A Thermolabile Phospholipase B From Talaromyces Marneffei GD-0079:Biochemical Characterization And Structure Dynamics Studies

Phospholipase B(EC 3.1.1.5)are a distinctive group of enzymes that catalyzes the hydrolysis of fatty acids esterified at the sn-1 and sn-2 positions forming free fatty acids and lysophospholipids.The structural features and catalytic mechanism showed that these enzyme contain C-terminal hydrophobic sequence with an extracellular domain and a signal peptide for secretion.They are usually glycosylated and termed as ecto-phospholipases.The serine residues in domain II has the phospholipase B activity.The GXSXG consensus sequence discovered in fungal phospholipase B have Arg,Ser,and Asp as catalytic triad.The structural features of Phospholipase B are fewer studied.Thermolabile Phospholipase-B can be employed in the Industry as they offer better environmental and economic benefits through energy savings in biocatalysis.In food Industry they are choice,because they avoid changes in food ingredients.In detergent Industry,such Phospholipase-B can help in the formulation of detergents because their addition allows to work at lower washing temperatures.Despite the enormous advantages of such Thermolabile Phospholipase B in biotechnology Industry,research in this area has not been developed as rapidly as in the case of other thermotolerant Phospholipase B.Keeping in view of these objectives,in the present study we reported a putative phospholipase B(TmPLB1)from Talaromyces marneffei GD-0079 synthesized by genome mining library.The gene(Tm Plb1)was expressed and the TmPLB1 was purified using E.coli shuffle T7 expression system.The putative TmPLB1 was purified by affinity chromatography with a yield of 13.5 %.The TmPLB1 showed optimum activity at 35 °C and p H 7.0.The TmPLB1 showed enzymatic activity using Lecithin(soybean >98% pure),and the hydrolysis of TmPLB1 by 31 P NMR showed phosphatidylcholine(PC)as a major phospholipid along with lyso-phospholipids(1-LPC and 2-LPC)and some minor phospholipids.The molecular modeling studies indicate that its active site pocket contains Ser125,Asp183 and His215 as the catalytic triad.The structure dynamics and simulations results explained that conformational changes associated with different environmental conditions.This is the first report on biochemical characterization and structure dynamics of TmPLB1 enzyme.The present study could be helpful to utilize TmPLB1 in food industry for the determination of food components containing phosphorus.Additionally,such enzyme could also be useful in Industry for the modifications of phospholipids.Researchers are nowadays,focusing on different kind of reaction media for non –aqueous enzymatic reactions since environmental toxicity,flammability and volatility is getting very high.In order to reduce the limitations of Ionic liquids(ILs),deep eutectic solvents were developed.They are mixtures of weak Lewis acids(hydrogen bond donor HBD)and bases Hydrogen bond acceptor HBA)and consists of anionic /cationic species(ammonium or phosphonium salt)and are pure.Choline chloride(Ch Cl)is the most commonly used cationic salt as it is biodegradable,has least cost and 98% purity.Further studies focused on “Deep eutectic solvents” to consider their importance as a solvent in various reactions systems.Deep eutectic solvents were prepared with different hydrogen bond donors with molar ratio at a concentration of 50 wt % and TmPLB1 activity was investigated at an optimum temperature(35oC)using PC(Lecithin >98 %)as substrate.The results with polyols based deep eutectic solvents showed that sorbitol proved to be a better DES that increased the activity to 80 %while ethylene glycol and glycerol showed slightly better activity for instance.TmPLB1 was also incubated at different time intervals in ten deep eutectic solvents used in this study.Higher stability results were also shown by polyols based DES solvents using xylitol,ethylene glycol and glycerol as hydrogen bond donors.Ethylene glycol showed 92 % at 12 hr,glycerol showed 70% at 12 hr and xylitol showed 92 % activity after 24 hr.The structural integrity of TmPLB1 was studied using circular dichroism.The secondary structure element was retained and preserved mostly by polyols.The polyols seems to mainly derive from the increased number of hydroxyl groups in their chemical structures and their synergistic effect.Further,effects of deep eutectic solvents were investigated for the synthesis of Phosphatidylserine(PS)by 31 P NMR.The reaction were terminated after 48 hours and results showed that polyols based deep eutectic solvents provided a better environment for PS synthesis.On the basis of bonding interaction of ligands and binding affinity,the molecular docking simulations results showed that TmPLB1 showed high activity and stability in the order Sorbitol>Acetamide> Sucrose > Malic acid.The overall studies provides an evidence that polyols are better solvents since they provided favorable environment for TmPLB1.The polyols are good hydrogen bond donors combined with choline chloride to increase the activity of enzyme when they are used in bio catalysis or cosolvents for enzyme catalyzed reactions.The reason may be that only integral molecule may diffuse into enzyme central space creating its denaturation by disturbing intramolecular hydrogen bonds.The study also demonstrates that the type of DES,its molar ratio and concentration are important for improving biocatalytic reactions performance.The results showed that better perspectives of the application of these solvents in Food(functional foods)or Biopharmaceutical Industry.This study concluded that TmPLB1 Phospholipase B is stable with conformational changes associated with different environmental conditions.