Acid-stable amperometric soybean peroxidase biosensor based on a self-gelatinizable grafting copolymer of polyvinyl alcohol and 4-vinylpyridine

A peroxidase was extracted from Chinese soybean seed coat, and its thermostability and acid-stability were characterized. This peroxidase was immobilized into a self-gelatinizable grafting copolymer of polyvinyl alcohol with 4-vinylpyridine(PVA-g-PVP) to construct an acid-stable hydrogen peroxide biosensor. The effect of pH was studied for optimum analytical performances by amperometric and spectro-photometric methods, also the K-m(app) and the stability of the soybean peroxidase-based biosensor are discussed. At pH 3.0, the soybean peroxidase maintained its bioactivity and the enzyme electrode had a linear range from 0.01 to 6.2 mM with a detection limit of 1.0 x 10(-7) M. In addition, the main characteristics of different hydrogen peroxide sensors were compared.

Sol-gel thin-film immobilized soybean peroxidase biosensor for the amperometric determination of hydrogen peroxide in acid medium

An acid-stable soybean-peroxidase biosensor was devel oped by immobilizing the enzyme in a sol-gel thin film. Methylene blue was used as a mediator because of its high electron-transfer efficiency. The sol-gel thin film and enzyme membrane were characterized by FT-IR, and the effects of pH, operating potential, and temperature were explored for optimum analytical performance by using the amperometric method. The H2O2 sensor exhibited a fast response (5 s), high sensitivity (27.5 mu A/mM), as well as good thermostability and long-term stability. In addition, the performance of the biosensor was investigated using flow-injection analysis (FIA).

Comparison of photobioreactors and optimal light regime for rapid vegetative propagation of transgenic Undaria pinnatifida gametophytes

BACKGROUND: Previously, tachyplesin gene (tac) has been successfully transferred into Undaria pinnatifida gametophytes using the method of microprojectile bombardment transformation. The objectives of this study were to compare and evaluate the performance of bubble-column and airlift bioreactors to determine a preferred configuration of bioreactor for vegetative propagation of transgenic U. pinnatifida gametophytes, and to then investigate the influence of light on vegetative propagation of these gametophytes, including incident light intensity, photoperiod and light quality to resolve the problems of rapid vegetative propagation within the selected bioreactor. RESULTS: Experimental results showed that final dry cell density in the airlift bioreactor was 12.7% higher than that in the bubble-column bioreactor under the optimal aeration rate of 1.2 L air min(-1) L-1 culture. And a maximum final dry cell density of 2830 mg L-1 was obtained within the airlift bioreactor using blue light at 40 mu mol m(-2) s(-1) with a light/dark cycle of 14/10 (h). Polymerase chain reaction (PCR) analysis indicated that genes (bar and tac) were not lost during rapid vegetative propagation within the airlift bioreactor. CONCLUSION: The airlift bioreactor was shown to be much more suitable for rapid vegetative propagation of transgenic U. pinnatifida gametophytes than the bubble-column bioreactor in the laboratory. The use of blue light allows improvement of vegetative propagation of transgenic U. pinnatifida gametophytes. (C) 2009 Society of Chemical Industry

Microprojectile bombardment of Laminaria japonica gametophytes and rapid propagation of transgenic lines within a bubble-column bioreactor

A human acidic fibroblast growth factor gene, hafgf, was successfully transferred into Laminaria japonica (kelp) gametophytes via microprojectile bombardment using the biolistic PDS-1000/He gene gun. Following phosphinothricin screening, PCR detection and Southern blot analysis, transgenic L. japonica gametophytes were cultivated in an illuminated bubble-column bioreactor to optimize growth conditions. A maximal final dry cell density of 1,695 mg l(-1) was obtained in a batch culture having an initial dry cell density of 129.75 mg l(-1). This was achieved using an aeration rate of 1.08 l air min(-1) l(-1) culture in a medium containing 1.5 mM inorganic nitrate and 0.15 mM phosphate. In addition, the relationship between different nitrogen sources and growth of transgenic gametophytes indicated that both urea and sodium nitrate were effective nitrogen sources for cell growth, while ammonium ions inhibited growth of these gametophytes.

Optimal light regime for the cultivation of transgenic Laminaria japonica gametophytes in a bubble-column bioreactor

Fluctuating light intensity had a more significant impact on growth of gametophytes of transgenic Laminaria japonica in a 2500 ml bubble-column bioreactor than constant light intensity. A fluctuating light intensity between 10 and 110 mu E m(-2) s(-1), with a photoperiod of 14 h:10 h light:dark, was the best regime for growth giving 1430 mg biomass l(-1).

Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l(-1) was obtained in batch culture with an initial dry cell weight of 126 mg l(-1) and with aeration rate of 1.2 l air min(-1) l(-1) culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 mu g g(-1) dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.

An electrochemical DNA sensor based on electrodepositing aluminum ion films on stearic acid-modified carbon paste electrode and its application for the detection of specific sequences related to bar gene and CP4 Epsps gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid-modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well-defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single-stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the Specific sequence related to the target bar gene with the dynamic range comprised between 1.0 X 10(-7) mol/L to 1.0 x 10(-4) mol/L. A detection limit of 2.25 x.10(-8) mol/L. of oligonucleotides can be estimated.

Rapid optimization of process conditions for cultivation of transgenic Laminaria japonica gametophyte cells in a stirred-tank bioreactor

Batch cultivation for transgenic kelp gametophyte cells was investigated in an online controlled 5 L stirred-tank photo-bioreactor to rapidly optimize the process conditions by monitoring the rate of increase of pH. The transgenic kelp gametophytes with heterologous gene encoding hepatitis B surface antigen (HBsAg) could rapidly grow in the bioreactor. Optimal temperature and agitation rate for bioreactor cultivation of gametophytes were 15 degrees C and 200 rpm. Optimal incident light intensities depended on the initial cell densities. (c) 2006 Elsevier B.V. All fights reserved.

Sensitive and Rapid Detection of Genetic Modified Soybean (Roundup Ready) by Loop-Mediated Isothermal Amplification

Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening.

Three phases of dark-recovery course from photoinhibition resolved by the chlorophyll fluorescence analysis in soybean leaves under field conditions

The chlorophyll fluorescence in soybean leaves was observed by a portable fluorometer CF-1000 under field conditions. On clear days, F-0 increased while F, and F-v/F-m decreased gradually in the morning. At midday F-O reached its maximum while F-v and F-v/F-m reached their minimum. The reverse changes occurred in the afternoon. At dusk these parameters could return to levels near those at dawn. Following exposure to a strong sunlight for more than 3 h, the dark-recovery process displayed three phases: (1) slow increases in F-0, F-v and F-v/F-m within the first hour; (2) a faster decrease in F-0 and faster increases in F-v and F-v/F-m within subsequent two hours; (3) a slow decrease in F-0 and slow increases in F-v and F-v/F-m within the fourth hour. In comparison with darkness, weak irradiance had no stimulating effect on the recovery from photoinhibition. Hence the photoinhibition in soybean leaves is mainly the reflection of reversible inactivation of some photosystem 2 reaction centres, but not the result of D1 protein loss.

Optimal light regime for the cultivation of transgenic Laminaria japonica gametophytes in a bubble-column bioreactor

Fluctuating light intensity had a more significant impact on growth of gametophytes of transgenic Laminaria japonica in a 2500 ml bubble-column bioreactor than constant light intensity. A fluctuating light intensity between 10 and 110 mu E m(-2) s(-1), with a photoperiod of 14 h:10 h light:dark, was the best regime for growth giving 1430 mg biomass l(-1).