Two new thermostable α-L-rhamnosidases from a novel thermophilic bacterium
Two new thermostable α-L-rhamnosidases from a novel thermophilic bacterium
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DOI：
10.1016/j.enzmictec.2003.12.012
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发表时间：
2004-05-01
影响因子：
3.4
通讯作者：
Mattiasson, B
中科院分区：
文献类型：
--
作者：
Birgisson, H;Hreggvidsson, GO;Mattiasson, B
从一株新的嗜热菌中克隆并表达了两种新的具有新底物水解型式的耐热α-L鼠李糖苷酶。在该细菌部分测序的基因组中鉴定出两个α-L鼠李糖苷酶基因片段，即rrmA和rrmB。用单一的特异性和非特异性的步行性引物分别扩增其侧翼序列，获得完整的基因序列。然后将回收的基因克隆到大肠杆菌中，并生产和纯化它们的酶。这两种酶都是二聚体和单体的相对分子质量。RhmA和RhmB分别为104 kDa和107 kDa。两种鼠李糖苷酶的最适温度均为70℃。RhmA的最适pH为7.9，RhmB的最适pH为5.0~6.9，RhmA的最适pH为5.0~8.7，RhmB的最适pH为4.0~7.9，活性均在50%以上。在60℃下孵育24小时后，两种酶的残余活力均在20%以上。在对-硝基苯基-α-L-鼠李糖侧，RhmA和RhmB的K值分别为0.46和0.66 mm，V-max分别为134和352U mg(-1)。两种鼠李糖苷酶在α-1，2-和α-1，6-连接到β-D-葡萄糖苷上都是活性的。(C)2004 Elsevier Inc.保留所有权利。
Two new thermostable alpha-L-rhamnosidases with novel substrate hydrolysis pattern were cloned and expressed from a new thermophilic bacterium. Fragments of the two alpha-L-rhamnosidase genes, rhmA and rhmB were identified in a partially sequenced genome of the bacterium. Whole genes were recovered by amplifying flanking sequences with single specific primers and nonspecific walking primers. The recovered Genes were then cloned into Escherichia coli and their enzymes produced and purified. Both enzymes were dimers and the MW of the monomers. were 104 and 107 kDa for RhmA and RhmB, respectively. Both rhamnosidases had a temperature optimum at 70degreesC. RhmA had pH optimum at 7.9 and RhmB had a broad pH optimum of 5.0 to 6.9 and RhmA had over 50% activity in the pH interval 5.0 to 8.7 and RhmB in the pH interval 4.0 to 7.9. Both enzymes had over 20% residual activity after 24-h incubation at 60degreesC. RhmA and RhmB had K values of 0.46 and 0.66 mM and V-max values of 134 and 352 U mg(-1) respectively, on p-nitrophenyl-alpha-L-rhamnopyrano side. Both rhamnosidases were active on both alpha-1,2- and alpha-1,6-linkages to beta-D-glucoside. (C) 2004 Elsevier Inc. All rights reserved.