Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005
Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005
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DOI：
10.1007/s11274-013-1385-2
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发表时间：
2013-05
影响因子：
4.1
通讯作者：
Yun Gao;Yue Xi;Xiaoling Lu;Heng Zheng;Bo Hu;Xiao-Yu Liu;B. Jiao
中科院分区：
文献类型：
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作者：
Yun Gao;Yue Xi;Xiaoling Lu;Heng Zheng;Bo Hu;Xiao-Yu Liu;B. Jiao
海藻糖合酶（TreS）催化麦芽糖和海藻糖的可逆相互转化。从海洋假单胞菌中克隆了一个新的treS基因，全长3,369 bp，编码1,122个氨基酸残基，预测分子量为126 kDa。P8005（CCTCC：M2010298）在大肠杆菌中表达。该新的TreS与其他已报道的TreS之间的氨基酸序列同源性相对较低。纯化的重组TreS显示最适pH和温度分别为7.2和37 °C。该酶在平衡过程中对麦芽糖转化为海藻糖的转化率高达70%，对麦芽糖的催化效率（kcat/Km）高于海藻糖，表明该酶可用于海藻糖的生产。除了麦芽糖和海藻糖外，这种酶还可以作用于蔗糖，尽管这种活性相对较低。突变研究表明，单独用丙氨酸取代D 78，Y81，H121，D219，E261，H331或D332，酶活性显著降低，这意味着这些残基可能在P8005-TreS中是重要的。使用同位素标记的底物的实验表明，[2 H2]海藻糖与未标记的海藻糖结合可转化为[2 H2]麦芽糖和麦芽糖，但没有产生任何[2 H]麦芽糖或[2 H]海藻糖，也没有将外源[2 H7]葡萄糖掺入该酶催化的转化过程中的二糖中。这一发现表明P8005-TreS催化麦芽糖和海藻糖的可逆相互转化涉及分子内机制。
Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A noveltreSgene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marinePseudomonassp. P8005 (CCTCC: M2010298) and expressed inEscherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (kcat/Km) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [2H2]trehalose combined with unlabeled trehalose was converted to [2H2]maltose and maltose, but without any production of [2H]maltose or [2H]trehalose and with no incorporation of exogenous [2H7]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.