Biochemical Characterization of a Marine Pseudoalteromonas citrea-Derived Fatty Acyl-AMP Ligase That Exhibits N-Acyl Amino Acid Synthetic Activity.

Activation of fatty acids as acyl-adenylates by fatty acid-AMP ligase (FAAL) is a well-established process contributing to the formation of various functional natural products. Enzymatic characterization of FAALs is pivotal for unraveling both the catalytic mechanism and its role in specific biosynthetic pathways. In this study, we recombinantly expressed and characterized a novel FAAL derived from marine Pseudoalteromonas citrea (PcFAAL). PcFAAL was a cold-adapted neutral enzyme, demonstrating optimal activity at 30 °C and pH 7.5. Notably, its specific activity relied on the presence of Mg2+; however, higher concentrations exceeding 10 mM resulted in inhibition of enzyme activity. Various organic solvents, especially water-immiscible organic solvents, demonstrated an activating effect on the activity of PcFAAL on various fatty acids. The specific activity exhibited a remarkable 50-fold increase under 4% (v/v) n-hexane compared to the aqueous system. PcFAAL displayed a broad spectrum of fatty acid substrate selectivity, with the highest specific activity for octanoic acid (C8:0), and the catalytic efficiency (kcat/Km) for octanoic acid was determined to be 1.8 nM-1·min-1. Furthermore, the enzyme demonstrated biocatalytic promiscuity in producing a class of N-acyl amino acid natural products, as verified by LC-ESI MS. Results indicated that the PcFAAL exhibits promiscuity towards 10 different kinds of amino acids and further demonstrated their potential value in the biosynthesis of corresponding functional N-acyl amino acids.

Crystal Structure of Fungal Nonspecific Phospholipase C Unveils a Distinct Catalytic Mechanism.

Nonspecific phospholipase C (NPC) plays a pivotal role in hydrolyzing phospholipids, releasing diacylglycerol─an essential second messenger. Extensive research has elucidated the structure and function of bacterial and plant NPCs, but our understanding of their fungal counterparts remains limited. Here, we present the first crystal structure of a fungal NPC derived from Rasamsonia emersonii (RePLC), unraveling its distinguishable features divergent from other known phospholipase C. Remarkably, the structure of RePLC contains solely the phosphoesterase domain without the crucial C-terminal domain (CTD) found in plant NPCs, although CTD is important for their activity. Through a comparative analysis of structural features among NPCs from diverse species combined with structure-based mutation analyses and bioinformatics methods, we propose a potential molecular mechanism that may universally underlie the catalysis of phospholipid hydrolysis in fungal NPCs. Furthermore, our study sheds light on the captivating evolutionary trajectory of enzymes across diverse species.

Association between cell-free DNA fetal fraction and pregnant character: a retrospective cohort study of 27,793 maternal plasmas.

To determine the association between cell-free DNA fetal fraction (cffDNA) and various prenatal characters to better guide the clinical application of noninvasive prenatal screening (NIPS), a retrospective cohort study of 27,793 women with singleton pregnancies was conducted. Results indicated that no significant difference on cffDNA between trisomy/sex chromosome aneuploidy (SCA) and non-trisomy groups was found. However, the fetal fraction (FF) in the T18 and T13 subgroups were significantly lower than that in the non-trisomy group, while the FF in the T21 group was significantly higher than the non-trisomy group. Pearson's correlation analysis revealed a positive correlation between √FF and gestational week in the T21, SCA, and non-trisomy groups. A negative correlation between maternal age and √FF in T21 and non-trisomy cases was found, but a positive correlation in SCA group. Compared to the decreasing trend in FF in the T21 group, no significant difference was observed in the SCA group. The √FF level was negatively correlated to maternal BMI in T21 and non-trisomy group, while a positive correlation in SCA group. FF was close related to the result of NIPS and related maternal factors. Though NIPS has increased accuracy, the complexity still should be recognized especially in clinical practice.

Distinct Lipids Profiles and Associations With Clinical Indicators and Gut Microbiota in Children With Prader-Willi Syndrome.

Lipid metabolism is closely linked to adiposity. Prader-Willi syndrome (PWS) is a typical genetic disorder causing obesity; however, the distinct lipidomic profiles in PWS children have not been thoroughly investigated. Herein, serum lipidomics analyses were simultaneously explored in PWS, simple obesity (SO), and normal children (Normal). Results indicated that the total concentration of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the PWS group were significantly deceased compared with both the SO and the Normal group. In contrast, compared with the Normal group, there was an overall significant increase in triacylglycerol (TAG) levels in both the PWS and the SO groups, with the highest found in SO group. Thirty-nine and 50 differential lipid species were screened among 3 groups: between obesity (PWS and SO) and the Normal group. Correlation analysis revealed distinct profiles in PWS that was different from other 2 groups. Notably, PC (P16:0/18:1), PE (P18:0-20:3), PE (P18:0-20:4)) showed significant negative correlation with body mass index (BMI) only in the PWS group. PE (P16:0-18:2) showed a negative association with BMI and weight in the PWS group, but significant positive correlation in the SO group; no statistically significant association was found in the Normal group. We also found a significant negative correlation between Blautia genus abundance and several significantly changed lipids, including LPC (14:0), LPC (16:0), TAG (C50:2/C51:9), TAG (C52:2/C53:9), TAG (C52:3/C53:10), and TAG (C52:4/C53:11), but no significant correlation in the Normal group and the SO group. Similarly, in the PWS group, the Neisseria genus was significantly negatively associated with acylcarnitine (CAR) (14:1), CAR (18:0), PE (P18:0/20:3), and PE (P18:0/20:4), and extremely positively associated with TAG (C52:2/C53:9); no obvious correlations were observed in the Normal group and the SO group.

Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 µM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.

Engineering the Thermostability of the Mono- and Diacylglycerol Lipase SMG1 for the Synthesis of Diacylglycerols.

Diacylglycerols (DAGs) display huge application prospectives in food industries. Therefore, new strategies to produce diacylglycerides are needed. Malassezia globose lipase (SMG1) could be used to synthesize DAGs. However, the poor thermostability of SMG1 seriously hampers its application. Herein, a rational design was used to generate a more thermostable SMG1. Compared with the wild type (WT), the M5D mutant (Q34P/A37P/M176V/G177A/M294R/ G28C-P206C), which contains five single-point mutations and one additional disulfide bond, displayed a 14.0 °C increase in the melting temperature (Tm), 5 °C in the optimal temperature, and 1154.3-fold in the half-life (t1/2) at 55 °C. Meanwhile, the specific activity towards DAGs of the M5D variant was improved by 3.0-fold compared to the WT. Molecular dynamics (MD) simulations revealed that the M5D mutant showed an improved rigid structure. Additionally, the WT and the M5D variants were immobilized and used for the production of DAGs. Compared with the WT, the immobilized M5D-catalyzed esterification showed a 9.1% higher DAG content and a 22.9% increase in residual activity after nine consecutive cycles. This study will pave the way for the industrial application of SMG1.

Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy.

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.

A New Phospholipase D from Moritella sp. JT01: Biochemical Characterization, Crystallization and Application in the Synthesis of Phosphatidic Acid.

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.

Acyl Chain Specificity of Marine Streptomyces klenkii PhosPholipase D and Its Application in Enzymatic Preparation of Phosphatidylserine.

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.

Enhancement of Phospholipid Binding and Catalytic Efficiency of Streptomyces klenkii Phospholipase D by Increasing Hydrophobicity of the Active Site Loop.

The mechanism of active site loops of Streptomyces phospholipase D (PLD) binding to the lipid-water interface for catalytic reactions still remains elusive. A flexible loop (residues 376-382) in the active site of Streptomyces klenkii PLD (SkPLD) is conserved within PLDs in most of the Streptomyces species. The residue Ser380 was found to be essential for the enzyme's adsorption to the interface and its substrate recognition. The S380V mutant showed a 4.8 times higher catalytic efficiency and nearly seven times higher adsorption equilibrium coefficient compared to the wild-type SkPLD. The monolayer film technique has confirmed that the substitution of Ser380 with valine in the loop exhibited positive interaction between the enzyme and PCs with different acyl chain lengths. The results of the interfacial binding properties indicated that the S380V mutant might display suitable phosphatidylserine synthesis activity. The present study will be helpful to explain the role of residue 380 in the active site loops of Streptomyces PLD.

Discovery, characterization and engineering of ligases for amide synthesis.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

An Effective Strategy for the Production of Lauric Acid-Enriched Monoacylglycerol via Enzymatic Glycerolysis from Black Soldier Fly (Hermetia illucens) Larvae (BSFL) Oil.

Here, we developed an efficient strategy for the production of lauric acid-enriched monoacylglycerol (MAG) via enzymatic glycerolysis using black soldier fly (Hermetia illucens) larvae (BSFL) oil. The effects of the substrate molar ratio, reaction temperature, type of immobilized lipase, and organic solvent on the MAG content and conversion degree of BSFL oil were optimized. The maximum substrate conversion rate (97.88%) and MAG content (70.84%) were obtained in a tert-butanol system at 50 °C with a glycerol/BSFL oil molar ratio of 4:1 by using immobilized MAS1 lipase as a catalyst. The MAG content in the purified product reached 97.7%, with lauric acid accounting for 50.2%. Improved oxidation stability was observed after glycerolysis. Overall, this study provides a new strategy for the preparation of lauric acid-enriched MAG from BSFL oil.

Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket.

Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure-function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between ß9 and α9 (aa228-246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272-275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between ß11 and ß12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.

A novel numerical model for protein sequences analysis based on spherical coordinates and multiple physicochemical properties of amino acids.

How to characterize short protein sequences to make an effective connection to their functions is an unsolved problem. Here we propose to map the physicochemical properties of each amino acid onto unit spheres so that each protein sequence can be represented quantitatively. We demonstrate the usefulness of this representation by applying it to the prediction of cell penetrating peptides. We show that its combination with traditional composition features yields the best performance across different datasets, among several methods compared. For the convenience of users, a web server has been established for automatic calculations of the proposed features at http://biophy.dzu.edu.cn/SNumD/.

Exploring the influence of phospholipid monolayer conformation and environmental conditions on the interfacial binding of Gibberella Zeae lipase.

The involvement of different parameters on Gibberella zeae lipase (GZEL) membrane binding were characterized by using monomolecular film technology and circular dichroism spectroscopy. Among four kinds of phospholipid monolayers, 1,2­dimyristoyl­sn­glycero­3­phosphoethanolamine have the highest maximum insertion pressure (MIP) value. Comparing the GZEL adsorption to phosphatidylcholine monolayers with different acyl chains in sn-1 and sn-2 positions, the higher MIP values were found for 1,2­dilauroyl­sn­glycero­3­phosphocholine. Significantly improvement between 1,2­dioleoyl­sn­glycero­3­phosphocholine and 1,2­distearoyl­sn­glycero­3­phosphocholine suggested that the presence of fatty acid unsaturation may affect protein adsorption by changing the chemical structure in each phospholipid. The MIP value was shown higher (48.6 mN m-1) at pH 5 and pH 6 (47.5 ±â€¯1.9 mN m-1) but decreased significantly (34.2 mN m-1) at pH 9. This may indicate that the proportion of helices in the protein decreases with the alteration of the catalytic center, thus affecting the binding of the protein to its substrate. The MIP values obviously decreased with increasing salt ion concentration, suggesting that excessive salt ion concentration may destabilize the secondary and tertiary structures of the protein, thereby affecting the characteristics of its adsorption at the interfaces. Present studies improve our understanding on the protein-membrane interaction of this enzyme.

Deletion the C-terminal peptides of Vibrio harveyi phospholipase D significantly improved its enzymatic properties.

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.

Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus.

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.

Function of C-terminal peptides on enzymatic and interfacial adsorption properties of lipase from Gibberella zeae.

BACKGROUND: The crystal structure of lipase from Gibberella zeae (GZEL) indicates that its C-terminal extension is composed of a loop and a α-helix. This structure is unique, possibly providing novel evidence on lipase mechanisms. METHODS: Two C-terminally truncated mutants (GZEL-Δ(α-helix) and GZEL-Δ(α-helix+loop)) were constructed. The role of these secondary structure segments on enzymatic activities and interfacial binding properties of GZEL was investigated by using conventional pH-stat method and monomolecular film techniques. In addition, inactive variants (Ser144Ala) of wild-type GZEL and two truncated mutants were constructed and produced specifically for interfacial binding experiments. RESULTS: Compared to the wild-type GZEL, lipase and phospholipase activities were significantly decreased in the two mutants. Deletion of the α-helix had great influence on the lipase activity of GZEL, resulting in residual 7.3% activity; the additional deletion of the loop led to 8.1% lipase activity. As for the phospholipase function, residual activities of 63.0% and 35.4% were maintained for GZEL-Δ(α-helix) and GZEL-Δ(α-helix+loop), respectively. Findings obtained with monomolecular film experiments further indicated that the reduction in phospholipase activity occurred with the anionic phospholipid as substrate, but was not seen with zwitterionic phospholipid. Results of the maximum insertion pressure, synergy factor and binding kinetic parameters documented that the α-helix structure of GZEL strongly influence the binding and insertion of enzyme to the phospholipid monolayer. Moreover, the interfacial binding function of α-helix was partly conformed by connecting to the C-terminal of Aspergillus oryzae lipase. GENERAL SIGNIFICANCE: Our results provide important information on the understanding of the structure-function relationship of GZEL.

Recombinant Lipase from Gibberella zeae Exhibits Broad Substrate Specificity: A Comparative Study on Emulsified and Monomolecular Substrate.

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.