Lactulose Production Using Immobilized Cells Including Thermostable Cellobiose 2-epimerase
Lactulose Production Using Immobilized Cells Including Thermostable Cellobiose 2-epimerase
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DOI：
10.4014/mbl.1609.09005
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发表时间：
2016-12-01
影响因子：
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通讯作者：
Lee, Hyeon-Cheol
中科院分区：
文献类型：
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作者：
Park, Ah-Reum;Koo, Bong-Seong;Lee, Hyeon-Cheol
乳果糖是一种合成二糖，因其作为益生元的作用而受到越来越多的关注，可以促进双歧杆菌和乳杆菌属的增殖。并增强钙、镁的吸收。虽然乳果糖的工业生产仍然主要通过乳糖在碱性介质中的化学异构化来实现，但该过程存在一些缺点，包括需要去除催化剂和副产物，以及高能量需求。最近，纤维二糖 2-差向异构酶 (CE) 的使用被认为是工业乳果糖生产的一个有趣的替代方案。在本研究中，为了开发使用 CE 进行酶促乳果糖生产的工艺，我们从易于出错的 PCR 技术生成的文库中筛选了改进的突变酶 (CS-(HCE)-C-R)。一种突变体的热稳定性得到增强，稳定性高达75℃，其乳果糖转化率比野生型CE提高了1.3倍。使用含有 CS35 (HCE)-C-R 表达质粒的重组大肠杆菌菌株，我们制备了固定在海藻酸钙基质上的细胞珠，并优化了其反应条件。在200 g/l乳糖溶液和固定化细胞珠的间歇反应中，乳糖在2 h内转化为乳果糖，转化率为43%。在重复的38次批次反应中，固定化细胞珠相对稳定，4个循环后保留了80%的原始酶活性。总之，我们开发了一种通过固定表达热稳定性 CE 的细胞来生产乳果糖的合理方法。需要进一步开发以在工业规模上应用这种方法。
Lactulose, a synthetic disaccharide, has received increasing interest because of its role as a prebiotic that can increase the proliferation of Bifidobacterium and Lactobacillus spp. and enhance the absorption of calcium and magnesium. While the industrial production of lactulose is still mainly achieved by the chemical isomerization of lactose in alkaline media, this process has drawbacks including the need to remove catalysts and by-products, as well as high energy requirements. Recently, the use of cellobiose 2-epimerase (CE) has been considered an interesting alternative for industrial lactulose production. In this study, to develop a process for enzymatic lactulose production using CE, we screened improved mutant enzymes (CS-(HCE)-C-R) from a library generated by an error-prone PCR technique. The thermostability of one mutant was enhanced, conferring stability up to 75., and its lactulose conversion yield was increased by 1.3-fold compared with that of wild-type CE. Using a recombinant Escherichia coli strain harboring a CS35 (HCE)-C-R expressing plasmid, we prepared cell beads immobilized on a Ca-alginate substrate and optimized their reaction conditions. In a batch reaction with 200 g/l lactose solution and the immobilized cell beads, lactose was converted into lactulose with a conversion yield of 43% in 2 h. In a repeated 38-plex batch reaction, the immobilized cell beads were relatively stable, and 80% of the original enzyme activity was retained after 4 cycles. In conclusion, we developed a reasonable method for lactulose production by immobilizing cells expressing thermostable CE. Further development is required to apply this approach at an industrial scale.