Heterologous expression, purification and characterization of bromelain from md2 pineapple
Bromelain is a complex mixture of proteases mainly found in the stems and fruits of pineapple (Ananas comosus). This enzyme belongs to a cysteine protease family member with a wide range of applications. Traditionally, bromelain is widely used as a meat tenderizer. The bromelain-based meat tenderize...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2020
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| Subjects: | |
| Online Access: | https://eprints.ums.edu.my/id/eprint/41286/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/41286/2/FULLTEXT.pdf |
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| Summary: | Bromelain is a complex mixture of proteases mainly found in the stems and fruits of pineapple (Ananas comosus). This enzyme belongs to a cysteine protease family member with a wide range of applications. Traditionally, bromelain is widely used as a meat tenderizer. The bromelain-based meat tenderizer products are commercially available which are produced through direct extraction and purification from pineapples. No recombinant bromelain products are so far available in the market. While the production of recombinant bromelain was available in many reports, none of them was successfully produced in fully soluble forms. Previous genomics study on MD2 pineapple revealed that this pineapple strain has 14 genes encoding bromelain with various sizes ranging from 19 kDa up to 211 kDa and therefore classified into small- (MD2-SBro; <20 kDa), medium- (MD2-MBro; 20-60 kDa) and large-sized (MD2-LBro; >60 kDa) bromelain. MD2-SBro primary structure has shown to have no N- or C-terminal additional sequence, meanwhile, MD2-MBro and MD2- LBro demonstrated to have both additional sequences with different sizes. It is therefore assumed that these two bromelains are structurally comparable and may represent each other. This study aims to: (1) determine expressibility and solubility of MD2-SBro and MD2-MBro from MD2-pineapple under heterologous expression using Escherichia coli; (2) compare the catalytic and structural properties of MD2- SBro and MD2-MBro overexpressed in E. coli; and (3) determine the effects of the selected recombinant MD2-bromelain on the meat tenderization and physicochemical properties. For this purpose, the gene encoding MD2-SBro and MD2-MBro were codon-optimized, chemically synthesized and cloned into pGS-21a and pET-32b(+) plasmids, respectively and transformed into Escherichia coli BL21-CodonPlus(DE3). MD2-SBro was expressed in an insoluble form, while MD2-MBro was successfully expressed in a soluble form. MD2-SBro and MD2-MBro were successfully purified to get 14 mg and 20 mg from 1L culture, respectively. Comparative analysis had shown that these bromelains had remarkable difference catalytic properties, whereby catalytic efficiency (kcat/KM) of MD2-SBro (213 mM-1s-1) was found to be lower than that of MD2-MBro (29.13 x 105 mM-1s-1). Comparative analysis on the threedimensional models of both proteins demonstrated that while both bromelains have conserved catalytic triads consisting of Cys-His-Asn, the distance of Cys-His in MD2- SBro was found to be not in the appropriate distance for the nucleophilic attack. Besides, the hydrophobicity of the substrate-binding cavity of these bromelains was also found to be different. These are believed to be the main factors causing the differences in their catalytic properties. Given its remarkable higher catalytic activity, MD2-MBro was further characterized and applied as a meat tenderizer. Far-UV circular dichroism of MD2-MBro showed that this protein was indeed in a properly folded state with the helical content of 59.2%. The thermal unfolding curve of this protein was also shown that MD2-MBro has a melting temperature of 54.93 + 0.2 oC. Further application of MD2-MBro indicated that MD2-MBro had shown to be able to tenderize the meat in a concentration-dependent manner. The shear-force values of the meat in the presence of MD2-MBro fall under the category of tender. The result also suggested that the concentration of 0.01% MD2-MBro was sufficient to tenderize the meat. Nevertheless, the ability of meat tenderizing by MD2-MBro was accompanied by some changes in physicochemical properties of the meat (pH, water holding capacity and cooking loss). Altogether, this study should contribute to the new knowledge on the catalytic properties of different types of bromelain and provide a platform for the production of recombinant bromelain as a meat tenderizer. |
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