Optimization Of Nattokinase Fermentation And The Study On Purification Of P450_BM-3 By Chromatography

Nattokinase(NK), which was found to be a strong fibrinolytic enzyme, was extracted from the Japanese food natto. 1 g.L-1 xylose added in seed medium can not only shortened the lag phase,accelerated the Bacillus subtilis HL-1 cell growth, but Nattokinase(NK) production was improved from 1314 IU.ml-1 to 1698 IU.mL-1 in 50/250mL shake flask. The fermentation of NK was scaled up in 3.7L KLF Bioengineering fermentator.The results indicated that dissolved oxygen and Bacillus subtilis density were very important parameters affecting the NK production. By adding 1 g.L-1xylose in seed medium,keeping dissolved oxygen between 30% and 40%, and fed-batch culture in the Bacillus subtilis density(OD66o) decreasing, Nattokinase(NK) production was increased from 1314 IU-mL-1 in shake flask without xylose added to 2348 IU-mL-1 in Fermentator.P450BM-3 (CYP102) isolated from Bacillus megaterium is a self-sufficient fattery acid monooxygenase,it catalyzes the ω-2 hydroxylation of long chain saturated fatty acids (C12-C120) and the hydroxylation and epoxidation of unsaturated fatty acids, its gene was cloned and expressed in E.coli. In this paper, the plasmid coding the gene of P450BM-3 was successfully transformed into E.coli DH5a, the culture condition and expression condition of the transformant was optimized and at last isolation and purification of P450BM-3 was finished.Firstly, the plasmid coding the gene of P450BM-3 was successfully transformed into E.coli DH5alpha.Ampicilin concentration and inoculum quantity affect host cell growth was investigated.The results showed that the inhibition of Ampicilin on the cell growth was strong and the lag phase in the cell growth course was prolonged with the decrease of inoculum volume. Then, the optimum culture condition of P450BM-3 was studied, the results showed that with inoculum quantity as 1% of the culture medium ,dissolved oxygen 20%,adding O.lmg/L FeCl3,inducing 5 hour in 42癈 when its OD578 reached 0.5-1.3, higher expression of P450BM-3 could be gained.Effective purification procedures of P450BM-3 were studyed by (NH4)2SO4 fraction precipitation and gel filtration chromatography.The optimal saturated concentration of (NH4)2SO4 was 35%-70%, the optimal conditions of gel filtrationchromatography were that the velocity 0.5ml/minl.After sequential use of ammonium sulfate fractionation precipitation, sephacryl S-200, the result shows the enzyme activity recovery is 49.8%, purification folds is 2.316.The results that anion exchange meida DEAE-Sepharose FF^ Resource Q and ANX purify P450BM-3 indicate anion exchange media could adsorb P450BM-3,but the purification folds are different, DEAE-Sepharose FF purification effectiveness is the lowest in three meida. After sequential use of Resource Q, sephacryl S-200, the result shows the activity recovery is 18.03%, purification folds is 3.4.The separation and purification of P450BM-3 was executed by hydrophobic interaction chromatography.By comparing the adsorption performance of several hydrophobic medium,we choice the Source 15ISO for the purification of P450BM-3-The partial optimal conditions of P450BM-3 purification by HIC on AKTA explorer system were loading buffer solutinrl.OM (NH4)2SO4, 0.1 mM Na2EDTA 0.1mM PMSF,50mM,pH 7.5-8.0 PBS,linear flow rate for buffer solutin:0.5~1.0mL/min,the gradient length :4~5CV.At last ,the purified P450BM-3 is obtained by sequential use of ammonium sulfate fractionation precipitation, source 15ISO hydrophobic interaction chromatograph and sephacryl S-200 size exclusion chromatograph.The result shows that the higher purified P450BM-5 is attained by fewer steps with 13.5% recovery,13.7 purification folds.