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The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6؋ His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6؋ His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml ؊1 could be achieved even with a nonoptimized cultivation procedure.

Aims: Construction of a transgenic Arxula adeninivorans strain that produces a high concentration of adenine deaminase and investigation into the application of the enzyme in the production of food with low purine content. Methods and Results: The A. adeninivorans AADA gene, encoding adenine deaminase, was expressed in this yeast under the control of the strong inducible nitrite reductase promoter using the Xplor â 2 transformation/ expression platform. The recombinant enzyme was biochemically characterized and was found to have a pH range of 5Á5-7Á5 and temperature range of 34-46°C with medium thermostability. A beef broth was treated with the purified enzyme resulting in the concentration of adenine decreasing from 70Á4 to 0Á4 mg l À1 .Conclusions: It was shown that the production of adenine deaminase by A. adeninivorans can be increased and that the recombinant adenine deaminase can be used to lower the adenine content in the food. Significance and Impact of the Study: Adenine deaminase is one component of an enzymatic system that can reduce the production of uric acid from food constituents. This study gives details on the expression, characterization and application of the enzyme and thus provides evidence that supports the further development of the system.

Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food.

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Peroxidases (POD) are used in textile decoloration and bleaching processes, but these enzymes are unfortunately inactivated rapidly at high hydrogen peroxide concentrations. A new concept has therefore been developed, which is based on a simultaneous application of glucose oxidase and peroxidase. Starting with glucose as a substrate for glucose oxidase (GOD), hydrogen peroxide was generated in situ. The freshly formed substrate H2O2 was immediately used by the POD oxidizing colored compounds in dyeing baths. For example, 20 mg of the dyestuff Sirius Supra Blue®FGG 200 % could be decolorized using 125 mg glucose which corresponds to 24 mg hydrogen peroxide. These experiments show that the enzyme cascade works in principle in homogeneous decoloration processes. The enzymes were not degraded by the oxidant, because under these conditions the stationary peroxide concentration is nearly zero over the whole reaction time. Moreover, experiments were carried out to check if this combined system with GOD, glucose and POD could be used even in heterogeneous systems such as the textile bleaching of natural cotton fibers. Starting from 55, a significant higher degree of whiteness (according to Berger) up to 66 could be obtained.

Aims: Isolation and characterization of xanthine oxidoreductase and its application in the production of food with low purine content. Methods and Results: The A. adeninivorans xanthine oxidoreductase is an inducible enzyme. The best inducers were identified by enzyme activity tests and real-time PCR and used to produce large amounts of the protein.Xanthine oxidoreductase was partially purified and biochemically characterized, showing pH and temperature optimum of 8Á5 and 43°C, respectively. The enzyme decreased xanthine and hypoxanthine concentrations in yeast extract and was active simultaneously with other purine-degrading enzymes so that all of the substrates for uric acid production were reduced in a single step. Conclusions: It was shown that induced A. adeninivorans can produce sufficient amount of xanthine dehydrogenase and that the enzyme is able to reduce xanthine and hypoxanthine content in food, and when used in conjunction with other enzymes of the pathway, uric acid concentration is significantly reduced. Significance and Impact of the Study: Reduction in dietary purines is recommended to people suffering from hyperuricemia. Elimination of most purine-rich foods may affect balanced nutrition. Food with lowered purine concentration will assist in controlling the disease. This study is a continuation of previous studies that characterized and overexpressed other enzymes of the purine degradation pathway.

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