Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Heterologous Expression and Characterization of a Novel Exo-Polygalacturonase from Aspergillus fumigatus Af293 and Its Application in Juice Extraction

  • Chengwei Yang (College of Food Science and Quality Engineering, Nanning University) ;
  • Ting Zhang (College of Food Science and Quality Engineering, Nanning University) ;
  • Jing Zhu (College of Food Science and Quality Engineering, Nanning University) ;
  • Yunyi Wei (College of Food Science and Quality Engineering, Nanning University) ;
  • Furong Zhu (College of Food Science and Quality Engineering, Nanning University) ;
  • Zhong Cheng (College of Food Science and Quality Engineering, Nanning University)
  • Received : 2022.11.02
  • Accepted : 2022.12.24
  • Published : 2023.04.28
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Abstract

Exo-polygalacturonase (exo-PG) hydrolyzes pectin acids and liberates mono-galacturonate, which plays an important role in juice extraction, and has rarely been reported. Exo-PG (AfumExoPG28A) from Aspergillus fumigatus belongs to the glycoside hydrolase 28 family. In this study, its gene was cloned and the protein was expressed and secreted in Pichia pastoris with a maximal activity of 4.44 U/ml. The optimal temperature and pH of AfumExoPG28A were 55℃ and 4.0, respectively. The enzyme exhibited activity over almost the entire acidic pH range (>20.0% activity at pH 2.5-6.5) and remained stable at pH 2.5-10.0 for 24 h. The Km and Vmax values of AfumExoPG28A were calculated by the substrate of polygalacturonic acid as 25.4 mg/ml and 23.6 U/mg, respectively. Addition of AfumExoPG28A (0.8 U/mg) increased the light transmittance and juice yield of plantain pulp by 11.7% and 9%, respectively. Combining AfumExoPG28A (0.8 U/mg) with an endo-PG (0.8 U/mg) from our laboratory, the enzymes increased the light transmittance and juice yield of plantain pulp by 45.7% and 10%, respectively. Thus, the enzyme's potential value in juice production was revealed by the remarkable acidic properties and catalytic activity in fruit pulp.

Keywords

Acknowledgement

This work was financially supported by the Doctoral Scientific Research Fund of Nanning University (2021DSRF01), Science Foundation of Nanning University (2022XJ18).

References

  1. Lara-Espinoza C, Carvajal-Millan E, Balandran-Quintana R, Lopez-Franco Y, Rascon-Chu A. 2018. Pectin and pectin-based composite materials: Beyond food texture. Molecules 23: 942.
  2. Samanta S. 2021. Microbial pectinases: a review on molecular and biotechnological perspectives. J. Microbiol. Biotechol. Food Sci. 2021: 248-266. https://doi.org/10.15414/jmbfs.2019.9.2.248-266
  3. Satapathy S, Rout JR, Kerry RG, Thatoi H, Sahoo SL. 2020. Biochemical prospects of various microbial pectinase and pectin: an approachable concept in pharmaceutical bioprocessing. Front. Nutr. 7: 117.
  4. Xu H, Zhang P, Zhang Y, Liu Z, Zhang X, Li Z, et al. 2020. Overexpression and biochemical characterization of an endo-α-1, 4-polygalacturonase from Aspergillus nidulans in Pichia pastoris. Int. J. Mol. Sci. 21: 2100.
  5. Ortiz GE, Ponce-Mora MC, Noseda DG, Cazabat G, Saravalli C, Lopez MC, et al. 2017. Pectinase production by Aspergillus giganteus in solid-state fermentation: optimization, scale-up, biochemical characterization and its application in olive-oil extraction. J. Ind. Microbiol. Biotechnol. 44: 197-211. https://doi.org/10.1007/s10295-016-1873-0
  6. Nathan VK, Rani ME, Rathinasamy G, Dhiraviam KN. 2017. Low molecular weight xylanase from Trichoderma viride VKF3 for bio-bleaching of newspaper pulp. BioResources 12: 5264-5278. https://doi.org/10.15376/biores.12.3.5264-5278
  7. Ning T, Chen C, Yi G, Chen H, Liu Y, Fan Y, et al. 2021. Changes in homogalacturonan metabolism in banana peel during fruit development and ripening. Int. J. Mol. Sci. 23: 243.
  8. Kashyap DR, Vohra PK, Chopra S, Tewari R. 2001. Applications of pectinases in the commercial sector: a review. Bioresour. Technol. 77: 215-27. https://doi.org/10.1016/S0960-8524(00)00118-8
  9. Hoondal G, Tiwari R, Tewari R, Dahiya NBQK, Beg Q. 2002. Microbial alkaline pectinases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 59: 409-418. https://doi.org/10.1007/s00253-002-1061-1
  10. Singh A, Yadav RD, Kaur A, Mahajan R. 2012. An ecofriendly cost effective enzymatic methodology for deinking of school waste paper. Bioresour. Technol. 120: 322-327. https://doi.org/10.1016/j.biortech.2012.06.050
  11. Huang D, Song Y, Liu Y, Qin Y. 2019. A new strain of Aspergillus tubingensis for high-activity pectinase production. Braz. J. Microbiol. 50: 53-65. https://doi.org/10.1007/s42770-018-0032-3
  12. Shanmugavel M, Vasantharaj S, Yazhmozhi A, Bhavsar P, Aswin P, Felshia C. 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
  13. Kassara S, Li S, Smith P, Blando F, Bindon K. 2019. Pectolytic enzyme reduces the concentration of colloidal particles in wine due to changes in polysaccharide structure and aggregation properties. Int. J. Biol. Macromol. 140: 546-555. https://doi.org/10.1016/j.ijbiomac.2019.08.043
  14. Demir N, Acar J, Sarioglu K, Mutlu M. 2001. The use of commercial pectinase in fruit juice industry. Part 3: Immobilized pectinase for mash treatment. J. Food Eng. 47: 275-280. https://doi.org/10.1016/S0260-8774(00)00127-8
  15. Yepes C, Estevez J, Arroyo M, Ladero M. 2022. Immobilization of an industrial β-glucosidase from Aspergillus fumigatus and its use for cellobiose hydrolysis. Processes 10: 1225.
  16. Thakur N, Patel SK, Kumar P, Singh A, Devi N, Sandeep K, et al. 2022. Bioprocess for hyperactive thermotolerant Aspergillus fumigatus phytase and its application in dephytinization of wheat flour. Catal. Lett. 152: 3220-3232. https://doi.org/10.1007/s10562-021-03886-0
  17. Zhou Z, Guo G, Li J, Yan H, Li F. 2022. Pretreatment of natural lignocellulose with inorganic salts improves ligninase production fermented by Aspergillus fumigatus. J. Polym. Environ. 30: 3633-3644. https://doi.org/10.1007/s10924-022-02456-8
  18. Lu B, Xian L, Zhu J, Wei Y, Yang C, Cheng Z. 2022. A novel endo-polygalacturonase from Penicillium oxalicum: gene cloning, heterologous expression and its use in acidic fruit juice extraction. J. Microbiol. Biotechnol. 32: 464-472. https://doi.org/10.4014/jmb.2112.12023
  19. 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. https://doi.org/10.1016/0003-2697(76)90527-3
  20. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  21. Cheng Z, Chen D, Wang Q, Xian L, Lu B, Wei Y, et al. 2017. Identification of an acidic endo-polygalacturonase from Penicillium oxalicum CZ1028 and its broad use in major tropical and subtropical fruit juices production. J. Biosci. Bioeng. 123: 665-672. https://doi.org/10.1016/j.jbiosc.2017.01.013
  22. Adedeji OE, Ezekie lOO. 2020. Chemical composition and physicochemical properties of mango juice extracted using polygalacturonase produced by Aspergillus awamori CICC 2040 on pretreated orange peel. LWT 132: 109891.
  23. Cheng Z, Chen D, Lu B, Wei Y, Xian L, Li Y, et al. 2016. A novel acid-stable endo-polygalacturonase from Penicillium oxalicum CZ1028: purification, characterization and application in the beverage industry. J. Microbiol. Biotechnol. 26: 989-998. https://doi.org/10.4014/jmb.1511.11045
  24. Van SY, Benen JA, Schroter KH, Kalk KH, Armand S, Visser J, et al. 1999. 1.68-A Crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. J. Biol. Chem. 274: 30474-30480. https://doi.org/10.1074/jbc.274.43.30474
  25. Li Y, Wang Y, Tu T, Zhang D, Ma R, You S, et al. 2017. Two acidic, thermophilic GH28 polygalacturonases from Talaromyces leycettanus JCM 12802 with application potentials for grape juice clarification. Food Chem. 237: 997-1003. https://doi.org/10.1016/j.foodchem.2017.06.037
  26. Peng XB, Chen GJ, Han ZG, Yang JK. 2019. High-level secretive expression of a novel achieved Talaromyces cellulolyticus endo-polygalacturonase in Pichia pastoris by improving gene dosage for hydrolysis of natural pectin. World J. Microbiol. Biotechnol. 35: 84.
  27. Tan H, Yang G, Chen W, Liu Q, Li K, Yin H. 2020. Identification and characterization of thermostable endo-polygalacturonase II B from Aspergillus luchuensis. J. Food Biochem. 44: e13133.
  28. Xu H, Zhang P, Zhang Y, Liu Z, Zhang X, Li Z, et al. 2020. Overexpression and biochemical characterization of an endo-α-1, 4-polygalacturonase from Aspergillus nidulans in Pichia pastoris. Int. J. Mol. Sci. 21: 2100.
  29. Abdulrachman D, Thongkred P, Kocharin K, Nakpathom M, Somboon B, Narumol N, 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.
  30. Wagschal KC, Rose SJ, Chan VJ, Jordan DB. 2017. Expression and characterization of hyperthermostable exopolygalacturonase RmGH28 from Rhodothermus marinus. Appl. Biochem. Biotechol. 183: 1503-1515. https://doi.org/10.1007/s12010-017-2518-0
  31. Tu T, Meng K, Bai Y, Shi P, Luo H, Wang Y, et al. 2013. High-yield production of a low-temperature-active polygalacturonase for papaya juice clarification. Food. Chem. 141: 2974-2981. https://doi.org/10.1016/j.foodchem.2013.05.132
  32. Osorio S, Castillejo C, Quesada MA, Medina-Escobar N, Brownsey GJ, Suau R, et al. 2008. Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca). Plant J. 54: 43-55. https://doi.org/10.1111/j.1365-313X.2007.03398.x
  33. De LDAR, Da STM, Maller A, Jorge JA, Terenzi HF, Polizeli MDLTDM. 2010. Purification and partial characterization of an exo-polygalacturonase from Paecilomyces variotii liquid cultures. Appl. Biochem. Biotechnol. 160: 1496-1507. https://doi.org/10.1007/s12010-009-8682-0
  34. Sakiyama CC, Paula EM, Pereira PC, Borges AC, Silva DO. 2001. Characterization of pectin lyase produced by an endophytic strain isolated from coffee cherries. Lett. Appl. Microbiol. 33: 117-121. https://doi.org/10.1046/j.1472-765x.2001.00961.x
  35. Lu X , Lin J , Wang C. 2016. Purification and characterization of exo-polygalacturonase from Zygoascus hellenicus V25 and its potential application in fruit juice clarification. Food Sci. Biotechnol. 25: 1379-1385. https://doi.org/10.1007/s10068-016-0215-3
  36. Pagnonceli J, Rasbold LM, Rocha GB, Silva J, Kadowaki MK, Simao R, et al. 2019. Biotechnological potential of an exo-polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices. J. Appl. Microbiol. 127: 1706-1715. https://doi.org/10.1111/jam.14426
  37. Amin F, Bhatti HN, Bilal M, Asgher M. 2017. Purification, kinetic, and thermodynamic characteristics of an exo-polygalacturonase from Penicillium notatum with industrial perspective. Appl. Biochem. Biotechnol. 183: 426-443. https://doi.org/10.1007/s12010-017-2455-y
  38. Byrne CE, Cavalitto SF, Voget CE. 2017. Purification and characterization of two inducible exopolygalacturonases from Aspergillus kawachii. Biocatal. Agric. Biotechol. 10: 38-45. https://doi.org/10.1016/j.bcab.2017.02.005
  39. Anand G, Yadav S, Yadav D. 2017. Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice. 3 Biotech 7: 122.
  40. Anand G, Yadav S, Yadav D. 2017. Purification and biochemical characterization of an exo-polygalacturonase from Aspergillus flavus MTCC 758. Biocatal. Agric. Biotechol. 10: 264-269. https://doi.org/10.1016/j.bcab.2017.03.018
  41. KoBAYAsHI T, Higaki N, Yajima N, SUzUMATsU A, Hagihara H, KAWAI S, et al. 2001. Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus. Biosci. Biotechol. Biochem. 65: 842-847. https://doi.org/10.1271/bbb.65.842
  42. Mei Y, Chen Y, Zhai R, Liu Y. 2013. Cloning, purification and biochemical properties of a thermostable pectinase from Bacillus halodurans M29. J. Mol. Catal. B-Enzym. 94: 77-81. https://doi.org/10.1016/j.molcatb.2013.05.004
  43. Yadav S, Maurya SK, Anand G, Dwivedi R, Yadav D. 2017. Purification, characterization and retting of Crotolaria juncea fibres by an alkaline pectin lyase from Fusarium oxysporum MTCC 1755. 3 Biotech 7: 136.
  44. Majaliwa N, Kibazohi O, Alminger M. 2021. Proteomic potential of East African Highland Bananas (EAHBs) for banana juice extraction: comparison between juice-producing and cooking cultivars. CyTA-J. Food 19: 370-377. https://doi.org/10.1080/19476337.2021.1909145
  45. Matsunaga E, Tanaka Y, Toyota S, Yamada H, Oka T, Higuchi Y, et al. 2021. Identification and characterization of β-D-galactofuranosidases from Aspergillus nidulans and Aspergillus fumigatus. J. Biosci. Bioeng. 131: 1-7. https://doi.org/10.1016/j.jbiosc.2020.09.006
  46. Song W, Tong Y, Li Y, Tao J, Li J, Zhou J, et al. 2021. Expression and characterization of a raw-starch glucoamylase from Aspergillus fumigatus. Process Biochem. 111: 97-104. https://doi.org/10.1016/j.procbio.2021.10.024
  47. Kaur M, Mehta A, Gupta R. Synthesis of methyl butyrate catalyzed by lipase from Aspergillus fumigatus. J. Oleo Sci. 68: 989-993.
  48. Damasio AR, Rubio MV, Goncalves TA, Persinoti GF, Segato F, Prade RA, et al. Xyloglucan breakdown by endo-xyloglucanase family 74 from Aspergillus fumigatus. Appl. Microbiol. Biotechnol. 101: 2893-2903.
  49. Carvalho AF, Neto PO, Zaghetto DAP, Bueno DSJ, Escaramboni B, Pastore GM. 2015, Screening of xylanolytic Aspergillus fumigatus for prebiotic xylooligosaccharide production using bagasse. Food Technol. Biotechnol. 53: 428.
  50. Latge JP, Chamilos G. 2019. Aspergillus fumigatus and Aspergillosis in 2019. Clin. Microbiol. Rev. 33: e00140-18.
  51. Hong G, Alby K, Ng S, Fleck V, Kubrak C, Rubenstein RC, et al. 2020. The presence of Aspergillus fumigatus is associated with worse respiratory quality of life in cystic fibrosis. J. Cyst. Fibros. 19: 125-130. https://doi.org/10.1016/j.jcf.2019.08.008