Construction and co-expression of polycistronic plasmids encoding thermophilic L-arabinose isomerase and hyperthermophilic β-galactosidase for single-step production of D-tagatose
Construction and co-expression of polycistronic plasmids encoding thermophilic L-arabinose isomerase and hyperthermophilic β-galactosidase for single-step production of D-tagatose
复制标题
编码嗜热 L-阿拉伯糖异构酶和超嗜热 β-半乳糖苷酶的多顺反子质粒的构建和共表达，用于一步生产 D-塔格糖
DOI：
10.1016/j.bej.2015.12.015
复制
发表时间：
2016-05-15
影响因子：
3.9
通讯作者：
Xu, Hong
中科院分区：
文献类型：
--
作者：
Xu, Zheng;Xu, Zhaoxian;Xu, Hong
塔格糖是一种天然存在的己酮糖。它在自然界中很少被发现，作为一种低热量的甜味剂和作为蔗糖替代品的潜力显示出优势。d -塔格糖的生物生产包括β -半乳糖苷酶水解乳糖和l -阿拉伯糖异构酶（AI）将释放的d -半乳糖异构化为d -塔格糖。在本研究中，两种耐热酶在大肠杆菌中共表达，包括来自发酵乳杆菌CGMCC2921的AI和来自嗜热热菌HB27的β -半乳糖苷酶。优化了两个基因的顺序、下游的Shine-Dalgarno （SD）序列和对齐间距（AS）长度，以平衡它们的表达水平。一株重组大肠杆菌以GAAGGAGA为下游SD序列，以10个核苷酸（nt）为as序列，显示出两种酶的平衡活性。在全细胞生物催化过程中，重组大肠杆菌细胞的最佳催化温度为70℃，pH为7.0℃。在添加硼酸盐的情况下，以廉价的乳糖为原料，在16 h内以101 g/L的浓度直接生产d -塔格糖，产率为20.2%，产率为6.3 g/Lh。本研究揭示了在嗜热生物转化系统中高效、经济的单步生产d -塔格糖。(C) 2015 Elsevier B.V.版权所有
D-Tagatose is a naturally occurred hexoketose. It is rarely found in nature and shows advantages as a low calorie sweetener and potentials as the substitute of sucrose. The biological production of D-tagatose involves hydrolysis of lactose by beta-galactosidase and isomerization of released D-galactose into D-tagatose by L-arabinose isomerase (AI). In this study, two thermostable enzymes including an AI from Lactobacillus fermentum CGMCC2921 and a beta-galactosidase from Thermus thermophilus HB27 were co-expressed in Escherichia coli. The order of the two genes, the downstream Shine-Dalgarno (SD) sequence and the aligned spacing (AS) length were optimized to balance their expression levels. One recombinant E. coli strain showed balanced activities for the two enzymes using GAAGGAGA as the downstream SD sequence and 10 nucleotides (nt) for AS. During whole cell bio-catalysis, the recombinant E. coli cells showed optimal catalytic temperature and pH at 70 degrees C and 7.0, respectively. With the addition of borate, D-tagatose was produced directly from cheap raw material lactose in 16 h in a concentration of 101 g/L, a yield of 20.2%, and a productivity of 6.3 g/Lh. This study shed light on the efficient and economical single-step production of D-tagatose in a thermophilic bio-conversion system. (C) 2015 Elsevier B.V. All rights reserved.