pH-Dependent Activation of Streptomyces hygroscopicus Transglutaminase Mediated by Intein
pH-Dependent Activation of Streptomyces hygroscopicus Transglutaminase Mediated by Intein
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内含肽介导的吸水链霉菌转谷氨酰胺酶的 pH 依赖性激活
DOI：
10.1128/aem.02820-13
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发表时间：
2013-11
期刊：
影响因子：
--
通讯作者：
Zhemin Zhou
中科院分区：
文献类型：
--
作者：
Kun Du;Zhongmei Liu;Cui Wenjing;Yi Liu;Guocheng Du;Jian Chen;Zhemin Zhou
链霉菌属的微生物转氨酶（MTG）是一种天然分泌的酶原（pro-MTG），它的N端前区被其它蛋白酶去除后被激活。内含肽是一种蛋白质间插序列，在没有辅因子的情况下催化蛋白质剪接。将pH依赖型集胞藻（Synechocystis sp.）PCC 6803 DnaB mini-intein（SDB）基因导入pro-MTG中，通过控制pH简化其激活过程，获得重组蛋白pro-SDB-MTG，并确定其体外激活过程在pH 7时需要24 h。为了研究MTG第一个残基对酶活性和切割时间的影响，表达了两种变体pro-SDB-MTG（D1 S）和pro-SDB-MTG（ΔD1），发现其激活时间分别为6 h和30 h。重组MTG及其两个变体的酶学性质和二级结构与野生型相似，表明mini-intein的插入并不影响MTG的功能。分子动力学模拟进一步说明了这种不显著的影响。本研究揭示了一种可调控的、有效的策略来调控由小内含肽介导的pro-MTG的活化过程，具有很大的工业化生产潜力。
ABSTRACT Microbial transglutaminase (MTG) from Streptomyces is naturally secreted as a zymogen (pro-MTG), which is then activated by the removal of its N-terminal proregion by additional proteases. Inteins are protein-intervening sequences that catalyze protein splicing without cofactors. In this study, a pH-dependent Synechocystis sp. strain PCC6803 DnaB mini-intein (SDB) was introduced into pro-MTG to simplify its activation process by controlling pH. The recombinant protein (pro-SDB-MTG) was obtained, and the activation process was determined to take 24 h at pH 7 in vitro. To investigate the effect of the first residue in MTG on the activity and the cleavage time, two variants, pro-SDB-MTG(D1S) and pro-SDB-MTG(ΔD1), were expressed, and the activation time was found to be 6 h and 30 h, respectively. The enzymatic property and secondary structure of the recombinant MTG and two variants were similar to those of the wild type, indicating that the insertion of mini-intein did not affect the function of MTG. This insignificant effect was further illustrated by molecular dynamics simulations. This study revealed a controllable and effective strategy to regulate the activation process of pro-MTG mediated by a mini-intein, and it may have great potential for industrial MTG production.
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影响因子：
5
作者：
Heck, Tobias;Faccio, Greta;Richter, Michael;Thoeny-Meyer, Linda
通讯作者：
Thoeny-Meyer, Linda
影响因子：
2.9
作者：
Mototaka Suzuki;K. Sakurai;Young-Ho Lee;T. Ikegami;K. Yokoyama;Y. Goto
通讯作者：
Mototaka Suzuki;K. Sakurai;Young-Ho Lee;T. Ikegami;K. Yokoyama;Y. Goto
影响因子：
4.4
作者：
Fu, RY;Chen, J;Li, Y
通讯作者：
Li, Y
影响因子：
4.1
作者：
Kalé, L;Skeel, R;Schulten, K
通讯作者：
Schulten, K
影响因子：
15
作者：
ALBERTY, RA
通讯作者：
ALBERTY, RA