| Mannanase is an important hemicellulase and has been widely used in many fields.In industrial production,not only the enzymes with excellent properties are needed,but also the production of enzymes is the key factor affecting the profit of the enterprise.Therefore,it is necessary to improve the secretion and expression of mannanase by genetic engineering to meet the needs of industrial applications.The Pichia pastoris expression system is a highly efficient exogenous protein expression system,which has been widely used to express and produce a variety of exogenous proteins.Signal peptides on expression vectors can well guide the secretion of exogenous proteins and facilitate the isolation and purification of expressed proteins.The ?-factor from Saccharomyces cerevisiae is the most commonly used signal peptide in the Pichia pastoris expression system.However,when different signal peptides mediate the secretion and expression of the same exogenous protein,the secretion and expression efficiency of exogenous protein varies greatly.Therefore,it is important to modify and screen the endogenous signal peptides of Pichia pastoris for improving the secretion and expression of exogenous protein in Pichia pastoris.In this study,five Pichia pastoris endogenous signal peptides GAS1,DSE4,SCW11,MSB2 and EXG1 were selected,which respectively mediated the expression of mannanase ManA(Accession No.KJ806637)in Pichia pastoris X33.The ?-factor was used as the reference,and the results of horizontal induction of shake flask expression showed that the extracellular enzyme activities of GAS1,DSE4,SCW11,MSB2,EXG1 and ?-factor mediation ManA secretion reached a maximum at 120 h,their enzymatic activities are 302 U/mL,329 U/mL,128 U/mL,88 U/mL,114 U/mL and 303 U/mL respectively.The corresponding intracellular enzyme activities at 120 h were 65 U/mL,27 U/mL,48 U/mL,96 U/mL,15 U/mL,and 40 U/mL respectively.In addition,the transcriptional levels of five signal peptides mediated with ManA expression were analyzed by real-time PCR.Compared with ?-factor,the transcriptional levels of GAS1,DSE4,SCW11,MSB2 and EXG1 mediated the expression of ManA were 1.03,2.12,0.32,0.21 and 0.60 times as much as that of ?-factor,respectively.The above results indicate that compared with SCW11,MSB2 and EXG1,DSE4 and GAS1 mediated the secretion and expression of ManA in Pichia pastoris X33 is more efficient and can effectively replace ?-factor.The changes of basic amino acid in the N-terminal region and the polar amino acid in the C-terminal region of the signal peptide have a certain effect on the secretion and expression of the foreign protein.For increasing the secretion expression of ManA in Pichia pastoris,a series of modifications is for GAS1 and DSE4 in this study,including the introduction of the basic amino acid R,K or H at the third amino acid at the N-terminus and the introduction of polar amino acids N,K,H,R,Q,D Or E at the penultimate amino acid at the C-terminus.When fermented to 120 h,compared with GAS1,the total enzyme activity of R(GAS1-3)introduced at the third amino acid of the N-terminus was increased by 3%,the supernatant enzyme activity increased by 5%,and the expression of RNA was 1.25 times higher than that of GAS1.The total enzyme activity of K(GAS1-2K)introduced into the penultimate amino acid of the C-terminal of GAS1 is increased by 20%,and the supernatant activity increased by 17%,and the mRNA expression was 2.64 times that of GAS1.Compared with DSE4,the total enzyme activity of R(DSE4-3R)introduced into the third amino acid of the N-terminus of DSE4 increased by 5%,and the supernatant enzyme activity increased by 3%,and the mRNA expression level was 1.25 times that of DSE4.GAS1-3,GAS1-2K and DSE4-3 can increase the secretory expression of ManA in Pichia pastoris to varying degrees through the above-mentioned modifications of two signal peptides,which provides a certain theoretical basis for efficient secretory expression of the foreign protein in Pichia pastoris. |
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