Expression Of Transglutaminase In Pichia Pastoris GS115 And Optimization Of Fermentation Conditions

Transglutaminase(TGase)is widely used in protein modification.It can change protein network structure,enhance water retention capacity and promote gel formation,and even improve the nutritional value of protein.It has important application value in food,medicine,textile and other fields.At present,the main source of TGase is microorganisms,because microorganisms grow fast,have large yields and high efficiency.Natural fermentation strains can directly obtain active TGase,but the fermentation cycle is long,the process is complicated and the cost is high.The heterologous recombinant expression process is simple,low cost and short cycle time.Pichia pastoris has a clear research background and is an ideal heterologous protein expression system.Therefore,choosing Pichia pastoris heterologous expression of transglutaminase is a feasible strategy.In this study,the transglutaminase gene of Streptomyces hydroscopicus 107.3 screened in the previous stage was expressed,ultrafiltration purification and enzymatic characterization were investigated in P.pastoris GS115.The fermentation conditions of the recombinant strain were optimized by response surface method,and the recombinant enzyme was expressed in a small 10 L fermentation tank to evaluate the fermentation performance of the recombinant strain.The main results are as follows:(1)The secretion vector of transglutaminase was successfully constructed and expressed in Pichia pastoris GS115.The secretion vector pPIC9K-pro-kex-TGase containing the three-part fusion gene of S.hygroscopicus propeptide pro,kex2 restriction site and mature enzyme(mTGase)was constructed and integrated into the genome of P.pastoris GS115 by electrotransformation to obtain positive clone transformants GS115/pPIC9K-pro-kex-TGase(hs-D6)can directly obtain the active recombinant transglutaminase after expression,and the enzyme activity of the recombinant enzyme reached 0.314 U/mL after dispase Ⅱ treatment.SDS-PAGE analysis showed that recombinant P.pastoris hs-D6 strain could express about 45 kDa protein bands,which were consistent with the size of the theoretical molecule of the enzyme without glycosylation.However,the mTGase protein band was not seen.The possible reason is that the kex2 protease of GS115 strain recognizes the kex2 site and cuts a small part of the expressed TGase zymogen.Therefore,the Pichia expression system of recombinant S.hygroscopicus TGase constructed in this study can not only produce a large amount of zymogen protein,but also directly produce mTGase with enzymatic activity,which to a certain extent achieves our design goal of directly obtaining active TGase and provides a new idea for the production and molecular transformation of transglutaminase.(2)The recombinant transglutaminase was purified by ultrafiltration and its enzymatic properties were characterized.The method of ultrafiltration can be used to realize the simple purification of the recombinant enzyme.The 10 kDa ultrafiltration tube can completely intercept the recombinant enzyme protein.The purified recombinant transaminase was used for the determination of enzymatic properties.The optimum temperature was 40℃ and the optimum pH was 7.The maximum velocity(Vmax)and kinetic constant(Km)of the recombinant enzyme determined under the optimal conditions were 16.58 μmol/(L·min)and 3.79 μmol/L,respectively.Na+ and K+ had certain promotion effect on enzyme activity,while Ca2+ had almost no effect.Mg2+,Mn2+,Fe2+,Zn2+ and Cu2+ showed inhibition effect on the recombinant TGase,and the inhibition effect of Fe2+,Zn2+ and Cu2+ on enzyme activity increased with the increase of concentration.Methanol and ethanol with a volume fraction of 10%had a certain positive promotion effect on the recombinant TGase,while glycerol had a small influence on the enzyme activity.Acetone and isopropanol had an inhibitory effect on the enzyme activity,especially acetone.Among the surfactants,SDS significantly inhibited the recombinant TGase,while PEG800,PEG8000 and Tween20 all had certain promoting effects on the activity of the recombinant TGase.The characteristics of transglutaminases from different sources are different,so it is of great significance to study their enzymatic properties for the practical production and application of enzymes.(3)The response surface method was used to optimize the fermentation conditions of transglutaminase,and the fermentation performance was evaluated by small-scale cultivation in a 10 L fermentor.The Plackett-Burman(PB)design and response surface methodology(RSM)were used to study five fermentation conditions at the shake flask level,including inoculation amount,induction temperature,time,initial pH and methanol concentration.The optimal enzyme production conditions of recombinant Pichia pastoris hs-D6 strain in shake flasks were:inoculum OD600 of 6,pH 7,methanol concentration of 2.0%,induction temperature of 23℃,and induction time of 72 h.After optimizing the fermentation conditions,the recombinant strain produced TGase enzyme activity in the shake flask up to 1.1 U/mL,which was 3.6 times the initial enzyme activity..The results of fermentation conditions optimized by response surface shake flasks were expanded and cultured in a 10 L fermentor to express recombinant enzymes.The results showed that under optimized conditions,the enzyme activity of recombinant strains producing transglutaminase was further improved,which not only could accumulate more recombinant strains larger biomass,but also can have a good result for enzyme production;Under the condition of high biomass and induced pH5.0,the activity of recombinant enzyme reached 2.54 U/mL at the highest,which is 8.5 times the initial enzyme activity,and the accumulated biomass can reach 30 g/L.The results showed that the recombinant P.pastoris hs-D6 had better fermentation performance under the optimal conditions of shake flask and fermenter.