Enzymatic synthesis of hydroxyl phthalic acid as a monomer for proton-conducting polymer

本研究以开发质子传导聚合物单体2-羟基邻苯二甲酸和3-羟基-4-甲基邻苯二甲酸的新型酶促合成方法为目的，分离得到3株具有2-羟基邻苯二甲酸脱羧酶活性的细菌，分别命名为WU-0402、WU-0403和WU-0404。对这些菌株的2-羟基邻苯二甲酸脱羧酶活性进行了研究。WU-0404的生长细胞通过2-羟基邻苯二甲酸的脱羧作用完全降解作为产物的3-羟基苯甲酸。另一方面，WU-0402和WU-0403是从2-羟基邻苯二甲酸化学计量积累的3-羟基苯甲酸。根据羟基苯甲酸脱羧酶的保守序列，从WU-0404菌株中克隆了2-羟基邻苯二甲酸脱羧酶基因。推导的氨基酸序列显示约40%的相似性对羟基苯甲酸脱羧酶，以前报道的。重组大肠表达该基因的大肠杆菌对2-羟基邻苯二甲酸和3-羟基苯甲酸均无脱羧活性。然而，该脱羧酶的N-末端氨基酸不能确定，因为N-末端可能被封闭。

This research purpose is the development of novel enzymatic synthesis method of 2-hydroxyphthalic acid and 3-hydroxy-4-methyl phthalic acid as a monomer for proton-conducting polymer.Three bacteria showing decarboxylase activity of 2-hydroxyphthalic acid were isolated and designated as WU-0402, WU-0403, and WU-0404, respectively. The 2-hydroxyphthalic acid decarboxylase activity of these strains wasinvestigated. The growing cell of WU-0404 completely degraded 3-hydroxybenzoic acid as a product by the decarboxylation of 2-hydroxyphthalic acid. On the other hand, WU-0402 and WU-0403 were stoichiometrically accumulated 3-hydroxybenzoic acid from 2-hydroxyphthalic acid. Although the carboxylation activity toward 3-hydroxybenzoic acid of three strains was investigated, no activity was shown.Based on the conserved sequences of hydroxyl benzoic acid decarboxylase, 2-hydroxyphthalic acid decarboxylase from WU-0404 was cloned. The deduced amino acid sequence showed approximately 40% similarity toward hydroxyl benzoic acid decarboxylases that were previously reported. The recombinant E. coli expressing this gene did not show both decarboxylating toward 2-hydroxyphthalic acid and carboxylating activity toward 3-hydroxybenzoic acid.The 2-hydroxyphthalic acid decarboxylase was purified from WU-0404 by 4 steps chromatography. However, N-terminus amino acid of this decarboxylase could not be determined, because N-terminus may be blocked.