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A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain

BACKGROUND: Several types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatid...

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Autores principales: Zhang, Haiyang, Chu, Wenqin, Sun, Jianan, Liu, Zhen, Huang, Wen-can, Xue, Changhu, Mao, Xiangzhao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530184/
https://www.ncbi.nlm.nih.gov/pubmed/31118018
http://dx.doi.org/10.1186/s12896-019-0519-5
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author Zhang, Haiyang
Chu, Wenqin
Sun, Jianan
Liu, Zhen
Huang, Wen-can
Xue, Changhu
Mao, Xiangzhao
author_facet Zhang, Haiyang
Chu, Wenqin
Sun, Jianan
Liu, Zhen
Huang, Wen-can
Xue, Changhu
Mao, Xiangzhao
author_sort Zhang, Haiyang
collection PubMed
description BACKGROUND: Several types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatidyl groups into various phosphatidyl alcohols to produce modified phospholipids. Transphosphatidylation is a useful biocatalytic method for the synthesis of functional phospholipids from lecithin or phosphatidylcholine (PC), which are both easily accessible. Different PLD coding genes have been cloned from various sources from viral, prokaryotic, and eukaryotic organisms. Despite the catalytic potential of PLD, their low productivity has hampered their practical applications, probably because PLD, which is highly toxic to the host cells, when transformation of the PLD genes into the host cells, degrade PLs in the cell membrane. In this study, we designed a novel two-step expression system to produce and secrete recombinant PLD in extracellular medium, cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins. RESULTS: The engineered BL21 (DE3) host strain, which harbored the final expression vector pET28a-PLD-CBD-araC-ESN, was induced by IPTG and L-arabinose, the cell density decreased rapidly over a 2 h period and the enzymes released into the extracellular medium accounts owned 81.75% hydrolytic activity. Scanning electron microscopy results showed that there were obvious structural changes on the cell surface. The extracellularly secreted PLD-CBD powder was used to catalyze the transphosphatidylation reaction synthesis of phosphatidylserine, 2.3 U enzymes reacted for 12 h, during which the conversion rate reached 99% with very few by-products being produced. When the fused protein PLD-CBD immobilized on microcrystalline cellulose, the enzymes can be cycle used five times with 26% conversion rate was preserved. CONCLUSIONS: This study introduced an effective method for use in the expression of recombinant proteins and their extracellular secretion that simplifies the steps of sonication and purification and demonstrates great potential in the industrial application of enzymes. Cellulose as the most abundant renewable biomass resources in nature, and the cost is low, used for PLD immobilization make it more simple, effective and sustainable.
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spelling pubmed-65301842019-05-28 A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain Zhang, Haiyang Chu, Wenqin Sun, Jianan Liu, Zhen Huang, Wen-can Xue, Changhu Mao, Xiangzhao BMC Biotechnol Research Article BACKGROUND: Several types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatidyl groups into various phosphatidyl alcohols to produce modified phospholipids. Transphosphatidylation is a useful biocatalytic method for the synthesis of functional phospholipids from lecithin or phosphatidylcholine (PC), which are both easily accessible. Different PLD coding genes have been cloned from various sources from viral, prokaryotic, and eukaryotic organisms. Despite the catalytic potential of PLD, their low productivity has hampered their practical applications, probably because PLD, which is highly toxic to the host cells, when transformation of the PLD genes into the host cells, degrade PLs in the cell membrane. In this study, we designed a novel two-step expression system to produce and secrete recombinant PLD in extracellular medium, cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins. RESULTS: The engineered BL21 (DE3) host strain, which harbored the final expression vector pET28a-PLD-CBD-araC-ESN, was induced by IPTG and L-arabinose, the cell density decreased rapidly over a 2 h period and the enzymes released into the extracellular medium accounts owned 81.75% hydrolytic activity. Scanning electron microscopy results showed that there were obvious structural changes on the cell surface. The extracellularly secreted PLD-CBD powder was used to catalyze the transphosphatidylation reaction synthesis of phosphatidylserine, 2.3 U enzymes reacted for 12 h, during which the conversion rate reached 99% with very few by-products being produced. When the fused protein PLD-CBD immobilized on microcrystalline cellulose, the enzymes can be cycle used five times with 26% conversion rate was preserved. CONCLUSIONS: This study introduced an effective method for use in the expression of recombinant proteins and their extracellular secretion that simplifies the steps of sonication and purification and demonstrates great potential in the industrial application of enzymes. Cellulose as the most abundant renewable biomass resources in nature, and the cost is low, used for PLD immobilization make it more simple, effective and sustainable. BioMed Central 2019-05-22 /pmc/articles/PMC6530184/ /pubmed/31118018 http://dx.doi.org/10.1186/s12896-019-0519-5 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Zhang, Haiyang
Chu, Wenqin
Sun, Jianan
Liu, Zhen
Huang, Wen-can
Xue, Changhu
Mao, Xiangzhao
A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain
title A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain
title_full A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain
title_fullStr A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain
title_full_unstemmed A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain
title_short A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain
title_sort novel autolysis system for extracellular production and direct immobilization of a phospholipase d fused with cellulose binding domain
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530184/
https://www.ncbi.nlm.nih.gov/pubmed/31118018
http://dx.doi.org/10.1186/s12896-019-0519-5
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