Preliminary investigations on a polygalacturonase from Aspergillus fumigatus in Chinese Pu’er tea fermentation

Abstract

Background

Polygalacturonase is one kind of pectinases which hydrolyze the alpha-1,4 glycosidic bond between galacturonic acid residue. Polygalacturonase has been widely used in the fields of food, biofuel, and textile industries, in which thermostable polygalacturonase is often demanded at high temperatures of 50–60 °C. Herein, we reported a thermostable polygalacturonase producing from Aspergillus fumigatus isolated from the pile fermentation of Pu’er tea in China.

Results

The thermophilic polygalacturonase-producing strain was identified as A. fumigatus L45 on basis of its morphology, physicochemical properties, and 18S rDNA analysis. The crucial fermentation parameters affecting polygalacturonase activity were optimized by response surface methodology (RSM); the optimum fermentation parameters were the following: inoculums concentration of 0.07 % (v/v), fermentation time of 36 h, pH of 5.0, and temperature of 45 °C. Under the optimized conditions, the highest polygalacturonase activity of 359.1 ± 10.1 U/mL was obtained. The polygalacturonase showed good thermostability and pH stability. The enzyme was activated by metal ions Zn²⁺ and Mg²⁺, but inhibited by K⁺. However, Na⁺ and Ca²⁺ showed little effects on its activity. K_m and V_max values were estimated to be 35.0 mg/mL and 7.69 μmol/mL/min, respectively.

Conclusions

A polygalacturonase from A. fumigatus L45 was preliminarily investigated, the crucial fermentation parameters were optimized by RSM, and the properties of polygalacturonase was examined. The polygalacturonase showed good thermostability and pH stability, which suggested the enzyme has potential applications in the biofuel and textile industries.

Keywords

Polygalacturonase / Aspergillus fumigatus / Identification / Response surface methodology (RSM) / Enzymatic properties

References

[1.] da Silva ME, Leite RSR, da Silva R, Gomes E. Production and characterization of polygalacturonase from thermophilic Thermoascus aurantiacus on submerged fermentation. Ann Microbiol, 2012, 62(3): 1199-1205.

[2.] Malvessi E, Silveira MM. Influence of medium composition and pH on the production of polygalacturonases by Aspergillus oryzae. Braz Arch Biol Technol, 2004, 47(5): 693-702.

[3.] Heerd D, Yegin S, Tari C, Fernandez-Lahore M. Pectinase enzyme-complex production by Aspergillus spp. in solid-state fermentation: a comparative study. Food Bioprod Process, 2012, 90(2): 102-110.

[4.] Phutela U, Dhuna V, Sandhu S, Chadha B. Pectinase and polygalacturonase production by a thermophilic Aspergillus fumigatus isolated from decomposting orange peels. Braz J Microbio, 2005, 36(1): 63-69.

[5.] Kaur A, Mahajan R, Singh A, Garg G, Sharma J. A novel and cost effective methodology for qualitative screening of alkalo-thermophilic cellulase free xylano-pectinolytic microorganisms using agricultural wastes. World J Microb Biotechnol, 2011, 27(2): 459-463.

[6.] Ouattara HG, Koffi BL, Karou GT, Sangare A, Niamke SL, Diopoh JK. Implication of Bacillus sp in the production of pectinolytic enzymes during cocoa fermentation. World J Microb Biotechnol, 2008, 24(9): 1753-1760.

[7.] Jayani RS, Saxena S, Gupta R. Microbial pectinolytic enzymes: a review. Process Biochem, 2005, 40(9): 2931-2944.

[8.] Kant S, Vohra A, Gupta R. Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323. Protein Expres Purif, 2013, 87(1): 11-16.

[9.] Oyeleke S, Oyewole O, Egwim E, Dauda B, Ibeh E. Cellulase and pectinase production potentials of Aspergillus niger isolated from corn cob. Bayero J Pure Appl Sci, 2012, 5(1): 78-83.

[10.] Sandri IG, Lorenzoni CMT, Fontana RC, da Silveira MM. Use of pectinases produced by a new strain of Aspergillus niger for the enzymatic treatment of apple and blueberry juice. LWT-Food Sci Technol, 2013, 51(2): 469-475.

[11.] Lan L, Zhou Q, Cai Q, Dong X, Wang P, Jiang D. Screening and identification of a high-yield pectinase Penicillium strain. J Zhejiang Normal Uni (Natural Sci), 2011, 4: 452-456.

[12.] Banu AR, Devi MK, Gnanaprabhal G, Pradeep B, Palaniswamy M. Production and characterization of pectinase enzyme from Penicillium chrysogenum. Indian J Sci Techn, 2010, 3(4): 377-381.

[13.] Tu T, Bai YG, Luo HY, Ma R, Wang YR, Shi PJ, . A novel bifunctional pectinase from Penicillium oxalicum SX6 with separate pectin methylesterase and polygalacturonase catalytic domains. Appl Microbiol Biot, 2014, 98(11): 5019-5028.

[14.] Qureshi AS, Bhutto MA, Chisti Y, Khushk I, Dahot MU, Bano S. Production of pectinase by Bacillus subtilis EFRL 01 in a date syrup medium. Afr J Biotechnol, 2012, 11: 12563-12570.

[15.] Rehman HU, Aman A, Silipo A, Qader SAU, Molinaro A, Ansari A. Degradation of complex carbohydrate: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support. Food Chem, 2013, 139(1): 1081-1086.

[16.] Tepe O, Dursun AY. Exo-pectinase production by Bacillus pumilus using different agricultural wastes and optimizing of medium components using response surface methodology. Environ Sci Pollut R, 2014, 21(16): 9911-9920.

[17.] Kittur FS, Vishu Kumar AB, Tharanathan RN. Low molecular weight chitosans—preparation by depolymerization with Aspergillus niger pectinase, and characterization. Carbohyd Res, 2003, 338(12): 1283-1290.

[18.] Singh SA, Appu Rao A. A simple fractionation protocol for, and a comprehensive study of the molecular properties of, two major endopolygalacturonases from Aspergillus niger. Biotechnol Appl Biochem, 2002, 35(2): 115-123.

[19.] Corredig M, Kerr W, Wicker L. Separation of thermostable pectinmethylesterase from marsh grapefruit pulp. J Agr Food Chem, 2000, 48(10): 4918-4923.

[20.] Mei Y, Chen Y, Zhai R, Liu Y. Cloning, purification and biochemical properties of a thermostable pectinase from Bacillus halodurans M29. J Mol Catal B Enzym, 2013, 94(0): 77-81.

[21.] Pakarinen A, Zhang J, Brock T, Maijala P, Viikari L. Enzymatic accessibility of fiber hemp is enhanced by enzymatic or chemical removal of pectin. Bioresource Technol, 2012, 107: 275-281.

[22.] Mathew A, Eldo AN, Molly A. Optimization of culture conditions for the production of thermostable polygalacturonase by Penicillium SPC-F 20. J Ind Microbiol Biotechnol, 2008, 35(9): 1001-1005.

[23.] Maller A, Damásio ARL, Silva TMd, Jorge JA, Terenzi HF, Polizeli MdLTdM (2011) Biotechnological potential of agro-industrial wastes as a carbon source to thermostable polygalacturonase production in Aspergillus niveus. Enzyme research. 2011:6. Article ID 289206, doi: 289210.284061/282011/289206

[24.] Martins ES, Leite RSR, da Silva R, Gomes E. Purification and properties of polygalacturonase produced by thermophilic fungus Thermoascus aurantiacus CBMAI-756 on solid-state fermentation. Enzyme research, 2013, 438645: 1.

[25.] Zeni J, Cence K, Grando CE, Tiggermann L, Colet R, Lerin LA, . Screening of pectinase-producing microorganisms with polygalacturonase activity. Appl Biochem Biotechnol, 2011, 163(3): 383-392.

[26.] Lin Q, Liu Y. A new marine microorganism strain L0804: taxonomy and characterization of active compounds from its metabolite. World J Microb Biotechnol, 2010, 26(9): 1549-1556.

[27.] Dong S, Huang Y, Zhang R, Lian Z, Wang S, Liu Y. Inclusion complexes of astaxanthin with hydroxypropyl-β-cyclodextrin: parameters optimization, spectroscopic profiles, and properties. Eur J Lipid Sci Technol, 2014, 116(8): 978-986.

[28.] Liu Y, Yan Y, Hu F, An Y, Wang Z, Wei F. Transesterification for biodiesel production catalyzed by combined lipases: optimization and kinetics. Aiche J, 2010, 56(6): 1659-1665.

[29.] Wang S, Sun Z, Dong S, Liu Y, Liu Y. Molecular interactions between (−)-epigallocatechin gallate analogs and pancreatic lipase. PLoS ONE, 2014, 9(11

[30.] Ghani M, Ansari A, Aman A, Zohra RR, Siddiqui NN, Qader SAU. Isolation and characterization of different strains of Bacillus licheniformis for the production of commercially significant enzymes. Pak J Pharm Sci, 2013, 26(4): 691-697.

[31.] Babavalian H, Amoozegar MA, Pourbabaee AA, Moghaddam MM, Shakeri F. Isolation and identification of moderately halophilic bacteria producing hydrolytic enzymes from the largest hypersaline playa in Iran. Microbiology, 2013, 82(4): 466-474.

[32.] Patil SR, Dayanand A. Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions. Bioresour Technol, 2006, 97(18): 2340-2344.

[33.] Taşkin E, Eltem R, Silva ES, Souza JVB. Screening of Aspergillus strains isolated from vineyards for pectinase production. Journal of food, agriculture & environment, 2008, 6: 412-414.

[34.] Fachin D, Smout C, Verlent I, Ly Nguyen B, Van Loey A, Hendrichx M. Inactivation kinetics of purified tomato polygalacturonase by thermal and high-pressure processing. J Agric Food Chem, 2004, 52: 2697-2703.

[35.] Tucker GA, Robertsons NG, Grierson D. The conversion of tomato-fruit polygalacturonase isoenzyme 2 into isoenzyme 1 in vitro. Eur J Biochem, 1981, 115: 87-90.

[36.] Ali ZM, Brady CJ. Purification and characterization of the polygalacturonases of tomato fruits. Aust J Plant Physiol, 1982, 9: 155-169.

[37.] Moshrefi M, Luh BS. Purification and characterization of two tomato polygalacturonase isoenzymes. J Food Biochem, 1984, 8: 39-54.

[38.] Siddiqui MA, Pande V, Arif M (2012) Production, purification, and characterization of polygalacturonase from Rhizomucor pusillus isolated from decomposting orange peels. Enzyme Res. 2012:8. Article ID 138634, doi:138610.131155/132012/138634

[39.] Baracat-Pereira MC, Vanetti MCD, de Araujo EF, Silva DO. Partial characterization of Aspergillus fumigatus polygalacturonases for the degumming of natural fibers. J Ind Microbiol, 1993, 11(3): 139-142.

[40.] Ezugwu A, Eze S, Chilaka F, Anyanwu C. Production and characterization of pectinases obtained from Aspergillus fumigatus in submerged fermentation system using pectin extracted from mango peels as carbon source. Plant Products Research Journal, 2013, 16(1): 47-53.

[41.] Rashad MM, Abdou HM, Shousha WG, Ali MM, El-Sayed NN. Purification and characterization of extracellular polygalacturonase from Pleurotus Ostreatus using citrus limonium waste. J Appl Sci Res, 2010, 6(1): 81-88.

[42.] Riou C, Freyssinet G, Fevre M. Purification and characterization of extracellular pectinolytic enzymes produced by Sclerotinia sclerotiorum. Appl Environ Microbiol, 1992, 58(2): 578-583.

[43.] Silva D, Martins ES, Rd S, Gomes E. Pectinase production by Penicillium viridicatum RFC3 by solid state fermentation using agricultural wastes and agro-industrial by-products. Braz J Microbio, 2002, 33: 318-324.

[44.] Martins E, Silva D, Da Silva R, Gomes E. Solid state production of thermostable pectinases from thermophilic Thermoascus aurantiacus. Process Biochem, 2002, 37(9): 949-954.

[45.] Damásio ARL, Maller A, Silva TM, Jorge JA, Terenzi HF, Polizeli MLT. Biotechnological potential of alternative carbon sources for production of pectinases by Rhizopus microsporus var. rhizopodiformis. Braz Arch Biol Technol, 2011, 54(1): 141-148.