Part:BBa_K4130011

Glucose Oxidase (A. niger) with 7 Stabilizing Mutations

This part encodes a mutated form of the enzyme glucose oxidase (GOx) from Aspergillus niger. 7 mutations, T10K, A36M, R145N, P192C, H201C, G274S, and E374Q, were introduced to improve thermostability and catalytic efficiency.

Biology

Glucose oxidase (GOx) catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, producing hydrogen peroxide. Known as the “Ferrari” of enzymes [3], GOx has a catalytic efficiency (kcat/Km) several orders of magnitude larger than other oxidases [4]. Accordingly, GOx is one of the most widely utilized enzymes with applications in bioelectronics and biosensors [1,2].

Originally derived from the fungi Aspergillus niger and Penicillium amagasakiense, the endogenous form of the protein is difficult to purify, with preparations often containing impurities such as catalase, cellulase, and amylase [6]. Subsequently, GOx is often recombinantly expressed in Saccharomyces cerevisiae, where purification is standardized. Recombinant expression in S. cerevisiae results in a highly glycosylated form of GOx. While this highly glycosylated form has improved thermostability [7], the addition of these post-translational modifications impedes the biosensing applications of the enzyme, likely by reducing electron tunneling and transfer to the electrodes [8]. Successful attempts at deglycosylation of purified GOx have been made, though the procedure is considered expensive and complicated [9]. Distinctly, prokaryotic organisms typically lack glycosylation machinery and can serve as a platform for the production of non-glycosylated proteoforms. Recombinant expression of glucose oxidase in Escherichia coli has resulted in the production of non-glycosylated forms better suited for biosensing applications [6].

Although deglycosylation results in improved catalytic properties for biosensing, it has a negative effect on thermostability [6]. To counteract this limitation, research has focused on the generation of thermostable mutants. In particular, Mu et. al (2019) employed a computational library design strategy (FRESCO) resulting in the generation of a mutant containing 5 amino acid substitutions [4]. Computational analyses posit that the 5 stabilizing mutations strengthen interactions between β-sheet A, β-sheet B and H1 helix as well as optimize the charge distribution on the protein surface. Additionally, Ittisoponpisan and Jeerapan (2021) computationally scanned the amino acid sequence of glucose oxidase to locate amino acid pairs that, when replaced with cysteines, would result in the formation of stabilizing disulfide bonds within the enzyme structure [5]. One such pair- Pro192 and His201- was selected because the mutations came from less conserved regions and thus were not expected to affect enzyme functionality.

Usage

Our project aims to utilize glucose oxidase to measure glucose in maple syrup. Therefore, we created a new GOx biobrick tailored for use in biosensing applications. We chose E. coli BL21 as the expression system, to prevent post-translational glycosylation. To mitigate the thermostability effects due to lack of glycosylation, we incorporated the 5 mutations discovered by Ittisoponpisan and Jeerapan as well as the 2 mutations discovered by Mu et. al. The “Combined Stabilizing Mutations” part contains both sets of mutations for a total of seven. A T7 promoter (BBa_I712074), strong ribosomal binding site (BBa_B0034), and double terminator (BBa_B0015) were incorporated for enhanced in vivo expression. Importantly, E. coli SHuffle was chosen as the expression strain for this biobrick; 2 of the 7 stabilizing mutations incorporated into the coding region are dependent upon the formation of cysteine disulfide bonds, which can only occur in a non-reducing cytoplasmic environment. The strain E. coli SHuffle is specifically designed for expression of T7 induced, disulfide bond-forming proteins, and was therefore used for expression of this part.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Mano, N. Engineering glucose oxidase for bioelectrochemical applications. Bioelectrochemistry 2019, 128, 218–240.

Khatami, S.H.; Vakili, O.; Ahmadi, N.; Soltani Fard, E.; Mousavi, P.; Khalvati, B.; Maleksabet, A.; Savardashtaki, A.; Taheri-Anganeh, M.; Movahedpour, A. Glucose oxidase: Applications, sources, and recombinant production. Biotechnol. Appl. Biochem. 2021.

Bauer JA, Zámocká M, Majtán J, Bauerová-Hlinková V. Glucose Oxidase, an Enzyme "Ferrari": Its Structure, Function, Production and Properties in the Light of Various Industrial and Biotechnological Applications. Biomolecules. 2022 Mar 19;12(3):472.

Mattevi A. To be or not to be an oxidase: Challenging the oxygen reactivity of flavoenzymes. Trends Biochem. Sci. 2006;31:276–283. doi: 10.1016/j.tibs.2006.03.003.

Ittisoponpisan, S., & Jeerapan, I. In Silico Analysis of Glucose Oxidase from Aspergillus niger: Potential Cysteine Mutation Sites for Enhancing Protein Stability. Bioengineering 2021. 8(11), 188.

Witt S, Singh M, Kalisz HM. Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli. Appl Environ Microbiol. 1998 Apr;64(4):1405-11. doi: 10.1128/AEM.64.4.1405-1411.1998. PMID: 9546178; PMCID: PMC106162.

Frederick K R, Tung J, Emerick R S, Masiarz F R, Chamberlain S H, Vasavada A, Rosenberg S, Chakraborty S, Schopfer L M, Massey V. Glucose oxidase from Aspergillus niger. J Biol Chem. 1990;265:3793–3802.

Kohen A, Jonsson T, Klinman J P. Effects of protein glycosylation on catalysis: changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction.

Kalisz H M, Hecht H-J, Schomburg D, Schmid R D. Crystallization and preliminary x-ray diffraction studies of a deglycosylated glucose oxidase from Aspergillus niger. J Mol Biol. 1990;213:207–209.