Expression, characterization and one step purification of heterologous glucose oxidase gene from Aspergillus niger ATCC 9029 in Pichia pastoris

Abstract

Glucose Oxidase (GOD), is a common flavoprotein from Aspergillus niger ATCC 9029 with a broad application in biotechnology, food and medical industries. In this study, GOD gene was cloned into the expression vector, pPIC9 and screened by the alcohol oxidase promoter. The enzyme production increased at 28 °C. GOD activity induced by 1.0% methanol and the highest level of GOD production was the result of shaking rate at 225 rpm. The highest enzyme activity obtained at a pH value ranged from 5 to 7 at 50 °C. The enzyme was stable at a broad pH range and temperature.

Engineering glucose oxidase for bioelectrochemical applications

“…[91] Alternatively, a glycosylated deficient strain S. cerevisiae ngd29 has been used leading to lower and more homogeneous glycosylation than the native strain. [92] Much lower glycosylation state (~10 wt%) could be attained for GOx from A. niger [93][94][95][96][97][98][99], from P. amagasakiense [63] and from Penicillium variable P16 [78] overexpressed in P. pastoris with an activity almost identical to the native enzyme. To avoid glycosylation, GOx from A. niger [100][101][102] and P. amagasakiense [103,104] were overexpressed in E. coli.…”

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

“…Gu et al (2015) reported recently that a yield of GOD reached 21.81 g/L, with an activity of 1972.9 U/mL, in P. pastoris S17 of which is a genetically modified strain by manipulating genes involved in protein folding machinery and abnormal folding stress responses. Belyad, Karkhanei & Raheb (2018) also cloned the GOD gene from A. niger ATCC 9029 and inserted into the pPIC9 vector for protein expression in P. pastoris GS115 by the alcohol oxidase promoter, but no the expression ability and enzyme activity were introduced. Although the above literatures has been reported the produce of GOD, there are no detailed determination on the activity of glucose oxidase in supernatant of the fermentation medium, the supernatant of the cell lysate, and the precipitation of cell lysate.…”

“…As Dal et al (2008) description, yeast could inhibit the gray mold growth, the novel viewpoint could be used to the development of bacteriostatic agent by genetic manipulations. Studies have found that most P. pastoris expression systems use methanol-induced ethanol oxidase promoters to express GOD ( Crognale et al, 2006 ; Yamaguchi et al, 2007 ; Belyad, Karkhanei & Raheb, 2018 ), and the concentration of methanol directly affects cell growth and protein expression ( Cereghino et al, 2002 ; Xiao, Wang & Chen, 2004 ; Daly & Hearn, 2005 ). Crognale et al (2006) described a genetically modified P. pastoris X 33 with the gene encoding the GOX from P. variabile P16, and the activity only reached at 50 U/mL.…”

“…Until 1963, Muller found that penicillin B and penatin were identical to notatin, and all of them belong to Glucose oxidase (GOD) (Bentley, 1963), which played the important roles in the inhibition of microbial growth. Thereafter, Glucose oxidase, referred to as an an ideal enzyme, has attracted the attention of researchers (Bentley, 1963;Park et al, 2000;Crognale et al, 2006;Belyad, Karkhanei & Raheb, 2018;Li et al, 2019;Tu et al, 2019).…”

“…As above-mentioned descriptions, GODs are prepared mainly from the fermentation of Aspergillus (Tu et al, 2019), Penicillium (Bodade, Khobragade & Arfeen, 2010;Khan et al, 2016), Bacillus sp. (Xu et al, 2018), Cladosporium neopsychrotolerans (Ge et al, 2020), transgenic Trichoderma reesei (Wu et al, 2017), transgenic P. pastoris (Park et al, 2000;Crognale et al, 2006;Yamaguchi et al, 2007;Rocha et al, 2010;Fang et al, 2015;Belyad, Karkhanei & Raheb, 2018), and directly used in industry (Wong, Wong & Chen, 2008) as a control agent against pathogenic microorganism (Hopkinsa et al, 2019;Lee et al, 2019;Li et al, 2019) or a key catalyst for bioelectrochemical applications (Visvanathan et al, 2018;Mano, 2019). However, very little information is available whether a glucose oxidase-secreting microbe could inhibit growth of its surrounding living things and become an ecological bacteriostatic agent.…”