Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg.
Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg.
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DOI：
10.1016/j.jbiotec.2013.06.017
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发表时间：
2013-09
影响因子：
4.1
通讯作者：
Dragana Despotović;Ljubica Vojcic;M. Blanusa;K. Maurer;M. Zacharias;M. Bocola;Ronny Martínez;U. Schwaneberg
中科院分区：
文献类型：
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作者：
Dragana Despotović;Ljubica Vojcic;M. Blanusa;K. Maurer;M. Zacharias;M. Bocola;Ronny Martínez;U. Schwaneberg
酶混杂性描述了生物催化剂催化其自然反应之外的转化的能力。促进副反应的酶工程对于合成和工业应用是有吸引力的。例如，枯草杆菌蛋白酶Carlsberg蛋白酶变体（T58 A/L216 W）除了其蛋白水解活性之外，还催化在过氧化氢存在下由相应的酯生成过氧羧酸。在目前的研究中，我们使用了一种半理性的设计方法来转移的特异性枯草杆菌蛋白酶嘉士伯对生产过氧羧酸。在其他确定的氨基酸取代，位置Gly 165在S1结合口袋中提供的见解枯草杆菌蛋白酶嘉士伯的混杂促进酯过水解。枯草杆菌蛋白酶Carlsberg对丙酸甲酯、丁酸甲酯和戊酸甲酯过水解的催化常数分别提高了3.5倍、5.4倍和5.5倍，而对N-琥珀酰-Ala-Ala-Pro-Phe-对硝基苯胺底物（suc-AAPF-pNA）的蛋白水解降低了100倍。
Enzyme promiscuity describes the ability of biocatalysts to catalyze conversions beyond their natural reactions. Enzyme engineering to promote side reactions is attractive for synthetic and industrial applications. For instance, a subtilisin Carlsberg protease variant (T58A/L216W) catalyzes in addition to its proteolytic activity the generation of peroxycarboxylic acids from corresponding esters in the presence of hydrogen peroxide. In the current study we used a semi-rational design approach to shift the specificity of subtilisin Carlsberg towards production of peroxycarboxylic acid. Among other identified amino acid substitutions, position Gly165 in the S1 binding pocket provided insights in subtilisin Carlsberg's promiscuity by promoting ester perhydrolysis. Catalytic constants of subtilisin Carlsberg for perhydrolysis of methyl-propionate, methyl-butyrate and methyl-pentanoate were increased up to 3.5-, 5.4- and 5.5-fold, respectively, while proteolysis was decreased up to 100-fold for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate (suc-AAPF-pNA).