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. 2022 Sep 16;27(18):6031.
doi: 10.3390/molecules27186031.

Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple

Rafida Razali  1 , Fikran Aranda Fahrudin  1 , Vijay Kumar Subbiah  1 , Kazufumi Takano  2 , Cahyo Budiman  1
Affiliations

Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple

Rafida Razali et al. Molecules. .

Abstract

Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (kcat/Km) of MD2-SBro were 3.56 ± 0.08 U mg-1 and 4.75 ± 0.23 × 10-3 µM-1 s-1, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC50 of 0.022 mg mL-1. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

Keywords: antioxidant; bromelain; catalytic activity; expression; metal ion; purification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression and solubilization check of MD2-SBro protein. Lane 1: Before IPTG induction; Lane 2: After IPTG induction; Lane 3: Soluble fraction obtained after the sonication; Lane 4: Insoluble fraction obtained after the sonication; Lane 5: Soluble fraction obtained after the solubilization; Lane 6: Insoluble fraction obtained after the solubilization. The band corresponding to MD2-SBro is indicated by the arrow.
Figure 2
Figure 2
Purification check of MD2-SBro protein. Lane P: Purified protein.
Figure 3
Figure 3
The proteolytic assay mixture of the N-CBZ-Gly-pNP substrate. Tube A is a blank (only substrate, without MD2-SBro and free GST protein). Tubes B and C are the mixture containing the substrate with MD2-Sbro and substrate with free GST protein, respectively. Tubes D, E, and F refer to the mixtures containing substrate, MD2-SBro, and free GST protein. The ratio of MD2-SBro and free GST protein were 1:1 (reaction D), 1:10 (reaction E), and 1:50 (reaction F).
Figure 4
Figure 4
(a) Michaelis–Menten curve and (b) Lineweaver–Burk double reciprocal plot of MD2-SBro.
Figure 4
Figure 4
(a) Michaelis–Menten curve and (b) Lineweaver–Burk double reciprocal plot of MD2-SBro.
Figure 5
Figure 5
Temperature-dependent activities of the purified MD2-SBro. The highest activity at 50 °C (19.77 × 10−3 U/mg) was adjusted as 100%.
Figure 6
Figure 6
The pH-dependent activities of the purified MD2-SBro. The highest activity at pH 8.0 (3.56 × 10−3 U/mg) was adjusted as 100%.
Figure 7
Figure 7
Inhibition of DPPH radical in the presence of different concentrations of ascorbic acid (control) and MD2-SBro.
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