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

**Rafida Razali <sup>1</sup> , Fikran Aranda Fahrudin <sup>1</sup> , Vijay Kumar Subbiah <sup>1</sup> , Kazufumi Takano <sup>2</sup> and Cahyo Budiman 1,\***


**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 <sup>±</sup> 0.08 U mg−<sup>1</sup> and 4.75 <sup>±</sup> 0.23 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>µ</sup>M−<sup>1</sup> 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 IC<sup>50</sup> of 0.022 mg mL−<sup>1</sup> . Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

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

#### **1. Introduction**

Bromelain is a member of the papain family that contains a complex and diverse natural mixture of proteases. It belongs to the family of sulfhydryl proteolytic enzymes and has a catalytic mechanism that involves the triad Cys-His-Asn/Glu [1–3]. This enzyme can be found in the pineapple plant (*Ananas comosus*). Depending on the source, it is usually classified as either fruit bromelain or stem bromelain. Apart from the stem and fruits, bromelain was also reported to be present in pineapple peel, core, crown, and leaves [4]. While bromelain is a unique cysteine protease name for pineapple, other cysteine proteases are widely distributed in plants and animals, including papain from papaya (*Carica papaya*) [5] and ficin from *Ficus insipida* [6]. In addition, cysteine protease was also found in viruses [7].

Bromelain possesses significant and notable therapeutic properties such as antiinflammatory, anti-thrombotic and fibrinolytic effects, inhibition of platelet aggregation, anti-cancer activity, immunomodulatory effects, enhanced wound healing and adsorption of drugs, particularly antibiotics, and cardiovascular and circulatory improvement [8,9]. It is also widely used in the food industry and considered a food supplement approved by the Food and Drug Administration of the United States of America, and is now freely available in the market [1,10]. It can be absorbed into the human intestines without degradation or losing biological activity [11–13]. Moreover, bromelain is also known to have the ability to

**Citation:** Razali, R.; Fahrudin, F.A.; Subbiah, V.K.; Takano, K.; Budiman, C. Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple. *Molecules* **2022**, *27*, 6031. https://doi.org/10.3390/ molecules27186031

Academic Editor: Smaoui Slim

Received: 14 June 2022 Accepted: 8 September 2022 Published: 16 September 2022

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hydrolyze meat proteins, particularly myofibril and connective fractions, and is considered a meat tenderizer that can be used traditionally [14].

Earlier, the whole-genome sequence of MD2 pineapple revealed the presence of 14 genes encoding cysteine proteases under the bromelain protease group [15]. Interestingly, these 14 genes encode various molecular weights of cysteine proteases ranging from 19 kDa to more than 200 kDa [15,16]. However, most of the studies on cysteine proteases of bromelain deal with medium-sized bromelain with a size of about 20–40 kDa [1,17–19]. So far, no study has reported on small-sized bromelain (20 kDa or less). Production of single cysteine protease bromelain for further applications as an enzyme-based bioactive compound is challenging due to a lengthy purification process. The use of the recombinant approach to producing active bromelain from a single gene is feasible yet challenging due to its solubility issue [1].

Our previous in silico study showed that the small-sized bromelain of MD2 pineapple (MD2-SBro) exhibited different structural features than the medium-sized bromelain of MD2 pineapple (MD2-MBro). Structurally, the Cys catalytic site of MD2-SBro is found to be located at the flexible loop, which is quite mobile and affects its proximity to the substrate [16]. In addition, both MD2-SBro and MD2-MBro also displayed differences in the hydrophobicity of the substrate-binding cavity. Earlier, we demonstrated that recombinant MD2-MBro produced under *Escherichia coli* (*E. coli)* was catalytically active with the specific activity and catalytic efficiency of 6.13 <sup>±</sup> 0.01 U mg−<sup>1</sup> and 5.64 <sup>±</sup> 0.02 <sup>×</sup> <sup>10</sup>–2 <sup>µ</sup>M−<sup>1</sup> s −1 , respectively [1].

This report provides the first experimental evidence on catalytic properties of recombinant MD2-SBro produced from *E. coli* host cells. We demonstrated that MD2-SBro was catalytically active with the activity modulated by pH, temperature, and metal ions. In addition, the antioxidant activity of this protein was also detectable.

#### **2. Materials and Methods**

#### *2.1. Gene Optimization, Synthesis and Expression System Construction*

The MD2-Sbro gene sequence was retrieved from NCBI with accession number OAY85828.1. The gene sequence was then optimized using OptimumGeneTM (Piscataway, NJ, USA) according to the codon usage preference of *E. coli*, and chemically synthesized under the GenScript outsource service (Piscataway, NJ, USA). The gene was provided in the pUC18 plasmid, designated as pUC18-SBro. Further, to construct the expression system for the gene, the SBro gene was amplified from the pUC18 plasmid using polymerase chain reaction (PCR) with a pair of specific primers. The sequences of the PCR primers used are as follows: 50 -CGAAGCTTATGGCGGAGTACGGTCGTGTG-30 (forward, with *Hind*III site) and 50 -GGCTCGAGGCCCCACCAGGAACCCCAGC-30 (reverse, with *Xho*I site). PCR was performed using KOD polymerase (Toyobo Co., Ltd., Kyoto, Japan) with the GeneAmp PCR system 2400 (Applied Biosystems, Tokyo, Japan). The amplicon was then digested with restriction enzymes of *Hind*III and *Xho*I and ligated into the pGS-21a expression vector using the DNA Ligation Kit, Mighty Mix (Takara, Japan). The success of ligation was confirmed using an insert check with restriction enzymes and nucleotides using the Prism 310 DNA sequencer (Applied Biosystems). The recombinant DNA of the MD2-SBro gene and pGS-21a is designated as an expression system of pGS21-SBro. This expression system allows MD2-SBro to be expressed in a fusion form to a glutathione S-transferase (GST) tag at its N-terminal. This expression system was then transformed into *E. coli* BL21(DE3) using the heat shock method based on Froger and Hall [20].

#### *2.2. Protein Expression*

Expression of recombinant MD2-SBro was performed based on Razali et al. [1] with some modifications. Briefly, the transformed cells were cultured in Luria Bertani (LB) media supplemented with 100 µg mL−<sup>1</sup> ampicillin and incubated at 37 ◦C at 180 rpm. The enzyme was induced by 1 mM IPTG (isopropyl β-D-1-thiogalactosidase (IPTG) once the OD<sup>600</sup> reached 0.7, followed by a prolonged incubation at 37 ◦C, 180 rpm for 5 h. The culture was harvested by centrifugation at 8000× *g* for 10 min at 4 ◦C. The cell pellet was then washed twice and resuspended in 20 mM phosphate buffer, pH 8.0, containing 100 mM NaCl, followed by cell lysis by a sonication in ice. The soluble fraction was then separated from the cell debris (pellet) by centrifugation at 35,000× *g* for 30 min at 4 ◦C. Soluble and pellet fractions were aliquoted for protein expression and solubility checking under 15% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). The whole fractions were also kept for further steps.

#### *2.3. Solubilization and Refolding of Insoluble Protein*

For the protein expressed in an insoluble form, solubilization and refolding steps were performed using urea treatment according to the method described by Kannan et al. [21] and Yamaguchi and Miyazaki [22], with some modifications. Briefly, the inclusion bodies (pellets) obtained after sonication were resuspended in 20 mM phosphate buffer (pH 8.0) containing 8 M urea, 2 mM DTT, and 100 mM NaCl. The sample solution was incubated at 4 ◦C overnight, followed by centrifugation at 35,000× *g*, 4 ◦C for 30 min. The supernatant was then collected for dialysis to refold the protein by removing the urea against 20 mM phosphate buffer (pH 8.0) at 4 ◦C overnight. Following the dialysis, the sample was centrifuged at 20,000× *g* for 15 min. The supernatant was collected and considered as solubilized MD2-SBro in a crude form, which was then used for solubility checking under 15% SDS-PAGE and further purification steps.

#### *2.4. Protein Purification*

Purification of a crude form of solubilized MD2-Sbro was performed according to [23]. Briefly, the GSTrap HP 5 mL (GE Healthcare; Chicago, IL, USA) column was firstly equilibrated with the binding buffer (20 mM phosphate buffer, pH 8.0 containing 100 mM NaCl). The crude form of solubilized MD2-Sbro, which was filtered previously using a syringe filter 0.22 µM membrane (Pall Life Sciences; Port Washington, NY, USA), was then loaded into the column. The sample was then eluted by linear gradient at gradual increments from 0% to 100% of an elution buffer containing 50 mM Tris-HCl (pH 8.0) with 10 mM reduced glutathione. The presence and purity of eluted MD2-Sbro were then checked from the fractions across the peak of interest in the chromatogram using 15% SDS-PAGE. Finally, the fractions containing MD2-Sbro in acceptable purity were pooled and dialyzed against 50 mM Tris-HCl, pH 8.0.

The purified protein concentration was determined by the NanoDrop™ 2000 (Thermo Fisher Scientific; Waltham, MA, USA) on the basis that the absorbance at 280 nm of 0.1% (1 mg mL−<sup>1</sup> ) solution is 1.69, as calculated based on Goodwin and Morton [24].
