The Synthesis Of Mannose-6-Phosphate By Biocatalyst

Mannose-6-phosphate (M6P), one of the most important phosphate compounds for cell protein modification, has great application in treating disease and cosmetic. Compared to the chemical method, the synthesis process of M6P by enzyme is more environment-friendly and safety, however, the high cost of ATP and kinase is limited to the M6P further applications. To overcome this weakness, a new catalyst polyphosphate dependent mannokinase was overexpressed in Escherichia coli for the synthesis of M6P with polyphosphate. The main work were as following:the construction of genetically engineered bacteria, the research of enzyme properties, the protein overexpression conditions and the optimization of reaction conditions.(1) Polyphosphate dependent mannokinase from Arthrobacter sp strain KM and Mycobacterium sp strain MCS were cloned, and the genetically engineered bacterias were constructed as:E.coli BL21(DE3)(pET28a-17) and E.coli BL21(DE3)(pET28a-mp). The fermentation broth were used for the synthesis of M6P.(2) Polyphosphate dependent mannokinase MP from Mycobacterium sp strain MCS properties were researched. The optimum temperature was 25 ?, the optimum pH was 8.5, MP with high affinity for hexametaphosphate, the optimum required bivalent metal ion Mg2+ amount was 5 mM. The Km values of the MP for glucose, mannose, ATP, and hexametaphosphate were determined to be 9.57,203.65,4.62,1.7 mM.(3) Polyphosphate dependent mannokinase L7 from Arthrobacter sp strain KM overexpression condition was optimized. The density of protein was 3 times higher, and the enzyme activity was increased 4.7-fold when with TB medium than LB medium (28 ?,0.2 mM IPTG,12 h). The yield of L7 rearched 2.25×106U under the 3 L fermentation.(4) The L7 reaction condition was optimized. When the mole ratio of mannose to hexametaphosphate being 1:3, L7 enzyme loading amount being 0.356 mg·mL-1,30 ?, pH 8.5 for 3h, the mannose conversion achieved the highest (99.8%),and the M6P concentration was 14 g·L-1.(5) When the mannose concentration being 40 g-L-1, the enzyme loading amount being 39.3 U·g-1 sugar in 500 mL system for the synthesis of M6P, 30 ?, pH 8.5,400 rpm for 8 h, the conversion of mannose reached 75% and the M6P concentration was 47 g·L-1.