A family of thermostable fungal cellulases created by structure-guided recombination
A family of thermostable fungal cellulases created by structure-guided recombination
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DOI：
10.1073/pnas.0901417106
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发表时间：
2009-04-07
影响因子：
11.1
通讯作者：
Arnold, Frances H.
中科院分区：
文献类型：
--
作者：
Heinzelman, Pete;Snow, Christopher D.;Arnold, Frances H.
SCHEMA结构引导重组的3种真菌II类纤维二糖水解酶（CBH II纤维素酶）产生了高度耐热CBH II嵌合体的集合。从6，561个可能的嵌合序列中取样的48个基因中的23个由酿酒酵母异源宿主以催化活性形式分泌。这些嵌合体中的五个在63摄氏度下的热灭活半衰期大于最稳定的亲本，来自嗜热真菌Humicola insolens的CBH II酶，这表明该嵌合体集合包含数百种高度稳定的纤维素酶。基于第一组嵌合体的热稳定性的数学建模，设计了25个新序列。这些序列中有10个以活性形式表达;所有10个都比H保留了更多的活性。insolens CBH II。总共15种经验证的热稳定CBH II酶具有高序列多样性，与其最接近的天然同源物在多达63个氨基酸位置上不同。选定的纯化的热稳定嵌合体水解磷酸溶胀纤维素在7至15摄氏度的温度高于亲本酶。这些嵌合体在长时间纤维素水解测定中也水解了与亲本CBH II酶一样多或更多的纤维素，并且具有与亲本酶一样宽或更宽的pH/活性曲线。产生这组多样的热稳定真菌CBH II嵌合体是建立稳定纤维素酶库存的第一步，从中可以配制用于生物质转化的优化酶混合物。
SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63 degrees C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63 degrees C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15 degrees C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.