Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli

Abstract

Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b(+) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its Nterminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg−1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10-2 µM−1 s−1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.

Isolation, identification, and characterization of phosphate solubilizing bacteria, Paenibacillus sp., from the soil of Danum Valley Tropical Rainforest, Sabah, Malaysia

“…Nevertheless, empirically, the purified enzyme should exhibit higher specific activity as compared to the unpurified one. Budiman et al (2011; and Razali et al (2021) reported that the increase of enzyme purity is accompanied by the increase in its specific activity. During purification, antagonist enzymes such as proteases are removed, allowing the protein of interest to be in the best possible state to produce its actual catalytic activity.…”

Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide

“…When the gene is heterologously expressed under E. coli as a host cell, Pk-ClpP was well expressed under the induction of 1 mM IPTG. The advantages of using E. coli for the heterologous expression of industrial and therapeutical proteins were widely reported [42][43][44]. Notably, k cat /K M of Pk-ClpP was found to be lower than Pf-ClpP [26].…”

“…Otherwise, this protein is unable to deliver its proper function in the cells. Further, it is also a common feature for serine protease to be active at neutral and alkaline pH, with an optimum between pH 7.0 and 11.0 [44,52].…”

“…The competitiveness of hyptolide was demonstrated by a comparable ∆G value with the substrate (only ±0.2 kcal/mol differences). Du et al [60] and Razali et al [44] implied that the free binding energy obtained from the docking simulation also reflects the complex's binding affinity. The competitive inhibition mechanism of hyptolide to Pk-ClpP is supported by the interaction pattern shown in Figure 9b,c, whereby most residues were involved in the interaction between Pk-ClpP and hyptolide or substrates are similar, with the uniqueness of more hydrophobic residues involved in a Pk-ClpP/hyptolide interaction.…”

Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5

“…The production of ClpP of L. plantarum IIA-1A5 is therefore considered a novel recombinant ClpP produced from LAB. In addition, protein production under recombinant technology is often halted by expression and solubility issues due to the incompatibility of the gene interest, the existence of rare codon, un-ideal GC ratio, or other related issues (Rosano & Ceccarelli, 2014;Razali et al, 2021). Accordingly, the codon optimization strategy was introduced a few decades ago in which the sequence of gene interest is changed to mimic the codon preferences of the host (Rosano & Ceccarelli, 2014).…”