Int. J. Biol. Macromol.202323012313610.1016/j.ijbiomac.2023.12313636621739
    [Google Scholar]
  16. KikaniB. PatelR. ThumarJ. BhattH. RathoreD.S. KoladiyaG.A. SinghS.P. Solvent tolerant enzymes in extremophiles: Adaptations and applications.Int. J. Biol. Macromol.202323812405110.1016/j.ijbiomac.2023.12405136933597
    [Google Scholar]
  17. ChettriD. VermaA.K. SarkarL. VermaA.K. Role of extremophiles and their extremozymes in biorefinery process of lignocellulose degradation.Extremophiles202125320321910.1007/s00792‑021‑01225‑033768388
    [Google Scholar]
  18. DuttaB. BandopadhyayR. Biotechnological potentials of halophilic microorganisms and their impact on mankind.Beni. Suef Univ. J. Basic Appl. Sci.20221117510.1186/s43088‑022‑00252‑w35669848
    [Google Scholar]
  19. DidariM. BagheriM. AmoozegarM.A. BouzariS. BabavalianH. TebyanianH. HassanshahianM. VentosaA. Diversity of halophilic and halotolerant bacteria in the largest seasonal hypersaline lake (Aran-Bidgol-Iran).J. Environ. Health Sci. Eng.202018296197110.1007/s40201‑020‑00519‑333312616
    [Google Scholar]
  20. TadeoX. López-MéndezB. TriguerosT. LaínA. CastañoD. MilletO. Structural basis for the aminoacid composition of proteins from halophilic archea.PLoS Biol.2009712e100025710.1371/journal.pbio.100025720016684
    [Google Scholar]
  21. De Lourdes MorenoM. PérezD. GarcíaM. MelladoE. Halophilic bacteria as a source of novel hydrolytic enzymes.Life (Basel)201331385110.3390/life301003825371331
    [Google Scholar]
  22. GhasemiY. Rasoul-AmiS. EbrahimineA. ZarriniG. KazemiA. Mousavi-KhS. GhoshoonM.B. RaeeM.J. Halotolerant Amylase Production by a Novel Bacterial Strain, Rheinheimera aquimaris.Res. Microbiol.20105
    [Google Scholar]
  23. PrakashB. VidyasagarM. MadhukumarM.S. MuralikrishnaG. SreeramuluK. Production, purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101.Process Biochem.200944221021510.1016/j.procbio.2008.10.013
    [Google Scholar]
  24. TanT.C. MijtsB.N. SwaminathanK. PatelB.K.C. DivneC. 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.2008378485287010.1016/j.jmb.2008.02.04118387632
    [Google Scholar]
  25. Kanthi KiranK. ChandraT.S. Production of surfactant and detergent-stable, halophilic, and alkalitolerant alpha-amylase by a moderately halophilic Bacillus sp. Strain TSCVKK.Appl. Microbiol. Biotechnol.20087751023103110.1007/s00253‑007‑1250‑z17999060
    [Google Scholar]
  26. HashimS.O. DelgadoO. Hatti-KaulR. MulaaF.J. MattiassonB. Starch hydrolysing Bacillus halodurans isolates from a Kenyan soda lake.Biotechnol. Lett.2004261082382810.1023/B:BILE.0000025885.19910.d715269555
    [Google Scholar]
  27. AmoozegarM.A. MalekzadehF. MalikK.A. Production of amylase by newly isolated moderate halophile, Halobacillus sp. strain MA-2.J. Microbiol. Methods200352335335910.1016/S0167‑7012(02)00191‑412531504
    [Google Scholar]
  28. DeutchC.E. Characterization of a salt-tolerant extracellular a-amylase from Bacillus dipsosauri.Lett. Appl. Microbiol.2002351788410.1046/j.1472‑765X.2002.01142.x12081555
    [Google Scholar]
  29. MevarechM. FrolowF. GlossL.M. Halophilic enzymes: proteins with a grain of salt.Biophys. Chem.2000862-315516410.1016/S0301‑4622(00)00126‑511026680
    [Google Scholar]
  30. MaghrabyY.R. El-ShabasyR.M. IbrahimA.H. AzzazyH.M.E.S. Enzyme Immobilization Technologies and Industrial Applications.ACS Omega2023865184519610.1021/acsomega.2c0756036816672
    [Google Scholar]
  31. DiCosimoR. McAuliffeJ. PouloseA.J. BohlmannG. Industrial use of immobilized enzymes.Chem. Soc. Rev.20034223436023
    [Google Scholar]
  32. LieseA. HilterhausL. Evaluation of immobilized enzymes for industrial applications.Chem. Soc. Rev.201342156236624910.1039/c3cs35511j23446771
    [Google Scholar]
  33. RazzaghiM. HomaeiA. MosaddeghE. Penaeus vannamei protease stabilizing process of ZnS nanoparticles.Int. J. Biol. Macromol.201811250951510.1016/j.ijbiomac.2018.01.17329382577
    [Google Scholar]
  34. AricaM.Y. BayramoǧluG. Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes.J. Mol. Catal.200427
    [Google Scholar]
  35. MohidemN.A. MohamadM. RashidM.U. NorizanM.N. HamzahF. MatH. Recent Advances in Enzyme Immobilisation Strategies: An Overview of Techniques and Composite Carriers.J. Compos. Sci.202371248810.3390/jcs7120488
    [Google Scholar]
  36. RodgersL.E. KnottR.B. HoldenP.J. PikeK.J. HannaJ.V. FosterJ.R. BarlettJ.R. Structural evolution and stability of sol–gel biocatalysts.Phys. B: Condens.2006385386
    [Google Scholar]
  37. da SilvaR.M. GonçalvesL.R.B. RodriguesS. Different strategies to co-immobilize dextransucrase and dextranase onto agarose based supports: Operational stability study.Int. J. Biol. Macromol.202015641141910.1016/j.ijbiomac.2020.04.07732302628
    [Google Scholar]
  38. XingM. ChenY. LiB. TianS. Highly efficient removal of patulin using immobilized enzymes of Pseudomonas aeruginosa TF-06 entrapped in calcium alginate beads.Food Chem.202237713197310.1016/j.foodchem.2021.13197334990945
    [Google Scholar]
  39. YilmazN. Bi̇ran AyS. Immobilization of Amylases via Adsorption on Agar-Coated Magnetic Nanoparticles.E-J. Sci. Technol.20223449650010.31590/ejosat.1083196
    [Google Scholar]
  40. GennariA. FührA.J. VolpatoG. Volken de SouzaC.F. Magnetic cellulose: Versatile support for enzyme immobilization - A review.Carbohydr. Polym.202024611664610.1016/j.carbpol.2020.11664632747279
    [Google Scholar]
  41. FilippovichS.Y. IsakovaE.P. GesslerN.N. DeryabinaY.I. Advances in immobilization of phytases and their application.Bioresour. Technol.202337912903010.1016/j.biortech.2023.12903037037335
    [Google Scholar]
  42. MohamedA. RamaswamyH.S. Characterization of Caseinate–Carboxymethyl Chitosan-Based Edible Films Formulated with and without Transglutaminase Enzyme.J. Compos. Sci.20226721610.3390/jcs6070216
    [Google Scholar]
  43. ChenS. LiZ. GuZ. BanX. HongY. ChengL. LiC. Immobilization of β-cyclodextrin glycosyltransferase on gelatin enhances β-cyclodextrin production.Process Biochem.202211321622310.1016/j.procbio.2022.01.005
    [Google Scholar]
  44. CaoL. Immobilised enzymes: science or art?Curr. Opin. Chem. Biol.20059221722610.1016/j.cbpa.2005.02.01415811808
    [Google Scholar]
  45. ShahM. HameedA. KashifM. MajeedN. MuhammadJ. ShahN. RehanT. KhanA. UddinJ. KhanA. KashtohH. Advances in agar-based composites: A comprehensive review.Carbohydr. Polym.202434612261910.1016/j.carbpol.2024.12261939245496
    [Google Scholar]
  46. ClarkA.H. Ross-MurphyS.B. Structural and Mechanical Properties of Biopolymer Gels.Biopolymers198783
    [Google Scholar]
  47. KhireJ.M. Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan.Lett. Appl. Microbiol.199419421021210.1111/j.1472‑765X.1994.tb00945.x
    [Google Scholar]
  48. MageswariA. SubramanianP. ChandrasekaranS. SiyashanmugamK. BabuS. GothandamK.M. Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns.JGEB2012102201208
    [Google Scholar]
  49. AnupamaA. JayaramanG. Detergent stable, halotolerant α-amylase from bacillus aquimaris vitp4 exhibits reversible unfolding.Int. J. Appl. Biol. Pharm. Technol.2011366376
    [Google Scholar]
  50. GóreckaE. JastrzebskaM. Immobilization techniques and biopolymer carriers.Biotechnol Food Sci2011751
    [Google Scholar]
  51. PrakashO. JaiswalN. Immobilization of a Thermostable -Amylase on Agarose and Agar Matrices and its Application in Starch Stain Removal.World Appl. Sci. J.2011572577
    [Google Scholar]
  52. ChaudharyM. RanaN. VaidyaD. GhabruA. RanaK. DiptaB. Immobilization of Amylase by Entrapment Method in Different Natural Matrix.Int. J. Curr. Microbiol. App. Sci.20198510971103
    [Google Scholar]
  53. SinghS. A comparative study on immobilization of alpha amylase enzyme on different matrices.Int J Plant Anim Environ Sci20144
    [Google Scholar]
  54. BiróE. NémethÁ. Sz.; Sisak, C.; Feczkó, T.; Gyenis, J., Preparation of chitosan particles suitable for enzyme immobilization.J. Biochem. Biophys. Methods200870
    [Google Scholar]
  55. VillalbaM. Verdasco-MartínC.M. dos SantosJ.C.S. Fernandez-LafuenteR. OteroC. Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction.Enzyme Microb. Technol.201690354410.1016/j.enzmictec.2016.04.00727241290
    [Google Scholar]
  56. BahamondesC. ÁlvaroG. WilsonL. IllanesA. Effect of enzyme load and catalyst particle size on the diffusional restrictions in reactions of synthesis and hydrolysis catalyzed by α-chymotrypsin immobilized into glyoxal-agarose.Process Biochem.20175317217910.1016/j.procbio.2016.12.004
    [Google Scholar]
  57. GerritsP.J. WillemanW.F. StraathofA.J.J. HeijnenJ.J. BrusseeJ. van der GenA. Mass transfer limitation as a tool to enhance the enantiomeric excess in the enzymatic synthesis of chiral cyanohydrins.J. Mol. Catal., B Enzym.2001154-611112110.1016/S1381‑1177(01)00014‑5
    [Google Scholar]
  58. SattarH. AmanA. QaderS.A.U. Agar-agar immobilization: An alternative approach for the entrapment of protease to improve the catalytic efficiency, thermal stability and recycling efficiency.Int. J. Biol. Macromol.201811191792210.1016/j.ijbiomac.2018.01.10529415415
    [Google Scholar]
  59. SharmaJ. MahajanR. GuptaV.K. Comparison and suitability of gel matrix for entrapping higher content of enzymes for commercial applications.Indian J. Pharm. Sci.201072222322810.4103/0250‑474X.6501020838527
    [Google Scholar]
  60. PervezS. NawazM. JamalM. MaqboolF. ShahI. AmanA. Ul QaderS. Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization.Carbohydr. Res.202043631683070
    [Google Scholar]
  61. RiazA. AnsariB. SiddiquiA. AhmedS. NaheedS. QaderS.A.U. Immobilization of α-amylase in operationally stable calcium-alginate beads: A cost effective technique for enzyme aided industrial processes.Int. J. Biotechnol. Res. (Chennai)20153
    [Google Scholar]
  62. KumarS. DwevediA. KayasthaA.M. Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications.J. Mol. Catal., B Enzym.2009581-413814510.1016/j.molcatb.2008.12.006
    [Google Scholar]
  63. SharmaM. SharmaV. MajumdarD. K. Entrapment of α-Amylase in Agar Beads for Biocatalysis of Macromolecular Substrate.Int Sch Res Notices.2014201493612910.1155/2014/936129
    [Google Scholar]
  64. MulagalapalliS. KumarS. KalathurR.C.R. KayasthaA.M. Immobilization of urease from pigeonpea (Cajanus cajan) on agar tablets and its application in urea assay.Appl. Biochem. Biotechnol.2007142329129710.1007/s12010‑007‑0022‑718025589
    [Google Scholar]
  65. NorouzianD. Enzyme immobilization: the state of art in biotechnology.Iran. J. Biotechnol.20031
    [Google Scholar]
  66. LiuQ. HuaY. KongX. ZhangC. ChenY. Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme.J. Mol. Catal., B Enzym.201395899810.1016/j.molcatb.2013.05.024
    [Google Scholar]
  67. AricaM.Y. HasirciV. AlaeddinoǧluN.G. Covalent immobilization of α-amylase onto pHEMA microspheres: preparation and application to fixed bed reactor.Biomaterials1995161076176810.1016/0142‑9612(95)99638‑37492706
    [Google Scholar]
  68. BayramogluZ. AkbulutU. SungurS. Immobilization of α-amylase into photographic gelatin by chemical cross-linking.Biomaterials1992131070470810.1016/0142‑9612(92)90131‑71420716
    [Google Scholar]
  69. DragomirescuM. VintilaT. PredaG. Influence of immobilization on biocatalytic activity of a microbial Bacillus amyloliquefaciens alpha-amylase.Rom. Biotechnol. Lett.201217
    [Google Scholar]
  70. AnwarA. QaderS.A. RiazA. IqbalS. AzharA. Calcium alginate: A support material for immobilization of proteases from newly isolated strain of Bacillus subtilis KIBGE-HAS.World Appl. Sci. J.20097
    [Google Scholar]
  71. DeyG. BhupinderS. BanerjeeR. Immobilization of a-amylase produced by Bacillus circulans GRS 313.Braz. Arch. Biol. Technol.2023167176
    [Google Scholar]
/content/journals/cp/10.2174/0115701646326690241127100453
Loading
/content/journals/cp/10.2174/0115701646326690241127100453
Loading

Data & Media loading...


  • Article Type:
    Research Article
Keyword(s): Agar-Agar; amylase; halotolerant; immobilization; micrococcus spp.; reusability
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test