Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Abstract

Phosphatidylserine (PS) has been widely used in the fields of food and medicine, among others, owing to its unique chemical structure and health benefits. However, the phospholipase D (PLD)-mediated enzymatic production of PS remains a challenge due to the low transphosphatidylation activity of PLD. Therefore, in the present study, we designed a maltose-binding protein (MBP) tag and a PLD co-expression method to achieve the expression of soluble PLD in Escherichia coli. A “reconstruct substrate pocket” strategy was then proposed based on the catalytic mechanism and molecular dynamics simulation, expanding the substrate pocket and manipulating the coordination of l-Ser within the active site. The best mutant (Sr MBPPLDMu6) exhibited a 2.04-fold higher transphosphatidylation/hydrolysis ratio than the wild-type Furthermore, under optimal conditions, Mu6 produced 58.6 g/L PS with 77.2% conversion, within 12 h on a 3 L scale, which demonstrates the potential of the proposed method for industrial application.

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004

“…Based on the catalytic mechanism and MD simulation, Qi et al used the “reconstituted substrate pocket” to expand the substrate pocket of phospholipase and regulate the coordination of l -Ser in the active site. Finally, the specific activity of the mutant was increased by 2.04 times . In the present study, a structural model suitable for SNDH activity was established and its substrate pocket was determined, providing a basis for the following rational design of the substrate pocket.…”

“…Finally, the specific activity of the mutant was increased by 2.04 times. 33 In the present study, a structural model suitable for SNDH activity was established and its substrate pocket was determined, providing a basis for the following rational design of the substrate pocket.…”

Reprogramming the Transition States to Enhance C–N Cleavage Efficiency of Rhodococcus opacus l-Amino Acid Oxidase

“…The expression plasmid pET-28a (+) and host strain E. coli BL21 (DE3) were purchased from Novagen (Madison, WI, USA). The construct containing the MBP tag strain, pET-28a-Sr MBP PLD/ BL21(DE3), 44 of existing strains in the laboratory; the DNA and protein sequences can be seen in the Supporting Information. Catalase, isopropyl β-D-1thiogalactopyranoside (IPTG), and the required antibiotics were purchased from BBI Life Sciences (Shanghai, China).…”

Enzyme engineering for functional lipids synthesis: recent advance and perspective

“…The resulting optimal mutant displayed a notable 2.04-fold increase in the transphosphatidylation/hydrolysis ratio compared to the WT. Under optimal conditions, the mutant Mu6 achieved a production of 58.6 g/L of phosphatidylserine with a 77.2% conversion within 12 h on a 3 L scale, showcasing its potential for industrial application (Qi et al 2022 ). In another case, a conserved flexible loop (residues 376–382) in the active site of Streptomyces klenkii PLD (SkPLD) was identified based on sequence conservation and amino acid analysis.…”