以蔗糖为底物利用重组大肠杆菌合成甘露醇

【目的】异型发酵乳酸菌可利用胞内产生的甘露醇脱氢酶将果糖高效转化为甘露醇,但果糖作为底物相对昂贵,不利于工业化生产。为了降低生产成本,必须选择廉价的底物。蔗糖相对便宜,并且大量存在于自然界中,能够被重组大肠杆菌利用产生甘露醇。蔗糖水解酶(Sucrose hydrolase)和甘露醇脱氢酶(Mannitol dehydrogenase)是发酵生产甘露醇中催化蔗糖转化成甘露醇的关键酶,构建蔗糖水解酶和甘露醇脱氢酶共表达菌株并进行相关研究是本文的主旨。【方法】利用PCR方法分别从植物乳杆菌(Lactobacillus plantarum)和布氏乳杆菌(Lactobacillus buchneri)基因组DNA中获得sac A和mdh基因,得到大小分别为1 502 bp和1 032 bp的目的基因,经序列分析后将其连接到表达载体p ET-28a(+)上,得到重组表达载体p ET28a-sac A-mdh。将重组质粒转化到大肠杆菌BL21(DE3)中,并用SDS-PAGE分析目的蛋白的表达情况并测定其酶活。【结果】SDS-PAGE显示表达蛋白的大小亚基分子量分别为55.1 k D和37.8 k D,与预期分子量一致,实现sac A和mdh基因的表达。蔗糖水解酶和甘露醇脱氢酶酶活分别为25.78 U/m L和14.56 U/m L。对重组菌株BL21(DE3)/p ET28a-sac A-mdh进行发酵条件优化,甘露醇质量浓度达到45.19 g/L,总糖转化率为37.66%。【结论】与乳酸菌利用蔗糖发酵生产甘露醇相比,产量提高了6倍,且具有发酵周期短、稳定性高等优点,菌株的成功构建为甘露醇工业化生产奠定了基础。

Mannitol biotransformation from sucrose with recombinant Escherichia coli

[Objective] Heterofermentative lactic acid bacteria (LAB) can convert fructose to mannitol efficiently using the intracellular mannitol dehydrogenase enzyme, but fructose as a substrate is relatively expensive and not suitable for industrial production. In order to reduce cost, inexpensive substrates must be selected. Sucrose, abundantly in nature, is relatively cheap and can be used by recombinant Escherichia coli to produce mannitol. Sucrose hydrolase and mannitol dehydrogenase are the key enzymes to convert sucrose into mannitol. This paper was focused on the construction of sucrose hydrolase and mannitol dehydrogenase co-expression strain. [Methods] The target genes sacA and mdh, 1 502 bp and 1 032 bp respectively, were derived from the Lactobacillus plantarum and Lactobacillus buchneri. The genes were cloned into the expression vector pET-28a(+), resulting in the generation of the recombinant expression vector pET-28a-sacA-mdh, which was transformed into the E. coli BL21. The expression of the target protein was analysed by SDS-PAGE and the enzyme activity measurement. [Results] SDS-PAGE showed that the molecular weight of the expressed protein was 55.1 kD and 37.8 kD, respectively, which was consistent with the expected molecular weight, demonstrating the expression of genes sacA and mdh. The enzyme activity of sucrose hydrolase and mannitol dehydrogenase was 25.78 U/mL and 14.56 U/mL, respectively. The concentration of mannitol reached 45.19 g/L and the conversion was 37.66% after optimizing the fermentation conditions. [Conclusion] Compared with the mannitol production by LAB fermentation using sucrose, the yield was increased six-fold, with the advantages of short fermentation period and high stability. The successful construction of recombinant strain laid the foundation for the industrial production of mannitol.