Arxula adeninivorans recombinant adenine deaminase and its application in the production of food with low purine content.

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(®) 2 transformation/expression platform. The recombinant enzyme was biochemically characterized and was found to have a pH range of 5.5-7.5 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 70.4 to 0.4 mg l(-1). 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.

Arxula adeninivorans xanthine oxidoreductase and its application in the production of food with low purine content.

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 8·5 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.

The use of yeast and moulds as sensing elements in biosensors.

Whole cell biosensors are able to provide information that sensors based on single and multiple types of molecules are unable to do. For example, broad-spectrum catabolite analysis, cell toxicity and genotoxicity are best detected in the context of a functioning cell. Most whole cell sensors have used bacterial cells as the sensing element. Fungal cells, however, can provide all of the advantages bacterial cells offer but in addition they can provide information that is more relevant to other eukaryote organisms. These cells are easy to cultivate, manipulate for sensor configurations and are amenable to a wide range of transducer methodologies. An overview of the use of yeast and filamentous fungi as the sensing element of some biosensors is presented here.

Detection of two distinct substrate-dependent catabolic responses in yeast cells using a mediated electrochemical method.

Mediated electrochemical detection of catabolism in prokaryotic cells is well documented; however, the application of this technique to eukaryotic cells has received less attention. Two catabolic substrate-dependent mediated electrochemical signals were detected in the yeast Saccharomyces cerevisiae. The signal using a single hydrophilic mediator (ferricyanide) is small whereas the response using a double mediator system comprising a hydrophilic and a lipophilic mediator (ferricyanide and menadione) is up to three orders of magnitude larger. The behaviour of each response during cell ageing is different: the single mediator response increases whereas the double mediator response decreases. This difference indicates that the two signals originate at different points in the catabolic pathways. In S. cerevisiae the double mediator response is proposed to originate from the reduction of the lipophilic mediator by NADPH produced in the pentose phosphate pathway. The single mediator signal arises from reduction of the hydrophilic mediator by an extracellular redox species produced in response to the presence of glucose.

Biochemical mediator demand--a novel rapid alternative for measuring biochemical oxygen demand.

The biochemical oxygen demand (BOD) test (BOD5) is a crucial environmental index for monitoring organic pollutants in waste water but is limited by the 5-day requirement for completing the test. We have optimised a rapid microbial technique for measuring the BOD of a standard BOD5 substrate (150 mg glucose/l, 150 mg glutamic acid/l) by quantifying an equivalent biochemical mediator demand in the absence of oxygen. Elevated concentrations of Escherichia coli were incubated with an excess of redox mediator, potassium hexacyanoferrate(III), and a known substrate for 1 h at 37 degrees C without oxygen. The addition of substrate increased the respiratory activity of the microorganisms and the accumulation of reduced mediator; the mediator was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. Catabolic conversion efficiencies exceeding 75% were observed for the oxidation of the standard substrate. The inclusion of a mediator allowed a higher co-substrate concentration compared to oxygen and substantially reduced the incubation time from 5 days to 1 h. The technique replicates the traditional BOD5 method, except that a mediator is substituted for oxygen, and we aim to apply the principle to measure the BOD of real waste streams in future work.