| Transglutaminase?EC 2.3.2.13,TGase?is a kind of acyltransferase,which can catalyze the cross-linking reaction between protein molecules,and has a wide application prospect in food,pharmaceutical,textile and other fields.In the previous work of our lab,a recombinant strain,Yarrowia lipolytica po1h/hpro-TGase,was constructed to express pro-TGase of Streptomyces mobaraensis.In order to improve the production efficiency of TGase,Y.lipolytica po1h/hpro-TGase was selected as the object in this paper.The fermentation conditions of Y.lipolytica po1h/hpro-TGase in 5 L tank were optimized,and the production strain of TGase activated protease?TAMEP?was constructed and immobilized to realize the efficient fermentation and activation of TGase.The main results are as follows:?1?Fermentation optimization of Y.lipolytica po1h/hpro-TGaseIn a 5 L fermentor,the effects of stirring speed,p H control and glycerol supplement on the fermentation of Y.lipolytica po1h/hpro TGase were investigated.The results showed that low stirring speed?600 rpm and 700 rpm?achieved higher the expression of pro-TGase than high speed?800 rpm and 900 rpm?did after fermentation after 80 hours fermentation.Then,the production of pro-TGase reached 6.8 U·mL-1 at 600 rpm after 120 hours fermentation,45%higher than that at 900 rpm.After depletion of glycerol,the pH of recombinant bacteria increased and pro TGase began to express.Therefore,the effect of pH 5?8 on pro-TGase fermentation was investigated.The results showed that the yield of pro-TGase reached 17.4 U·m L-11 when pH was kept at 7,which was 156%higher than that without pH control.To increase the concentration of cell,10-40 g·L-1glycerol was supplemented after the glycerol in the medium was exhausted.The results showed that the cell concentrations of 10,20,30 and 40 g·L-1 glycerol were increased by 35%,83%,107%and 142%,resulting in the production of pro-TGase was decreased by 17%,34%,25%and 32%,respectively.Finally,the batch fermentation conditions of 5 L fermentor were determined as follows:the stirring speed was 600 rpm;the pH 7 was controlled at the pH rising stage;the fermentation time was 120 h.?2?Expression of TAMEP in E.coli BL21?DE3?and analysis of its enzymatic propertiesIn order to prepare pro-TGase activation protease,TAMEP from S.mobaraensis was secreted and expressed in E.coli BL21?DE3?.The optimization of signal peptide showed that extracellular TAMEP mediated by the natural signal peptide of TAMEP was 92.6%higher than that done by pelB signal peptide.A random synonymous mutation within first 10 residues of the N-terminus of TAMEP signal peptide were conducted,screening a mutant with a 30%increase in TAMEP production.Through single factor optimization,the conditions for efficient secretion of TAMEP were determined as follows:induction temperature,IPTG,OD600 at induction initiation,glucose content were set at 20?,10?M,1.5,1.0%,respectively.Under these conditions,the activity of extracellular TAMEP reached 186.3 U·m L-1,which was 5.9 times higher than that before optimization.The specific activity of TAMEP that purified using Nickle affinity column reached437 U·mg-1.The analysis of enzyme properties showed that the optimal reaction temperature and pH of TAMEP were 55?and 7.0,respectively;Around 50%residual activity was obtained after the incubation of the TAMEP at 30-50?for 1 hour,but it was completely inactivated after treating at 60?for 1 hour;TAMEP was more stable in alkaline environment than in acidic environment.These properties are like those of native S.mobaraensis TAMEP.Activation analysis showed that0.133?M TAMEP could transform 6.91?M pro-TGase into active TGase in 10 minutes,and it could not degrade the activated TGase continuously.?3?Immobilization of TAMEP and optimization of catalytic conditions for the activation of pro TGase mediated by the fixed TAMEPTAMEP was immobilized to chitosan microspheres with glutaraldehyde as crosslinking agent.The effects of glutaraldehyde concentration?1-4%?,chitosan activation time?10-240 min?,TAMEP concentration?1.77-14.17?M?and crosslinking time?8-48 h?on immobilization were investigated.The immobilization conditions of TAMEP were as follow:1%?v/v?glutaraldehyde,chitosan activation for 3 h,10.63?M TAMEP,and crosslinking reaction for 8 h.Under this condition,the immobilization rate,enzyme recovery and specific activity of immobilized TAMEP reached 76.0%,36%,42.6 U·g-1,respectively.After incubation for 1 h at 50 and 60?,the residual enzyme activity of immobilized TAMEP increased by 26%and 19.3%in contrast to the free TAMEP,respectively;the immobilized TAMEP also showed 39.7%and 14.7%increases residual enzyme activities,respectively,after the incubation for 1 h at pH 5 and 10.These results show that the immobilized TAMEP has higher thermal stability and pH stability than the free TAMEP.In addition,immobilization shifted the optimal pH of TAMEP from 7 to 9.TGase activity analysis showed that 10 immobilized TAMEP spheres could completely activate 1.40 U of TGase at 37?in 10 minutes,and the enzyme activity remained 75%after 10 times of repeated reaction. |
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