Heterologous Expression,Fermentation Optimization,and Immobilization Of A Thermophilic Sarcosine Oxidase From Archaea

Sarcosine oxidase（SOX）can catalyze the N-methyl of sarcosine and produce formaldehyde,hydrogen peroxide and glycine.It has been applied in pharmaceutical and food industries and has great potential economic value.Due to the relatively poor thermostability and low expression,the large-scale application of this type of enzyme is limited.The predictive SOX of Archaea HR32 was selected for this study,the main research work involves the heterologous expression and purification of r SOX,characterization of enzymatic properties,fermentation optimization and immobilization of r SOX.The results are as follows:（1）By changing different expression systems,r SOX was expressed in Bacillus subtilis WB600 in soluble form for the first time.Then r SOX was purified by heat treatment and nickel-column chromatography and further characterized of the enzymatic properties.The purified r SOX was identified with a size of approx.42 k Da and the secondary structure of it was observed to be composed of 35.0%α-helix,11.1%β-sheet,23.3%β-turn,and 31.6%random coil.The melting temperature（T_m）and the denaturation enthalpy（ΔH）were verified as 92.13℃and 1070 k J/mol,respectively.When having sarcosine as the substrate,the optimal p H and temperature were characterized as 8.0 and 70℃.With the half-time of 12 h at 80℃,the K_m,k_catand k_cat/K_m of r SOX were measured as 1.17 mmol·L^-1,74.59 min^-1 and 63.78 L·（mmol·min）^-1,respectively.r SOX was also observed to exhibit resistance to normal organic solvents,whilst metal ions were found to have either promotion or inhibition effect on the enzymatic activity.In addition,the current research verified the catalytic degradation capability of r SOX to different types of N-methyl compounds,in particular the chiral selectivity against the L-type substrates.（2）After single factor optimization and orthogonal experiments,the optimal fermentation medium was determimrd.By optimizing the fermentation conditions in shaking flask,the maximal enzyme activity(17.2 U·L^-1)was obtained at 37℃,200 r·min^-1,initial medium p H7.5,liquid loading 35 m L,inoculation amount 10%,xylose addition 1.5%at the sixth hour,fermentation for 24 h.And the enzyme activity after optimization was 5.9 times higher than that before.After heat treatment,the total enzyme activity of crude enzyme increased by 96%.14.7 mg pure enzyme could be obtained from 1 L fermentation broth,and the specific enzyme activity was 2.3 U·mg^-1.（3）To further improve the expression of r SOX,the fermentation process and feeding conditions were optimized in 5 L fermentor.The results showed that,when dissolved oxygen was 30%,p H was controlled at 7.0 in the first 10h then controlled at 8.0 in the later stage,glucose was added at flow Iing rate of 3.5 g·（L·h）^-1 from 6h to 26h,the enzyme activity reached93.9 U·L^-1,which was 5.46 times of the shaking flask.After heat treatment,the enzyme activity reached 184.0 U·L^-1,which was 63 times of that before optimization.After purification of 1 L fermentation broth,80.2 mg pure enzyme was obtained,the expression was greatly improved.（4）The immobilization methods were screened to determine that the optimal immobilization carrier was magnetic chitosan microspheres.After optimizing the preparation and immobilization conditions,the maximum enzyme loading was 40 mg·g^-1 and the maximum enzyme activity retention was 44.3%.The enzymatic properties of immobilized enzyme showed that the optimal reaction temperature was 10℃lower than that of free enzyme,and the thermal stability was also reduced.The optimal p H shifted to alkaline,and the p H adaptability was broader than that of free enzyme.After immobilization,the affinity to sarcosine decreased,and about 85%of the enzyme activity can be retained after repeated use for 15 times,indicating good operational stability.