Part:BBa_K2094002

beta-agarase YM01-3

This enzyme hydrolyzes the β-1,4-glycosidic linkages of agarose.

Contribution

• Group: iGEM Team Heidelberg 2021
• Author: Franziska Giessler
• Summary: The Part BBa_K2094002 was used for our project and further characterized by enzyme activity measurements.

Background

Figure1: Enzyme activity of the β-agarases

Agar is often used in the food industry as a thickening agent or as a vegan alternative to gelatin. It is a complex polysaccharide consisting of alternating 3-O-linked β-D-galactopyranose and 4-O-linked α-L-galactopyranose. Agar cannot be degraded by most microorganisms, but there are some bacteria that metabolize agar as a carbon and energy source. They are mainly found in marine environments, where food resources are limited and agar is abundant in the form of the cell wall of some algae [1], [2].

The idea is to use the ability of agar degradation as a selection advantage for specific bacteria in order to overcome the established antibiotic selection used in the laboratory.

One of the enzymes present in agarolytic bacteria is the β-Agarase that hydrolyzes the β-(1,4) glycosidic bonds (see Figure 1).

Experiments and Results

Cloning

The DNA was synthesized using the sequence from part BBa_K2094002. Amplification was performed via PCR. The DNA was digested with BamHI and NdeI restriction enzymes and after that ligated with a T4 ligase into a pet15b backbone. This construct includes a T7 promoter, lac operator and an ampicillin resistance. The construct was transformed into competent E. coli BL21 via heat shock.

Culturing

Transformed E. coli BL21 were cultured on LB agar plates with carbenicillin for antibiotic selection and isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the expression of β-agarase. Agarolytic activity was confirmed by pit formation on the agar plates.

Figure 2: Pit formation on LB agar plates. Both images show the same LB agar plate with E. coli BL21 with β-agarase. With the human eye the pit formation due to agarolytic activity is easily detectable, but hard to visualize in a 2D picture. The arrow shows the colony with the most prominent pit. A) Top view of the plate. B) side view of the plate.

Experiment 1: Assay of enzyme activity

A solution containing 4% agarose was melted and then solidified in 50 mL Erlenmeyer flasks.

To the flasks with agar either the supernatant or the cell suspension of E. coli BL21 with β-agarase or E. coli BL21 with mCherry was added (Fig. 3).

Figure 3: Experimental setup agarase assay. The yellow substance in the Erlenmeyer flasks represents cured 4 % agar. The positive control with β-agarase expresses agarase and should be able to degrade the agar into reducing sugars As a negative control the same E. coli strain with the same plasmid but with a different insert (mCherry) was chosen. From both E. coli cultures either the supernatant or a diluted cell culture was used and incubated for 12 h before the DNS assay was done. Created with BioRender.com

Overnight cultures of E. coli BL21 with pet15b-β-agarase and E. coli BL21 with pet15b-mcherry were grown at 37° in LB medium. To the overnight cultures as well as to the in vivo experiments, carbenicillin was added for selection and IPTG was added to induce expression. Samples were incubated for 12h at 37°C with 70 rpm shaking. E. coli BL21 with pet15b-mcherry were used as a negative control to confirm that the occurrence of reducing sugars is due to the β-agarases and not to other metabolic pathways. By using bacteria having the same plasmid but with another insert, a possible influence of the pet15b vector could also be ruled out.

Agarase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) [3].

Figure 4: Agarase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) [3]. In the presence of reducing sugars like D-galactose and a temperature of 100 °C DNS reacts and as a result, the solution changes from yellow to red.

Briefly, 1.5 mL of sample solution was mixed with 0.5 mL of DNS reagent, the reaction was heated in boiling water for 5 min and then placed on ice for 5 min. Absorbance was measured at a wavelength of 540 nm, a standard curve of D-galactose dissolved in LB medium was used to determine the total amount of reducing sugars.

Samples were measured as described in table 1. We tested both the supernatant (hereafter in vitro) and the cell culture (hereafter in vivo) to find out whether or not β-agarase is secreted in the medium.

Table 1: Samples of the β-agarase assay. A brief description of each sample's content and the abbreviated form used in figure 4 are written in the first two columns. The absorbance value is the measurement we got when measuring the absorption at 540 nm with the TECAN and the total amount of reducing sugars was calculated using the standard curve and absorption value.

Sample	Sample name Figure 4	Absorbance Value	total amount of reducing sugars(mg/mL)
ß-Agarase supernatant without agar	agarase no agar	0.209400	0.057727
in vivo positive control	agarase in vivo	0.963100	0.658203
in vitro positive control	agarase in vitro	0.609800	0.376728
mcherry supernatant without agar	neg. control no agar	0.200600	0.050716
in vivo negative control	neg. control in vivo	0.188600	0.041156
in vitro negative control	neg. control in vitro	0.210600	0.058683

Figure 5: Results β-agarase activity
A) Samples after the DNS assay was performed from left to right: agarase no agar, agarase in vitro, agarase in vivo, neg. control no agar, neg. control in vitro, neg. control in vivo. The more reducing sugars were present before the experiment, the more red the final solution is.
B) Absorbance measurements of the samples with and without agarase in vitro and in vivo plotted on the D-galactose standard curve. Measurements without agar and without agarase are all around 0.2 absorption and therefore cover each other. The absorption was measured at 540 nm.

As can be seen in the presented table the absorption and the corresponding concentration of reducing sugars is the highest in the in vivo measurement with 0.658 mg/mL. This suggests that most β-agarase activity is found in our in vivo experiment. The in vitro experiment also shows the presence of reducing sugars with a concentration of 0.376728mg/mL suggesting that our enzyme is to be found in the supernatant and must therefore be secreted.

The very few concentrations of reducing sugars for the negative control and the ß-agarase supernatant without agar indicate that the presence of reducing sugars is not attributable to other enzymes produced by the bacteria itself but only to the combination of the ß-agarase and agar as its substrate.

Experiment 2: The influence of IPTG activation on the enzyme activity

As our Plasmid possesses a lac operator we wanted to test the enzyme activity in dependence of expression induction with IPTG.

A solution containing 4% agarose was melted and then solidified in 50 mL Erlenmeyer Flasks.

To the flasks was added:

Figure 6: Experimental setup. Two overnight cultures (50mL) of E. coli BL21 with pet15b-β-agarase were grown at 37° in LB medium. To one of them, 700µl of IPTG (100mM) was added. Samples were incubated for 12h at 37°C with 70 rpm shaking. Created with BioRender.com

Agarase activity was determined as described above.

Samples were measured as described in table 2. We tested both the supernatant (hereafter in vitro) and the cell culture (hereafter in vivo)(values for “in vivo” and “in vitro” were taken from experiment 1)

Table 2: The influence of IPTG activation on the enzyme activity assay. A brief description of each sample's content and the abbreviated form used in figure 6 are written in the first two columns. The absorbance value is the measurement we got after measuring the absorption at 540 nm with the TECAN and the total amount of reducing sugars was calculated using the standard curve and absorption value.

Sample	Sample name Figure 6	Absorbance Value	total amount of reducing sugars(mg/mL)
supernatant +IPTG without agar	+IPTG supernatant	0.209400	0.057727
in vivo +IPTG overnight culture +IPTG dilution	in vivo	0.963100 (experiment 1)	0.658203 (experiment 1)
in vitro +IPTG overnight culture	in vitro	0.609800 (experiment 1)	0.376728 (experiment 1)
in vitro +IPTG overnight culture	+IPTG in vitro	0.919100 (experiment 2)	0.627809 (experiment 2)
in vivo +IPTG overnight culture −IPTG dilution	+IPTG in vivo	0.5334	0.33032
supernatant -IPTG without agar	-IPTG supernatant	0.223100	0.068602
in vitro −IPTG overnight culture	−IPTG in vitro	0.520500	0.307733
in vivo −IPTG overnight culture −IPTG dilution	−IPTG in vivo	0.402900	0.213204

Figure 7: Influence of IPTG activation on the enzyme activity. A) Samples from left to right: +IPTG supernatant , +IPTG in vitro, +IPTG in vivo, −IPTG supernatant, −IPTG in vitro, −IPTG in vivoB) Measurements: values for “in vitro” from experiment 1 and “in vitro + IPTG” differ noticeably even if experimental conditions were the same.

The results in Table 2 and Figure 7b show that the values for in vitro measurements are unexpected as the calculated concentration for +IPTG in vitro (0.628mg/mL) is high compared to the previous experiment in vitro (0.377mg/mL) while having the same experimental conditions. Nevertheless, a notable influence of IPTG on β-agarase expression can be observed by comparing the results from the in vivo measurements. The β-agarase activity, determined by the amount of reducing sugars produced, is the highest when IPTG is added to the overnight culture as well as to the dilution (in vivo) with a concentration of 0.658 mg/mL. The second-highest concentration (0.330 mg/mL) can be observed in the overnight culture where IPTG was added, but lacking in the dilution (−IPTG in vivo). The lowest concentration (0.213 mg/mL) can be observed (0.213 mg/mL) when there is no IPTG neither in the overnight culture nor in the dilution (−IPTG in vivo).

Discussion

With our experimental setup, we were able to prove that the ß-agarase expressed by E. coli is able to break down solid agar into reducing sugars. Furthermore, the positive results of the in vitro experiment show that the enzymatic reaction takes place in the supernatant separated from bacteria. Therefore it can be assumed that ß-agarase is secreted by E. coli.

Pursuant to publications, agar degradation by ß-agarase produces neoagarooligosaccharides with different degrees of polymerization having galactose residues at their reducing ends. These include, for example, neoagarotetraose, neoagarohexaose and neoagarooctaose [1]. For a more detailed characterization of our ß-agarase and the resulting products, an analysis of the supernatant, for example by mass spectroscopy, would be necessary.

It would also be necessary to find out whether agar can be cleaved by E. coli to the monomeric α-galactose-6-sulfate and galactose in order to be used for metabolism, or whether further enzymes would be necessary for the use of agar as a carbon source.

Furthermore, the positive results of the in vitro experiment show that the enzymatic reaction takes place in the supernatant separated from bacteria. Therefore it can be assumed that ß-agarase is secreted by E. coli.

The results of experiment 2 indicate that the repression of expression in the lac operator does not function with one hundred percent efficiency. The values for reducing sugars without the addition of IPTG are 0.213 mg/mL, which is significantly higher than those of the negative control (0.0412 mg/mL). Enzyme activity can be measured even without an inducer, i.e. the β-agarase gene is expressed even without the addition of IPTG, but to a significantly smaller extent. This suggests leakage in the plasmid system.

Reference

[1]Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Agar degradation by microorganisms and agar-degrading enzymes. Applied microbiology and biotechnology, 94(4), 917–930. https://doi.org/10.1007/s00253-012-4023-2

[2]Su, Q., Jin, T., Yu, Y., Yang, M., Mou, H., & Li, L. (2017). Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber. AMB Express, 7(1), 220. https://doi.org/10.1186/s13568-017-0525-8

[3]G. L. Miller. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry. Vol. 31(3):426-428. DOI: 10.1021/ac60147a030

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 794
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 810
  Illegal AgeI site found at 847
  
• 1000
  COMPATIBLE WITH RFC[1000]