Skip to content
2000
  1. Home
  2. A-Z Publications
  3. Current Proteomics
  4. Fast Track Articles
  5. Fast Track Article
image of Agar-Agar Matrix-Mediated Immobilization for Enhanced Catalytic Behavior and Stability of 1,4-α-D-Glucan Glucanohydrolase Obtained from Halotolerant Micrococcus spp. K11

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.

Method

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 in 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.

© 2025 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/cp/10.2174/0115701646326690241127100453
2025-01-03
2025-04-14

  • From This Site

    /content/journals/cp/10.2174/0115701646326690241127100453
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. Sahu P.K. Singh R. Shrivastava M. Darjee S. Mageshwaran V. Phurailtpam L. Rohatgi B. Microbial production of α-amylase from agro-waste: An approach towards biorefinery and bio-economy. Energy Nexus 2024 14 100293
    [Google Scholar]
  2. Riaz A. Ahmad S. Siddiqui A. Jabeen F. Tariq F. Ul Qader S.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. 2023 39 e39009 10.14393/BJ‑v39n0a2023‑62426
    [Google Scholar]
  3. Putri A.Z. Nakagawa T. Microbial α-Amylases in the Industrial Extremozymes. Rev. Agric. Sci 2020
    [Google Scholar]
  4. Ahmad A. Rahamtullah; Mishra R. Structural and functional adaptation in extremophilic microbial α-amylases. Biophys. Rev. 2022 ••• 14
    [Google Scholar]
  5. Farooq M.A. Ali S. Hassan A. Tahir H.M. Mumtaz S. Mumtaz S. Biosynthesis and industrial applications of α-amylase: a review. Arch Microbiol. 2021 203 4 1281 1292
    [Google Scholar]
  6. Ullah I. Khan M.S. Khan S.S. Ahmad W. Zheng L. Shah S.U.A. Ullah M. Iqbal A. Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil. Saudi J. Biol. Sci. 2021 28 1 970 979 10.1016/j.sjbs.2020.11.017 33424389
    [Google Scholar]
  7. Hussaina M. Jabbara B. Applications of Alpha (α)-Amylase in Biotechnology: A Review. GU. J. Phytosci. 2021 98 105
    [Google Scholar]
  8. Rohban R. Amoozegar M.A. Ventosa A. Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. Iran. J. Ind. Microbiol. Biotechnol. 2008 ••• 36 19037673
    [Google Scholar]
  9. Saxena R.K. Dutt K. Agarwal L. Nayyar P. A highly thermostable and alkaline amylase from a Bacillus sp. PN5. Bioresour. Technol. 2007 98 2 260 265 10.1016/j.biortech.2006.01.016 16524725
    [Google Scholar]
  10. Mohapatra B.R. Banerjee U.C. Bapuji M. Characterization of a fungal amylase from Mucor sp. associated with the marine sponge Spirastrella sp. J. Biotechnol. 1998 60 1-2 113 117 10.1016/S0168‑1656(97)00197‑1
    [Google Scholar]
  11. Mehta D. Satyanarayana T. Bacterial and Archaeal α-Amylases: Diversity and Amelioration of the Desirable Characteristics for Industrial Applications. Front. Micrbiol 2016
    [Google Scholar]
  12. Zhu D. Qaria M.A. Zhu B. Sun J. Yang B. Extremophiles and extremozymes in lignin bioprocessing. Renew. Sustain. Energy Rev. 2022 ••• 157
    [Google Scholar]
  13. Sarmiento F. Peralta R. Blamey J.M. Cold and hot extremozymes: industrial relevance and current trends. Front. Bioeng. Biotechnol. 2015 3 148 10.3389/fbioe.2015.00148 26539430
    [Google Scholar]
  14. Rai R. Samanta D. Goh K.M. Chadha B.S. Sani R.K. Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1. Int. J. Biol. Macromol. 2024 257 Pt 2 128679 10.1016/j.ijbiomac.2023.128679 38072346
    [Google Scholar]
  15. Ghattavi S. Homaei A. Marine enzymes: Classification and application in various industries. Int. J. Biol. Macromol. 2023 230 123136 10.1016/j.ijbiomac.2023.123136 36621739
    [Google Scholar]
  16. Kikani B. Patel R. Thumar J. Bhatt H. Rathore D.S. Koladiya G.A. Singh S.P. Solvent tolerant enzymes in extremophiles: Adaptations and applications. Int. J. Biol. Macromol. 2023 238 124051 10.1016/j.ijbiomac.2023.124051 36933597
    [Google Scholar]
  17. Chettri D. Verma A.K. Sarkar L. Verma A.K. Role of extremophiles and their extremozymes in biorefinery process of lignocellulose degradation. Extremophiles 2021 25 3 203 219 10.1007/s00792‑021‑01225‑0 33768388
    [Google Scholar]
  18. Dutta B. Bandopadhyay R. Biotechnological potentials of halophilic microorganisms and their impact on mankind. Beni. Suef Univ. J. Basic Appl. Sci. 2022 11 1 75 10.1186/s43088‑022‑00252‑w 35669848
    [Google Scholar]
  19. Didari M. Bagheri M. Amoozegar M.A. Bouzari S. Babavalian H. Tebyanian H. Hassanshahian M. Ventosa A. Diversity of halophilic and halotolerant bacteria in the largest seasonal hypersaline lake (Aran-Bidgol-Iran). J. Environ. Health Sci. Eng. 2020 18 2 961 971 10.1007/s40201‑020‑00519‑3 33312616
    [Google Scholar]
  20. Tadeo X. López-Méndez B. Trigueros T. Laín A. Castaño D. Millet O. Structural basis for the aminoacid composition of proteins from halophilic archea. PLoS Biol. 2009 7 12 e1000257 10.1371/journal.pbio.1000257 20016684
    [Google Scholar]
  21. De Lourdes Moreno M. Pérez D. García M. Mellado E. Halophilic bacteria as a source of novel hydrolytic enzymes. Life (Basel) 2013 3 1 38 51 10.3390/life3010038 25371331
    [Google Scholar]
  22. Ghasemi Y. Rasoul-Ami S. Ebrahimine A. Zarrini G. Kazemi A. Mousavi-Kh S. Ghoshoon M.B. Raee M.J. Halotolerant Amylase Production by a Novel Bacterial Strain, Rheinheimera aquimaris. Res. Microbiol. 2010 ••• 5
    [Google Scholar]
  23. Prakash B. Vidyasagar M. Madhukumar M.S. Muralikrishna G. Sreeramulu K. Production, purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101. Process Biochem. 2009 44 2 210 215 10.1016/j.procbio.2008.10.013
    [Google Scholar]
  24. Tan T.C. Mijts B.N. Swaminathan K. Patel B.K.C. Divne C. 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. 2008 378 4 852 870 10.1016/j.jmb.2008.02.041 18387632
    [Google Scholar]
  25. Kanthi Kiran K. Chandra T.S. Production of surfactant and detergent-stable, halophilic, and alkalitolerant alpha-amylase by a moderately halophilic Bacillus sp. Strain TSCVKK. Appl. Microbiol. Biotechnol. 2008 77 5 1023 1031 10.1007/s00253‑007‑1250‑z 17999060
    [Google Scholar]
  26. Hashim S.O. Delgado O. Hatti-Kaul R. Mulaa F.J. Mattiasson B. Starch hydrolysing Bacillus halodurans isolates from a Kenyan soda lake. Biotechnol. Lett. 2004 26 10 823 828 10.1023/B:BILE.0000025885.19910.d7 15269555
    [Google Scholar]
  27. Amoozegar M.A. Malekzadeh F. Malik K.A. Production of amylase by newly isolated moderate halophile, Halobacillus sp. strain MA-2. J. Microbiol. Methods 2003 52 3 353 359 10.1016/S0167‑7012(02)00191‑4 12531504
    [Google Scholar]
  28. Deutch C.E. Characterization of a salt-tolerant extracellular a-amylase from Bacillus dipsosauri. Lett. Appl. Microbiol. 2002 35 1 78 84 10.1046/j.1472‑765X.2002.01142.x 12081555
    [Google Scholar]
  29. Mevarech M. Frolow F. Gloss L.M. Halophilic enzymes: proteins with a grain of salt. Biophys. Chem. 2000 86 2-3 155 164 10.1016/S0301‑4622(00)00126‑5 11026680
    [Google Scholar]
  30. Maghraby Y.R. El-Shabasy R.M. Ibrahim A.H. Azzazy H.M.E.S. Enzyme Immobilization Technologies and Industrial Applications. ACS Omega 2023 8 6 5184 5196 10.1021/acsomega.2c07560 36816672
    [Google Scholar]
  31. DiCosimo R. McAuliffe J. Poulose A.J. Bohlmann G. Industrial use of immobilized enzymes. Chem. Soc. Rev. 2003 ••• 42 23436023
    [Google Scholar]
  32. Liese A. Hilterhaus L. Evaluation of immobilized enzymes for industrial applications. Chem. Soc. Rev. 2013 42 15 6236 6249 10.1039/c3cs35511j 23446771
    [Google Scholar]
  33. Razzaghi M. Homaei A. Mosaddegh E. Penaeus vannamei protease stabilizing process of ZnS nanoparticles. Int. J. Biol. Macromol. 2018 112 509 515 10.1016/j.ijbiomac.2018.01.173 29382577
    [Google Scholar]
  34. Arica M.Y. Bayramoǧlu G. Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes. J. Mol. Catal. 2004 ••• 27
    [Google Scholar]
  35. Mohidem N.A. Mohamad M. Rashid M.U. Norizan M.N. Hamzah F. Mat H. Recent Advances in Enzyme Immobilisation Strategies: An Overview of Techniques and Composite Carriers. J. Compos. Sci. 2023 7 12 488 10.3390/jcs7120488
    [Google Scholar]
  36. Rodgers L.E. Knott R.B. Holden P.J. Pike K.J. Hanna J.V. Foster J.R. Barlett J.R. Structural evolution and stability of sol–gel biocatalysts. Phys. B: Condens. 2006 385 386
    [Google Scholar]
  37. da Silva R.M. Gonçalves L.R.B. Rodrigues S. Different strategies to co-immobilize dextransucrase and dextranase onto agarose based supports: Operational stability study. Int. J. Biol. Macromol. 2020 156 411 419 10.1016/j.ijbiomac.2020.04.077 32302628
    [Google Scholar]
  38. Xing M. Chen Y. Li B. Tian S. Highly efficient removal of patulin using immobilized enzymes of Pseudomonas aeruginosa TF-06 entrapped in calcium alginate beads. Food Chem. 2022 377 131973 10.1016/j.foodchem.2021.131973 34990945
    [Google Scholar]
  39. Yilmaz N. Bi̇ran Ay S. Immobilization of Amylases via Adsorption on Agar-Coated Magnetic Nanoparticles. E-J. Sci. Technol. 2022 34 496 500 10.31590/ejosat.1083196
    [Google Scholar]
  40. Gennari A. Führ A.J. Volpato G. Volken de Souza C.F. Magnetic cellulose: Versatile support for enzyme immobilization - A review. Carbohydr. Polym. 2020 246 116646 10.1016/j.carbpol.2020.116646 32747279
    [Google Scholar]
  41. Filippovich S.Y. Isakova E.P. Gessler N.N. Deryabina Y.I. Advances in immobilization of phytases and their application. Bioresour. Technol. 2023 379 129030 10.1016/j.biortech.2023.129030 37037335
    [Google Scholar]
  42. Mohamed A. Ramaswamy H.S. Characterization of Caseinate–Carboxymethyl Chitosan-Based Edible Films Formulated with and without Transglutaminase Enzyme. J. Compos. Sci. 2022 6 7 216 10.3390/jcs6070216
    [Google Scholar]
  43. Chen S. Li Z. Gu Z. Ban X. Hong Y. Cheng L. Li C. Immobilization of β-cyclodextrin glycosyltransferase on gelatin enhances β-cyclodextrin production. Process Biochem. 2022 113 216 223 10.1016/j.procbio.2022.01.005
    [Google Scholar]
  44. Cao L. Immobilised enzymes: science or art? Curr. Opin. Chem. Biol. 2005 9 2 217 226 10.1016/j.cbpa.2005.02.014 15811808
    [Google Scholar]
  45. Shah M. Hameed A. Kashif M. Majeed N. Muhammad J. Shah N. Rehan T. Khan A. Uddin J. Khan A. Kashtoh H. Advances in agar-based composites: A comprehensive review. Carbohydr. Polym. 2024 346 122619 10.1016/j.carbpol.2024.122619 39245496
    [Google Scholar]
  46. Clark A.H. Ross-Murphy S.B. Structural and Mechanical Properties of Biopolymer Gels. Biopolymers 1987 ••• 83
    [Google Scholar]
  47. Khire J.M. Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan. Lett. Appl. Microbiol. 1994 19 4 210 212 10.1111/j.1472‑765X.1994.tb00945.x
    [Google Scholar]
  48. Mageswari A. Subramanian P. Chandrasekaran S. Siyashanmugam K. Babu S. Gothandam K.M. Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns. JGEB 2012 10 2 201 208
    [Google Scholar]
  49. Anupama A. Jayaraman G. Detergent stable, halotolerant α-amylase from bacillus aquimaris vitp4 exhibits reversible unfolding. Int. J. Appl. Biol. Pharm. Technol. 2011 ••• 366 376
    [Google Scholar]
  50. Górecka E. Jastrzebska M. Immobilization techniques and biopolymer carriers. Biotechnol Food Sci 2011 75 1
    [Google Scholar]
  51. Prakash O. Jaiswal N. Immobilization of a Thermostable -Amylase on Agarose and Agar Matrices and its Application in Starch Stain Removal. World Appl. Sci. J. 2011 ••• 572 577
    [Google Scholar]
  52. Chaudhary M. Rana N. Vaidya D. Ghabru A. Rana K. Dipta B. Immobilization of Amylase by Entrapment Method in Different Natural Matrix. Int. J. Curr. Microbiol. App. Sci. 2019 8 5 1097 1103
    [Google Scholar]
  53. Singh S. A comparative study on immobilization of alpha amylase enzyme on different matrices. Int J Plant Anim Environ Sci 2014
    [Google Scholar]
  54. Biró E. Németh Á. Sz.; Sisak, C.; Feczkó, T.; Gyenis, J., Preparation of chitosan particles suitable for enzyme immobilization. J. Biochem. Biophys. Methods 2008 ••• 70
    [Google Scholar]
  55. Villalba M. Verdasco-Martín C.M. dos Santos J.C.S. Fernandez-Lafuente R. Otero C. Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction. Enzyme Microb. Technol. 2016 90 35 44 10.1016/j.enzmictec.2016.04.007 27241290
    [Google Scholar]
  56. Bahamondes C. Álvaro G. Wilson L. Illanes A. 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. 2017 53 172 179 10.1016/j.procbio.2016.12.004
    [Google Scholar]
  57. Gerrits P.J. Willeman W.F. Straathof A.J.J. Heijnen J.J. Brussee J. van der Gen A. Mass transfer limitation as a tool to enhance the enantiomeric excess in the enzymatic synthesis of chiral cyanohydrins. J. Mol. Catal., B Enzym. 2001 15 4-6 111 121 10.1016/S1381‑1177(01)00014‑5
    [Google Scholar]
  58. Sattar H. Aman A. Qader S.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. 2018 111 917 922 10.1016/j.ijbiomac.2018.01.105 29415415
    [Google Scholar]
  59. Sharma J. Mahajan R. Gupta V.K. Comparison and suitability of gel matrix for entrapping higher content of enzymes for commercial applications. Indian J. Pharm. Sci. 2010 72 2 223 228 10.4103/0250‑474X.65010 20838527
    [Google Scholar]
  60. Pervez S. Nawaz M. Jamal M. Maqbool F. Shah I. Aman A. Ul Qader S. Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization. Carbohydr. Res. 2020 ••• 436 31683070
    [Google Scholar]
  61. Riaz A. Ansari B. Siddiqui A. Ahmed S. Naheed S. Qader S.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) 2015 ••• 3
    [Google Scholar]
  62. Kumar S. Dwevedi A. Kayastha A.M. Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications. J. Mol. Catal., B Enzym. 2009 58 1-4 138 145 10.1016/j.molcatb.2008.12.006
    [Google Scholar]
  63. Sharma M. Sharma V. Majumdar D. K. Entrapment of α-Amylase in Agar Beads for Biocatalysis of Macromolecular Substrate. Int Sch Res Notices. 2014 2014 936129 10.1155/2014/936129
    [Google Scholar]
  64. Mulagalapalli S. Kumar S. Kalathur R.C.R. Kayastha A.M. Immobilization of urease from pigeonpea (Cajanus cajan) on agar tablets and its application in urea assay. Appl. Biochem. Biotechnol. 2007 142 3 291 297 10.1007/s12010‑007‑0022‑7 18025589
    [Google Scholar]
  65. Norouzian D. Enzyme immobilization: the state of art in biotechnology. Iran. J. Biotechnol. 2003 ••• 1
    [Google Scholar]
  66. Liu Q. Hua Y. Kong X. Zhang C. Chen Y. Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme. J. Mol. Catal., B Enzym. 2013 95 89 98 10.1016/j.molcatb.2013.05.024
    [Google Scholar]
  67. Arica M.Y. Hasirci V. Alaeddinoǧlu N.G. Covalent immobilization of α-amylase onto pHEMA microspheres: preparation and application to fixed bed reactor. Biomaterials 1995 16 10 761 768 10.1016/0142‑9612(95)99638‑3 7492706
    [Google Scholar]
  68. Bayramoglu Z. Akbulut U. Sungur S. Immobilization of α-amylase into photographic gelatin by chemical cross-linking. Biomaterials 1992 13 10 704 708 10.1016/0142‑9612(92)90131‑7 1420716
    [Google Scholar]
  69. Dragomirescu M. Vintila T. Preda G. Influence of immobilization on biocatalytic activity of a microbial Bacillus amyloliquefaciens alpha-amylase. Rom. Biotechnol. Lett. 2012 ••• 17
    [Google Scholar]
  70. Anwar A. Qader S.A. Riaz A. Iqbal S. Azhar A. Calcium alginate: A support material for immobilization of proteases from newly isolated strain of Bacillus subtilis KIBGE-HAS. World Appl. Sci. J. 2009 ••• 7
    [Google Scholar]
  71. Dey G. Bhupinder S. Banerjee R. Immobilization of a-amylase produced by Bacillus circulans GRS 313. Braz. Arch. Biol. Technol. 2023 ••• 167 176
    [Google Scholar]
/content/journals/cp/10.2174/0115701646326690241127100453
Loading
Agar-Agar Matrix-Mediated Immobilization for Enhanced Catalytic Behavior and Stability of 1,4-α-D-Glucan Glucanohydrolase Obtained from Halotolerant Micrococcus spp. K11
Bentham Science Publishers ; https://doi.org/10.2174/0115701646326690241127100453
/content/journals/cp/10.2174/0115701646326690241127100453
/content/journals/cp/10.2174/0115701646326690241127100453
Loading

Data & Media loading...


  • Article Type:     Research Article
Keywords: micrococcus spp. ; amylase ; immobilization ; halotolerant ; reusability ; Agar-Agar

Most Cited Most Cited RSS feed

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