Cloning And Expression Of N - Acetyl - D - Glucosamine 2 - Epimerase And Its Application

N-acetyl-D-glucosamine(GlcNAc)2-epimerase(AGE)is one of the two key enzymes for synthesis of N-acetyl-D-neuraminic acid(Neu5Ac).Dual-enzymatic synthesis of Neu5Ac has been basically established,and the N-acetylneuraminate lyase(NAL)from E.coli source which can meet the requirement of large-scale production has been commercialized already as well.However the protein,which was originated from the AGE gene from porcine kidneys cloned into E.coli,is expressed mainly in the form of inclusion bodies due to differences in species.Therefore, developping a high efficient AGE is imminent.The contents and achievements of this research include:Firstly,AGE gene from pig kidneys and Anabaena respectively was amplified by RT-PCR and PCR method,and cloned into the pMD T-18 vector.The nucleotide sequencing results were compared to the reported sequences from pig kidneys and Anabaena,and it was proved entirely correct.Then they were recombined into the expression vector pET30a,and two new plasmid pLY9 and pLY11 were constructed and expressed in E.coli BL21(DE3).Secondly,the activity of AGE was detected by PMP derivatization method and N-acyl-D-mannosamine(ManNAc)was used as substrate.The optimal reaction conditions of the recombinant Anabaena epimerase were pH 8.0 and 40℃,respectively.The results based on the comparison between the two epimerases gene expressed in E.coli BL21(DE3)showed that in the same conditions,the output of Anabaena epimerase activity was 16-fold higher than that of the pig kidneys.Finally,three different methods were tried to prepare Neu5Ac in this study.(1).The genetically engineered bacterium co-expressing NAL and AGE plasmid pLY12(pET30a-based)was constructed.After the transformation of pLY12 into E.coli BL21(DE3),the bacterium could produce NAL and AGE after inducing by lactose.The purpose of using this method was to reduce the time of fermentation.It was proved that the crude enzyme prepared by this method was active, and could be used to prepare Neu5Ac.(2).Recombinant E.coli BL21(DE3)/pLY11 and E.coli BL21(DE3)/pLY3,which carry AGE gene and NAL gene individually,were harvested respectively after induced by lactose.Then the two whole cells were used to prepare Neu5Ac.Experimental results showed that the yield of Neu5Ac is very low.(3).Crude enzyme of NAL and AGE was prepared respectively by inducing E.coli BL21(DE3)/pLY3 and E.coli BL21(DE3)/pLY11,and the two crude enzymes were then used to prepare Neu5Ac.The conversion conditions such as substrate concentration,proportion of enzyme,reaction temperature,pH,reaction time were investigated. Optimized conditions for the best transformation were:sodium pyruvate 250 mmol;GlcNAc 550 mmol;NAL 1.28 U/ml;AGE 0.48 U/ml,pH 8.0,temperature 25℃;transformation time 12 h.