A novel phospholipase D that originate from marine Vibrio harveyi (VhPLD) was recombinant expressed and biochemically characterized. Moreover, effects of C-terminal peptides on catalytic and interfacial binding properties of VhPLD were investigated by constructing two truncated mutants (VhPLD-Δ(472-483) and VhPLD-Δ(437-483)). Optimal reaction temperature and pH value for wild-type VhPLD (VhPLD-WT) was 45 °C and pH 8.0. However, optimal reaction temperature of VhPLD-Δ(437-483) increased to 50 °C. Meanwhile, catalytic efficiency (kcat/KM) of VhPLD-Δ(472-483) and VhPLD-Δ(437-483) to the 1,2-Dioctanoyl-sn-glycero-3-phosphatidyl-p-nitrophenol (PpNP) was 12.9 and 14.2 times higher than that of VhPLD-WT. However, when compare the catalytic efficiency between VhPLD-Δ(472-483) and VhPLD-Δ(437-483), no significant change can be found between the two mutants. These results strongly indicated that the C-terminal 12 amino acids (472-483) have important role on the activity of VhPLD. Effects of C-terminal peptides on the interfacial binding properties of VhPLD to different phospholipid monolayers were also investigated by using monolayer film technology. Results of the maximum insertion pressure (MIP) indicated that deletion the C-terminal segment of VhPLD improved its interfacial binding properties to different phospholipid monolayers.

Mining of phospholipase D (PLD) with altered acyl group recognition except its head group specificity is also useful in terms of specific acyl size phospholipid production and as diagnostic reagents for quantifying specific phospholipid species. Microbial PLDs from Actinomycetes, especially Streptomyces, best fit this process requirements. In the present studies, a new PLD from marine Streptomyces klenkii (SkPLD) was purified and biochemically characterized. The optimal reaction temperature and pH of SkPLD were determined to be 60 °C and 8.0, respectively. Kinetic analysis showed that SkPLD had the relatively high catalytic efficiency toward phosphatidylcholines (PCs) with medium acyl chain length, especially 12:0/12:0-PC (67.13 S-1 mM-1), but lower catalytic efficiency toward PCs with long acyl chain (>16 fatty acids). Molecular docking results indicated that the different catalytic efficiency was related to the increased steric hindrance of long acyl-chains in the substrate-binding pockets and differences in hydrogen-bond interactions between the acyl chains and substrate-binding pockets. The enzyme displayed suitable transphosphatidylation activity and the reaction process showed 26.18% yield with L-serine and soybean PC as substrates. Present study not only enriched the PLD enzyme library but also provide guidance for the further mining of PLDs with special phospholipids recognition properties.

A new phospholipase D from marine Moritella sp. JT01 (MsPLD) was recombinantly expressed and biochemically characterized. The optimal reaction temperature and pH of MsPLD were determined to be 35 °C and 8.0. MsPLD was stable at a temperature lower than 35 °C, and the t1/2 at 4 °C was 41 days. The crystal structure of apo-MsPLD was resolved and the functions of a unique extra loop segment on the enzyme activity were characterized. The results indicated that a direct deletion or fastening of the extra loop segment by introducing disulfide bonds both resulted in a complete loss of its activity. The results of the maximum insertion pressure indicated that the deletion of the extra loop segment significantly decreased MsPLD's interfacial binding properties to phospholipid monolayers. Finally, MsPLD was applied to the synthesis of phosphatidic acid by using a biphasic reaction system. Under optimal reaction conditions, the conversion rate of phosphatidic acid reached 86%. The present research provides a foundation for revealing the structural-functional relationship of this enzyme.

The effects of N-terminal (1⁻34 amino acids) and C-terminal (434⁻487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor "a" indicated that the loop structure (1⁻25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26⁻34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434⁻487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434⁻469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

Mining of Phospholipase D (PLD) with high activity and stability has attracted strong interest for investigation. A novel PLD from marine Moritella sp. JT01 (MsPLD) was biochemically and structurally characterized in our previous study; however, the short half-life time (t1/2) under its optimum reaction temperature seriously hampered its further applications. Herein, the disulfide bond engineering strategy was applied to improve its thermostability. Compared with wild-type MsPLD, mutant S148C-T206C/D225C-A328C with the addition of two disulfide bonds exhibited a 3.1-fold t1/2 at 35 °C and a 5.7 °C increase in melting temperature (Tm). Unexpectedly, its specific activity and catalytic efficiency (kcat/Km) also increased by 22.7% and 36.5%, respectively. The enhanced activity might be attributed to an increase in the activation entropy by displacing more water molecules by the transition state. The results of molecular dynamics simulations (MD) revealed that the introduction of double disulfide bonds rigidified the global structure of the mutant, which might cause the enhanced thermostability. Finally, the synthesis capacity of the mutant to synthesize phosphatidic acid (PA) was evaluated. The conversion rate of PA reached about 80% after 6 h reaction with wild-type MsPLD but reached 78% after 2 h with mutant S148C-T206C/D225C-A328C, which significantly reduced the time needed for the reaction to reach equilibrium. The present results pave the way for further application of MsPLD in the food and pharmaceutical industries.

Using the classical emulsified system and the monomolecular film technique, the substrate specificity of recombinant Gibberella zeae lipase (rGZEL) that originates from Gibberella zeae was characterized in detail. Under the emulsified reaction system, both phospholipase and glycolipid hydrolytic activities were observed, except for the predominant lipase activity. The optimum conditions for different activity exhibition were also determined. Compared with its lipase activity, a little higher ratio of glycolipid hydrolytic activity (0.06) than phospholipase activity (0.02) was found. rGZEL preferred medium chain-length triglycerides, while lower activity was found for the longer-chain triglyceride. Using the monomolecular film technique, we found that the preference order of rGZEL to different phospholipids was 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS) > 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (PG) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) > l-α-phosphatidylinositol (PI) > cardiolipin (CL) > 3-sn-phosphatidic acid sodium salt (PA) > l-α-phosphatidylethanolamine (PE), while no hydrolytic activity was detected for sphingomyelin (SM). Moreover, rGZEL showed higher galactolipase activity on 1,2-distearoyimonoglactosylglyceride (MGDG). A kinetic study on the stereo- and regioselectivity of rGZEL was also performed by using three pairs of pseudodiglyceride enantiomers (DDGs). rGZEL presented higher preference for distal DDG enantiomers than adjacent ester groups, however, no hydrolytic activity to the sn-2 position of diglyceride analogs was found. Furthermore, rGZEL preferred the R configuration of DDG enantiomers. Molecular docking results were in concordance with in vitro tests.