摘要
Various combinations of the four domains of the multifunctional mannanase from Caldicellosiruptor saccharolyticus have been cloned and expressed in Escherichia coli. The four domains comprise two catalytic domains (1 and 4), and two putative cellulose binding domains (2 and 3). Each of the six gene products (Man1, Man123, Man1234, Man23, Man234 and Man4) was partially purified by heat treatment.
The enzymes Man1234, Man123 and Man1 exhibited activity on mannans, and Man1234, Man234 and Man4 exhibited activity on xylan and carboxymethylcellulose (CMC). For the complete enzyme (Man1234) all activities were of the same order of magnitude. Activities were additive against a mixture of mannan and xylan or mannan and CMC (but not xylan and CMC). The expression product Man23 exhibited activity on none of the substrates tested, nor did its presence influence thermostability or significantly reduce the Km value for any of the substrates. However, when expressed in combination with domains 1 or 4 it greatly increased their activity.
We conclude that domain 1 catalyses mannan hydrolysis and domain 4 catalyses xylan and CMC hydrolysis at the same active site: domains 2 and 3 have no obvious function, since they do not reduce substrate Km nor affect thermostability. However, their effect on rates of substrate hydrolysis may indicate a role influencing the conformation of the adjacent catalytic domains.
摘要译文
已经克隆了来自Caldicellosiruptor saccharolyticus的多功能甘露聚糖酶的四个结构域的各种组合并在大肠杆菌中表达。四个结构域包含两个催化结构域（1和4），以及两个推定的纤维素结合结构域（2和3）。通过热处理部分纯化六种基因产物（Man1，Man123，Man1234，Man23，Man234和Man4）中的每一种。酶Man1234，Man123和Man1对甘露聚糖表现出活性，Man1234，Man234和Man4对木聚糖和羧甲基纤维素（CMC）表现出活性。对于完整的酶（Man1234），所有活性都具有相同的数量级。对甘露聚糖和木聚糖或甘露聚糖和CMC（但不是木聚糖和CMC）的混合物添加活性。表达产物Man23对所测试的底物均未显示出活性，其存在也不影响热稳定性或显着降低任何底物的K m值。然而，当与结构域1或结构4组合表达时，它大大增加了它们的活性。我们得出结论，结构域1催化甘露聚糖水解，结构域4在相同的活性位点催化木聚糖和CMC水解：结构域2和3没有明显的功能，因为它们不降低底物K m也不影响热稳定性。然而，它们对底物水解速率的影响可能表明影响相邻催化结构域构象的作用。
TracyFrangos[a];DeniseBullen[a];PeterBergquist[b];RoyDaniel[a];. Hemicellulolytic and cellulolytic functions of the domains of a β-mannanase cloned from Caldicellosiruptor saccharolyticus[J]. The International Journal of Biochemistry & Cell Biology, 1999,31(8): 853-859