Expression And Optimization Of The Novel Bifunctional Glutathione Synthetase In Pichia Pastoris And Escherichia Coli

Glutathione is a thiol-containing active tripeptide which is synthesized by the condensation of glutamic acid,cysteine and glycine.It has two forms:reductive(G-SH)and oxidative(G-S-S-G),only the former has activity.Glutathione is widely used in food,cosmetics and pharmaceutical industries because it contains active thiol and has many physiological functions.With the deepening understanding of GSH,the market demand of GSH is also increasing.Therefore,the industrial production of GSH is particularly important.At present,GSH production methods mainly include solvent extraction,chemical synthesis,microbial fermentation and enzymatic method.Among them,enzymatic method refers to the use of enzymes to catalyze the synthesis of GSH from three amino acids.In most cells and Gram-negative bacteria,the enzymatic synthesis of GSH consists of two steps:?-glutamate cysteine synthase(?-GCS,encoded by gene gshA in Escherichia coli and gshI in yeast)and y-glutathione synthase(GS,encoded by in Escherichia coli and gshll in yeast).However,when the content of GSH is too high,it will synthesize with y-glutathione.Enzyme binding results in the accumulation of intermediate products,which reduces GSH production.In recent years,a novel ATP-dependent enzyme,GshF,has been found in some bacteria.It is reported that GshF from most bacteria is insensitive to GSH.Therefore,the main contents of this study are as follows:1)Two pairs of primers were designed according to the sequence of gshFSA in NCBI and the polyclonal loci on pPIC9K and pET-28a.The target gene gshFSA was cloned from Streptococcus agalatiae by high fidelity PCR.Then the recombinant expression vectors pPIC9K-gshFSA and pET28a-gshFSA were constructed and transformed into GS115 and BL21 respectively.High-copy transformants were screened by G418 resistance,and then induced to express.The GshF in fermentation broth was detected by SDS-PAGE.The total protein content in crude enzyme broth was determined by Bradford method.The total protein content in pPIC9K-gshFSA/GS1 15 fermentation broth was 0.46 mg/mL,and that in pET28a-gshFSA/BL21 fermentation broth was 1.46 mg/mL.2)The methanol induction concentration,induction temperature and fermentation broth pH of recombinant yeast pPIC9K-gshFSA/GS1 15 were optimized by single factor gradient.The expression of GshF was the highest when the induction temperature was 30 C and the concentration of methanol was 2%.When the induction pH was 6.0,the expression of GshF was the highest.Then the orthogonal experiment was designed to determine the best combination of these factors in order to obtain the maximum yield of GSH.The results showed that when the induction concentration of methanol was 2.5%,the induction temperature was 300C and the induction pH was 6.0,the expression of GshF by recombinant yeast was the highest.The highest activity of GshF was 9.1U/mL.3)The temperature,timing and concentration of inducer of recombinant engineering bacteria pET28a-gshFSA/BL21 were optimized.The highest activity of GshF was 12.23U/mL after 5.5 hours of culture and 7 hours of induction with IPTG.The highest activity of GshF was 12.02U/mL when the induction temperature was 340C.The highest activity of GshF was 3.91U/mL when the concentration of inducer was 0.3 mmol/L.The bacteria with the highest enzyme activity were also selected for ultrasonic wall-breaking treatment.It was found that the protein content and enzyme activity increased by four times and about three times after wall-breaking.