The Construction And Fermenting Condition S’ Optimization Of The Engineering Bacteria That Express The Endo-1,4-β-mannosidase From Thermotoga Maritima

Over the last twenty years, the extreme microorganism and its enzyme have become a hotspot in many fields. Glucosidase is an indispensable enzyme in industrial production. The endo-1,4-β-mannosidase has more important value for industrial application in food, pharmaceutical, feed, fiber manufacture and paper industry. To develop thermophilic endo-1,4-β-mannosidase for industrial production has great significance. Thermoto gamaritima is a kind of thermophilic microorganism. The growth conditions of this bacteria are very strict, and very difficult for large-scale fermentation culture in industrial production. For these reasons, using genetic engineering technology to clone and express the enzymes is a main way for the use of Thermotoga maritima.This study is about the endo-1,4-β-mannosidase from Thermotoga maritima. The genetically engineered bacteria which can express endo-1,4-β-mannosidase from Thermotoga maritima was obtained by molecular biology methods. Most impurity can be removed by heat treatment, so the protein can be easy to purify. No sense either it would be, in addition fermentation conditions for production of enzyme were optimized” or “in addition fermentation conditions for production of enzyme should be optimized” check again.The main research results are as follows:1. The endo-1,4-β-mannosidase gene fragment(MSB8-898257) from Thermotoga maritima was selected and used p ET-28a(+) vector to construct recombinant plasmid with MSB8-898257 gene to express in E.coli BL21. Then got the genetic engineering bacteria of endo-1,4-β-mannosidase from Thermotoga maritima.2. Using IPTG as the inducer to induce protein, and detecting protein expression by polyacrylamide gel electrophoresis, Coomassie blue staining and Western blotting, and the protein was appeared in 77 k Da band.3. Analysis of physical and chemical properties by bio-informatics software. DNS assay was used to detect the enzyme activity. Ni-NTA resin was used to purify the target protein to obtain more pure protein.And also optimizing the induction conditions.4. The fermentation conditions of BL21-898257 genetically engineered bacteria and optimized fermentation conditions by Shake flask experiments. Mainly studied five factors: temperature, p H, inducer concentration, induction time, induction of OD600. The results showed that the expression of protein was significantly influenced by temperature, time and initial OD600. According to the experimental results, the orthogonal testing scheme was designed to optimize the fermentation conditions of 1 L fermentation broth. The optimal fermentation conditions were: 32℃, initial OD600=1, induction time was 7 h, 5 mg of enzyme can be obtained per liter fermentation broth.5. The application of the recombinant enzyme was studied through the preliminary analysis of degumming. The results showed that degumming of the recombinant enzyme was significant, and it has a good application value in the fiber manufacturing industry.