| Prolyl aminopeptidase(PAP;E.C.3.4.11.5)is an exopeptidase which catalyzes the hydrolysis of the N-terminus proline residue of proteins or peptides.PAP has an important application in protein hydrolysates debittering of food processing industry and the preparation of bioactive peptides,and it also has great application prospect in the fields of biomedicine and detection.In this study,the cDNA of PAP from A.oryzae was as a template,then constructed a Bacillus subtilis strain that expressed PAP with his tag successfully.The medium was optimized by single factor and orthogonal experiment,and the flask culture medium consists of(per liter)20 g glucose,60 g yeast extract,18.75 g peptone form fish,6.25 g NH4Cl,12.54 g K2HPO4,2.31 g KH2PO4.The PAP activity reached 103.1 U·mL-1 under the optimized medium.0.1%(v/v)phytic acid was added into optimized medium,the PAP activity was raised to 111.1 U·mL-1.The optimized shake-flashing culture conditions were initial pH 7.0,4%of inoculation amount,50 mL of loading volume,culture temperature at 33 ℃,shaker speed at 220 r·min-1 and culture for 24 h.Moreover,0.5 mmol·L-1 SDS and 0.2%of Triton x-100 were added into optimized medium to improve the yield of extracellular PAP,the PAP activity in the fermentation broth increased from 8.4 U·mL-1 to 31.3 U·mL-1,and the total PAP activity was 116.8 U·mL-1,basically consistent with the previous.Batch fermentation conditions(5 L fermentor),including the speed of agitator,pH and temperature were systematically optimized based on analysis of fermentation kinetics.Finally,the speed of agitator was controlled at 200 rpm in 0～6 h,400 rpm in 6～12 h,500 rpm in 12～28 h,and 400 rpm after 28 h,respectively.The pH was not adjusted in the first 12 h and during 16～28 h,and fixed at 7.0 in 12～16 h and after 28 h.The fermentation temperature was controlled at 40℃ in the first 8 h,then at 35 ℃ in 8～12 h,and at 33 ℃ after 12 h.The yield of PAP was reached 174.8 U·mL-1(included the 52.6 U·mL-1 of extracellular PAP activity)under the optimized conditions.In addition,glucose was fed at 12 h in the fermentation process,4 h as a cycle of substrate feeding and repeated 8 times,maintained the reducing sugar concentration at 15 g·L-1.In this setting,the total PAP activity was raised to 227.1 U·mL-1,included the 73.6 U·mL-1 of extracellular PAP activity.The extracellular PAP was purified by 40%～50%ammonium sulfate fractionation and Ni2+ affinity chromatography,the specific activity of the purified PAP was 115.8 U·mg-1.The purification factor and recovery were 3.6,38.0%,respectively.The recombinant B.subtilis was disrupted by lysozyme and repeated freezing and thawing,the bacterial lysate was purified by Ni2+ affinity chromatography.The specific activity of the purified PAP was 135.3 U·mg-1.The purification factor and recovery were 3.9,27.2%,respectively.Both of the purified PAP reached electrophoretically pure that were identified by SDS-PAGE,and the enzymatic properties were basically consistent with the previous that without his-tag.The stability of PAP during the freeze-drying process was increased significantly and its activity holds steady when 3%sucrose,3%mannitol and 1%glycine were added as protectants.The retention rate of enzyme activity of the lyophilized PAP with protectants remained above 90%when it was stored at-20 ℃ for six months,and the storage stability was increased significantly.PAP cooperated with alkaline protease and leucine aminopeptidase was employed to hydrolyze rice protein.The free amino acids including hydrophobic amino acids in the hydrolysate were increased significantly,which contribute to the reducing of bitter taste.Hydrophilic peptides content increased in hydrolysate and the main distribution of the peptides molecular weight was below 1 kDa,and the proportion of small peptides that below 180 Da were 44.7%. |
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