Revision as of 16:39, 20 October 2021

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 was added:

Figure3: Experimental setup 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 can also be ruled out. Created with BioRender.com

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

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 was used to determine the total amount of reducing sugars.

Samples were measured as follows:

Figure 4:results β-agarase activity A) Samples 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 B) Measurements

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

As our Plasmid possesses an lac operator we wanted to test the enzyme activity in dependence from 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 5: 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 follows (values for “in vivo” and “in vitro” were taken from experiment 1):

Figure 6: Results 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.

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.

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 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.

Although the values for in vitro measurements are a bit unexpected, there can be seen a notable influence of IPTG on β-agarase expression 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 0.658mg/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).

This shows that the repression of expression in the lac operator does not function with one hundred percent efficiency. The values for reducing sugars without 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.

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