Purification and Characterization of Mannanase from Aspergillus awamori for Fruit Juice Clarification

Ikram ul Haq; Sheeba       Shakoor; Ali       Nawaz; Yesra       Arshad; Hamid       Mukhtar

doi:10.2174/0929866527666200916142305

Abstract

Background: Fruit juice clarification is a challenging aspect of beverage industry which needs to be addressed for economical and hygienic production of fruit juices.

Objective: Current study is focused on the complete purification, characterization and thermodynamic analysis of an efficient mannanase enzyme to analyze its applicability in biological clarification fruit juice.

Methods: Mannanase production using Aspergillus awamori IIB037 in a 25 L stirred fermenter at pre optimized reaction conditions was carried out. Enzyme purification was carried out via series of steps. Characterization of enzyme along with kinetics and thermodynamic studies was conducted. Purified and characterized enzyme was assessed for its applicability in fruit juice clarification through clarification experiments on fresh apple juice.

Results: Purification fold of 3.98 was obtained along with 86.80% purification yield of mannanase with specific activity of 158.16 U/mg. The molecular size of purified enzyme was determined as 66 kDa. The enzyme depicted 56% residual activity at 60°C after 8 hrs. Thermodynamic studies of an enzyme revealed enthalpy of activation (ΔH) and activation energy (Ea) as 30.53KJ/mol, 27.76KJ/mol, respectively. The enzyme activity increased in the presence of ß-mercaptoethanol surprisingly. On the other hand, methyl alcohol, ethanol, Hg2+ and Cu2+ inhibited enzyme activity. The enzyme showed Km and Vmax values of 11.07 mM and 19.08 μM min-1 for Locust Bean Gum (LBG) under optimal conditions. Juice treated with mannanase showed decrease in absorbance and increase in reducing sugar content.

Conclusion: The current study demonstrated that mannanase from Aspergillus awamori in its purified form has significant characteristics to be employed industrially for juice clarification.

Keywords: Thermodynamics, clarification, depolymerization, kinetics, characterization, catalysis.

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[1] 
Bharate, S.S.; Bharate, S.B. Non-enzymatic browning in citrus juice: chemical markers, their detection and ways to improve product quality. J. Food Sci. Technol.,  2014, 51(10), 2271-2288.
[http://dx.doi.org/10.1007/s13197-012-0718-8] [PMID: 25328169] 
[2] 
Pezzella, C.; Guarino, L.; Piscitelli, A. How to enjoy laccases. Cell. Mol. Life Sci.,  2015, 72(5), 923-940.
[http://dx.doi.org/10.1007/s00018-014-1823-9] [PMID: 25577278] 
[3] 
Sharma, H.P.; Patel, H; Sharma, S. Enzymatic extraction and clarification of juice from various fruits-a review. Postharvest Biol. Tec,  2014, 2(1), 1-14.
[4] 
Chauhan, P.S.; Jaiswar, S. Molecular dynamic simulation studies of bacterial thermostable mannanase unwinding the enzymatic catalysis. Biocatal. Agric. Biotechnol.,  2017, 9, 41-47.
[http://dx.doi.org/10.1016/j.bcab.2016.11.003] 
[5] 
Huang, Y.; Wang, L.; Chao, Y.; Nawawi, D.S.; Akiyama, T.; Yokoyama, T.; Matsumoto, Y. Relationships between hemicellulose composition and lignin structure in woods. J. Wood Chem. Technol.,  2016, 36(1), 9-15.
[http://dx.doi.org/10.1080/02773813.2015.1039543] 
[6] 
Harnpicharnchai, P.; Pinngoen, W.; Teanngam, W.; Sornlake, W.; Sae-Tang, K.; Manitchotpisit, P.; Tanapongpipat, S. Production of high activity Aspergillus niger BCC4525 β-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis. Biosci. Biotechnol. Biochem.,  2016, 80(12), 2298-2305.
[http://dx.doi.org/10.1080/09168451.2016.1230003] [PMID: 27648762] 
[7] 
Monclaro, A.V.; Aquino, E.N.; Faria, R.F.; Ricart, C.A.O.; Freitas, S.M.; Midorikawa, G.E.O.; Miller, R.N.G.; Michelin, M.; Polizeli, M.L.T.M. Characterization of multiple xylanase forms from Aspergillus tamarii resistant to phenolic compounds. Mycosphere,  2016, 7(10), 1554-1567.
[http://dx.doi.org/10.5943/mycosphere/si/3b/7] 
[8] 
Xie, Y.; Huang, C.; Li, X.; Lai, C.; Yu, S.; Yong, Q. Effect of agitation speed and aeration rate on fermentation synthesis of β-mannanase with Trichoderma reesei. Linye Gongcheng Xuebao,  2017, 2(6), 92-96.
[9] 
Liang, D.; Gong, L.; Yao, B.; Xue, X.; Qin, X.; Ma, R.; Luo, H.; Xie, X.; Su, X. Implication of a galactomannan-binding GH2 β-mannosidase in mannan utilization by Caldicellulosiruptor bescii. Biochem. Biophys. Res. Commun.,  2015, 467(2), 334-340.
[http://dx.doi.org/10.1016/j.bbrc.2015.09.156] [PMID: 26433124] 
[10] 
Anita, U.E.; Stephen, N.U.; Onilude, A.A.; Ibanga, I.A. Studies of the nutritional, environmental effects and repressive nature of simple sugars on the production of endo-β-mannanase by Aspergillus flavus PT7 on solid state fermentation. J. Adv. Biol. Biotechnol.,  2019, 21(4), 1-12.
[http://dx.doi.org/10.9734/jabb/2019/v21i430101] 
[11] 
Yang, M.; Cai, J.; Wang, C.; Du, X.; Lin, J. Characterization of endo-β-mannanase from Enterobacter ludwigii MY271 and application in pulp industry. Bioprocess Biosyst. Eng.,  2017, 40(1), 35-43.
[http://dx.doi.org/10.1007/s00449-016-1672-z] [PMID: 27534412] 
[12] 
Dey, T.B.; Banerjee, R. Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification. Braz. J. Microbiol.,  2014, 45(1), 97-104.
[http://dx.doi.org/10.1590/S1517-83822014000100014] [PMID: 24948919] 
[13] 
Suryawanshi, R.K.; Jana, U.K.; Prajapati, B.P.; Kango, N. Immobilization of Aspergillus quadrilineatus RSNK-1 multi-enzymatic system for fruit juice treatment and mannooligosaccharide generation. Food Chem.,  2019, 289, 95-102.
[http://dx.doi.org/10.1016/j.foodchem.2019.03.035] [PMID: 30955678] 
[14] 
Ichinose, H.; Suzuki, K.; Michikawa, M.; Sato, H.; Yuki, M.; Kamino, K.; Ogasawara, W.; Fushinobu, S.; Kaneko, S. Purification, cloning, functional expression, structure, and characterization of a thermostable β-mannanase from Talaromycestrachyspermus B168 and its efficiency in production of mannooligosaccharides from coffee wastes. J. Appl. Glycosci.,  2017, 65(2), 13-21.
[15] 
Sharma, K.; Dhillon, A.; Goyal, A. Insights into structure and reaction mechanism of β-mannanases. Curr. Protein Pept. Sci.,  2018, 19(1), 34-47.
[PMID: 27739373] 
[16] 
El-refai, M.A.; Khattab, O.K.H.; Ismail, S.A.; Hashem, A.M.; Abo-Elnasr, A.A.; Nour, S.A. Improved mannanase production from Penicillium humicola and application for hydrolysis property. Egypt. Pharmaceut. J.,  2014, 13(2), 160.
[http://dx.doi.org/10.4103/1687-4315.147102] 
[17] 
Olaniyi, O.O.; Ekundayo, T.C.; Igbe, O.F.; Fagbohungbe, Y.D. Influence of cultural and nutritional factors on β-mannanase production by Penicillium italicum under submerged state fermentation. Br. Microbiol. Res. J.,  2015, 5(6), 481-489.
[http://dx.doi.org/10.9734/BMRJ/2015/6887] 
[18] 
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.,  1976, 72(1-2), 248-254.
[http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051] 
[19] 
Lee, C.R.; Park, Y.H.; Min, H.; Kim, Y.R.; Seok, Y.J. Determination of protein phosphorylation by polyacrylamide gel electrophoresis. J. Microbiol.,  2019, 57(2), 93-100.
[http://dx.doi.org/10.1007/s12275-019-9021-y] [PMID: 30706340] 
[20] 
Siddiqui, K.S.; Rashid, M.H.; Ghauri, T.M.; Durrani, I.S.; Rajoka, M.I. Purification and characterization of an intracellular β-glucosidase from Cellulomonas biazotea. World J. Microbiol. Biotechnol.,  1997, 13, 245-247.
[http://dx.doi.org/10.1023/A:1018510418900] 
[21] 
Adiguzel, A.; Nadaroglu, H.; Adiguzel, G. Purification and characterization of β-mannanase from Bacillus pumilus (M27) and its applications in some fruit juices. J. Food Sci. Technol.,  2015, 52(8), 5292-5298.
[http://dx.doi.org/10.1007/s13197-014-1609-y] [PMID: 26243955] 
[22] 
Miller, D.L. Use of Dinitrosalicylic acid as reagent of reducing sugars. Anal. Chem.,  1959, 31, 426-428.
[http://dx.doi.org/10.1021/ac60147a030] 
[23] 
Tang, S.Z.; Lin, F.L.; Zheng, J.; Zhou, H.B. Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris.  J. Cent. South Univ. T.,  2019, 26(1), 184-195.
[24] 
Bhoria, P.; Singh, G.; Hoondal, G.S. Optimization of mannanase production from streptomyces sp. in submerged fermentation. Biol. Res.,  2009, 4(3), 1130-1138.
[25] 
Arotupin, D.J.; Olaniyi, O.O. Screening and identification of mannanase-producing fungi isolated from selected agricultural wastes. Br. Microbiol. Res. J.,  2013, 3(4), 635-644.
[http://dx.doi.org/10.9734/BMRJ/2013/4218] 
[26] 
Zine, A.S.; Peshwe, S.A. Isolation screening and identification of fungal mannanase producer. Int. J. Curr. Res.,  2018, 7(02), 10931097.
[27] 
Marques, N.P.; de Cassia Pereira, J.; Gomes, E.; da Silva, R.; Araújo, A.R.; Ferreira, H.; Rodrigues, A.; Dussán, K.J.; Bocchini, D.A. Cellulases and xylanases production by endophytic fungi by solid state fermentation using lignocellulosic substrates and enzymatic saccharification of pretreated sugarcane bagasse. Ind. Crops Prod.,  2018, 122, 66-75.
[http://dx.doi.org/10.1016/j.indcrop.2018.05.022] 
[28] 
Karahalil, E.; Germec, M.; Karaoglan, M.; Yatmaz, E.; Coban, H.B.; Inan, M.; Turhan, I. Partial purification and characterization of a recombinant β-mannanase from Aspergillus fumigatus expressed in Aspergillus sojae grown on carob extract. In: Biomass. Convers. Bioref; , 2020; 10, pp. 1189-1205.
[29] 
Wang, C.; Zhang, J.; Wang, Y.; Niu, C.; Ma, R.; Wang, Y.; Bai, Y.; Luo, H.; Yao, B. Biochemical characterization of an acidophilic β-mannanase from Gloeophyllum trabeum CBS900.73 with significant transglycosylation activity and feed digesting ability. Food Chem.,  2016, 197(Pt A), 474-481.
[http://dx.doi.org/10.1016/j.foodchem.2015.10.115] [PMID: 26616977] 
[30] 
Blibech, M.; Ghorbel, R.E.; Fakhfakh, I.; Ntarima, P.; Piens, K.; Bacha, A.B.; Chaabouni, S.E. Purification and characterization of a low molecular weight of β-mannanase from Penicillium occitanis Pol6. Appl. Biochem. Biotechnol.,  2010, 160(4), 1227-1240.
[http://dx.doi.org/10.1007/s12010-009-8630-z] [PMID: 19418261] 
[31] 
Cheng, L.; Duan, S.; Feng, X.; Zheng, K.; Yang, Q.; Liu, Z. Purification and characterization of a thermostable β-mannanase from Bacillus subtilis BE-91: potential application in inflammatory diseases. BioMed Res. Int.,  2016, 2016, 6380147.
[http://dx.doi.org/10.1155/2016/6380147] [PMID: 27868067] 
[32] 
de Marco, J.; Neto, G.; Castro, C.; Michelin, M.; Polizeli, M. Partial purification and characterization of a thermostable β-mannanase from Aspergillus foetidus. App. Sci.,  2015, 5(4), 881-893.
[http://dx.doi.org/10.3390/app5040881] 
[33] 
Ahirwar, S.; Soni, H.; Rawat, H.K.; Ganaie, M.A.; Pranaw, K.; Kango, N. Production optimization and functional characterization of thermostable β-mannanase from Malbranchea cinnamomea NFCCI 3724 and its applicability in mannotetraose (M4) generation. J. Taiwan Inst. Chem. Engrs.,  2016, 63, 344-353.
[http://dx.doi.org/10.1016/j.jtice.2016.03.033] 
[34] 
Nadaroglu, H.; Adiguzel, G.; Adiguzel, A.; Sonmez, Z. A thermostable-endo-β-(1, 4)-mannanase from Pediococcus acidilactici (M17): purification, characterization and its application in fruit juice clarification. Eur. Food Res. Technol.,  2017, 243(2), 193-201.
[http://dx.doi.org/10.1007/s00217-016-2735-8] 
[35] 
Koivisto, E.; Zevenhoven, R. Membrane separation of ammonium bisulfate from ammonium sulfate in aqueous solutions for CO2 mineralisation. Geosci.,  2018, 8(4), 123.
[http://dx.doi.org/10.3390/geosciences8040123] 
[36] 
Naganagouda, K.; Salimath, P.V.; Mulimani, V.H. Purification and characterization of endo-β-1,4 mannanase from Aspergillus niger gr for application in food processing industry. J. Microbiol. Biotechnol.,  2009, 19(10), 1184-1190.
[PMID: 19884778] 
[37] 
Liao, H.; Li, S.; Zheng, H.; Wei, Z.; Liu, D.; Raza, W.; Shen, Q.; Xu, Y. A new acidophilic thermostable endo-1,4-β-mannanase from Penicillium oxalicum GZ-2: cloning, characterization and functional expression in Pichia pastoris. BMC Biotechnol.,  2014, 14(1), 90.
[http://dx.doi.org/10.1186/s12896-014-0090-z] [PMID: 25348022] 
[38] 
El-Sharouny, E.E.; El-Toukhy, N.M.; El-Sersy, N.A.; El-Gayar, A.A.E.A. Optimization and purification of mannanase produced by an alkaliphilic-thermotolerant Bacillus cereus N1 isolated from Bani Salama Lake in Wadi El-Natron. Biotechnol. Biotechnol. Equip.,  2015, 29(2), 315-323.
[http://dx.doi.org/10.1080/13102818.2014.995932] [PMID: 26019646] 
[39] 
Chai, S.Y.; Bakar, F.D.A.; Mahadi, N.M.; Murad, A.M.A. A thermotolerant Endo-1, 4-β-mannanase from Trichoderma virens UKM1: Cloning, recombinant expression and characterization. J. Mol. Catal., B Enzym.,  2016, 125, 49-57.
[http://dx.doi.org/10.1016/j.molcatb.2015.12.011] 
[40] 
Benech, R.O.; Li, X.; Patton, D.; Powlowski, J.; Storms, R.; Bourbonnais, R.; Paice, M.; Tsang, A. Recombinant expression, characterization, and pulp prebleaching property of a Phanerochaetechrysosporiumendo-β-1,4-mannanase. Enzyme Microb. Technol.,  2007, 41, 740-747.
[http://dx.doi.org/10.1016/j.enzmictec.2007.06.012] 
[41] 
Chen, X.; Cao, Y.; Ding, Y.; Lu, W.; Li, D. Cloning, functional expression and characterization of Aspergillus sulphureus beta-mannanase in Pichia pastoris. J. Biotechnol.,  2007, 128(3), 452-461.
[http://dx.doi.org/10.1016/j.jbiotec.2006.11.003] [PMID: 17194495] 
[42] 
Ismail, S.A.; Khattab, O.K.H.; Nour, S.A.; Awad, G.E.; Abo-Elnasr, A.A.; Hashem, A.M. A thermodynamic study of partially-purified penicillium humicola β-mannanase produced by statistical optimization. J. Biol. Sci.,  2019, 12(2), 209-217.
[43] 
Unsworth, L.D.; van der Oost, J.; Koutsopoulos, S. Hyperthermophilic enzymes--stability, activity and implementation strategies for high temperature applications. FEBS J.,  2007, 274(16), 4044-4056.
[http://dx.doi.org/10.1111/j.1742-4658.2007.05954.x] [PMID: 17683334] 
[44] 
Olaniyi, O.O.; Arotupin, D.J.; Akinyele, B.J.; Bamidele, O.S. Kinetic properties of purified β-mannanase from Penicillium italicum. Br. Microbiol. Res. J.,  2014, 4, 1092-1104.
[http://dx.doi.org/10.9734/BMRJ/2014/6555] 
[45] 
Verma, D.; Kawarabayasi, Y.; Miyazaki, K.; Satyanarayana, T. Cloning, expression and characteristics of a novel alkalistable and thermostable xylanase encoding gene (Mxyl) retrieved from compost-soil metagenome. PLoS One,  2013, 8(1), e52459.
[http://dx.doi.org/10.1371/journal.pone.0052459] [PMID: 23382818] 
[46] 
Soni, H.; Rawat, H. K.; Pletschke, B. I.; Kango, N. Purification and characterization of β-mannanase from Aspergillus terreus and its applicability in depolymerization of mannans and saccharification of lignocellulosic biomass.   3 Biotech,  2016, 6(2), 136.
[47] 
Chivero, E.T.; Mutukumira, A.N.; Zvauya, R. Partial purification and characterisation of a xylanase enzyme produced by a micro-organism isolated from selected indigenous fruits of Zimbabwe. Food Chem.,  2001, 72(2), 179-185.
[http://dx.doi.org/10.1016/S0308-8146(00)00216-8] 
[48] 
Gonçalves, G.A.; Takasugi, Y.; Jia, L.; Mori, Y.; Noda, S.; Tanaka, T.; Ichinose, H.; Kamiya, N. Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse. Enzyme Microb. Technol.,  2015, 72, 16-24.
[http://dx.doi.org/10.1016/j.enzmictec.2015.01.007] [PMID: 25837503] 
[49] 
Yin, L.J.; Tai, H.M.; Jiang, S.T. Characterization of mannanase from a novel mannanase-producing bacterium. J. Agric. Food Chem.,  2012, 60(25), 6425-6431.
[http://dx.doi.org/10.1021/jf301944e] [PMID: 22694324] 
[50] 
Wu, F.; Su, L.; Yu, P.; Mao, L. Role of organic solvents in immobilizing fungus laccase on single-walled carbon nanotubes for improved current response in direct bioelectrocatalysis. J. Am. Chem. Soc.,  2017, 139(4), 1565-1574.
[http://dx.doi.org/10.1021/jacs.6b11469] [PMID: 28052671] 
[51] 
Ge, J.; Du, R.; Zhao, D.; Song, G.; Jin, M.; Ping, W. Kinetic study of a β-mannanase from the Bacillus licheniformis HDYM-04 and its decolorization ability of twenty-two structurally different dyes. Springerplus,  2016, 5(1), 1824.
[http://dx.doi.org/10.1186/s40064-016-3496-3] [PMID: 27818862] 
[52] 
Vu, T.T.; Quyen, D.T.; Dao, T.T.; Nguyen, Sle.T. Cloning, high-level expression, purification, and properties of a novel endo-β-1,4-mannanase from Bacillus subtilis G1 in Pichia pastoris. J. Microbiol. Biotechnol.,  2012, 22(3), 331-338.
[http://dx.doi.org/10.4014/jmb.1106.06052] [PMID: 22450788] 
[53] 
Hemant, S.; Hemant, K. R.; Brett, P.; Naveen, K. Purification and characterization of β-mannanase from Aspergillus terreus and its applicability in depolymerization of mannans and saccharification of lignocellulosic biomass.  3 Biotech.,  2016, 6(2), 136.
[54] 
Jana, U.K.; Suryawanshi, R.K.; Prajapati, B.P.; Soni, H.; Kango, N. Production optimization and characterization of mannooligosaccharide generating β-mannanase from Aspergillus oryzae. Bioresour. Technol.,  2018, 268, 308-314.
[http://dx.doi.org/10.1016/j.biortech.2018.07.143] [PMID: 30092484] 

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DOI https://dx.doi.org/10.2174/0929866527666200916142305	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305

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Purification and Characterization of Mannanase from Aspergillus awamori for Fruit Juice Clarification

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