An Oxidation Method Using Whole Cells as Catalysts
A whole-cell, catalyst technology, applied in microorganism-based methods, biochemical equipment and methods, chemical recycling, etc., can solve the problems of regulatory influence, difficult intracellular cofactor levels, etc., achieving strong applicability, saving reaction costs, cost saving effect
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Embodiment 1
[0028]The optimization of embodiment 1 mannitol dehydrogenase gene
[0029] Analysis of rare codons: The rare codons were mainly analyzed by the software E.coli Codon Usage Analysis 2.0 to obtain the codons whose relative frequency of use in Escherichia coli was lower than the threshold (10‰).
[0030] Optimize rare codons: optimize the codons whose usage frequency is lower than the threshold according to the usage frequency of different codons in E. coli. The optimization results are shown in Table 1:
[0031] Table 1 Comparison of codons before and after optimization
[0032]
[0033]
[0034] This patent application optimizes the mannitol dehydrogenase gene sequence according to the codon preference of Escherichia coli, and the optimized sequence is shown in SEQ ID NO:1. After comparison with DNAMAN software, it was found that the homology between the modified sequence (MtDH) and the original sequence (MtDH_Ag) was 73.35%. For details, see figure 1 .
Embodiment 2
[0035] Example 2 Construction of recombinant Escherichia coli MtDH-BL21 expressing mannitol dehydrogenase
[0036] Cloning the codon-optimized mannitol dehydrogenase gene MtDH of Example 1 into the pET-28a vector to obtain the recombinant plasmid pET-28a-MtDH, and then transforming the recombinant plasmid into E.coli.BL21 to obtain overexpression of the MtDH gene Recombinant Escherichia coli MtDH-BL21.
[0037] Among them, the vector plasmid pET-28a was purchased from Youbao Biology; E.coli BL21 was purchased from Takara Company.
[0038] The recombination of the gene MtDH and the pET-28a vector, and then transformation into E.coli.BL21, and the preparation of related medium were carried out according to the third edition of the Molecular Cloning Experiment Guide (translated by Huang Peitang et al., China, Science Press, 2002); The required primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
Embodiment 3
[0039] Embodiment 3 expression of recombinant escherichia coli MtDH-BL21
[0040] The recombinant Escherichia coli MtDH-BL21 constructed in Example 2 was inoculated in a Erlenmeyer flask containing 200mL LB liquid medium by 1% by volume, and cultivated to OD at 37°C at 200rpm 600 When it reaches 0.6-0.8, add IPTG to the culture medium, the final concentration of the IPTG is 0.6mM, then induce at 25°C and 200rpm for 12-16h, and obtain wet cells of recombinant Escherichia coli after centrifugation.
[0041] The formula of LB liquid medium is as follows: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L.
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