CLONING AND EXPRESSION OF AspPecA PECTINASE GENE  FROM Aspergillus niger M13 IN Pichia pastoris X33

Phan Thị Thanh Diễm; Nguyễn Đức Huy; Hoàng Tấn Quảng; Phạm Thị Ngọc Lan; Tạ Đặng Ý Vi; Trần Quốc Dung

doi:10.34238/tnu-jst.12613

CLONING AND EXPRESSION OF AspPecA PECTINASE GENE FROM Aspergillus niger M13 IN Pichia pastoris X33

About this article

Received: 17/04/25 Revised: 09/05/25 Published: 09/05/25

Authors

1. Phan Thi Thanh Diem, 1) University of Science - Hue University, 2) Quang Nam University
2. Nguyen Duc Huy, Institute of Biotechnology - Hue University
3. Hoang Tan Quang, Institute of Biotechnology - Hue University
4. Pham Thi Ngoc Lan, University of Science - Hue University
5. Ta Dang y Vi, University of Education - Hue University
6. Tran Quoc Dung , University of Education - Hue University

Abstract

This study aims to investigate the expression gene encoding pectinase (AspPecA) from Aspergillus niger M13 in Pichia pastoris X33 and the application of recombinant pectinase in fruit juice processing. A. niger M13 strain isolated from Da Xanh pomelo (Citrus maxima) peel was revived by culturing in Potato Dextrose Broth (PDB) at 35 ℃ for 18-20 h. The AspPecA gene was cloned into vector pPICZαA and expressed in P. pastoris X33 under the control of the AOX1 promoter. Purified rAspPecA had a molecular weight of approximately 42 kDa, as evidenced by SDS-PAGE. P. pastoris X33 transformants expressing recombinant pectinase were selected on an agar plate, and their ability to produce the pectinase was evaluated in flask cultures. Recombinant pectinase rAspPecA can clarify apple juice. Compared with controls, rAspPecA at a concentration of 2,400 U/L reduced apple juice turbidity by 29%. The results obtained in this work provide a basis for the development of applications of recombinant pectinase in various biotechnological fields.

Keywords

Aspergillus niger; AspPecA gene; Juice clarification; Pichia pastoris; Recombinant pectinase

Full Text:

PDF

References

[1] Y. Guan, D. Wang, C. Lv et al., "Archives of microbiology: screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential," Archives of Microbiology, vol. 202, no. 5, pp. 1005-1013, 2020.

[2] S. Haile, C. Masi, and M. Tafesse, "Isolation and characterization of pectinase-producing bacteria (Serratia marcescens) from avocado peel waste for juice clarification," BMC Microbiol., vol. 22, no. 1, 2022, doi: 10.1186/s12866-022-02536-8.

[3] S. Shrestha, M. S. Rahman, and W. Qin, "New insights in pectinase production development and industrial applications," Applied Microbiology and Biotechnology, vol. 105, no. 24, pp. 9069-9087, 2021.

[4] V. Priya and V. Sashi, "Pectinase enzyme producing microorganisms," International Journal of Scientific and Research Publications, vol. 4, no. 3, pp. 1-4, 2014.

[5] B. Saoudi, A. Habbeche, B. Kerouaz, et al., "Purification and characterization of a new thermoalkaliphilic pectate lyase from Actinomadura keratinilytica Cpt20," Process Biochemistry, vol. 50, no. 12, pp. 2259-2266, 2015.

[6] P. Thakur and G. Mukherjee, "Utilization of agro-waste in pectinase production and its industrial applications," in Recent Developments in Microbial Technologies, R. Prasad, V. Kumar, J. Singh, et al., ed., Singapore: Springer Nature Singapore, 2021, pp. 145-162.

[7] S. Shrestha, C. Chio, X. Zhang, et al., “Cloning and expression of pectinase gene from two forest soil bacteria, Streptomyces sp.,” The Microbe, vol. 1, pp. 1-8, 2023.

[8] C. Zhou, Y. Xue, and Y. Ma, "Cloning, evaluation, and high-level expression of a thermo-alkaline pectate lyase from alkaliphilic Bacillus clausii with potential in ramie degumming," Applied Microbiology and Biotechnology, vol. 101, no. 9, pp. 3663-3676, 2017.

[9] Y. Yuan, X. Y. Zhang, Y. Zhao, et al., "A novel PL9 pectate lyase from Paenibacillus polymyxa KF-1: Cloning, expression, and its application in pectin degradation," Int. J. Mol. Sci., vol. 20, pp.1-17, 2019.

[10] T. X. Ngo, T. H. Mai, and V. T. Vu, "Cloning and over Expression of an Aspergillus niger XP Phytase Gene (phyA) in Pichia pastoris," World Academy of Science, Engineering and Technology, vol. 32, pp. 383-386, 2009.

[11] S. Srivastava and S. K. Tyagi, "Effect of enzymatic hydrolysis on the juice yield from apple fruit (Malus domestica) pulp," International Journal of Biotechnology and Bioengineering Research, vol. 4, pp. 299-306, 2013.

[12] Z. Zhang, J. Dong, D. Zhang, et al., "Expression and characterization of a pectin methylesterase from Aspergillus niger ZJ5 and its application in fruit processing," Journal of Bioscience and Bioengineering, vol. 126, no. 6, pp. 690-696, 2018.

[13] L. Zhong, X. Wang, L. Fan, et al., "Characterization of an acidic pectin methylesterase from Paenibacillus xylanexedens and its application in fruit processing," Protein Expr. Purif., vol. 179, 2021, Art. no. 105798, 10.1016/j.pep.2020.105798.

[14] S. X. Xu, X. Qin, B. Liu, et al., "An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification," Lett. Appl. Microbiol., vol. 60, no.2, pp. 181-187, 2015.

[15] D. Abdulrachman, P. Thongkred, K. Kocharin, et al., "Heterologous expression of Aspergillus aculeatus endo-polygalacturonase in Pichia pastoris by high cell density fermentation and its application in textile scouring," BMC Biotechnol., vol. 17, no.1, 2017, doi: 10.1186/s12896-017-0334-9.

[16] A. C. Famotemi, S. O. Kareem, F. Oluwafemi, et al., "Purification and characterization of pectinase from Aspergillus niger strain F7-02 mutant," International Journal of Advanced Research in Biological Sciences, vol. 8, pp. 45-57, 2021.

[17] A. Dhayalan, N. Thillainathan, B. Velramar, et al., "Pectinase from a fish gut bacterium, Aeromonas guangheii (SS6): Production, cloning and characterization," The Protein Journal, vol. 41, no.6, pp. 572-590, 2022.

[18] A. M. Moharram, A-N. A. Zohri, A. E-L Hesham, et al., "Production of cold-active pectinases by three novel Cladosporium species isolated from Egypt and application of the most active enzyme," Scientific Reports, vol. 12, no.1, 2022, Art. no. 15599.

[19] K. Sudeep, J. Upadhyaya, D. R. Joshi, et al., "Production, characterization, and industrial application of pectinase enzyme isolated from fungal strains," Fermentation, vol. 6, no. 2, pp. 1-10, 2020.

[20] T. H. Phan and T. T. S. Nguyen, "Study on application of pectinase enzyme in the production of bottled apricot juice," in Proc. of the Scientific Conf., Food Technology Division, University of Food Industry, Ho Chi Minh City, pp. 124-134, 2015.

"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:

PT-BR;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'>

style='mso-element:field-end'>

DOI: https://doi.org/10.34238/tnu-jst.12613

Refbacks

There are currently no refbacks.



Remember me