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
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spelling	my-ump-ir.313132023-02-16T03:53:02Z Isolation, gene expression and structural analysis of fruit bromelain from Ananas comosus cultivar MD 2 2020-01 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 http://umpir.ump.edu.my/id/eprint/31313/ 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 pdf en public masters Universiti Malaysia Pahang Faculty of Industrial Sciences and Technology Aizi Nor Mazila, Ramli
institution	Universiti Malaysia Pahang Al-Sultan Abdullah
collection	UMPSA Institutional Repository
language	English
advisor	Aizi Nor Mazila, Ramli
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
qualification_level	Master's degree
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
granting_institution	Universiti Malaysia Pahang
granting_department	Faculty of Industrial Sciences and Technology
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
_version_	1783732164523720704

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

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