Production and characterization of recombinant glucose oxidase from Aspergillus niger expressed in Pichia pastoris

Abstract

Significance and Impact of Study: Glucose oxidase is a very important enzyme produced by several species. However, large-scale applications have always been postponed by its complexity in fermentation and purification. Our research focused on developing new purification strategy of recombinant GOD from A. niger expressed in P. pastoris. Here, we described this novel one-step purification method and subsequent research in the characteristics of rGOD which showed different results from previous work. These can open new opportunities to increase its application. AbstractRecombinant glucose oxidase from Aspergillus niger expressed in Pichia pastoris by fed-batch fermentation was purified and assessed with 1Á26 purification fold to homogeneity using Q-Sepharose F.F. chromatography. The enzyme was determined by SDS-PAGE and gradient PAGE, which showed a dimeric form of 150 kDa. The purified rGOD was proved to be a glycoprotein, and the content of which was estimated to be 36Á7 and 25Á14% by phenol-sulfuric acid and anthrone-sulfuric acid methods. Characteristics demonstrated that the highest activity was in pH 6Á0 at 40°C and was stable at a broad pH range from 4Á0 to 9Á0 at 55°C or below. The optimum substrate for this enzyme was D-glucose, and the K m was 21Á06 mmol l À1 as well as the V max was 359 lmol min À1 mg À1 .rGOD possessed high resistance to various chemicals except for Hg 2+ , Fe 2+, Ag + , Cu 2+ , 1,4-dithiothreitol, sodium dodecyl sulfate and ascorbic acid. In addition, the inhibitors also exhibited intensive fluorescence quenching effect on rGOD.

Biomimetic selenocystine based dynamic combinatorial chemistry for thiol-disulfide exchange

“…Glucose oxidase (β-D-glucose:oxygen-oxidoreductase, EC 1.1.3.4) from Aspergillus niger is a flavoprotein that catalyzes the oxidation of β-D-glucose to D-glucono-δ-lactone and hydrogen peroxide, using oxygen as an electron acceptor. GOx is well suited for DCC exploration, it is a robust and wellcharacterized protein with two identical subunits (molecular weight: 160 KDa) [47][48][49] . Besides, there are relatively few ligands reported for GOx being one of the most widely used enzymes.…”

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

“…Furthermore, large-scale GOD application is also troublesome because there are problems concerning production cost, purification difficulty, and low productivity. In order to overcome these obstacles, heterologous expression system has been suggested by various researchers [8] . Until now, GOD production using different expression systems – such as E-coli, H. polymorpha, Saccharomyces cerevisiae, and Pichia pastoris − have been studied [9] , [10] , [11] , [12] , [13] , [14] .…”

Optimization and kinetic characterization of recombinant 1,3-β-glucanase production in Escherichia coli K-12 strain BL21/pETSD10 - a bioreactor scale study

“…The structure of hemicelluloses is not easy to decompose and requires a very sophisticated enzymatic complex for hydrolysis. Therefore, an intense search for a new source of enzymes, more frequently obtained with the tools of molecular biology, is currently a worldwide trend (Suo and Leung 2001;Wang et al 2008;Meng et al 2013).…”