Application Of Extradiol Dioxygenase (BphC) In Catecholic Biosensors

To date, many biosensors for the detection of catecholic compounds have been reported based on the utilization of tyrosinase, horseradish peroxidase, or laccase as recognition unit. However, none of these hydroxylases show a specific selectivity in the detection of catecholic compounds because many phenolic compounds can be catalyzed by these enzymes. Therefore, it is necessary to explore novel enzymes in realizing highly selective, stable and sensitive determination of catecholics to meet the requirements in the fields of environmental monitoring and biological toxicology. Ring-cleavage enzymes, containing intradiol dioxygensases and extradiol dioxygenases, can catalyze the ring-cleavage reaction of catecholic compounds. Compared with those reported enzymes used in biosensors, these ring-cleavage enzymes possess more excellent specificity and selectivity for catecholic compounds. The purpose of this study is to fabricate catecholic biosensors utilizing an extradiol dioxygenase,2,3-dihydroxybiphenyl1,2-dioxygenase (BphC), for the sensitive, rapid, and specific detection of catecholic compounds. Moreover, it should give a guide for the application of other available enzymes as sensitive elements in biosensors. The main contents of this study are as follows:Firstly, two statistical experimental designs were employed to screen and optimize the factors in the expression of BphC from Escherichia coli BL21(DE3). Ten important factors were evaluated by Plackett-Burman design (PBD, N=12), and four most significant ones, i.e. inducing temperature, pH for seed medium, seed age, and inoculation amount were selected and optimized by Response surface methodology (RSM, N=30). According to the analytical results, the optimal conditions were obtained as follows:inducing temperature35℃, pH6.5for seed medium, seed age9h, and inoculation amount of0.95%. Under the optimal contiditons, the maximal specific activity of BphC was about0.59U/mg protein using catechol as substrate, which was nearly three times of that of before. SDS-PAGE was used to confirm the optimal results. The optimized BphC showed high activity to catechol, which made it more suitable for the construction of catechol biosensor.Secondly, the feasibility of utilizing BphC as sensitive element in the biosensor was evaluated by immobilizing BphC on different materials, such as ZnO nanorods, TiO2nanotubes, polyaniline (PANI), ZnO sol-gel, and so on. Electrostatic bonding, adsorption, cross-linking and embedding methods were applied for the immobilization. It was suggested that BphC crude extracts could be used as the sensitive element of biosensor. However, biosensors based on these methods mentioned above can not meet requirements for practical application. For example, the immobilization of BphC on the ZnO nanorods/Zn electrode by electrostatic bonding was not stable enough for the further study; TiO2nanotubes/BphC modified Ti electrode showed poor signal to substrates; BphC immobilized on the undoped PANI modified electrode kept better activity than that immobilized on the ionic liquid or poly(aminophenol) modified electrodes, but the repeatability of these enzyme electrodes was bad; ZnO sol-gel modified BphC enzyme electrode can keep the activity of BphC, but the enzyme membrane was easy to crackle, resulting in the poor performance. Therefore, further study is still needed to improve the common enzyme immobilization methods and to develop novel methods for the fabrication of enzyme electrodes.Thirdly, a BphC biosensor for the catechol detection was constructed based on the PVA modified SiO2sol-gel method. BphC embedded in SiO2gel maintained its bioactivity well and presented good eletrocatalytical response to catechol. For catechol detection, the linear amperometric response range of this biosensor was0.002～0.8mM (R2=0.9978). And the sensitivity was1.268mA/(mM·cm) with a detection limit of0.428μM (S/N=3). Furthermore, compared with bare glassy carbon electrode, BphC modified electrode showed better selectivity for catechol in the mixtures of catechol and phenol. The protocol of this enzyme biosensor will open up a new avenue for the application of key enzymes during the biodegradation in environmental monitoring.Moreover, a new type of fluorescent biosensing system for catechol and2,3-dihydroxybiphenyl (DHB) was described based on the inner filter effect (IFE) of CdTe quantum dots (QDs) with the combination of BphC, which belonged to dioxygenases during the aerobic biodegradation of aromatic compounds. The QD-BphC system required no complicated surface modification of QDs and no enzyme immobilization, and kept the activity of BphC well. In addition, the QD-BphC system presented good performances for the detection of catecholic compounds, and the detection limits for catechol and DHB were0.02and2μM, respectively. High selectivity to either catechol or DHB can also be observed in the mixtures of other phenolic compounds, such as phenol, hydroquinone and resorcinol. However, when catechol and DHB coexisted, the QD-BphC system could not separate the signals of these two compounds from each other due to the inherent substrate range of BphC.At last, a fluorescent gold nanoclusters (AuNCs) in-situ synthesis by five crude enzymes from genetic engineering microorganism in this study. Furthermore, a method for pH-dependent dopamine sensing was proposed based on the quenching of AuNCs fluorescence resulted from the oxidized dopamine-quinone under alkaline conditions. Several factors showed great effects on the synthesis of BphC-AuNCs, including pH, the amounts of enzyme and HAuCl4. The fluorescence of BphC-AuNCs can remain stable towards temperature changes. Moreover, the quantum yields of these five crude enzymes-based AuNCs (BphAB-AuNCs, BphC-AuNCs, BphD-AuNCs, MfphA-AuNCs and E. coli-AuNCs) were all much higher than that of bovine serum albumin (BSA)-based one. Under basic conditions, dopamine can cause fluorescence quenching of these five AuNCs, and the quenching effects enhanced when dopamine was increased.