Isolation, gene expression and structural analysis of fruit bromelain from ananas comosus cultivar MD 2 / Pang Wei Cheng

Faculty of Industrial Sciences & Technology

Thesis (Master of Science ) -- Universiti Malaysia Pahang – 2020

Includes bibliographical references

Ananas comosus, or commonly known as pineapple, is one of the most popular tropical fruits in the world. There are various pineapples cultivars with distinct characteristics. A. comosus cultivar MD 2 is the most successful pineapple hybrid with its attractive gold colour, super sweet taste and superior quality. Pineapple is rich in proteolytic enzymes such as fruit bromelain. In this study, fruit bromelain was characterised and compared between ripe and unripe A. comosus cultivar MD 2. Besides that, fruit bromelain transcripts were also isolated by conventional PCR and cloning. The extracted fruit bromelain sequence information was used in tertiary structure modelling followed by thermostability analysis. To investigate the gene expression level and proteolytic activity of fruit bromelain of A. comosus cultivar MD 2, quantitative Polymerase Chain Reaction (PCR) and casein enzymatic assay were conducted. Meanwhile, the sequence information of fruit bromelain was analysed using several bioinformatic tools including BioEdit, BLAST, InterProScan and ProtParam. The tertiary structure of the selected fruit bromelain sequences was modelled using MODELLER and refined via molecular dynamics simulation. Subsequently, the thermostability of fruit bromelain was evaluated using casein enzymatic assay and molecular dynamics simulation. The result revealed that fruit bromelain was down-regulated by 90 % in ripe A. comosus cultivar MD 2. It was also found that unripe A. comosus cultivar MD 2 has higher fruit bromelain activity (1.91 ± 0.08 U/mL) than ripe A. comosus cultivar MD 2 (1.13 ± 0.09 U/mL). On the other hand, three fruit bromelain sequences namely OAY62650.1, OAY68270.1 and OAY85858.1 were selected as sequences of interest to be isolated and cloned. The sequencing result demonstrated several nucleotides alteration in the isolated sequences. Moreover, Verify 3D, ERRAT and PROCHECK showed that the generated fruit bromelain models have high stereochemical quality. Structural analysis revealed interactions between the pro-domain and catalytic domain. Binding subsites of fruit bromelain His160, S2 Trp183, Glu19 and Asn159 were also identified. The His160, Trp183 and Glu19 were found conserved between fruit bromelain and papain. In addition, the thermal profile of MD 2 fruit bromelain was constructed. The optimum temperature of fruit bromelain was identified at 60 °C which is within the expected range. As the temperature raises, the stability of fruit bromelain was disrupted. The simulation result showed that fruit bromelain structures destruction may be due to flexible secondary loops as well as the presence of non-charged and hydrophilic amino acids residues. The information obtained from this study is expected to improve the applications of fruit bromelain in the future.