Biocatalytic synthesis of lactosucrose using a recombinant thermostable β-fructofuranosidase from Arthrobacter sp. 10138
Biocatalytic synthesis of lactosucrose using a recombinant thermostable β-fructofuranosidase from Arthrobacter sp. 10138
复制标题
使用来自节杆菌属的重组热稳定β-呋喃果糖苷酶生物催化合成乳果糖。
DOI：
10.1080/21655979.2020.1739404
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发表时间：
2020-01-01
期刊：
影响因子：
4.9
通讯作者：
Liu, Long
中科院分区：
文献类型：
--
作者：
Chen, Chunmei;Deng, Jieying;Liu, Long
低聚乳果糖作为一种益生元，在维持人体胃肠道内环境稳定方面起着重要作用。本研究从6株β-呋喃果糖苷酶产生菌中筛选出一株产低聚乳果糖的节杆菌10138耐热酶，并将其编码基因在大肠杆菌中进行异源表达。对重组β-呋喃果糖苷酶进行了纯化和MALDI-TOFMS生化分析。经HPLC和LC-MS分析，该重组酶的果糖基转移产物为低聚乳蔗糖，而不是其他寡糖或多糖。通过信号肽和诱导条件的优化，实现了β-呋喃果糖苷酶的高效胞外分泌。结果表明，在信号肽torT的作用下，胞外酶活最高可达111.01 U/mL，是信号肽OmpA的38.4倍。在最佳条件下（pH6.0，温度50 ℃，加酶量40 μ g/ml，蔗糖150 g/L，乳糖150 g/L），低聚乳果糖产量为109 g/L，摩尔转化率为49.3%。节杆菌10138耐热β-呋喃果糖苷酶可用于低聚乳蔗糖的高效合成，为低聚乳蔗糖的工业化生产奠定了基础。[图形]。
As a prebiotics, lactosucrose plays an important role in maintaining human gastrointestinal homeostasis. In this study, a thermostable enzyme from Arthrobacter sp. 10138 was screened from six beta-fructofuranosidase-producing strains for the lactosucrose production and the coding gene was heterologously expressed in Escherichia coli for efficient expression. Recombinant beta-fructofuranosidase was purified and biochemically characterized by MALDI-TOFMS spectrometry. The transfructosylation product by this recombinant enzyme was determined to be lactosucrose rather than other oligosaccharides or polysaccharides by HPLC and LC-MS. Efficient extracellular secretion of beta-fructofuranosidase was achieved by the optimization of signal peptide and induction conditions. It was found that with the signal peptide torT, the highest extracellular activity reached 111.01 U/mL, which was 38.4-fold higher than that with the OmpA signal peptide. Under the optimal conditions (pH 6.0, temperature 50 degrees C, enzyme amount 40 mu g/ml, sucrose 150 g/L and lactose 150 g/L), 109 g/L lactosucrose was produced with a molar conversion ratio of 49.3%. Here the thermostable beta-fructofuranosidase from Arthrobacter sp. 10138 can be used for efficient synthesis of lactosucrose, and this provides a good startpoint for the industrial production of lactosucrose in the future.[GRAPHICS].