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

ABSTRACT: 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.

SUBMITTER: Zhang H

PROVIDER: S-EPMC6530184 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Zhang Haiyang H Chu Wenqin W Sun Jianan J Liu Zhen Z Huang Wen-Can WC Xue Changhu C Mao Xiangzhao X

BMC biotechnology 20190522 1

<h4>Background</h4>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 lecithi ...[more]

PMID: 31118018

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Project description:Staphylococcus epidermidis is a common bacterial colonizer of human skin and mucous membranes, yet it has emerged as an important nosocomial pathogen largely due to its ability to form biofilms. Tobacco smoke has been demonstrated as a contributor to various infection diseases by improving the biofilm formation of multiple bacterial species; however, the association between tobacco smoke and S. epidermidis biofilm is still unclear. In this study, we tested the effect of nicotine, one of the most active components of tobacco, on S. epidermidis biofilm formation, and we studied the underlying mechanisms. Our results showed that nicotine promoted the biofilm formation of S. epidermidis 1457 strain (SE1457) and enhanced its initial attachment to a polyethylene surface as well as polysaccharide intercellular adhesin (PIA) production. In addition, an increased extracellular DNA release and a higher autolysis rate of SE1457 was detected after nicotine treatment, which was consistent with the increased ratio of dead cells in nicotine-treated SE1457 biofilm observed with confocal laser-scanning microscopy. Furthermore, the effect of nicotine on several autolysis-related and biofilm-related gene knockout mutants of SE1457 was tested. It showed that in ΔsaeRS, ΔlytSR, and ΔsceD, nicotine induced increase in biofilm formation was similar to that in SE1457; but in ΔarlRS, ΔatlE, and ΔicaC, the effect was obviously impaired. Consistently, the increase of the bacterial autolysis rate in ΔarlRS and ΔatlE induced by nicotine was not as significant as that in SE1457. Meanwhile, the growth inhibition of nicotine on SE1457 was observed, and it was much less on ΔarlRS and restored by the arlRS complementation. The arlRS transcription in SE1457 was inhibited by nicotine during cultivation as indicated by a promoter reporter assay using green fluoresent protein. Taken together, our study indicates that nicotine improves S. epidermidis biofilm formation by promoting its initial attachment and intercellular accumulation; the arlRS, atlE, and ica genes mediating bacterial autolysis and PIA production play an important role in this process.

Dataset Information

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

Publications

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

Similar Datasets

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