Bacheva A V, Baĭbak O V, Beliaeva A V, Lysogoskaia E N, Oksenoĭt E S, Lozinskiĭ V I, Filippova I Iu
Faculty of Chemistry, Moscow State University, Vorob'evy gory, Moscow, 119992 Russia.
Bioorg Khim. 2003 Sep-Oct;29(5):551-8. doi: 10.1023/a:1026013912147.
The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.
研究了天然枯草杆菌蛋白酶、其与聚丙烯酸的非共价复合物以及共价固定在聚乙烯醇冷冻凝胶中的枯草杆菌蛋白酶在低含水量有机溶剂混合物中的肽偶联反应中的催化效率，该效率取决于介质组成、反应时间和生物催化剂浓度。结果表明，在二甲基甲酰胺（DMF）含量>80%的介质中，修饰后的枯草杆菌蛋白酶的合成酶活性高于天然枯草杆菌蛋白酶。发现在由天然和固定化枯草杆菌蛋白酶催化的酶促合成过程中，在有机溶剂中使用具有游离羧基的N-酰基肽是可行的。在固定化酶存在的情况下，在DMF-乙腈中获得了一系列通式为Z-Ala-Ala-Xaa-Yaa-pNA的四肽对硝基苯胺（其中Xaa为亮氨酸或谷氨酸，Yaa为苯丙氨酸或天冬氨酸），产率为70-98%，无需羧基组分的任何活化，也无需保护多官能氨基酸的侧链离子基团。
