Expression of Glucose Oxidase Gene from Aspergillus niger in Pichia pastoris and Optimization of Enzyme Production Conditions

doi:10.13560/j.cnki.biotech.bull.1985.2020-1353

Abstract

Abstract:

Glucose oxidase gene(GOD)was cloned from Aspergillus niger and highly expressed in Pichia pastoris GS115. The GOD gene was amplified by designing degenerate primers,and the gene was connected to the plasmid pPICZαA and expressed in P. pastoris. The optimized induction conditions for enzyme production were optimized according to the shaking flask level. The fermentation scale-up experiment was carried out in a 30 L bioreactor. The His4 was co-expressed and the methanol feed strategy of DO-STAT was used for high-density fermentation. The results showed that the GOD enzyme activity in the supernatant of the fermentation broth reached 32.25 U/mL after the recombinant P. pastoris GS115/pPICZ-GOD was induced by 0.5% methanol at the shake flask level for 96 h. The optimal fermentation conditions were obtained as follows:under the condition of 30℃,pH 6.0 and 250 r/min,the activity of GOD enzyme induced by 1.0% methanol for 96 h reached 50.1 U/mL,which was 55.3% higher than that of the initial fermentation. Through the scale-up experiment in a 30 L bioreactor,the GOD enzyme activity reached 307.52 U/mL,which increased by 5.1 times. Co-expression of the His4 increased the wet cell weight by 72.5% and the GOD enzyme activity reached 461 U/mL,which was an increase of 50.2% compared to the original recombinant yeast. SDS-PAGE analysis showed that the molecular weight of GOD was about 90 kD. Recombinant GOD can be highly expressed in P. pastoris with high purity and few miscellaneous proteins,which is beneficial to purification.

Key words: Pichia pastoris, glucose oxidase, heterologous expression, optimization of enzyme production

LIAO Zhao-min, CAI Jun, LIN Jian-guo, DU Xin, WANG Chang-gao. Expression of Glucose Oxidase Gene from Aspergillus niger in Pichia pastoris and Optimization of Enzyme Production Conditions[J]. Biotechnology Bulletin, 2021, 37(6): 97-107.

Figures/Tables 13

Fig.1 Alignment of glucose oxidase amino acid sequences from partial A. niger

Fig.2 Agarose gel electrophoresis of whole genome extraction from Aspergillus niger M: DNA marker. 1: A. niger genomic DNA

Fig. 3 Agarose gel electrophoresis of PCR products of degenerate primers Z1 and Z2 M: DNA marker. 1: PCR products of degenerate primer Z1 and Z2

Fig.4 PCR amplification of glucose oxidase M: DNA marker. 1: PCR amplification of glucose oxidase

Fig.5 Construction of recombinant plasmid pPICZαA-GOD and identification by double restriction endonuclease digestion A: The plasmid map of the recombinant expression vector. B: The results of double restriction endonuclease digestion. M: DNA marker. 1: The product of double restriction endonuclease digestion of pPICZαA-GOD

Fig.6 Agarose gel electrophoresis of the linearized products of pPICZαA-GOD and pPICZαA M: DNA marker. 1: pPICZαA-GOD; 2: pPICZαA-GOD linearization product;3: pPICZαA; 4: pPICZαA linearization product

Fig.7 Screening of high concentration zeocin resistance of yeast transformants A:500 μg/mL. B:1 000 μg/mL. C:2 000 μg/mL

Fig.8 Activity curve of glucose oxidase expressed by trans-formants with high zeocin resistance in yeast

Fig.9 GS115/pPICZαA-GOD and GS115/pPICZαA-GOD/His4 shake flask level induction results

Fig.10 Optimization of fermentation conditions for GOD production by recombinant yeast

Fig.11 High-density fermentation curve of recombinant P. pastoris GS115/pPICZαA-GOD

Fig.12 SDS-PAGE of glucose oxidase protein expressed in recombinant P. pastoris in 30 L bioreactor M: Protein Maker; 1-9: the expression of glucose oxidase was induced by methanol at 0, 12, 24, 36, 48, 60, 72, 84 and 96 h

Fig.13 High density fermentation curve of recombinant P. pastoris GS115/pPICZαA-GOD/His4

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