Abstract
The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml−1 (2.5 g protein l−1) in a 3 l fermentor—410% higher than GOD-w (148 U ml−1), and thus is a low-cost alternative for the bread baking industry.
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References
Bonet A, Rosell CM, Caballero PA, Gómez M, Pérez-Munuera I, Lluch MA (2006) Glucose oxidase effect on dough rheology and bread quality: a study from macroscopic to molecular level. Food Chem 99:408–415
Chang S, Lee G, Shaw J (2006) Codon optimization of Candadi rugosa lip1 for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J Agric Food Chem 3:815–822
Crognale S, Pulci V, Brozzoli V, Petruccioli M, Federici F (2006) Expression of Penicillium variabile P16 glucose oxidase gene in Pichia pastoris and characterization of the recombinant enzyme. Enzym Microb Technol 39:1230–1235
Graham G, Mayers P, Henry R (1994) A simplified method for the preparation of fungal genomic DNA for PCR and RAPD analysis. Biotechniques 16:48–50
Guo Y, Lu FX, Zhao HZ, Tang YC, Lu ZX (2010) Cloning and heterologous expression of glucose oxidase gene from Aspergillus niger Z-25 in Pichia pastoris. Appl Biochem Biotechnol 162:498–509
Hatzinikolaou DG, Hansen OC, Macris BJ, Tingey A, Kekos D, Goodenough P et al (1996) A new glucose oxidase from Aspergillus niger characterization and regulation studies of enzyme and gene. Appl Microbiol Biotechnol 46:371–381
Hodgkins M, Sudbery P, Mead D, Ballance DJ, Goodey A (1993) Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations. Yeast 9:625–635
Huang HQ, Yang PL, Luo HY, Tang HG, Shao N, Yuan TZ et al (2008) High-level expression of a truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation. Appl Microbiol Biotechnol 78:95–103
Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681
Park EH, Shin YM, Lim YY, Kwon TH, Kim DH, Yang MS (2000) Expression of glucose oxidase by using recombinant yeast. J Biotechnol 81:35–44
Rando D, Kohring GW, Giffhorn F (1997) Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum. Appl Microbiol Biotechnol 48:34–40
Rasiah IA, Sutton KH, Low FL, Lin HM, Gerrard JA (2005) Crosslinking of wheat dough proteins by glucose oxidase and the resulting effects on bread and croissants. Food Chem 89:325–332
Simpson C (2005) Isolation, purification and characterization of a novel glucose oxidase from Penicillium canescens Tt42. Masters Thesis, Rhodes University
Standardization Administration of the People’s Republic of China (2008) Inspection of grain and oils: Bread baking test of wheat flour. Sponge-dough method. GB/T 14612-2008, pp 1–8
Valdivieso-Ugarte M, Ronchel C, Bañuelos O, Velasco J, Adrio JL (2006) Expression of an Aspergillus niger glucose oxidase in Saccharomyces cerevisiae and its use to optimize fructo-oligosaccharides synthesis. Biotechnol Prog 22:1096–1101
Vemulapalli V, Hoseney RC (1998) Glucose oxidase effects on gluten and water solubles. Cereal Chem 75:859–862
Wong C, Wong K, Chen X (2008) Glucose oxidase: natural occurrence, function, properties and industrial applications. Appl Microbiol Biotechnol 78:927–938
Xiong AS, Yao QH, Peng RH, Zhang Z, Xu F, Liu JG et al (2006) High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris. Appl Microbiol Biotechnol 72:1039–1047
Yamaguchi M, Tahara Y, Nakano A, Taniyama T (2007) Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris. Protein Expr Purif 55:273–278
Yin B, Yuan R, Chai YQ, Chen SH, Cao SR, Xu Y et al (2008) Amperometric glucose biosensors based on layer-by-layer assembly of chitosan and glucose oxidase on the Prussian blue-modified gold electrode. Biotechnol Lett 30:317–322
Zhao X, Huo KK, Li YY (2000) Synonymous codon usage in Pichia pastoris. Chin J Biotechnol 16:308–311
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This research was supported by the Fundamental Research Funds for the Central Research Institutes, CAAS-BRI (Grant no. 2011-08).
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Zhaowei Gao and Zhuofu Li have contributed equally.
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Gao, Z., Li, Z., Zhang, Y. et al. High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization. Biotechnol Lett 34, 507–514 (2012). https://doi.org/10.1007/s10529-011-0790-6
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DOI: https://doi.org/10.1007/s10529-011-0790-6