Screening And Fermentation Condition Optimization Of Glucose-1-phosphate High-producing Strain

Glucose-1-phosphate (G-1-P) is one of the important intermediates in glucose metabolism, produced by the phosphorylase catalyzing the phosphorolysis of polysaccharide in vivo, then catalyzed into glucose-6-phosphate by phosphoglucomutase to enter the glycolytic pathway, so it is an important energy matter. As an important starting reactant, glucose-1-phosphate is used for the biosynthesis of important substances, such as diphosphate glucose nucleotide, trehalose and maltoligosaccharide. In addition, glucose-1-phosphate has important medicinal value, which is the material for the preparation of cell proliferation, antibiotics and anticancer drug. At present, the main method for glucose-1-phosphate preparation is enzymatic synthesis, and there are free enzyme and immobilized enzyme two ways. Despite enzymatic synthesis of glucose-1-phosphate made great progress, but there are still many deficiencies, such as isolation and purification of phosphorylase cumbersome and the phosphorylase easy to inactivate, substrate utilization rate and product such conversion rate are not ideal, so that glucose-1-phosphate prices are still very expensive. For early realization of the industrialization of glucose-1-phosphate and its application, this paper attempts to use microbial direct fermentation methods to produce glucose-1-phosphate. The experiment screened one strain which can directly produced extracellular glucose-1-phosphate by fermentation; then a mutant strain high-yielding glucose-1-phosphate was obtained by UV mutagenesis; finally the fermentation conditions of the high-yielding strain were optimized, the output of glucose-1-phosphate improved further. Specific research contents and results are as follows:1. A strain high-yielding extracellular glucose-1-phosphate (G-1-P) with maltose as substrate was isolated from organic-rich soil by plate screening, flask rescreening with TLC and HPLC-MS detection. Through morphological, physiological and biochemical characteristics and 16S rDNA gene sequence analysis, it was identified as Bacillus subtilis, named Bacillus sublitis XH-13.2. Ultraviolet(UV) mutation breeding of Bacillus sublitis XH-13, a glucose-1-phosphate (G-1-P) producing strain screened by myself was studied in this paper. By plate screening, flask rescreening with HPLC-MS detection method, after three rounds of UV mutagenesis,obtained a strain Bacillus subtilis XH-13_UV3-8 high-yielding glucose-1-phosphate, the glucose-1-phosphate production of which was 6.85mg/mL,1.54 times of that of the original strain. After 6 subculturings, it still performanced a good genetic stability.3. Response surface methodology was used to optimize the fermentation condition of glucose-1-phosphate (G-1-P) production by Bacillus subtilis XH-13_UV3-8. After single factor test, a Plackett-Burman design was firstly used to evaluate the influence of 12 related factors.Yeast extract powder and K2HPO4·3H2O influenced glucose-1-phosphate production positively while pH did negatively.The path of steepest ascent was used to approach the optimal region of the fermentation condition subsequently.Box-Behnken design was used to optimize the three critical factors mentioned above, the optimum levels and the relationships among these factors were found out by quadratic regression model equation with Design-Expert statistic methods, the optimal levels of the variables were determined as:Yeast extract powder 0.8%, K2HPO4·3H2O 0.7% and pH 6.6. The optimized condition allowed the glucose-1-pho-sphate production to be increased from 6.85mg/mL to 8.56mg/mL.