Abstract
In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml−1, 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l−1 inulin, an enzyme concentration of 40 U g−1 substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.
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References
Angov E (2011) Codon usage: nature’s roadmap to expression and folding of proteins. Biotech J 6:650–659
Cereghino JL, Cregg JM (2006) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66
Chen DL, Tong X, Chen SW, Chen S, Wu D, Fang SG, Wu J, Chen J (2010) Heterologous expression and biochemical characterization of α-glucosidase from Aspergillus niger by Pichia pastroris. J Agr Food Chem 58:4819–4824
Chen HQ, Chen XM, Li Y, Wang J, ** ZY, Xu XM, Zhao JW, Chen TX, **e ZJ (2009) Purification and characterisation of exo-and endo-inulinase from Aspergillus ficuum JNSP5-06. Food Chem 115:1206–1212
Chen XM, Xu XM, ** ZY, Chen HQ (2012) Expression of an endoinulinase from Aspergillus ficuum JNSP5-06 in Escherichia coli and its characterization. Carbohyd Polym 88:748–753
Chi ZM, Zhang T, Cao TS, Liu XY, Cui W, Zhao CH (2011) Biotechnological potential of inulin for bioprocesses. Bioresour Technol 102:4295–4303
Chi Z, Zhang T, Liu G, Yue L (2009) Inulinase-expressing microorganisms and applications of inulinases. Appl Microbiol Biotechnol 82:211–220
Cho YJ, Yun JW (2002) Purification and characterization of an endoinulinase from Xanthomonas oryzae No. 5. Process Biochem 37:1325–1331
Jeung Cho Y, Sinha J, Pil Park J, Won Yun J (2001) Production of inulooligosaccharides from inulin by a dual endoinulinase system. Enzyme Microb Tech 29:428–433
** ZY, Wang J, Jiang B, Xu XM (2005) Production of inulooligosaccharides by endoinulinases from Aspergillus ficuum. Food Res Int 38:301–308
Kango N (2008) Production of inulinase using tap roots of dandelion (Taraxacum officinale) by Aspergillus niger. J Food Eng 85:473–478
Kango N, Jain SC (2011) Production and properties of microbial inulinases: recent advances. Food Biotechnol 25:165–212
Chul KH, ** KH, Bong CW, Wan NS (2006) Inulooligosaccharide production from inulin by Saccharomyces cerevisiae strain displaying cell-surface endoinulinase. J Microbiol Biotechnol 16:360–367
Kumar GP, Kunamneni A, Prabhakar T, Ellaiah P (2005) Optimization of process parameters for the production of inulinase from a newly isolated Aspergillus niger AUP19. World J Microb Biotechnol 21:1359–1361
Li Y, Liu GL, Wang K, Chi ZM, Madzak C (2011) Overexpression of the endo-inulinase gene from Arthrobacter sp. S37 in Yarrowia lipolytica and characterization of the recombinant endo-inulinase. J Mol Catal B-Enzym 74:109–115
Liu X, Wu D, Wu J, Chen J (2012) Optimization of the production of Aspergillus niger α-glucosidase expressed in Pichia pastoris. World J Microb Biotechnol 29:533–540
Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Nakamura T, Shitara A, Matsuda S, Matsuo T, Suiko M, Ohta K (1997) Production, purification and properties of an endoinulinase of Penicillium sp. TN-88 that liberates inulotriose. J Ferment Bioeng 84:313–318
Nguyen QD, Rezessy-Szabó JM, Czukor B, Hoschke Á (2011) Continuous production of oligofructose syrup from Jerusalem artichoke juice by immobilized endo-inulinase. Process Biochem 46:298–303
Ohta K, Akimoto H, Moriyama S (2004) Fungal inulinases: enzymology, molecular biology and biotechnology. J Appl Glycosci 51:247–254
Park JP, Bae JT, You DJ, Kim BW, Yun JW (1999) Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp. Biotechnol Lett 21:1043–1046
Yun JW, Kim DH, Uhm TB, Song SK (1997) Production of high-content inulo-oligosaccharides from inulin by a purified endoinulinase. Biotechnol Lett 19:935–938
Wang JH, Teng D, Yao Y, Yang YL, Zhang F (2004) Expression of Aspergillus niger 9891 endoinulinase in Pichia pastoris. High Tech Lett 10:52–56
Zhang L, Zhao C, Zhu D, Ohta Y, Wang Y (2004) Purification and characterization of inulinase from Aspergillus niger AF10 expressed in Pichia pastoris. Protein Expres Purif 35:272–275
Zhou JW, Liu H, Li J, Du GC, Chen J (2012) Production of α-cyclodextrin glycosyltransferase in Bacillus megaterium MS941 by systematic codon usage optimization. J Agr Food Chem 60:10285–10292
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This work was financially supported by the Fundamental Research Funds for the Central Universities (JUSRP211A05 and JUSRP51304A), the National Natural Science Foundation of China (31271813), and the 111 Project (111-2-06).
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He, M., Wu, D., Wu, J. et al. Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production. J Ind Microbiol Biotechnol 41, 105–114 (2014). https://doi.org/10.1007/s10295-013-1341-z
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DOI: https://doi.org/10.1007/s10295-013-1341-z