Study On Synthesis Process Of Mannsoe-6-Phosphate

Mannose-6-phosphate(M6P)is involved in many metabolic pathways in life and has important applications in disease treatment.The main purpose of this paper is to develop a new enzyme-catalyzed synthesis of mannose-6-phosphate,and to create a cost-effective enzyme-catalyzed synthesis method for synthesizing M6P using polyphosphate-dependent mannose kinase from Arthrobacter.This synthesis uses polyphosphate to replace ATP,which is expensive,and is greener and safer than chemical synthesis.The research includes the construction of genetically engineered bacteria,the influence of single factor variables on the synthesis of mannose-6-phosphate,the optimization of the synthesis reaction by response surface methodology,and the pilot scale-up of the process.The specific work and research results are as follows:(1)The subject successfully constructed an expression vector,cloned the polyphosphate-dependent mannose kinase gene from Arthrobacter sp,and realized the heterologous expression of polyphosphate-dependent kinase in E.coli.This research successfully achieved the synthesis of mannose-6-phosphate in vitro.(2)Use single-factor experiments to explore the effects of various conditions on conversion rate,and optimize the synthesis reaction.It is found that when the temperature is 30℃,the content of Mg2+>10 mM,the addition amount of mannose was 2%,and the addition amount of sodium hexametaphosphate was 20 g/L,the enzyme-catalyzed reaction had a high conversion.At the same time,it is found that Mg2+and Mn2+ are more suitable as cofactors,sodium hexametaphosphate and ATP are more suitable as phosphate donors,and the rest of the polyphosphates cannot basically participate in the reaction as phosphate donors.(3)Select the key factors,use the Box-Behnken design(BBD)design of the response surface method,build a model to further optimize the synthesis reaction,and the conversion after optimization can reach 99.17%.(4)Based on the above research,a pilot scale-up of the synthesis process of mannose-6-phosphate was carried out,opening up the whole process from recombinant E.coli fermentation to product preparation,which laid the foundation for the establishment of a synthetic production line of mannose-6-phosphate.