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.

Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis

“…The hydrolysis activity of the recombinant fusion enzyme was detected using an enzyme-linked colorimetric assay. In particular, using PC as the substrate, the rate of the formation of choline was obtained by measuring the choline oxidase activity coupled with peroxidase activity as described in our previous report . One unit (U) of enzyme activity was defined as the amount of enzyme required for the release of 1 μmol of choline per minute under the assay conditions.…”

“…In particular, using PC as the substrate, the rate of the formation of choline was obtained by measuring the choline oxidase activity coupled with peroxidase activity as described in our previous report. 31 One unit (U) of enzyme activity was defined as the amount of enzyme required for the release of 1 μmol of choline per minute under the assay conditions. The kinetic parameters of recombinant proteins were calculated using different concentrations of PC (0.5−5.0 mM) as the substrate.…”

Progress & Prospect of Enzyme-Mediated Structured Phospholipids Preparation

“…To increase the production and enhance the catalytic performance, genetic engineering and protein engineering on phospholipases have been put into effect [46,47]. Zhang, et al [48] designed a novel two-step expression system to produce and secrete recombinant PLD in the extracellular medium, and cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins, demonstrating great potential in industrial applications. Damnjanovic, et al [49] altered the sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysophospholipids (LPL) by protein engineering, to discriminate between PLs and LPL.…”

Enzymatic production of N-acetylneuraminic acid: advances and perspectives

“…If efficient extracellular expression is effective, the cost of enzyme expression and purification will be significantly lower than the existing cost. Effective strategies to obtain extracellular enzymes are mainly based on increasing permeability or rupturing the double membrane barrier in E. coli: use of Triton X-100 or glycine, use of cell wall free strain, and co-expression of lysis proteins [98][99][100][101]. In addition to employing these strategies for NeuAc synthesis enzymes using E. coli as a microbial cell factory, the expression of these enzymes can be optimized by selecting other hosts (such as Bacillus, Saccharomyces, Mold, and Trichoderma).…”