Construction Of Pickering Emulsion Biphasic Catalysis System Based On Lipase/Chitosan Polyelectrolyte Complex And Its Mechanism

Lipases possess multiple catalytic activities.They are highly efficient and environmentally friendly and have been widely used in multiple fields.Lipases are water-soluble,while their substrate are usually water insoluble.Hence,measures to improve the efficiency of lipases in biphasic systems deserve great interests.Lipases exhibit the unique interfacial activation property;hence,the increase of oil/water interfacial area is an effective strategy to increase their catalytic activities.Pickering emulsion stabilized by lipase-loaded colloidal particles is becoming an innovative platform for biphasic biocatalysis in recent years.In this system,each emulsion droplet can act as a microreactor,which leads to the outstanding advantages of tremendous oil/water interfacial area,sufficient lipase-substrate contact,high catalytic efficiency,easy lipase recovery,environmentally friendly,and simple product purification.Pickering emulsions stabilized by lipase-loaded inorganic particles have been successfully used in the construction of Pickering emulsion-based biphasic catalytic systems,but inorganic particles could cause safety concerns.The protein-polysaccharide polyelectrolyte complexes have the obvious advantages of excellent safety,easy preparation,and good environmental response.The complexes have been proposed as promising Pickering emulsion stabilizers and hence are potential in the construction of Pickering emulsion-based biphasic catalytic systems.Lipases are ampholyte and often negatively charged in catalysis,while chitosan is the only positively charged polysaccharide in nature,making the latter a potential lipase carrier.In this work,the lipase from Aspergillus oryzae（AOL）was selected and used to prepare polyelectrolyte complex with chitosan（CS）through complex coacervation.The resultant AOL-CS complex was then used to construct a Pickering emulsion-based biphasic catalysis system.In this case,the catalytic performance of AOL was evaluated and the assembly of the complex in the oil/water interface was explored.Meanwhile,the variation of emulsion property during hydrolysis was monitored and the application of the system in plant oil hydrolysis was investigated.Finally,the interaction between AOL and CS was simulated through molecular dynamic analysis.The main conclusions of the work are as follows:（1）The optimum conditions for the interaction between AOL and CS as well as the property variation of the coacervated AOL were elucidated.The results indicated that the optimum coacervation conditions were pH 5.5,AOL to CS mass ratio 5:1,and temperature 25℃ in the absence of Na Cl,which conferred an AOL loading efficiency of up to 95.48%and activity recovery of 69.60%.The formed polyelectrolyte complexes were highly porous in structure.The coacervation with CS increased the activity of AOL and decreased its optimal pH from 7.0 to 6.0,but exerted no effect on its optimum temperature（45℃）.Thermal deactivation kinetics analysis revealed that the coacervated AOL was more thermal stable,while the Michaelis-Menten kinetics analysis indicated that coacervation with CS increased the V_max of AOL by 2.4 folds,but decreased its substrate affinity by 3.6 folds.Hence,the coacervation with CS was a feasible way to fabricate AOL-loaded particles and the resultant complexes are potential in the construction of Pickering emulsion-based lipase catalysis systems.（2）The interaction pattern between AOL and CS was unveiled and the resultant complexes were characterized.QCM-D analysis revealed that the association in pH 5.5generated a viscoelastic hydrated adlayer with the largest thickness.ITC analysis indicated that the interaction between AOL and CS was driven by enthalpy and the highest one-site binding stoichiometry was recorded in pH 5.5,which was 1.3 and 7.9times higher than in pHs 5.0 and 6.0,respectively.Both electrostatic attraction and hydrophobic interaction played dominant role in the formation of the AOL-CS complexes.Compared with free AOL,the interaction with CS reduced itsα-helix content,increased itsβ-sheet content,and changed its tertiary structure;besides,the AOL-CS polyelectrolyte complexes assembled in pH 5.5 exhibited the highest contact angle up to 85.5°.Hence,the AOL-CS polyelectrolyte complexes could be used to construct novel biphasic catalysis system based on O/W Pickering emulsions.（3）The ability of the AOL-CS complex to stabilized Pickering emulsions and their adsorption behavior in the oil/water interface were explored and the stability of the resultant emulsions were evaluated.The results indicated that AOL-CS polyelectrolyte complexes was an effective Pickering stabilizer.When the complex was assembled in mass ratio 5:1,up to 90%AOL could be retained in the emulsion interface.QCM-D analysis suggested that the complex spontaneously absorbed to the oil-water interface;interfacial adsorption dynamics revealed that the adsorption was driven by diffusion accompanied by rapid structural rearrangement;interfacial dilatational rheology demonstrated the formation of an elastic film in oil-water interface.The Pickering emulsions were pseudoplastic and that in oil fraction 0.6 exhibited the elastic behavior in contrast to the viscous behavior in oil fractions 0.2 and 0.4.The Pickering emulsion exhibited excellent stability against storage for up to 28 d,pHs 2.0-12.0,heating at 25-90℃,and up to 500 mmol/L Na Cl,and corresponding interfacial AOL retentions exceeded 80%during exposure to these conditions.Hence,AOL-CS polyelectrolyte complexes could be used as a stabilizer to construct Pickering emulsion-based biphasic catalysis systems.（4）The conditions of the Pickering emulsion-based catalytic system were optimized using p-nitrophenol palmitate（p-NPP）as the model substrate.The results indicated that the optimum conditions were pH 6.0,temperature 45℃,water phase fraction 0.6 and p-NPP concentration 3mmol/L.Under such conditions,the conversion rate of p-NPP reached 80.25%in reaction time 300 min,which was higher than 62.10%of the traditional biphasic system.As the hydrolysis proceeded,the average particle size of emulsion droplets increased and the zeta potential decreased,while the endogenous fluorescence of AOL and the surface hydrophobicity of emulsion droplets increased,indicating that the hydrophobic amino acid residues of AOL got more exposed.Water distribution and rheological measurements revealed that the emulsion viscoelasticity declined,fluidity increased,and stability decreased with hydrolysis proceeding.The AOL-CS complex exhibited good reusability and more than 80%activity was maintained after 6 cycles.（5）The application of the Pickering emulsion-based catalytic system in the hydrolysis of plant oils were investigated.Pickering emulsions were prepared with plant oils consisting of C₁₂-C₁₈chains and their hydrolysis was monitored.The results indicated that the oil containing C18 chains was more susceptible to hydrolysis.When the reaction time was set to 360 min,the hydrolysis rates of coconut oil,palm oil,flaxseed oil,soybean oil and olive oil in the traditional biphasic system were 40.34%,42.45%,44.13%,48.89%,and 53.89%,respectively,while the Pickering emulsion system demonstrated better performance and the hydrolysis rates were 62.63%,70.19%,77.03%,82.10%,and 85.89%,respectively.After hydrolysis,the AOL-CS complexes could be recovered by centrifugation.After 6 cycles,the Pickering emulsion maintained78.27%,80.34%,80.17%,82.19%,and 88.90%activity towards the hydrolysis of coconut oil,palm oil,flaxseed oil,soybean oil,and olive oil still reached of the initial activity,respectively.（6）The molecular dynamic for the interaction between AOL and CS was simulated.The results indicated that AOL and CS could form stable polyelectrolyte complexes and their minimum binding energy was-6.2 kcal/mol.The AOL-CS complex had higher R_g value,indicating good folding stability and relatively low density.The surface area slightly increased with minor fluctuations,but remained constant during simulation,indicating the stable conformation during the simulation process.Compared with free AOL,the interaction markedly changed the secondary structure contents.Besides,the interaction between AOL and CS mainly relied on van der Waals forces and electrostatic interaction.Most amino acids could interact with CS through van der Waals forces,of which Trp:97,Gly:119,Phe:120 and other amino acids had weaker binding forces with CS,while Thr:69 and Thr:102 had weaker electrostatic forces with CS.In conclusion,the Pickering emulsion stabilized by the AOL-CS polyelectrolyte complex can be used as a novel system for the catalytic reaction of lipases.This work is of great significance to promote the application of lipase in the food industry and expand the application of food-grade Pickering emulsions.