Applications of luminous bacteria enzymes in ecotoxicology
Historically, the application of bacterial luminescence in toxicology began with the usage of luminous bacteria for ecological monitoring and they are still widely used (Girotti et al., 2008; Fernández-Piñas et al., 2014). These methods made it possible to determine environmental pollution by comparing the light emission intensity of luminous bacteria in control with samples. As opposed to other test objects such as paramecia, algae, crustaceans, and so on, the bioluminescent assay is faster (typically < 30 min). However, as with other living organisms, living luminous bacteria is petulant. The failure to maintain the stable state of bacterial culture during measurements and storage results in low accuracy of measurement, a clear disadvantage of this method caused by the “petulance”. The bacteria react to the appearance of toxic substances either by decreasing or by increasing the luminous intensity, often leading to ambiguous interpretation of results. Because of these shortcomings the assay based on luminous bacteria didn’t show very good results in ecological laboratories. To overcome those difficulties it was suggested to use enzymes of luminous bacteria NAD(P)H:FMN-oxidoreductase and luciferase in soluble and immobilized forms (Kratasyuk, 1990; Esimbekova et al., 2013). Since 1990, bioluminescent enzymatic toxicity assay has been developed (Kratasyuk, 1990), and is nowadays actively used in ecology, medicine, agriculture, and other areas (Esimbekova et al., 2014).
The bacterial coupled enzyme system: NAD(P)H:FMN-oxidoreductase + luciferase (Red + Luc) involves two reactions:
FMN·H2 + RCHO + O2 -------> FMN + RCOOH + H2O + hν, (1)
NAD(P)H + FMN + H+ ---------> NAD(P)+ + FMN·H2 (2)
In Reaction 1, luciferase catalyzes the oxidation of long-chain aliphatic aldehydes involving reduced flavin mononucleotide. One of the products of this reaction is a quantum of light in the blue-green spectrum. To provide luciferase with reduced flavin mononucleotide, the luciferase reaction is coupled with the reaction catalyzed by NAD(P)H:FMN-oxidoreductase (Reaction 2) (Shimomura, 2006).
Application of bioluminescent enzymatic toxicity assays is justified by the fact that Red as a part of enzymatic assays is present in all living organisms, leading to good correlation between the effect of toxic substances on living organisms and that on the coupled enzyme system of luminous bacteria. The main principle of the bioluminescent toxicity enzymatic assays is inhibition of Red and/or Luc activities by the toxic components of analyzed samples.
The principles of bioluminescent enzymatic toxicity assay were successfully used for the analysis of aquatic environments (Kratasyuk et al., 2001; Kratasyuk et al., 1999; Vetrova et al., 2002) as well as air and soil pollutions (Rimatskaya et al., 2012, 2014).
This work was done using the lyophilized preparations of highly purified enzymes produced in the laboratory of nanobiotechnology and bioluminescence of the Institute of biophysics SB RAS (Krasnoyarsk, Russia). Each vial of the lyophilized preparation of enzymes contained 0.5 mg luciferase EC 220.127.116.11 from the recombinant strain E. coli and 0.15 units of NADH:FMN-oxidoreductase EC 1.5.1 (Bezrukikh et al., 2014) from Vibrio fischeri culture collection IBSO 836. To prepare the enzymes solutions 5 mL of potassium-phosphate buffer was added to the vial with enzymes.
The immobilized multi-component reagents "Enzymolum" were produced by Prikladnye Biosistemy Ltd. (Krasnoyarsk, Russia). The reagents contain enzymes (Red + Luc) co-immobilized with substrates (NADН and myristic aldehyde) into 3% (w/v) starch gel or 1% (w/v) gelatin gel (Kratasyuk and Esimbekova, 2011; Bezrukikh et al., 2014).
FMN (Serva); NADН (Gerbu) and tetradecanal (Merck) were used as the substrates of Red and Luc. 0.0025 % (v/v) solution of myristic aldehyde was prepared by mixing 50 µL of 0.25 % (v/v) ethanol solution of aldehyde and 5 mL of 0.05 М potassium-phosphate buffer (рН 6.9). NADН solution was prepared in 0.05 М potassium-phosphate buffer (рН 6.9).
Principle of bioluminescent enzymatic toxicity assays
Bioluminescent enzymatic toxicity assay can be carried out using different schemes (Figure 1). The first scheme places a cuvette with all the necessary components of the bacterial coupled enzyme system (enzymes, their substrates and buffer solution) into a bioluminometer, register the maximum light emission intensity Ic (control), add the sample or pollutant solution into the cuvette, and finally registers the maximum light emission intensity Iexp again (Figure 1A). This approach is the quickest and has demonstrated good repeatability of results.
Figure 1. (A) Bioluminescent assay scheme; (B) modified scheme of bioluminescent assay.
When analyzing toxicity of the water samples, the luciferase index (LI) or toxicity coefficient (TC) are calculated according to the formulas:
LI =(Iexp/ Ic) ·100 %.
TC = [(Ic - Iexp)/ Ic] ·100 %.
TC = 100 - LI.
LI and TC are the residual luminescence and the degree of inhibition of the bacterial coupled enzyme system Red + Luc in the presence of analyzed sample, respectively. The criterion of toxicity is a 50 % decrease in the maximum of light emission for the bacterial coupled enzyme system Red + Luc after the analyzed sample is added, as compared to the control. To estimate toxicity of individual substance values of EC50 and EC20 are calculated. They constituted 50% and 20% of the loss of luminescence for the coupled enzyme system Red + Luc. The decay constant kd is also estimated according to the following formula: kd = [ln(I2/I1)]/∆t, where I1 is the peak of bioluminescence intensity, I2 is the bioluminescence intensity at the certain moment of time after reaching the bioluminescence maximum, and ∆t is the time needed for I1 to reach I2.
The second scheme involves testing of the control sample (usually distilled water or buffer solution) and analyzed sample in different cuvettes. This approach is possible to achieve higher sensitivity of the assays to the toxic substances. The results are also calculated by the values of TC and kd. But in that case, it is possible to use one more parameter - the time when the coupled enzyme system reached the luminescence maximum (Tmax; Figure 1B).
The set of bioluminescent enzymatic toxicity assays
Bioluminescent enzymatic toxicity assay provides an instrument to solve a problem of complex evaluation of environmental toxicity. It is well-known that to estimate environmental toxicity it is necessary to use the battery of bioassays. Usually they represent different levels of organisms such as cells, organs, organisms and ecosystems. Due to the coupling with bacterial luciferase, it is possible to design new enzymatic bioassays in toxicology and combine them into a set to provide the toxicity control at the enzymatic level (Kratasyuk and Gitelson, 1987). The set includes enzymes of different classes, or key enzymes of metabolic processes in living organisms. The bacterial luciferase may be the terminal enzyme in coupling chains for more than 100 enzymes including such as lactate dehydrogenase, trypsin, glucose-6-phosphate dehydrogenase, and others, making it possible to measure the enzyme activities according to the light emission intensity.
To develop the set of bioluminescent enzymatic toxicity bioassays different enzyme interaction mechanisms were suggested (Figure 2). For example, in research by Kratasyuk et al. (2001) to estimate toxicity of water samples two enzymes were chosen: alcohol dehydrogenase (ADH) and trypsin, because they belong to different classes (oxidoreductases and hydrolases), and secondly, because they interact differently with bacterial luciferase, providing sensitivity to the different toxic substances (Kudryasheva et al., 1999, 2003).
Figure 2. Examples of coupling of the enzymatic reactions. (A) The sequence of enzymes in the triple enzyme system: ADH + Red + Luc (Petushkov et al., 1987); (B) interaction of enzymes in the triple enzyme system: trypsin + Red + Luc (Njus et al., 1974).
The effect of toxic substances on the activities of the triple enzyme system with ADH and trypsin were measured using the bioluminescence decay constant. Moreover, it became possible to regulate the sensitivity of enzymatic toxicity assays. For example, it was shown that the sensitivity of enzymatic assays to the toxic substances may be increased by extending the coupling chain of enzymatic reactions (Kudryasheva et al., 1999).
The set of bioluminescent enzymatic toxicity assays was used for monitoring natural and laboratory aquatic ecosystems (Kratasyuk et al., 2001) and for studying the seasonal dynamics of zooplankton non consumptive mortality (Dubovskaya et al., 2002), as well as for toxicity analysis of pesticides (Vetrova et al., 2007) and sanitary assessment of natural polymers polyhydroxyalkanoates (Shishatskaya et al., 2002).
Enzymatic Reagents for Bioluminescent Analysis
Widespread use of the available bioluminescent enzymatic toxicity bioassays is limited by the instability of the enzymes during use, limited shelf-life of enzymes–reagents, the need to control ambient conditions (i.e., pH, temperature, etc.), high manufacturing cost, and other factors. These problems can be solved by using immobilized enzymes that possess high catalytic activity and stability for long-term storage and successfully serve as biological modules of biosensors.
For the last 30 years immobilization has been widely used for production of stable reagents for bioluminescent analysis based on various bioluminescent systems: luminous bacteria, and bacterial and firefly luciferases. Many of the available immobilized reagents are successfully used in analytic measurements and in biosensors, because they simplify the analysis procedure, sometimes enabling full automation. At present, there are more than 40 different methods of immobilizing luminous organisms and enzymes (Kratasyuk and Esimbekova, 2003). An important advantage of immobilized enzymes is the possibility to control the enzyme stability to physical and chemical factors by way of choosing a suitable microenvironment. The optimal microenvironment for bacterial luciferase is natural polymer gels such as gelatin or starches (potato, rice, or corn). By varying gel concentration, time, and mode of drying of immobilized enzymes it is possible to make reagents with different enzymatic activity (Esimbekova et al., 2007, 2015).
It was shown that coupled enzyme system Red + Luc immobilized in starch or gelatin gel, saves its activity for 2 years (Lonshakova-Mukina et al., 2015). Moreover, immobilization in these gels leads to a considerable stabilization of the coupled enzyme system with regard to denaturation treatment: pH optimum of the enzymes expands both to the acid and alkaline areas; high enzyme activity is maintained at increased salt concentration; thermal stability increases essentially, especially in case of starch gel immobilization (Esimbekova et al., 2009; Bezrukikh et al., 2014).
Several substrates of bacterial bioluminescent reaction can be co-immobilized together with the coupled enzyme system to make the final test much simpler. For example, homogeneous multicomponent reagent named Enzymolum contains the enzymes Red and Luc, their substrates (myristic aldehyde and NADH) and buffer salts, co-immobilized in starch or gelatin gel (Kratasyuk and Esimbekova, 2011). The reagent is currently produced in flake form and can be used in the cuvette bioluminometer. The multicomponent reagent Enzymolum is a flake of dried gel, diameter 6–7 mm; dry weight 1.5 ± 0.2 mg.
The advantages of enzymatic assays using Enzymolum are their rapidity (the time of analysis does not exceed 3–5 min), high sensitivity, one-step measuring procedure and possibility of automation of analysis.
Thus, the new approach to develop the bioluminescent enzymatic biosensors, toxicity bioassays and reagents has been described. To solve the problem of how to detect, identify, and measure the contents of the numerous chemical compounds in environmental monitoring, food product monitoring, and medical diagnostics, the bioluminescent enzymatic toxicity assays were proposed, wherein the bacterial coupled enzyme system NAD(P)H:FMN-oxidoreductase-luciferase substitutes for living organisms. The immobilized reagent Enzymolum was introduced to facilitate and accelerate the development of cost-competitive enzymatic systems for use in biosensors for toxicological assays. The reagent is easy to use and convenient to be applied not only in toxicology studies but also in course of education, mainly in ecological and enzymological practical courses. Prototype biosensors offer cost advantages, versatility, high sensitivity, rapid response, extended shelf and flexible storage conditions.
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Applications of luminous bacteria enzymes in ecotoxicology
Valentina Kratasyuk, Elena Esimbekova
The principle and applications of bioluminescent enzymatic toxicity bioassays is described. This type of assays uses bacterial coupled enzyme systems: NADH:FMN-oxidoreductase and luciferase to replace living organisms in developing cost-competitive biosensors for environmental, medical and industrial applications. These biosensors instantly signal chemical and biological hazards and allow for detecting a great amount of toxic compounds with advantages associated with fast results, high sensitivity, simplicity, low cost and safety of the procedure.