Heterologous Expression And Preparation Of Calcium-independent Phospholipase A₁

Phospholipase A₁（EC 3.1.1.32,PLA₁）is a hydrolysis enzyme which specific hydrolysis the sn-1 easter bond of phospholipid.PLA₁ has a wide application in food industry,including vegetable oil degumming,diary,baking,phospholipid modification,etc.At present,it has been found that a variety of microorganisms can secrete PLA₁.However,most of them are pathogenic bacteria such as Serratia.Using these pathogenic wild bacteria to ferment and produce PLA₁ not only has many impurities in the fermentation broth but also has potential food safety risk.Thus,relevant studies mostly prepare PLA₁ by constructing genetically engineered bacteria for heterologous expression.However,due to the toxicity of PLA₁ to host cells,the reported heterologous expression level of PLA₁ is generally low.In addition,most of the reported PLA₁ need calcium ion to activate their activity.The catalytic activity of these PLA₁ will be significantly reduced with chelating agent existing.Thus,these shortcomings will not only decrease the enzymatic hydrolysis efficiency but also limit the application fields of PLA₁.Therefore,it is necessary to study the heterologous expression and preparation of calcium ion-independent PLA₁.In this study,calcium ion-independent phospholipase A₁（saPLA₁）from Streptomyces albidoflavus was selected.Firstly,by studying the recombinant expression of saPLA₁ in E.coli and the renaturation of inclusion bodies,an efficient method for the preparation of saPLA₁ was developed;Then,an affinity ligand that can specifically bind to the catalytic pocket of saPLA₁ was designed and synthesized,and saPLA₁ with high purity was obtained by one-step purification;Next,the hybrid nanoflowers immobilization of saPLA₁ and the modification of saPLA₁ based on the fusion of functional short peptide were studied.Finally,the composite enzyme composed of saPLA₁ and phospholipase C was applied in oil degumming,trying to develop a kind of PLA₁ which is suitable for industrial application.The main results are as follows:（1）The inclusion body expression was applied as the best preparation method of saPLA₁in E.coli,and the enzymatic properties of the recombinant enzyme were studied.The gene of saPLA₁ was expressed in the intracellular and periplasmic space of E.coli BL21（DE3）by expression vector p ET-28a（+）and p ET-22b（+）,respectively.The enzyme activity of 1.1 U·m L^-1 was obtained from the cell disruption supernatant by intracellular soluble expression of saPLA₁.The recombinant saPLA₁ was secreted to extracellular medium during the periplasmic expression and the enzyme activity of 0.2U·m L^-1 was obtained.This might be due to the damage of saPLA₁ to the cell membrane,resulting in the leakage of recombinant enzyme to the outside of the cell.Cells of 1 m L fermentation broth yielded0.3 mg of inclusion body protein,which represented 39%of the total cellular protein.After refolding,the enzyme activity of 12 U·m L^-1 was obtained.The specific enzyme activity of saPLA₁was 1380U?mg^-1.These results indicated that the expression of saPLA₁ by inclusion body could obtain a high amount of recombinant enzyme and avoid the cell toxicity of saPLA₁.At last,the study of enzymatic properties of saPLA₁showed that the optimal p H and temperature of the enzymatic reaction was p H6.5 and 60℃,respectively,and the recombinant saPLA₁ was indeed a calcium independent enzyme.Moreover,substrate spectrum analysis showed that the recombinant enzyme preferred to hydrolysis phosphatidylinositol,phosphatidylglycerol and phosphatidylserine containing hydrophilic groups,preferred to hydrolyze phospholipids containing saturated fatty acid chains of hexadecanoic acid.Surprisingly,the enzyme was discovered that had lipase activity at low temperature.（2）The optimal conditions of dilution renaturation were determined,and the continuous dilution renaturation strategy was used to realize the efficient preparation of saPLA₁.At first,the optimal conditions of dilution refolding were tested and the results are as follows:p H 8.0,GSSG and GSH concentrations of 0.2 mmol?L^-1 and 0.8 mmol?L^-1,urea concentration of 1.6 mol?L^-1,protein concentration of 75μg?m L^-1.Under the optimal renaturation conditions,the enzyme activity increased faster in the first 4 h of renaturation and the highest enzyme activity was obtained by 16 h renaturation,which was 29.6 U?m L^-1 and 2.4 times higher than that before optimization.The refolding yield was obtained at 28.6%.Then,the effect of protein amount and supplementation times on continuous dilutions refolding was investigated.It was indicated that the optimal strategy was to add 75μg?m L^-1 denatured protein to the refolding solution every 4 h for five consecutive additions.By using the optimal strategy,enzyme activity of the refolding solution was achieved at 155 U?m L^-1,which was5.2 times higher than that of direct dilution refolding and 1.7 times higher than the enzyme activity expressed in Streptomyces lividans.（3）Based on molecular docking analysis,an affinity ligand that can specifically bind to saPLA₁substrate catalytic pocket was designed,and high-purity recombinant enzyme was obtained by one-step purification.Firstly,the ligand was synthesized and composed to Sepharose CL-4B through epichlorohydrin by a four-step reaction.The density of the ligand on Sepharose beads was 22.5μmol?g^-1 wet gel.Secondly,adsorption analysis indicated the maximum adsorption(Q_max)and the desorption constant（K_d）of the sorbent was 10.7 mg?g^-1 and 426.6μg?m L^-1.Finally,saPLA₁ with purity of 96.6%was obtained by linear elution with 100 mmol?L^-1 acetic acid.The purification multiple was 7.6 times and the recovery was 52.5%.（4）The optimal conditions for the preparation of hybrid nanoflower immobilized enzyme by recombinant enzyme saPLA₁ with Mn²⁺,Cu²⁺,Al³⁺and Co²⁺were determined.The changes of stability,substrate specificity and organic solvent tolerance of the recombinant enzyme before and after immobilization were compared.Firstly,under the optimal preparation conditions,the encapsulation yield（EY）and specific enzyme activity of immobilized enzyme saPLA₁-Mn was 72%and 1.23 U·mg^-1.The EY and specific enzyme activity of immobilized enzyme saPLA₁-Cu was 69%and 2.59 U·mg^-1.The EY and specific enzyme activity of immobilized enzyme saPLA₁-Al was 82%and 1.23 U·mg^-1.The EY and specific enzyme activity of immobilized enzyme saPLA₁-Co was 100%and 4.8 U·mg^-1.Then,the differential scanning calorimeter（DSC）test showed that the Tm values of the immobilized enzyme were higher than the free enzyme.The thermal stability of the immobilized enzyme formed with three metal ions(Cu²⁺,Al³⁺,and Co²⁺)was obviously improved,and about 70%of the enzyme activity remained after incubation at 50°C for 30 min.The immobilized enzyme also showed significantly better storage stability than the free enzyme that more than 60%enzyme remained after 10 days storage at 4°C.At last,the tolerance of saPLA₁ to organic solvents test indicated that the tolerance ability of the immobilized enzyme to dimethyl sulfoxide,methanol,ethanol,acetone,isopropanol,dioxane,n-butanol and n-hexane was significantly improved.（5）Based on the fusion of amphiphilic peptide NKC and coiled coil peptide MAT,the affinity of saPLA₁ to substrate was improved.The reaction kinetic parameters of saPLA₁ and its fusion enzyme suggested that NKC-saPLA₁-MAT displayed the highest k_cat/K_m and the lowest K_m,which indicated the catalytic efficiency and substrate affinity of NKC-saPLA₁-MAT were better than others.Then,according to the results of previous degumming experiments,saPLA₁,NKC-saPLA₁-MAT and phospholipase A₁ from Serratia liquefaciens（sl PLA₁）were compounded with phospholipase C（PLC）,respectively.Next,the determination of the optimum degumming conditions of the three composite enzymes showed that the optimum reaction temperature was 50℃,the optimum reaction time was 3 h.Moreover,the optimum degumming p H of the composite enzyme composed of saPLA₁and NKC-saPLA₁-MAT with PLC was 6.5,and that of the composite degumming enzyme composed of saPLA₁ and PLC was 7.0.Finally,the enzyme addition of saPLA₁ and NKC-saPLA₁-MAT in degumming application was only 2070 U·kg^-1,much lower than sl PLA₁(13240 U·kg^-1).Moreover,under the same conditions,the results of degumming using the fusion enzyme was better than that of the original enzyme.