Cloning And Functional Analysis Of Mannose Isomerase Gene Of E.coli

Mannose isomerase (MI,EC 5.3.1.7) can catalyse fructose to mannose and it is the key enzyme on the biological preparation of mannose. It is also the key enzyme on preparation of mannitol by chemical catalysis to reduce the sorbitol, byproduct of mannitol.Many microorganisms can produce mannose isomerase including Pseudomonas saccharophila, Xanthomonas rubrilineans, Streptomyces aerocolorigenes, Mycobacterium smegmatis, Agrobacte aerogenes and so on. Industrially, mannose isomerase is mainly extracted from the producing strain at present, but the yield is low. The best way is to obtain MI high-yield bacteria by genetic engineering to overcome the problem.In this research, the mannose isomerase gene(mi) was cloned from E.coli JM109, and can be expressed in E. coli BL21(DE3) and Pichia pastoris X33. The results were as follows:(1) The mi was cloned by PCR from E.coli JM109. The gene size is 1242 bp, 99% identity with E.coli str. K-12 substr. MG1655(U00096), E.coli str. K12 substr. DH10B (CP000948), E.coli str. K12 substr. W3110 DNA (CP009048). The amino acids sequence of MI has 99% identity with the aboved strains;(2) The expression vector, pET-mi, was constructed and transformed to E.coli BL21 (DE3). The induced protein molecular weight was 51.4kD screened by SDS-PAGE gel when induced by 1.0mmol/L IPTG. The results of enzyme activity analysis showed that the activity of MI were 10.34±0.087U/mg and 5.64±0.23U/mg when mannose and fruose as the substrate respectively at 37℃for 1h;(3) The mi gene was cloned into pGAPZαA vector to construct pGAP-mi and transformed to P. pastoris X33 by lithium chloride transformation method. The MI could be secreted into the medium when the transformed Pichia was cultivated in YPD medium with glycerol instead of glucose after 96h. The MI activity was 5.94±0.07U/mg when fruose as the substrate;(4) The optimized condition was built for increasing the solubale MI and to overcome inclusion protein producing in E.coli. The results showed that more soluble MI was induced at 18℃, 150r/min after the addition of 10mmol/L lactose at OD600≈1.0;(5) Some properties of MI was also studied. Results revealed that the MI has the maximum activity at 37℃, pH7.5. The preferred substrate was mannose compared with fructose;(6) The renaturation of MI inclusion and influencing factors were investigated. The MI inclusion was renatured by dilution by degrees with renaturation buffer. The renatured MI was purified and concentrated by DEAE-Sepharose Fast Flow column. The best renatured condition was 2mol/L urea, 12h renaturation time and 0.5mmol/L GSH. The molar ratio of GSH to GSSG was 3. The recovered activity of MI was 3.14±0.11U and 54.7±0.13U/mg, respectively. The recycle efficiency of MI was 20.6%.