A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay

Abstract

A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

Introduction

Lysine decarboxylase (LDC; E.C. 4.1.1.18) is an enzyme that converts lysine to cadaverine [13]. Cadaverine is produced in metabolic pathways to protect the cells from the drop of pH [4] and is widely used as chemical intermediates for drugs, and it can also be used as the monomer of polyamides such as Nylon 510 [10]. Normally, to detect decarboxylase activity, pH indicators [3], enzyme coupling [7], and high-performance liquid chromatography (HPLC) are commonly used [5,12]. Among these, HPLC generally gives the best results with repetitive and stable data; however, it takes tens of minutes to analyze a single sample and sometimes requires derivatization of analytes [11]. For the quick detection of decarboxylases, pH indicators can be employed to monitor the progress of enzyme-catalyzed reactions simply, and these colorimetric methods can be used to measure enzymatic activity on the basis of a color change as the reaction proceeds to release or consume protons [1]. The color change can be easily detected by a spectrophotometer, and thus allows quantitative as well as qualitative measurements. In the case of lysine decarboxylase, a pH indicator-based assay has been previously reported on solid agar with pH indicator [6]. However, this method was only used for plate-based screening and qualitative purpose, and it is inappropriate for the monitoring of samples in a short period. As a result, the solid-based assay needs to be modified, and a feasibility test of a liquid-based assay with pH indicator to lysine decarboxylase will be very helpful.

Here, we applied pH indicators for the quick detection of liquid-based biotransformation systems with quantitative data and applied it for practical purposes. It could be applied to monitoring of lysine decarboxylase for repetitive enzyme reaction, and quick quantification of activity for optimization of lysine decarboxylase overexpression with different IPTG concentrations. This assay is expected to be applied for monitoring and quantifying liquid-based enzyme reactions in biotransformation of decarboxylase in a high-throughput way

 

Materials and Methods

Chemical Reagents

The chemical reagents used in this study, such as cadaverine, lysine, bromocresol purple (BCP), bromothymol blue (BTB), phenol red (PR), iso-propyl-ß-D-thiogalactopyranoside (IPTG), kanamycin, and Tris–HCl, were purchased from Sigma Aldrich (St. Louis, MO, USA). Bacto-Agar and LB medium were purchased from Difco (Detroit, MI, USA).

Colorimetric Assay with pH Indicator

A stock solution of BTB was prepared by dissolving 134 mg of BTB in 1 ml of 100% ethanol and diluted to 1 ml with water. A stock solution of BCP was prepared by dissolving 162 mg of BCP in 1 ml of 100% ethanol and diluted to 1 ml with water. PR was prepared by dissolving 105 mg of PR to 1 ml with water. For color assay, 180 μl of 500 mM sodium acetate buffer (pH 6.0), 10 μl of reaction sample, and 10 μl of pH indicator stock solution were used. All measurements were performed in a 96-well microplate format and the most common 96-well (8 by 12 matrix) microplate was used with a typical reaction volume of 200 μl per well in this experiment. The absorbance at 595 nm of the pH indicator was recorded with a UV-Vis spectrophotometer (TECAN Sunrise, Tokyo, Japan). For the pH measurements, as ISTEK EcoMet p15 pH meter equipped with a combined glass calomel electrode was used.

Immobilization of Whole Cells

For immobilized strains, the resulting recombinant plasmid pET24ma-cadA was transformed into E. coli BL21(DE3) [8], and E. coli BL21(DE3)/pET24ma-cadA cells were grown at 37℃ with shaking at 200 rpm in 100 ml of LB broth containing 50 mg kanamycin l-1. The cells were grown to an optical density (OD) of 0.6 at 600 nm, after which 0.1 mM IPTG was added to the cell broth to induce recombinant protein expression. The cells were harvested following 16 h of induction at 30℃ and resuspended in 25 mM Tris–HCl buffer (pH 8.0) [2]. Cells grown in 50 ml of LB broth were mixed with an equal volume (1:1 (v/v)) of sodium alginate solution and stirred for 5min. The mixed solution obtained was then placed in a syringe and allowed to drop into a sterile 0.2 M CaCl2 solution that was stirred continuously. Alginate drops were solidified upon contact with CaCl2, forming beads and thus entrapping bacteria cells. The beads were washed two times with sterile distilled water and used for the production of cadaverine. For the reusability test, each cycle for free and immobilized cells was performed for 1 h with fresh 100mM L-lysine, 0.1 mM pyridoxal-5-phosphate, and 500 mM sodium acetate buffer (pH 6.0) at 37℃ in a water bath. After the reactions, the cells were centrifuged and detection of lysine decarboxylase activity was performed in a 96-well microplate. This procedure was repeated for 18 cycles.

Expression of Various Lysine Decarboxylases

In order to construct vectors containing ldcC from Burkholderia thailandensis, Enterobacter aerogenes, and E. coli, ldcC was amplified by PCR using the upstream primers ldcC (HindIII)-F and ldcC (XhoI)-R, and the PCR fragments were cloned into pET24ma (Table 1). The resulting recombinant plasmids were transformed into E. coli BL21(DE3), and E. coli BL21(DE3)/pET24ma-ldcC cells were grown at 30℃ and 200 rpm on a shaking incubator (Han-Beak Science Co., Korea). The first pre-cultures (5 ml of LB medium in 14 ml round-bottom tube) were inoculated with single colonies from agar plates, made by adding Bacto-Agar (Difco) of a concentration up to 2% and incubating for 16 h. Cells were harvested by centrifugation (13,000 ×g for 2 min at 4℃), washed with distilled water, and used as inoculums for the main cultivation. This was then carried out in 50 ml of LB medium, in a 250 ml baffled flask containing 50 mg kanamycin l-1. The cells were grown to an optical density of 0.6 at 600 nm, after which various concentrations of IPTG were added to the cell broth to induce recombinant protein expression. The cells were harvested following 15 h of induction at 30℃ and resuspended in 25 mM Tris–HCl buffer (pH 8.0). The cultures were harvested by centrifugation (13,000 ×g, 5 min, 4℃) and washed twice with distilled water. The enzymatic reaction was performed at 37℃ in a total volume of 500 μl, containing 20 μl of lysine decarboxylase-overexpressed whole cells, 500 mM sodium acetate buffer (pH 6.0), 10 mM L-lysine, and 0.1 mM pyridoxal-5-phosphate as final concentration. The reaction was stopped after 2 h by boiling for 5 min at 95℃. For color assay, 180 μl of sodium acetate buffer (50 mM), 10 μl of reaction sample, and 10 μl of BCP stock solution were used.

Table 1.Strains and plasmids used in this study.

 

Results and Discussion

Screening of pH Indicator for Lysine Decarboxylase

Before several pH indicators were examined, variations of pH with cadaverine and lysine concentration were examined (Fig. 1). The pH change by lysine concentration was negligible, and the pH remained around 5.8 regardless of lysine concentration. However, when cadaverine was examined, the pH change of cadaverine moved from 5.8 to 10 depending on the concentration of cadaverine. Linear regression showed that pH was correlated to the concentration of cadaverine (R2 = 0.9155).

Fig. 1.Change of pH with different concentration of cadaverine and lysine.

To screen out good indicators, a pH range for the lysine decarboxylase reaction reaching from 6 to 10 was considered and several candidates were selected based on previous reports and pH range. Among them, BCP, BTB, and PR were selected and examined further. Then 20 mg/ml of BCP, BTB, and PR and different concentrations of cadaverine were added to final concentrations of 1.25, 2.5, 5, 10, and 20 mM with 50 mM sodium acetate buffer (pH 6.0) in 96-well plates and the OD595 was measured (Fig. 2). Depending on the pH and indicator, the change of colors was different. BCP changed from pale yellow to bright blue and BTB changed from transparency to blue. However, PR showed yellow with low pH and pink at high pH. When the linearity was examined at different concentrations, BCP (R2 = 0.9754) showed better linearity than BTB (R2 = 0.8094) and PR (R2 = 0.8721). BCP was better than a regression of pH with different cadaverine concentrations (R2 = 0.9155). When the sensitivity and response were compared, BCP showed 3.5-fold to BTB and 2.1-fold to PR based on the slope of linear regression curve (Fig. 2). Based on these results, BCP was used for further experiments for lysine decarboxylase activity.

Fig. 2.Standard curves of cadaverine with bromocresol purple (A), bromothymol blue (B) and phenol red (C) as indicator (duplicated).

Application to Monitor the Repetitive Lysine Decarboxylase Reaction in 96-Well Microplate

One advantage for this liquid-based colorimetric assay is its quick and fast analysis of many samples. When we apply HPLC analysis, it needs several preparation steps for samples and takes some time to prepare the analytical system and perform chromatography [9]. Specifically, when we need continuous monitoring of repetitive reactions, it is very hard to know when the reaction should be stopped and new enzymes are introduced. Fig. 3 shows the reusability test of cadA-overexpressed whole cell (free cell) and cadA-overexpressed whole cell immobilized to Ca-alginate beads (immobilized cell). The cadA-overexpressed whole cells were prepared for the reusability test as explained in Materials and Methods. Free cells lost 50% of their productivity after 10 cycles with 100 mM of lysine as a substrate, and it was clearly monitored by the decrease of color change rate from yellow to purple after 10 cycles. By this result, we could know when our enzyme lost its activity and when we need to stop this reaction. In contrast, when immobilized whole cells were applied, the immobilized whole-cell enzyme reaction showed repeated color change until 18 cycles, meaning it kept its activity until 18 cycles.

Fig. 3.Monitoring of repetitive enzyme reactions with free and immobilized cells by colorimetric assay.

Optimization of Enzyme Expression with Different Concentrations of IPTG on Different Enzymes

Normally, optimization of enzyme expression is laborous because it needs many repetitive experiments to optimize and sometimes several enzyme assays and SDS-PAGEs, which could not give quantitative data easily, are needed. Based on previous difficulties on optimzation of enzyme overexpression, the liquid-based colorimetric assay was applied to optimize lysine decarboxylase overexpression with different concentrations of IPTG. We applied this to find the optimal IPTG concentration for newly cloned lysine decarboxylase from B. thailandensis, E. aerogenes, and E. coli ldcCs in E. coli. Once cells were induced, the same amount of cells was moved to 1.5 ml Eppendorf tubes and 10 mM of lysine and 0.74 mM of BCP were mixed in, and after 1 h, the OD was measured at 595 nm (Fig. 4A). By doing this, we compared different IPTG effects on different strains and compared the absorbance as different expression level effects on the enzyme activity of lysine decarboxylases. Among the tested strains, 0.1 mM of IPTG and 0.001 mM of IPTG with E. aerogenes ldcC were applied to SDS-PAGE, confirming that our system works for optimization of lysine decarboxylase overexpression (Fig. 4B).

Fig. 4.Optimization of enzyme expression with different concentrations of IPTG.

In conclusion, in this study, we applied a simple and convenient bromocresol purple-based colorimetric method for fast detection of lysine decarboxylase activity. This method seems quite useful for monitoring the lysine decarboxylase reaction in biotransformation, meaning one substrate like lysine is changed to one product like cadaverine. It will be especially useful for the screening of lysine decarboxylases from a library, because these experiments need a lot of samples and experiments for the detection of enzyme reaction.