Abstract
Objectives
A novel, high-level expression, thermostable mannan endo-1,4-beta-mannosidase is urgently needed for industrial applications.
Results
The mannan endo-1,4-β-mannosidase gene (MAN) from Aspergillus niger CBS 513.88 was optimized based on the codon usage bias in Pichia pastoris and synthesized by overlapping PCR to produce MAN-P. It was expressed in P. pastoris GS115 from a constitutive expression vector pHBM-905 M. MAN-P reached 594 mg/l in shake-flasks after 192 h induction. On production in a 5 l fermenter, the yield of MAN-P reached ~3.5 mg/ml and the enzyme activity was 1612 U/ml. The enzyme exhibited a maximum activity of 3049 U/ml at 80 °C and retained 60 % enzyme activity at 80 °C for 2 h. The pH optimum was 4.5 and the enzyme was stable over the pH range 1.5–11.
Conclusion
The thermostability of MAN-P is higher than other known fungal mannanases and the expression and thermophilic properties make MAN-P useful for industrial applications.
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References
Akita M, Takeda N, Hirasawa K et al (2004) Crystallization and preliminary X-ray study of alkaline mannanase from an alkaliphilic Bacillus isolate. Acta Crystallogr D Biol Crystallogr 60:1490–1492
Chauhan PS, Puri N, Sharma P, Gupta N (2012) Mannanases: microbial sources, production, properties and potential biotechnological applications. Appl Microbiol Biotechnol 93:1817–1830
Couturier M, Roussel A, Rosengren A et al (2013) Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserine reveal differences upon manno-oligosaccharide catalysis. J Biol Chem 288:14624–14635
Dhawan S, Kaur J (2007) Microbial mannanases: an overview of production and applications. Crit Rev Biotechnol 27:197–216
Do BC, Dang TT, Berrin JG et al (2009) Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01. Microb Cell Fact 8:59
Fu L, Zhai C, Kang LX et al (2012) Cloning of the gene encoding endo-1,4-β-mannosidase from Bacillus subtilis HB002 and expression in Pichia pastoris. Sciencepaper Online.http://www.paper.edu.cn/releasepaper/content/201211-323. Accessed 19 Nov 2012
Moreira LR, Filho EX (2008) An overview of mannan structure and mannan-degrading enzyme systems. Appl Microbiol Biotechnol 79:165–178
Puchart V, Vrsanská M, Svoboda P et al (2004) Purification and characterization of two forms of endo-beta-1,4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Biochim Biophys Acta 1674:239–250
Qiao J, Rao Z, Dong B et al (2010) Expression of Bacillus subtilis MA139 beta-mannanase in Pichia pastoris and the enzyme characterization. Appl Biochem Biotechnol 160(5):1362–1370
Van Zyl WH, Rose SH, Trollope K, Gorgens JF (2010) Fungal beta-mannanases: mannan hydrolysis, heterologous production and biotechnological applications. Process Biochem 45:1203–1213
Wang C, Luo H, Niu C et al (2015) Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity. Appl Microbiol Biotechnol 99:1217–1228
Zhang J, He M, He Z (2002) Operational and storage stability of neutral β-mannanase from Bacillus Licheniformis. Biotechnol Lett 24:1611–1613
Zhao W, Zheng J, Zhou H (2011) A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS513.88: constitutive overexpression and high-density fermentation in Pichia pastoris. Bioresour Technol 102:7538–7547
Acknowledgments
This study was supported by the National Natural Science Foundation of China (Nos. 31100057, 31172320 and 31340036); the National 973 Program of China (No. 2013CB910801); the Nature Science Fund for Creative Research Groups of Hubei Province of China (No. 2012FFA034); and the Foundation for High and New Technology Industrial Innovative Research Groups of the Wuhan Science and Technology Bureau’s Department (No. 2014070504020239).
Supporting information
Supplementary Fig. 1—Effect of Endo H on the activity (filled circle) at different temperatures of MAN-P. MAN-P was incubated with Endo H at 37 °C for 3 h, diluted in a buffer of 0.1 M citric acid and 0.2 M Na2HPO4 (pH 5.0), and then incubated at different temperatures for 5 min. The enzyme activity at 80 °C was taken as 100 %. The absolute enzyme activity corresponding to 100 % was 334.8 U/ml. Values are the means of three independent experiments ± standard deviations.
Supplementary Table 1-Primers used for synthesis of the MAN-P gene.
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Shi Yu and Zhezhe Li have contributed equally to this work.
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Yu, S., Li, Z., Wang, Y. et al. High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris . Biotechnol Lett 37, 1853–1859 (2015). https://doi.org/10.1007/s10529-015-1848-7
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DOI: https://doi.org/10.1007/s10529-015-1848-7