Synergism Of Oleate Hydratase With Yarrowia Lipolyticafor Conversion Of Oleic Acidto ?-lactone

With the development of the perfume industry,the demand for natural flavors is increasing.?-decalactone?GDL?is an aromatic compound naturally found in some fruits and fermented products.It plays an irreplaceable role in the flavor production and pharmaceuticals.However,chemically synthesized GDL is an optically active racemate and is not stable and safe.On the other hand,the GDL obtained by biological methods is optically active,and more natural,similar to that obtained from fruits and plants.Therefore,the biological production of GDL has a broader development prospect.In this study,the oleate hydratase gene derived from Lactococcus garvieae was expressed in Escherichia coli BL21.The induction conditions of the recombinant bacterialstrain and the enzymatic properties of the recombinant enzyme were studied.Further,the oleate hydratase and Yarrowia lipolytica were combined to convert oleic acid to?-decalactone and investigate the effect of medium condition optimization on GDL yield.The specific content has the following aspects:?1?The oleate hydratase gene derived from L.garvieae was cloned,ligated to the pET28a vector,and transformed into E.coli DH5?.The sequence analysis confirmed the correct gene sequence with a total number of 1764 bp.The recombinant oleate hydratase from L.garvieaewas successfully induced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?analysis,and its theoretical protein molecular weight was approximated to be 67.2 kDa.The recombinant strain ZJLgohA was induced to culture in Lauria-bertani?LB?medium supplemented with 4 mg/mL arabinose,1.0 mM IPTG was added,and induced at37°C for 8 h,the maximum protein content was 1.2 mg/mL.?2?The bacterial liquid was concentrated 10 times for ultra-sonication to release the recombinase.The ultrasonic working conditions were:ultrasonic power17%,ultrasonic 1 s pause 4s,and working time of 40 min.The purified protein was isolated by C-terminal 6×His tag and eluted with 50 mM imidazole to obtain pure enzyme.After SDS-PAGE electrophoresis analysis,a pure single band was obtained with a protein concentration of 2.5 mg/mL.The enzyme purification titer was 2.05 times,and the enzyme activity was 152 U/mL.The Michaelis constant Km and Vmax values were recorded to be 0.69 g/L and 0.96×10^-33 g/L/min,respectively.The enzyme was preserved in 10%glycerol and stored at-20°C for40 days;the residual enzyme activity was about 86%.Mg²⁺has a good effect on the oleate hydratase activity,which can increase the enzyme activity by 3.5times.The optimal reaction conditions for the purified enzyme were as follows:in the pH 7.5 citric acid-sodium citrate buffer system,the ethanol concentration was4%,the substrate oleic acid concentration was 30 g/L,and the reaction was carried out at 30°C for 8 h to convert to 10-hydroxystearic acid was 2.3 g/L,conversion oleic acid to 10-hydroxystearic acid was 7.2%.?3?Oleate hydratase synergizes with Yarrowia lipolytica to convert oleic acid to?-decalactone.Oleate hydratase and oleic acid were intermittently added in the fermentation 24 h and 36 h of Y.lipolytica.At this time,the GDL yield was the highest,and the GDL yield reached a maximum of 3.7 g/L.The single factor test and the response surface test were conducted to study the addition amount of Tween-80,soybean powder,and ammonium nitrate,and ferrous sulfate.Under the optimum medium conditions of Tween-80 0.23%,soy flour 3.46%,nitric acid,mmonium 1.35%,FeSO₄ 0.18%,the?-decalactone yield 4.72 g/L,and oleic acid conversion to GDL was 26.15%.