Expression,Characterization And Fermentation Optimization Of A Cold-Adapted Leudh

In recent years,the discovery and utilization of special function genes are a hot topic research of extreme environmental microbiology.In Antarctica,the unique extreme geographical environment has created a low-temperature and high-salt sea-ice environment.Thus,sea-ice microorganisms have formed unique physiological and biochemical metabolic characteristics.Due to the special enzymatic properties and molecular structures,Antarctic cold-adapted enzymes have a high catalytic efficiency at low-temperature,reduce the activation energy of the catalytic reaction,and have strong specificity for substrates and products.Therefore,they can save energy and have a wide range of application,which have attracted widespread attention from scholars at domestic and abroad.Leucine dehydrogenase?LeuDH?is one of the special cold-adapted enzymes,and it is widely used in the synthesis of chiral auxiliaries,protease inhibitors,anticancer drugs,anti-AIDS and ligands.Therefore,we used Antarctic sea-ice bacteria as materials to systematically study the cloning,expression and enzymatic properties of leudh gene,and recombinant LeuDH fermentation process,which will obtain anti-reverse gene resources with independent intellectual property rights.Meanwhile,the theoretical basis for the development and utilization of cold-adapted enzymes from the Antarctica source was provided.In this study,the full-length sequence of leudh gene was amplified from the Antarctic microbe Pseudoalteromonas sp.ANT178 by molecular biology,and named psleudh.Bioinformatics analysis of PsLeuDH showed that the gene was 1 209 bp,which encoded 402 aa protein,and the theoretical pI was 5.08.Through multiple sequence alignment analysis,PsLeuDH had a conserved Phe site,a NAD⁺coenzyme binding domain containing the conformation of the???-fold.PsLeuDH was a member of the Glu/Leu/Phe/Val dehydrogenase family.The psleudh gene was connected with the pET-28a?+?and then introduced into the E.coli BL21.The positive recombinant strain,named pET-leudh,was selected and induced by low temperature culture inducer.The expressed recombinat rPsLeuDH was purified by Ni-NTA and detected by SDS-PAGE.Specific activity of purified protein was 275.13 U/mg with a yield 24.73%.On the basis of purification,the enzymatic properties of rPsLeuDH were systematically studied:the optimum temperature of the enzyme was 30�C,and retained at least 40%of its initial activity even at 0 ^oC.The optimum pH was 9.0.Futhermore,the relative activity of the enzyme was the highest when the salt concentration was 2.0 M.Substrate specificity and kinetic studies of rPsLeuDH demonstrated that L-leucine was the most suitable substrate,and the catalytic activity at low temperatures was ensured by maintaining a high k_cat.Using homology modeling analysis,compared with thermophilic enzyme,the results indicated that rPsLeuDH showed more glycine residues,reduced arginine and proline residues,as well as salt bridges.These structural features imparted high flexibility and low thermal stability to protein molecules,making them highly active at low temperatures.The experimental conditions of Plackett-Burman?PB?and Central Composte Design?CCD?were used to optimize the fermentation conditions of rPsLeuDH strains.NaCl,L-leucine and the induction temperature were the signaficant factors affecting the yield of r PsLeuDH.The multiple regression models for production of rPsLeuDH were simulated and the optimum fermentation conditions were finally determined.Through further verification experiments,it was found that the model of rPsLeuDH production had a good fit.Its optimal enzyme yield was 75.90 U/mg,and its production was significantly increased by SDS-PAGE.In this study,a fermentation culture model for producing rPsLeuDH was established,which made production 2.61 times higher than before optimization.These results will provide important parameters for the large-scale fermentation of the recombinant protein.