使用来自植物细胞壁的特定底物检测反式对香豆酰酯酶。

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls.

作者信息

Borneman W S, Hartley R D, Himmelsbach D S, Ljungdahl L G

机构信息

Richard B. Russell Agricultural Research Center, U.S. Department of Agriculture, Athens, Georgia 30613.

出版信息

Anal Biochem. 1990 Oct;190(1):129-33. doi: 10.1016/0003-2697(90)90145-y.

DOI:10.1016/0003-2697(90)90145-y

PMID:2285139

Abstract

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.

摘要

用多糖水解酶处理海岸狗牙根（狗牙根）的细胞壁，以释放O-[5-O-(反式对香豆酰基)-α-L-阿拉伯呋喃糖基]-(1→3)-O-β-D-吡喃木糖基-(1→4)-D-吡喃木糖（PAXX），通过液相色谱法分离该物质。通过1H NMR测定，分离得到的PAXX纯度大于95%，并用作反式对香豆酰酯酶灵敏测定的底物。PAXX被厌氧真菌新美鞭菌属MC-2的培养滤液水解。通过反相高效液相色谱法测定酶促水解释放的反式对香豆酸，最低可测定100 ng酸。酸释放的稳态速度符合米氏动力学。在pH 7.5和30℃下，Vmax测定为1.17 μmol min-1 mg-1，Km为13.2 μM。