Optimization Of Fermentation Conditions And Kinetics For Secretory Expression Of Recombinant Levansucrase By Pichia Pastoris

Levansucrase belongs to the glycoside hydrolase family 68 and can catalyze the hydrolysis and polymerization.Levansucrase can synthesize levan by transfructosylation using sucrose as substrate.Levan is widely used in various fields.Enzymatic synthesis of levan has great advantages in preparation of large-scale industrial production.In our previous work a high-level secretory expression system for levansucrase was constructed.In this study,fermentation conditions was optimized.It is significant to lay the foundation for industrial application of enzyme for the production of levan.The research of conplete secretion levansucrase by P.pastoris is very rare in our best know.The recombinant P.pastoris GS115/p PIC9K-lev(α-mf)was constructed in our laboratory.This strain was able to complete secretion and expression the recombinant levansucrase from Bacillus subtilis by shake flask fermentation in BMGY/BMMY.In this work,The conditions for secretory expression of recombinant levansucrase were optimized,and study fermentation kinetics in 5-L automatic fermenter,to provide theoretical guidance for future production research.Firstly,we compared the result of expressing recombinant enzymes in FM22,BSM and BMGY/BMMY,Shake flask fermentation results showed that FM22 was more suitable for high-density expression of levansucrase,and its expression of levansucrase was 69% higher than BSM and the cost is lower than BMGY/BMMY.Next,one factor optimization and high cell density cultivation of fermentor were studied based on FM22.The fermentation conditions were optimized by single factor optimization in shake flask,the expressed enzyme activity induced for 7 d under optimized condition was about 15.23 ± 0.12 U/m L,which was about 45.7% higher than the initial condition.Through the expressive conditions were optimized by response surface experiment,Plackett-Burman test design was carried out for five factors by one factor optimization,screening out three main effect factors for Box-Behnken experiment,the optimal conditions were obtained as initial p H 5.9,0.1%addition of PTM4 and 1.72% addition of induced methanol.Under the optimized conditions,Inducted expression of shake flask fermentation,the highest extracellular enzyme activity of levansucrase was 17.56 ± 0.29 U/m L,which was 68% higher than initial condition and 15.3% higher than one factor optimization.The comparative analysis of SDS-PAGE,intracellular and extracellular enzyme activities showed that the recombinant enzyme could maintain nearly complete secretion and expression at higher level.According to the optimized experimental conditions of shaking flask,amplification to 5-L scale of fermentor to carry out the process study of fed fermentation to express recombinant enzyme.The highest extracellular enzyme activity 93.39 ± 5.75 U/ml,which was 8.9 times higher than shake flask fermentation before optimization,At the same time,almost no enzyme activity was detected in the cell,The results showed that the recombinant P.pastoris could still keep superior character of almost complete secretion of recombinant enzyme during high density fermentation in fermentor.The supernatant of fermentation broth removed the cells by centrifuged was directly detected by SDS-PAGE,and the results showed that the recombinant enzyme in the supernatant was close to the electrophoretic pure,impurity protein content was very low,which was very helpful to simplify the subsequent production and purification process.According to the fermentation process curve in the 5-L fermenter,a variety of equations were used to fit,and the best fitting equation was selected to construct the growth kinetics,product generation and substrate consumption kinetics models,and kinetic model parameters of μm,C0,Cm,a,b,Yx/s,Yp/s and m were determined.The substrate consumption kinetics model of methanol induction stage,R2 is only 0.725,the relative RMSE is between 10% and 20%,because the pulse feeding of methanol has a certain effect on the biological reproducibility,but it still has a good reference significance.Other equation R2 is higher than 0.9,and the relative RMSE was lower than 10%,which indicated that the fitting results were well and had important guiding significance for fermentation control.This paper firstly to studied the high-density fermentation of Pichia pastoris complete secretion and expression recombinant Bacillus subtilis levansucrase.The highest enzyme production in a 5-L fermentor can reached93.39 ± 5.75 U/m L,levansucrase almost complete secretion outside the cell,which was 11 times higher than the shake flask fermentation.The fermentation supernatant was used as enzyme preparation and reacted for 24 h under certain conditions.The maximum yield of levan reached 89.6 ± 1.33 g/L and the highest conversion rate was 39.84 ± 1.54%,which indicated the enzyme had good application potential.This work lay a basement for preparation large-scale enzymatic production of levan and have a good application prospect.