Wong Dominic W S, Chan Victor J, McCormack Amanda A, Hirsch Ján, Biely Peter
Western Regional Research Center, USDA-ARS, Albany, CA 94710, USA.
Biotechnol Res Int. 2012;2012:951267. doi: 10.1155/2012/951267. Epub 2012 Jul 4.
The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K(m) 0.25 mM, V(max) 16.3 μM·min(-1), and k(cat) 9.27 s(-1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.
编码裂褶菌葡糖醛酸酰基酯酶的基因在基因组的支架17中被鉴定出来，该基因含有两个分别为50 bp和48 bp的内含子，转录序列为1179 bp。该基因被合成并克隆到毕赤酵母表达载体pGAPZα中，以实现重组酶以可溶性活性形式的组成型表达和分泌。纯化后的蛋白质经糖基化后为53 kD，酸性等电点为3.7。对几种糖醛酸及其衍生物的活性分析表明，该酶仅识别4-O-甲基-D-葡糖醛酸衍生物的酯，即使带有4-硝基苯基糖苷配基，但不水解D-半乳糖醛酸的酯。以4-硝基苯基2-O-（甲基4-O-甲基-α-D-吡喃葡糖醛酸基）-β-D-吡喃木糖苷为底物时，动力学值为K(m) 0.25 mM，V(max) 16.3 μM·min(-1)，k(cat) 9.27 s(-1)。
