<b><i>Arxula adeninivorans</i></b> Recombinant Guanine Deaminase and Its Application in the Production of Food with Low Purine Content

Abstract

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods.

A novel enzymatic approach in the production of food with low purine content usingArxula adeninivoransendogenous and recombinant purine degradative enzymes

“…[10][11][12][13] The expression of all genes was monitored over time with different concentration of inducers using SYBR Green fluorescent dye for double-stranded DNA quantification. The mRNA accumulation was strongly enhanced by the presence of hypoxanthine and adenine in cultivation medium (2 times higher mRNA level for hypoxanthine than adenine in case of adenine deaminase, guanine deaminase and xanthine oxidoreductase); uric acid also strongly induced the urate oxidase gene (Fig.…”

“…Only in case of urate oxidase was the induction of its gene equally good with adenine as it was with hypoxanthine. [10][11][12][13] Clearly, the mRNA relation between every inducer is reflected here in the enzyme activity, however, the amount of accumulated transcript does not translate into the amount protein synthesized. The very high (up to 3,500, Fig.…”

“…3,4 To provide patients with better control of the disease with reduced symptoms and frequency of gout attacks, a novel enzyme system decreasing the purine concentration of food products has been developed. [10][11][12][13] This system is an enzyme mixture that, at present, consists of 4 purine-degrading enzymes: adenine deaminase, guanine deaminase, xanthine oxidoreductase and urate oxidase, which simultaneously break down purines to a water-soluble 5-hydroxyisourate. The goal of this article is to give an overview of the composition and application of this enzyme system.…”

“…The products of those genes, adenine deaminase (Aadap), guanine deaminase (Agdap) and urate oxidase (Auoxp), were successfully synthesized in transgenic A. adeninivorans cells using the Xplor Ò 2 transformation/expression platform as described by Jankowska et al 10 and TrautweinSchult et al 12,13 The productivity of each transformant strain was optimized by modification of the medium, temperature and carbon source concentration. The highest productivity for guanine deaminase and urate oxidase was obtained in yeast minimal medium supplemented with 1% glucose after 24 h of cultivation at 30 C whereas for adenine deaminase the conditions were yeast minimal medium with 2% glucose for 5 h at 37 C. Eventually, the activity attained was up to 15,000 U g -1 dcw (dry cell weight) for adenine deaminase, up to 73 U g -1 dcw for guanine deaminase and up to 25 U g -1 dcw for urate oxidase.…”

“…For determining the optimal temperature, the enzymes and other assay solution were equilibrated in chosen temperatures for 5 min before start of the reaction. The pH and thermal stabilities were assessed after incubation of enzymes under given conditions for set times (described by Jankowska et al, 10,11 Trautwein-Schult et al 12,13 ). The optimal assay conditions and pH and temperature stabilities were similar enough to enable the enzymes to work well when mixed in a single solvent of fixed pH and temperature (pH 8.0 and 40 C - Table 1).…”