Gene Expression And Fermentation Process Optimization Of Marine Microbial Malate Dehydrogenase

Malate dehydrogenase(MDH)is an oxidoreductase,which can catalyze the oxidation of L-malic acid and coenzyme NAD + to form oxaloacetic acid.The reverse reaction catalyzes the synthesis of L-malic acid by oxaloacetate and coenzyme NADH.At the same time MDH in food,clinical medicine and agricultural production and other fields,have a very important application.As the current domestic MDH enzyme preparation is expensive,and mostly from the animal heart,there are complex and easy to cause environmental pollution and other issues,so from the rich marine micro-organisms,the extraction of MDH is of great significance.Unique marine environment to create a salt,heat,cold,pressure and acid,alkali and other characteristics of the extreme enzyme.Marine microorganisms in the new genes,new protein development and other aspects of the great potential for the application of marine plants from the mining of MDH gene,and the construction of the corresponding engineering strains,the following experiments:(1)Screening of target strains.A strain of Pseudomonas beteli with high MDH activity was screened from 14 strains of marine strains fed by the State Oceanic Administration's third institute,which were stored in the laboratory.(2)Construction of recombinant E.coli expressing recombinant MDH.The MDH gene was cloned from the Pseudomonas beteli genome and ligated into vector pET28a,transformed into E.coli BL21(DE3)and E.coli Rosetta(DE3)to obtain MDH-expressing engineering bacteria.(3)The optimum conditions and stability of enzymatic reaction of MDH were investigated.The recombinant strain Rosetta-pET28a-MDH was induced to produce the target protein MDH and purified by nickel column.The specific activity of the purified MDH was 31.3U/mg.The optimal temperature of MDH was 60?,and incubated at 40? for 1 h.The activity of enzyme was kept above 60%.The optimal enzymatic pH was 8.0,incubated at 40 ? for 1 h,and MDH was the most stable at pH = 7.0.(4)medium optimization.The results showed that the optimum medium composition(g/L)was optimized by using PB experiment and homogeneous design and the DPS data processing system to optimize the culture medium composition of the maltate dehydrogenase strain Rosetta-pET28a-MDH as the fermentation strain:glucose 10,yeast powder 37.5,dipotassium hydrogen phosphate 8.0,sodium dihydrogen phosphate 2.5,ammonium sulfate 1.4,EDTA0.5,trace element 1.1.(5)multi-stage regulation of 50L high-density fermentation.Maintain the speed before running 200rpm and dissolved oxygen value of about 40%.The first stage:after inoculation,the use of ventilation coupled with the method to adjust the speed to maintain the same ventilation,speed up to 500rpm,set the dissolved oxygen value of 30%,temperature 37 ?.The second stage:the use of ventilation coupled with the method to adjust the ventilation,speed maintained at 500 rpm the same,the system automatically adjust the ventilation,set the dissolved oxygen value of 30%,temperature 37 ?.The third stage:When the OD value of the fermentation broth reaches 10?11,add IPTG,induction,maintain the speed of 500 rpm,set the dissolved oxygen value of 30%,temperature 20 ?.Fermentation process,the use of constant pH feeding method,every 1?2h collection of 30ml fermentation broth.The enzyme activity and crude enzyme activity of malic acid dehydrogenase were 67.5U/mg and 41.0 U/ml,respectively.The fermentation broth of 1L contained 41000U of enzyme activity.