| Glucose isomerase（GI,EC 5.3.1.5）is an isomerase that can catalyze the isomerization of D-glucose to D-fructose.Most of the glucose isomerases produced in China have low catalytic activity and poor thermal stability,so domestic companies producing high fructose syrup and crystalline fructose rely on importing glucose isomerase produced by foreign enterprises,so it is of great research significance to mine glucose isomerase with high catalytic performance,good thermal stability and independent property rights.In this project,virtual probes were designed to mine from the Gene Bank database to find glucose isomerases derived from Caldicellu Losiruptor acetigenus（CAGI）,Thermoanaerobacter thermocopriae（TTGI）and Thermotoga petrophila（TPGI）.derived thermophilic glucose isomerase and successfully heterologous expression in E.coli BL21（DE3）-star,characterization of enzymatic properties of purified glucose isomerase;alanine scan of glucose isomerase TTGI and study of its subunit binding site combined with kinetic simulation;immobilization study of glucose isomerase TTGI.The main studies are as follows.（1）Whole protein energy scan of glucose isomerase based on Fold X and Rosetta,gene mining from Gene Bank database by designing virtual probes with conserved sequences,and successful screening of CAGI,TTGI and TPGI.glucose isomerase was isolated and purified and characterized enzymatically.Among them,TTGI has an optimum p H of 8.0,an optimum temperature of>95℃and a half-life of>24 h at 90℃,which is typical of thermophilic enzymes.The enzymatic kinetic parameters of glucose isomerase TTGI were:Km was 103.4 m M,vmax was 72.8 U/mg,and kcat was 35.3 s-1.（2）The four-level structure of glucose isomerase TTGI was investigated,and the alanine scan of its subunit interface site revealed that the flexibility of the C-terminal loop region of glucose isomerase has a large effect on glucose isomerase activity,and the saturation mutation of site 336 was found to enhance the 65℃conversion rate mutant TTGI-N336T according to the alanine scan results.The enzymatic properties and kinetic parameters of TTGI-N336T were determined.9.7%conversion was enhanced at 65℃and the optimum p H was 7.5-8.0.（3）The crude enzyme solution of glucose isomerase TTGI was immobilized on modified diatomaceous earth using the combination of embedding-crosslinking and biomimetic mineralization method.After rinsing for three times,titanium di（2-hydroxypropionic acid）diammonium hydroxide was added for mineralization.The enzyme activity of immobilized enzyme was 106.4 U/g,and 55.1%high-fruit syrup was obtained within 2 h after the use of immobilized glucose isomerase. |
