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Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2

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 super...

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Main Author: Pang, Wei Cheng
Format: Thesis
Language:English
Published: 2020
Subjects:
Q Science (General)
T Technology (General)
Online Access:http://umpir.ump.edu.my/id/eprint/31313/1/Isolation%2C%20gene%20expression%20and%20structural%20analysis%20of%20fruit%20bromelain%20from%20ananas%20comosus%20cultivar%20MD%202.wm.pdf
http://umpir.ump.edu.my/id/eprint/31313/
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spelling my.ump.umpir.313132023-02-16T03:53:02Z http://umpir.ump.edu.my/id/eprint/31313/ Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2 Pang, Wei Cheng Q Science (General) T Technology (General) 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. 2020-01 Thesis NonPeerReviewed pdf en http://umpir.ump.edu.my/id/eprint/31313/1/Isolation%2C%20gene%20expression%20and%20structural%20analysis%20of%20fruit%20bromelain%20from%20ananas%20comosus%20cultivar%20MD%202.wm.pdf Pang, Wei Cheng (2020) Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2. Masters thesis, Universiti Malaysia Pahang (Contributors, Thesis advisor: Aizi Nor Mazila, Ramli).
institution Universiti Malaysia Pahang
building UMP Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Pahang
content_source UMP Institutional Repository
url_provider http://umpir.ump.edu.my/
language English
topic Q Science (General)
T Technology (General)
spellingShingle Q Science (General)
T Technology (General)
Pang, Wei Cheng
Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2
description 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.
format Thesis
author Pang, Wei Cheng
author_facet Pang, Wei Cheng
author_sort Pang, Wei Cheng
title Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2
title_short Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2
title_full Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2
title_fullStr Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2
title_full_unstemmed Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2
title_sort isolation, gene expression and structural analysis of fruit bromelain from ananas comosus cultivar md 2
publishDate 2020
url http://umpir.ump.edu.my/id/eprint/31313/1/Isolation%2C%20gene%20expression%20and%20structural%20analysis%20of%20fruit%20bromelain%20from%20ananas%20comosus%20cultivar%20MD%202.wm.pdf
http://umpir.ump.edu.my/id/eprint/31313/
_version_ 1758578229055062016
score 13.211869

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