ascorbic-acid and alcohol-oxidase

An Aspergillus nidulans cell factory was genetically engineered to produce an aryl alcohol oxidase (AAO). The cell factory initiated production of melanin when growth-limited conditions were established using stationary plates and shaken flasks. This phenomenon was more pronounced when the strain was cultured in a trickle bed reactor (TBR). This study investigated different approaches to reduce melanin formation in fungal mycelia and liquid medium in order to increase the enzyme production yield. Removal of copper from the medium recipe reduced melanin formation in agar cultures and increased enzyme activities by 48% in agitated liquid cultures. Copper has been reported as a key element for tyrosinase, an enzyme responsible for melanin production. Ascorbic acid (0.44g/L) stopped melanin accumulation, did not affect growth parameters and resulted in AAO activity that was more than two-fold greater than a control treatment with no ascorbic acid.

Topics: Alcohol Oxidoreductases; Ascorbic Acid; Aspergillus nidulans; Bioreactors; Melanins; Monophenol Monooxygenase

Utilizing a simple fluidic structure, we demonstrate the improved performance of oxidase-based enzymatic biosensors. Electrolysis of water is utilized to generate bubbles to manipulate the oxygen microenvironment close to the biosensor in a fluidic channel. For the proper enzyme reactions to occur, a simple mechanical procedure of manipulating bubbles was developed to maximize the oxygen level while minimizing the pH change after electrolysis. The sensors show improved sensitivities based on the oxygen dependency of enzyme reaction. In addition, this oxygen-rich operation minimizes the ratio of electrochemical interference signal by ascorbic acid during sensor operation (i.e., amperometric detection of hydrogen peroxide). Although creatinine sensors have been used as the model system in this study, this method is applicable to many other biosensors that can use oxidase enzymes (e.g., glucose, alcohol, phenol, etc.) to implement a viable component for in-line fluidic sensor systems.

Topics: Alcohol Oxidoreductases; Ascorbic Acid; Biosensing Techniques; Creatinine; Electrochemical Techniques; Electrolysis; Glucose Oxidase; Hydrogen Peroxide; Hydrogen-Ion Concentration; Monophenol Monooxygenase; Oxidoreductases; Oxygen

We investigated the signaling role of hydrogen peroxide (H(2)O(2)) in regulating the ascorbate (AsA) level after exogenous methanol (MeOH) application. The endogenous H(2)O(2) and AsA levels as well as the expression of related genes were monitored after MeOH treatment of cultures of Oncidium protocorm-like bodies (PLB). A high MeOH concentration was deleterious and caused irreversible consumption of endogenous AsA. However, a low MeOH concentration (50mM) triggered the synthesis of H(2)O(2) and was effective in enhancing the expression of AsA-biosynthetic genes of the Smirnoff-Wheeler and galacturonate (GalUA) pathways. The increased expression of these genes could be blocked by the addition of hydroxylamine, an inhibitor of alcohol oxidase (EC: 1.1.3.13), and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase (EC: 1.6.3.1). Thus, the H(2)O(2) generated by MeOH application is a product of MeOH detoxification through alcohol oxidase and NADPH oxidase activation. In this chain, H(2)O(2) acts as a secondary messenger for the activation of AsA-related genes. Our results reveal the signaling function of H(2)O(2) and cellular AsA homeostasis in Oncidium orchids in response to MeOH stimulation. A mechanism for the MeOH effect on AsA production is suggested.

Topics: Alcohol Oxidoreductases; Ascorbic Acid; Gene Expression Regulation, Plant; Genes, Plant; Hexuronic Acids; Hydrogen Peroxide; Hydroxylamine; Methanol; NADPH Oxidases; Onium Compounds; Orchidaceae; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction