Part:BBa_K5216000

AgaA (β-agarase)

This part focuses on the synthesis of a β-agarase (AgaA). AgaA specifically cleaves the β-1,4 glycosidic bond between D-galactose and 3,6-anhydro-α-L-galactose residues in agarose, generating neoagarooligosaccharides (NAOS). The gene sequence of AgaA is derived from Marinimicrobium sp. H1 and has been codon-optimized for the E. coli host to enhance expression efficiency in a heterologous expression system[1].

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1129
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 198
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 339
  Illegal AgeI site found at 577
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 384
  Illegal BsaI site found at 985
  Illegal SapI site found at 89
  Illegal SapI site found at 221
  Illegal SapI site found at 284
  Illegal SapI.rc site found at 1066
  

Introduction

The goal of our project is to develop a novel antioxidant that can remove excess ROS produced in the skin due to environmental factors such as air pollution, UV radiation, and pathogenic microorganisms. This antioxidant will be applied in skincare products to help the skin resist oxidative stress, thereby slowing down aging. To achieve this goal, we have constructed a basic component, AgaA (BBa_K5216000). AgaA efficiently hydrolyzes agarose to generate NAOS, which exhibits high antioxidant activity and is a potential ingredient for skincare products.

Contents

1. Introduction
2. Design
3. Characterization
   3.1 Protein expression
   3.2 Enzyme activity verification
   3.3 Analysis of oxidation resistance of AgaA hydrolysate
4. Conclusion 
5. Reference

Design

We designed a prokaryotic expression system to express β-agarase AgaA (BBa_K5216000) (Fig.1). In our experiment, we constructed the AgaA_pET-32a(+) prokaryotic expression vector using double-enzyme digestion techniques. After successfully constructing the vector, the recombinant expression vector was transformed into E. coli Rosetta for subsequent protein expression(Fig.2).

Fig.1 Plasmid map of AgaA_pET-32a(+).

Fig.2 AgaA allogeneic expression diagram.

Characterization

Protein expression

AgaA_pET-32a (+) plasmid was transformed into E. coli Rosetta for expression. We induced expression at 16°C for 18 hours with IPTG at a final concentration of 0.5 mM. Protein expression was detected using SDS-PAGE, and Fig. 5A demonstrates that the target protein was successfully expressed and that the soluble protein can be used for subsequent experiments. The concentration of AgaA before purification was determined using the Bradford assay, resulting in a final protein concentration of 3.09 mg/mL and a total yield of 61.8 mg, which will be used for subsequent production of the neoagaroligosaccharides NAOS. The expression strain was then scaled up to 100 mL to express the protein, which was purified using Ni-NTA affinity chromatography (Fig. 5B). The figure indicates that the target protein can be purified using this method, with the wash buffer being chosen as 100 mM imidazole buffer and the elution buffer as 300 mM imidazole buffer.

Fig. 3 Expression and purification of AgaA.

(A) AgaA Protein Expression. M: marker; 1, E. coli Rosetta AgaA_pET-32a(+) culture (without IPTG); 2, E. coli Rosetta AgaA_pET-32a(+) culture (with IPTG at a final concentration of 0.8 mM); 3, E. coli Rosetta AgaA_pET-32a(+) soluble protein; 4, E. coli Rosetta AgaA_pET-32a(+) inclusion body. (B) AgaA Protein Purification. M: marker; AgaA (0.5 mM): 1, E. coli Rosetta AgaA_pET-32a(+) culture (without IPTG); 2, supernatant; 3, 0 mM imidazole; 4, 50 mM imidazole; 5, 100 mM imidazole; 6, 150 mM imidazole; 7, 200 mM imidazole; 8, 300 mM imidazole.

Enzyme activity verification

As observed in Fig. 6A, depressions appeared on the LB solid medium where the E. coli Rosetta AgaA_pET-32a(+) strain was cultured, indicating that this expression strain is capable of degrading agar, thereby demonstrating agarase activity. After inducing expression in this strain, the AgaA enzyme was obtained, and further enzyme activity verification is required. A mixture of 200 µl of AgaA and 200 µl of agarose (2%) was incubated in a 55°C metal bath for 30 minutes. The reaction was then terminated by heating at 95°C for 10 minutes. Next, 200 µl of the reaction mixture was transferred to a new 2 mL EP tube, and 200 µl of DNS reagent was added and mixed. The mixture was heated in a 95°C metal bath for 5 minutes (the control group used pre-inactivated AgaA). The color change between the control group and the experimental group was observed. As shown in Fig. 6B, the color of the experimental group significantly darkened, indicating the production of reducing sugars, specifically NAOS, in the reaction. This experimental result confirms that the heterologously expressed AgaA in E. coli has biological activity.

(A)Growth of the AgaA expression strain on LB agar plates. (B) AgaA enzyme activity assay using the DNS method (left: experimental group; right: control group).

The agarose hydrolysis products are displayed in Fig. 7. The standard sample consisted of a mixture of neoagarobiose (NA2), neoagarotetraose (NA4), and neoagarohexaose (NA6), with retention times of 6.75 minutes, 12.4 minutes, and 23.5 minutes, respectively. Upon comparing the chromatograms of the experimental group with the control group, new product peaks appeared at 6.75 minutes and 12.4 minutes following hydrolysis by the AgaA enzyme, indicating that the primary products of this reaction are NA2 and NA4. Earlier experiments revealed that AgaA hydrolyzes agarose into NA2, NA4, and NA6, but over time, NA6 is progressively broken down into NA2 and NA4. This demonstrates that the enzyme reaction was quite thorough, as NA6 was fully hydrolyzed into NA2 and NA4.

Fig. 5 HPLC Analysis of the AgaA Reaction Product

Analysis of oxidation resistance of AgaA hydrolysate

In this experiment, the antioxidant properties of NAOS, the hydrolysis products of AgaA, were evaluated using the ABTS assay. ABTS, when oxidized, forms a stable blue-green cation radical known as ABTS+, which is soluble in aqueous or acidic ethanol solutions and has a maximum absorbance at 734 nm. When an antioxidant is introduced to the ABTS+ solution, it reacts with the radical, causing a reduction in color intensity. The decrease in absorbance at 734 nm is measured to quantify the antioxidant capacity of the tested substance. As illustrated in Fig. 8, the antioxidant detection reagent containing the AgaA hydrolysis products exhibited a significant decolorization effect compared to the control group. This demonstrates that the NAOS generated from the AgaA-mediated hydrolysis of agarose possess significant antioxidant activity.

Fig. 6 Antioxidant activity analysis of AgaA hydrolysate.

Conclusion

The plasmid containing AgaA gene was successfully constructed and the active agarase was successfully expressed in E. coli Rosetta. The enzyme can effectively hydrolyze agarose to produce NAOS, and the product shows good antioxidant activity.

Reference

[1] Zhao, X., Li, X., He, L., Wang, L., & Zhang, Y. (2019). Production of neoagarobiose from agar through a dual-enzyme and two-stage hydrolysis strategy. Bioresource Technology, 278, 346-351. https://doi.org/10.1016/j.biortech.2019.01.06.