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Enhancing Soluble Expression of Phospholipase B for Efficient Catalytic Synthesis of L-Alpha-Glycerylphosphorylcholine.
- Source :
-
Catalysts (2073-4344) . Jun2022, Vol. 12 Issue 6, pN.PAG-N.PAG. 14p. - Publication Year :
- 2022
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Abstract
- Phospholipase B (PLB) harbors three distinct activities with broad substrate specificities and application fields. Its hydrolyzing of sn-1 and sn-2 acyl ester bonds enables it to catalyze the production of L-alpha-glycerylphosphorylcholine (L-α-GPC) from phosphatidylcholine (PC) without speed-limiting acyl migration. This work was intended to obtain high-level active PLB and apply it to establish an efficient system for L-α-GPC synthesis. PLB from Pseudomonas fluorescens was co-expressed with five different molecular chaperones, including trigger factor (Tf), GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), GroELS and DnaKJE, or GroELS and Tf or fused with maltose binding protein (MBP) in Escherichia coli BL21(DE3) to improve PLB expression. PLB with DnaKJE-assisted expression exhibited the highest catalytic activity. Further optimization of the expression conditions identified an optimal induction OD600 of 0.8, IPTG concentration of 0.3 mmol/L, induction time of 9 h, and temperature of 25 °C. The PLB activity reached a maximum of 524.64 ± 3.28 U/mg under optimal conditions. Subsequently, to establish an efficient PLB-catalyzed system for L-α-GPC synthesis, a series of organic-aqueous mixed systems and surfactant-supplemented aqueous systems were designed and constructed. Furthermore, the factors of temperature, reaction pH, metal ions, and substrate concentration were further systematically identified. Finally, a high yield of 90.50 ± 2.21% was obtained in a Span 60-supplemented aqueous system at 40 °C and pH 6.0 with 0.1 mmol/L of Mg2+. The proposed cost-effective PLB production and an environmentally friendly PLB-catalyzed system offer a candidate strategy for the industrial production of L-α-GPC. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 20734344
- Volume :
- 12
- Issue :
- 6
- Database :
- Academic Search Index
- Journal :
- Catalysts (2073-4344)
- Publication Type :
- Academic Journal
- Accession number :
- 157915265
- Full Text :
- https://doi.org/10.3390/catal12060650