Abstract
The present study discusses the purification, characterization and application of pectinase from Aspergillus terreus FP6 in fruit pigment extraction. By the four-step purification involving precipitation, dialysis, ion-exchange chromatography, gel filtration chromatography, a 20.85-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the pectinase was 47 kDa, as found by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum activity of the enzyme was recorded at pH 6.0 and 50 °C. The enzyme retained 80.3% and 79.1% residual activity, respectively at pH 6.0 and 50 °C for 90 min. The pectinase was best functional in the presence of toluene and retained its activity for 30 min. Cu2+ and Co2+ acted as enzyme activators, while Ca2+, β-mercaptoethanol, dimethyl sulfoxide and ethylenediaminetetraacetic acid proved to be the inhibitors. The Km and Vmax values of the pectinase with pectin as substrate were 0.002 mM and 27.39 U/mL, respectively thus indicating the high enzyme affinity towards the substrate. After 30-min treatment of the grape skin with the partially purified enzyme, microscopic observation revealed that a short time of the enzymatic treatment resulted in substantial loss of pigment and shrinkage of the grape skin cells thereby highlighting the high efficiency of the pectinase. The current study implies that the A. terreus FP6 pectinase may be applied as a bio-agent in the food and beverage industries and has the potential to replace harmful solvents by promoting a greener approach to extract plant pigments.
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The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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GraphPad Prism software (version 9.2.0); R software (version 4.0.2).
References
Abdulrachman D, Thongkred P, Kocharin K et al (2017) Heterologous expression of Aspergillus aculeatus endo-polygalacturonase in Pichia pastoris by high cell density fermentation and its application in textile scouring. BMC Biotechnol 17:15. https://doi.org/10.1186/s12896-017-0334-9
Adalberto P, Golfeto C, Moreira A, Almeida F, Ferreira D, Cass Q, Souza D (2016) Characterization of an exopolygalacturonase from Leucoagaricus gongylophorus, the symbiotic fungus of Atta sexdens. Adv Enzyme Res 4:7–19. https://doi.org/10.4236/aer.2016.41002
Ahmed A, Sohail M (2019) Characterization of pectinase from Geotrichum candidum AA15 and its potential application in orange juice clarification. J King Saud Univ Sci 32:955–961. https://doi.org/10.1016/j.jksus.2019.07.002
Ajsuvakova OP, Tinkov AA, Aschner M, Rocha J et al (2020) Sulfhydryl groups as targets of mercury toxicity. Coord Chem Rev 417:213343. https://doi.org/10.1016/j.ccr.2020.213343
Anand G, Yadav S, Yadav D (2016) Purification and characterization of polygalacturonase from Aspergillus fumigatus MTCC 2584 and elucidating its application in retting of Crotalaria juncea fiber. 3 Biotech 6:201–208. https://doi.org/10.1007/s13205-016-0517-4
Calderón-Oliver M, Ponce-Alquicira E (2021) Environmentally friendly techniques and their comparison in the extraction of natural antioxidants from green tea, rosemary, clove, and oregano. Molecules 26:1869. https://doi.org/10.3390/molecules26071869
Carrasco M, Rozas JM, Alcaíno J (2019) Pectinase secreted by psychrotolerant fungi: Identification, molecular characterization and heterologous expression of a cold-active polygalacturonase from Tetracladium sp. Microb Cell Fact 18:45. https://doi.org/10.1186/s12934-019-1092-2
de Medeiros Bezerra Jácome MC, de Araújo Padilha CE, do Nascimento Arrais MR et al (2020) Valorization of mangaba residue (Hancornia speciosa Gomes) for polygalacturonase production from Aspergillus niger IOC 4003 and fabrication of active chitosan films. Biomass Conv Bioref. https://doi.org/10.1007/s13399-020-01102-4
Dogan N, Tari C (2008) Characterization of three-phase partitioned exopolygalacturonase from Aspergillus sojae with unique properties. Biochem Eng J 39:43–50. https://doi.org/10.1016/j.bej.2007.08.008
D’Souza DH, Bhattacharya S, Das A (2020) Fibrinolytic protease from Bacillus cereus S46: Purification, characterization, and evaluation of its in vitro thrombolytic potential. J Basic Microbiol 60:661–668. https://doi.org/10.1002/jobm.202000148
El Enshasy HA, Elsayed EA, Suhaimi N, Malek RA, Esawy M (2018) Bioprocess optimization for pectinase production using Aspergillus niger in a submerged cultivation system. BMC Biotechnol 18:71. https://doi.org/10.1186/s12896-018-0481-7
Fratebianchi D, Cavello IA, Cavalitto SF (2017) Purification and biochemical and kinetic properties of an endo-polygalacturonase from the industrial fungus Aspergillus sojae. J Mol Microbiol Biotechnol 27:102–109. https://doi.org/10.1159/000460296
Ghazala I, Sayari N, Romdhane MB, Ellouz-Chaabouni S, Haddar A (2015) Assessment of pectinase production by Bacillus mojavensis I4 using an economical substrate and its potential application in oil sesame extraction. J Food Sci Technol 52:7710–7722. https://doi.org/10.1007/s13197-015-1964-3
Karimi M, Ignasiak M, Chan B et al (2016) Reactivity of disulfide bonds is markedly affected by structure and environment: implications for protein modification and stability. Sci Rep 6:38572. https://doi.org/10.1038/srep38572
Kaur A, Soni SK, Vij S, Rishi P (2021) Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces. AMB Expr 11:22. https://doi.org/10.1186/s13568-021-01183-y
Khatri BP, Bhattarai T, Shrestha S, Maharjan J (2015) Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal. Springerplus 4:488. https://doi.org/10.1186/s40064-015-1286-y
Kumar A, Dhar K, Kanwar SS, Arora PK (2016) Lipase catalysis in organic solvents: advantages and applications. Biol Proced Online 18:2. https://doi.org/10.1186/s12575-016-0033-2
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275. https://doi.org/10.1016/S0021-9258(19)52451-6
Magro MD, Silveira VCC, de Menezes EW, Benvenutti EV, Nicolodi S, Hertz PF, Klein MP, Rodrigues RC (2018) Magnetic biocatalysts of pectinase and cellulase: Synthesis and characterization of two preparations for application in grape juice clarification. Int J Biol Macromol 115:35–44. https://doi.org/10.1016/j.ijbiomac.2018.04.028
Mehmood T, Saman T, Asgher M, Irfan M, Anwar Z, Nadeem F, Siddiqa A (2019) Optimization of cultural parameters for pectin methylestrase and polygalacturonase production from Schizophyllum commune in solid state fermentation. Bangladesh J Bot 48:65–74. https://doi.org/10.3329/bjb.v48i1.474179
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428. https://doi.org/10.1021/ac60147a030
Mondal S, Jyoti Prakash Soren JP, Joy Mondal J, Rakshit S, Suman Kumar Halder SK, Mondal KC (2020) Contemporaneous synthesis of multiple carbohydrate debranching enzymes from newly isolated Aspergillus fumigatus SKF-2 under solid state fermentation: A unique enzyme mixture for proficient saccharification of plant bioresources. Ind Crops Prod 150:112409. https://doi.org/10.1016/j.indcrop.2020.112409
Munir M, Abdullah R, Haq IU, Kaleem A, Iqtedar M, Ashraf S (2020) Purification, characterization, kinetics and thermodynamic analysis of polygalacturonase from Aspergillus tamarii for industrial applications. Rev Mex Ing Quim 19:293–304. https://doi.org/10.24275/rmiq/Bio1753
Nandi S, Parui S, Halder R, Jana B, Bhattacharyya K (2018) Interaction of proteins with ionic liquid, alcohol and DMSO and in situ generation of gold nano-clusters in a cell. Biophys Rev 10:757–768. https://doi.org/10.1007/s12551-017-0331-1
Nath P, Kaur C, Rudra SG, Varghese E (2016) Enzyme-assisted extraction of carotenoid-rich extract from red capsicum (Capsicum annuum). Agric Res 5:193–204. https://doi.org/10.1007/s40003-015-0201-7
Okonji RE, Itakorode BO, Ovumedia JO, Adedeji OS (2019) Purification and biochemical characterization of pectinase produced by Aspergillus fumigatus isolated from soil of decomposing plant materials. J App Biol Biotech 7:1–8. https://doi.org/10.7324/JABB.2019.70301
Oumer OJ, Abate D (2017) Characterization of pectinase from Bacillus subtilis strain Btk 27 and its potential application in removal of mucilage from coffee beans. Enzyme Res 2017:1–8. https://doi.org/10.1155/2017/7686904
Palanivelu P (2006) Polygalacturonases: Active site analyses and mechanism of action. Indian J Biotechnol 5:148–162
Patidar MK, Nighojkar A, Nighojkar S, Kumar A (2017) Purification and characterization of polygalacturonase produced by Aspergillus niger AN07 in solid state fermentation. Can J Biotech 1:11–18. https://doi.org/10.24870/cjb.2017-000102
Poondla V, Chikati R, Kallubai M, Chennupati V, Subramanyam R, Obulam V (2017) Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae. 3 Biotech 7:285. https://doi.org/10.1007/s13205-017-0912-5
Prejanò M, Alberto ME, Russo N, Toscano M, Marino T (2020) The effects of the metal ion substitution into the active site of metalloenzymes: A theoretical insight on some selected cases. Catalysts 10:1038. https://doi.org/10.3390/catal10091038
Radha S, Sridevi A, Prasad NBL, Narasimha G (2018) Statistical and kinetic studies of acid protease by Aspergillus spp. isolated from soil contaminated with Abattoir waste. Int J Pharm Pharm Sci 10:72–79. https://doi.org/10.22159/ijpps.2018v10i2.23165
Robin T, Reuveni S, Urbakh M (2018) Single-molecule theory of enzymatic inhibition. Nat Commun 9:779. https://doi.org/10.1038/s41467-018-02995-6
Robinson PK (2015) Enzymes: principles and biotechnological applications. Essays Biochem 59:1–41. https://doi.org/10.1042/bse0590001
Roosdiana A, Prasetyawan S, Mahdi C, Sutrisno S (2013) Production and characterization of Bacillus firmus pectinase. J Pure App Chem Res 2:35–41. https://doi.org/10.21776/ub.jpacr.2013.002.01.111
Şakar D, Karaoğlan GK, Gümrükçü G, Özgür MÜ (2008) Determination of anthocyanins in some vegetables and fruits by derivative spectrophotometric method. Rev Anal Chem 27:235–250. https://doi.org/10.1515/REVAC.2008.27.4.235
Samanta S (2021) Microbial pectinases: A review on molecular and biotechnological perspectives. J Microbiol Biotechnol Food Sci 9:248–266. https://doi.org/10.15414/jmbfs.2019.9.2.248-266
Sethi BK, Nanda PK, Sahoo S (2016) Enhanced production of pectinase by Aspergillus terreus NCFT 4269.10 using banana peels as substrate. 3 Biotech 6:36. https://doi.org/10.1007/s13205-015-0353-y
Shanmugavel M, Vasantharaj S, Yazhmozhi A, Bhavsar P, Aswin P, Felshia C, Mani U, Ranganathan B, Gnanamani A (2018) A study on pectinases from Aspergillus tamarii: toward greener approach for cotton bioscouring and phytopigments processing. Biocatal Agric Biotechnol 15:295–303. https://doi.org/10.1016/j.bcab.2018.06.013
Sharma AK, Singh SP (2016) Effect of amino acids on the repression of alkaline protease synthesis in haloalkaliphilic Nocardiopsis dassonvillei. Biotechnol Rep 12:40–51. https://doi.org/10.1016/j.btre.2016.10.004
Siddiq M, Dolan KD, Perkins-Veazie P, Collins JK (2018) Effect of pectinolytic and cellulytic enzymes on the physical, chemical, and antioxidant properties of blueberry (Vaccinium corymbosum L.) juice. LWT-Food Sci Technol 92:127–132. https://doi.org/10.1016/j.lwt.2018.02.008
Sommer S, Cohen SD (2018) Comparison of different extraction methods to predict anthocyanin concentration and colour characteristics of red wines. Fermentation 4:39. https://doi.org/10.3390/fermentation4020039
Wang Y, Wang G, Moitessier N, Mittermaier AK (2020) Enzyme kinetics by isothermal titration calorimetry: allostery, inhibition, and dynamics. Front Mol Biosci 7:583826. https://doi.org/10.3389/fmolb.2020.583826
Yu P, Xu C (2018) Production optimization, purification and characterization of a heat-tolerant acidic pectinase from Bacillus sp. ZJ1407. Int J Biol Macromol 108:972–980. https://doi.org/10.1016/j.ijbiomac.2017.11.012
Zehra M, Syed MN, Sohail M (2020) Banana peels: A promising substrate for the coproduction of pectinase and xylanase from Aspergillus fumigatus MS16. Pol J Microbiol 69:19–26. https://doi.org/10.33073/pjm-2020-002
Zhou HX, Pang X (2018) Electrostatic interactions in protein structure, folding, binding, and condensation. Chem Rev 118:1691–1741. https://doi.org/10.1021/acs.chemrev.7b00305
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We extend our sincere gratitude to the management of JAIN (Deemed-to-be University) for providing the research facilities. The authors are grateful to Ms. Meghna Chakraborty, Research Scholar, Department of Microbiology, JAIN (Deemed-to-be University) for the statistical analysis.
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SB designed the study and interpreted the data. AD contributed to the enzyme characterization studies. RB, DM and NVK performed the experiments. All authors have seen and approved the final manuscript and its contents, and are aware of the responsibilities connected to the authorship.
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The accession number (Genbank accession number MZ068227) for the Aspergillus terreus isolate FP6 registered in NCBI database has been provided in the manuscript. Link for the same is https://www.ncbi.nlm.nih.gov/nuccore/MZ068227.
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Bhattacharyya, R., Mukhopadhyay, D., Nagarakshita, V.K. et al. Thermostable and organic solvent-tolerant acid pectinase from Aspergillus terreus FP6: purification, characterization and evaluation of its phytopigment extraction potential. 3 Biotech 11, 487 (2021). https://doi.org/10.1007/s13205-021-03033-x
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DOI: https://doi.org/10.1007/s13205-021-03033-x