Abstract
Polygalacturonase (PG) catalyses the hydrolysis of pectin substances and is commonly used in the textile and food industries. Herein, PG was purified from Arabian balsam using three techniques (ammonium sulfate precipitation, ion exchange chromatography, and gel filtration) with a recovery of 11.2% and tenfold purification. The molecular weight of the purified PG was estimated to be 75.5 kDa using a Sephadex G-150 column. To improve the stability and reusability of the purified enzyme, a novel method to immobilize PG through calcium alginate-coated polypyrrole/silver nanocomposite was described. The immobilized PG was characterized by FTIR, TGA, SEM, EDX, and Raman spectroscopy. The immobilization efficiency was 84.4%. Excellent long-term storage stability of the immobilized PG was demonstrated with 83% of the initial activity preserved after 60 days. The immobilized PG was highly reusable, showing high activity (91% and 68%) after five and ten cycles. The immobilized PG showed improved stability to temperature and pH relative to that of the free enzyme. The Km and Vmax were determined to be 0.368 mg/mL and 5.33 µmol/mL for the immobilized PG and 0.667 mg/mL and 7.38 µmol/mL for free PG, respectively. Improved storage stability, catalytic efficiency (Vmax/Km), and reusability of the immobilized PG make it ideal for biotechnological and industrial applications.
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Abbreviations
- FTIR:
-
Fourier transform infrared spectroscopy
- TGA:
-
Thermogravimetric analysis
- SEM:
-
Scanning electron microscopy
- EDX:
-
Energy-dispersive X-ray spectroscopy
References
Gupta R, Beg Q, Khan S, Chauhan B (2002) An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol 60:381–395
Mohamed SA, AL-Malki AL, Kumosani TA (2009) Characterization of a polygalacturonase from Trichoderma harzianum grown on citrus peel with application for apple juice. Aust J Basic Appl Sci 3:2770–2777
Tochi BN, Wang Z, Xu SY, Zhang W (2009) The influence of pectinase and pectinase/hemicellulases enzyme preparations on percentage pineapple juice recovery, particulates and sensory attributes. Pak J Nutr 8:1184–1189
Jayani RS, Saxena S, Gupta R (2005) Microbial pectinolytic enzymes: a review. Process Biochem 40:2931–2944
Kobayashi T, Higaki N, Yajima N, Suzumatsu A, Hagihara H, KAWAI S, ITo S (2001) Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus. Biosci Biotechnol Biochem 65:842–847
Fontana RC, da Silveira MM (2012) Production of polygalacturonases by Aspergillus oryzae in stirred tank and internal- and external-loop airlift reactors. Bioresour Technol 123:157–163
Tepe O, Dursun AY (2014) Exo-pectinase production by Bacillus pumilus using different agricultural wastes and optimizing of medium components using response surface methodology. Environ Sci Pollut Res 21:9911–9920
Khan M, Nakkeeran E, Umesh-Kumar S (2013) Potential application of pectinase in developing functional foods. Annu Rev Food Sci Technol 4:21–34
Caffall KH, Mohnen D (2009) The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydr Res 344:1879–1900
Mohamed SA, Al-Malki AL, Khan JA, Kabli SA, Al-Garni SM (2013) Solid state production of polygalacturonase and xylanase by Trichoderma species using cantaloupe and watermelon rinds. J Microbiol 51:605–611
Niture SK, Pant A (2004) Purification and biochemical characterization of polygalacturonase II produced in semi-solid medium by a strain of Fusarium moniliforme. Microbiol Res 159:305–314
Kashyap DR, Soni SK, Tewari R (2003) Enhanced production of polygalacturonase by Bacillus sp.DT7 using solid state fermentation. Bioresour Technol 88:251–254
Srivastava S, Nighojkar A, Kumar A (1994) Multiple forms of pectin methylesterase from Cuscuta reflexa filaments. Phytochemistry 37:1233–1236
Sheldon RA, Schoevaart R, Van Langen LM (2005) Cross-linked enzyme aggregates (CLEAs): a novel and versatilemethod for enzyme immobilization (a review). Biocatal Biotransform 23:141–147
Bilal M, Asgher M, Iqbal HMN, Hu H, Zhang X (2016) Gelatin-immobilized manganese peroxidase with novelcatalytic characteristics and its industrial exploitation for fruitjuice clarification purposes. Catal Lett 146:2221–2228
Bilal M, Iqbal HMN, Hu H, Wang W, Zhang X (2017) Enhanced bio-catalytic performance and dye degradation potential of chitosan-encapsulated horseradish peroxidase in a packed bed reactor system. Sci Total Environ 575:1352–1360
Abdulaal WH, Almulaiky YQ, El-Shishtawy RM (2020) Encapsulation of HRP enzyme onto a magnetic Fe3O4 Np–PMMA film via casting with sustainable biocatalytic activity. Catalysts 10:181
Almulaiky YQ, Aqlan FM, Aldhahri M, Baeshen M, Khan TJ, Khan KA, Alayafi AA (2018) α-Amylase immobilization on amidoximated acrylic microfibres activated by cyanuric chloride. R Soc Open Sci 5:172164
Al-Najada AR, Almulaiky YQ, Aldhahri M, El-Shishtawy RM, Mohamed SA, Baeshen M, Al-Harbi SA (2019) Immobilisation of α-amylase on activated amidrazone acrylic fabric: a new approach for the enhancement of enzyme stability and reusability. Sci Rep 9:1–9
Jia YJ, Feng BZ, Sun WX, Zhang XG (2009) Polygalacturonase, pectate lyase and pectin methylesteraseactivity in pathogenic strains of Phytophthora capsici incubatedunder different conditions. J Phytopathol 157:585–591
Gan J, Bagheri AR, Aramesh N, Gul I, Franco M, Almulaiky YQ, Bilal M (2020) Covalent organic frameworks as emerging host platforms for enzyme immobilization and robust biocatalysis–A review. Int J Biol Macromol 167:502–515
Brayner R, Vaulay M-J, Fiévet F, Coradin T (2007) Alginate-mediated growth of Co, Ni, and CoNi nanoparticles: influence of the biopolymer structure. Chem Mater 19:1190–1198
Krueger KM, Al-Somali AM, Mejia M, Colvin VL (2007) The hydrodynamic size of polymer stabilized nanocrystals. Nanotechnology 18:475709
Otari SV, Patil RM, Waghmare SR, Ghosh SJ, Pawar SH (2013) A novel microbial synthesis of catalytically active Ag-alginate biohydrogel and its antimicrobial activity. Dalton Trans 42:9966–9975
Sayo K, Deki S, Hayashi S (1999) A novel method of preparing nano-sized gold and palladium particles dispersed in composites that uses the thermal relaxation technique. Eur Phys J D 9:429–432
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Contreras-Esquivel JC, Hours RA, Voget CE, Mignone CF (1999) Aspergillus kawachii produces acidic pectin releasing enzyme activity. J Biosci Bioeng 88:48–52
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analyt chem 31:426–428
Omastová M, Mosnáčková K, Fedorko P, Trchová M, Stejskal J (2013) Polypyrrole/silver composites prepared by single-step synthesis. Synth Met 66:57–62
Al-Harbi SA, Almulaiky YQ (2020) Purification and biochemical characterization of Arabian balsam α-amylase and enhancing the retention and reusability via encapsulation onto calcium alginate/Fe2O3 nanocomposite beads. Int J Biol Macromol 160:944–952
Almulaiky YQ, Al-Harbi SA (2019) A novel peroxidase from Arabian balsam (Commiphora gileadensis) stems: its purification, characterization and immobilization on a carboxymethylcellulose/Fe3O4 magnetic hybrid material. Int J Biol Macromol 133:767–774
Köktepe T, Altın S, Tohma H, Gülçin I, Köksal E (2017) Purification, characterization and selected inhibition properties of peroxidase from haricot bean (Phaseolus vulgaris L.). Int J Food Prop 20:1944–1953
Mohamed SA, Abulnaja KO, Ads AS, Khan JA, Kumosani TA (2011) Characterisation of an anionic peroxidase from horseradish Cv. Balady Food Chem 128:725–730
Wingfield PT (Ed.) (2016) Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci, 84(1), A-3F.
Welinder KG (1992) Superfamily of plant, fungal and bacterial peroxidases. Curr Opin Struc Biol 2:388–393
Pedrolli DB, Carmona EC (2010) Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures. J Ind Microbiol Biot 37:567–573
Rha E, Park HJ, Kim MO, Chung YR, Lee CW, Kim JW (2001) Expression of exo-polygalacturonases in Botrytis cinerea. FEMS Microbiol Lett 201:105–109
Gomes E, Leite RSR, Da Silva R (2009) Purification of an exopolygalacturonase from Penicillium viridicatum RFC3 produced in submerged fermentation. Int J Microbiol. https://doi.org/10.1155/2009/631942
Rahman M, Choi YS, Kim YK, Park C, Yoo JC (2019) Production of novel polygalacturonase from Bacillus paralicheniformis CBS32 and application to depolymerization of ramie fiber. Polymers 11:1525
Ernest V, Gajalakshmi S, Mukherjee A (2014) Enhanced activity of lysozyme-AgNP conjugate with synergic antibacterial effect without damaging the catalytic site of lysozyme. Artif Cells Nanomed Biotechnol 42:336–343
Mohamed SA, Al-Harbi MH, Almulaiky YQ, Ibrahim IH, Salah HA, El-Badry MO, El-Shishtawy RM (2018) Immobilization of Trichoderma harzianum α-amylase on PPyAgNp/Fe3O4-nanocomposite: chemical and physical properties. Artif Cells Nanomed Biotechnol 46:201–206
Almulaiky YQ, El-Shishtawy RM, Aldhahri M, Mohamed SA, Afifi M, Abdulaal WH, Mahyoub JA (2019) Amidrazone modified acrylic fabric activated with cyanuric chloride: a novel and efficient support for horseradish peroxidase immobilization and 401 phenol removal. Int J Biol Macromol 140:949–958
Lin SY, Li MJ, Cheng WT (2007) FT-IR and Raman vibrational microspectroscopies used for spectral biodiagnosis of human tissues. Spectroscopy 21:1–30
Liu YC, Hwang BJ (2000) Identification of oxidized polypyrrole on Raman spectrum. Synth Met 113:203–207
Buga ML, Ibrahim S, Nok AJ (2010) Physico-chemical characteristics of immobilized polygalacturonase from Aspergillus niger (SA6). Artif Cells Nanomed Biotechnol 9:8934–8943
Anand G, Yadav S, Yadav D (2017) Purification and biochemical characterization of an exo-polygalacturonase from Aspergillus flavus MTCC 7589. Biocatal Agric Biotechnol 10:264–269
Almulaiky YQ, Khalil NM, El-Shishtawy RM, Altalhi T, Algamal Y, Aldhahri M, Mohammed MM (2021) Hydroxyapatite-decorated ZrO2 for α-amylase immobilization: toward the enhancement of enzyme stability and reusability. Int J Biol Macromol 167:299–308
Mazlan SZ, Hanifah SA (2017) Effects of temperature and pH on immobilized laccase activity in conjugated methacrylate-acrylate microspheres. Int J Polym Sci. https://doi.org/10.1155/2017/5657271
Rehman HU, Aman A, Nawaz MA, Karim A, Ghani M, Baloch AH, Qader SA (2016) Immobilization of pectin depolymerising polygalacturonase using different polymers. Int J Boil Macromol 82:127–133
Aldhahri M, Almulaiky YQ, El-Shishtawy RM, Al-Shawafi WM, Salah N, Alshahrie A, Alzahrani HA (2020) Ultra-thin 2D CuO nanosheet for HRP immobilization supported by encapsulation in a polymer matrix: characterization and dye degradation. Lett Catal. https://doi.org/10.1007/s10562-020-03289-7
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Almulaiky, Y.Q., Al-Harbi, S.A. Preparation of a Calcium Alginate-Coated Polypyrrole/Silver Nanocomposite for Site-Specific Immobilization of Polygalacturonase with High Reusability and Enhanced Stability. Catal Lett 152, 28–42 (2022). https://doi.org/10.1007/s10562-021-03631-7
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DOI: https://doi.org/10.1007/s10562-021-03631-7