Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli

PeerJ. 2020 Aug 4:8:e9010. doi: 10.7717/peerj.9010. eCollection 2020.

Abstract

The gene encoding glucose oxidase from Aspergillus niger ZM-8 was cloned and transferred to Pichia pastoris GS115, a transgenic strain P. pastoris GS115-His-GOD constructed. The growth curve of P. pastoris GS115-His-GOD was consistent with that of Pichia pastoris GS115-pPIC9K under non-induced culture conditions. Under methanol induction conditions, the growth of the GOD-transgenic strain was significantly lowered than P. pastoris GS115-pPIC9K with the induced-culture time increase, and the optical densities of GOD-transgenic strain reached one-third of that of the P. pastoris GS115-pPIC9K at 51 h. The activity of glucose oxidase in the cell-free supernatant, the supernatant of cell lysate, and the precipitation of cell lysate was 14.3 U/mL, 18.2 U/mL and 0.48 U/mL, respectively. The specific activity of glucose oxidase was 8.3 U/mg, 6.52 U/mg and 0.73 U/mg, respectively. The concentration of hydrogen peroxide formed by glucose oxidase from supernatant of the fermentation medium, the supernatant of the cell lysate, and the precipitation of cell lysate catalyzing 0.2 M glucose was 14.3 μg/mL, 18.2 μg/mL, 0.48 μg/mL, respectively. The combination of different concentrations of glucose oxidase and glucose could significantly inhibit the growth of Agrobacterium and Escherichia coli in logarithmic phase. The filter article containing supernatant of the fermentation medium, supernatant of the cell lysate, and precipitation of cell lysate had no inhibitory effect on Agrobacterium and E. coli. The minimum inhibitory concentration of hydrogen peroxide on the plate culture of Agrobacterium and E. coli was 5.6 × 103 μg/mL and 6.0 × 103 μg/mL, respectively.

Keywords: Antimicrobial activity; Aspergillus niger; Glucose oxidase; Pichia pastoris; Transgenic.

Grants and funding

This study was financially supported by Chinese National Natural Science Foundation (Nos. 31760028 and 31460032), the Fundamental Research Funds for Key Laboratory of Drug Screening and Deep Processing for Traditional Chinese and Tibetan Medicine of Gansu Province (No. KZZY20180605), and the Youth Talent Support Program of Lanzhou University of Technology (No. 2018). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.