林生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究

以林生山黧豆为材料，利用硫酸按分段盐析，丙酮沉淀，DEAE-SepharoseFF离子交换柱层析，SephacrylS300凝胶过滤柱层析及FPLC-MonoQ柱层析技术，以聚酰胺薄膜层析荧光定量法为酶活力检测手段，分离纯化了谷氨酸脱羧酶，达到电泳银染纯．纯化后的林生山黧豆谷氨酸脱羧酶活力达375．09U·mp-1，纯化倍数38．2倍，经SDS-PAGE测定，其亚基分子量为70kD，经梯度PAGE确定，天然分子量为140kD，表明该酶是由两个亚基组成的二聚体．酶学研究表明，纯化的林生山黧豆谷氨酸脱羧酶的最适pH值为5．4，对谷氨酸的Km值为1．62×10-3mol·L-1，酶的最适温度为40℃，酶特异性地使谷氨酸脱羧，不能使天门冬氨酸等其它氨基酸脱羧．

Studies on the Purification and Properties of glutamic Acid Decarboxylase from Flat Pea

Aprocedure to purify glutamic acid decarboxylase(GAD) was developed by (NH4)2 SO4 precipitation, acetone precipitation, DEAE-Sepharose FF, Sephacry1 5300, FPLC-Mono Q column chromatography with dansy1-C1 (DNS)method and polyamide thin-layer chromatography as the enzyme activity assay. The final preparation of GAD from flat pea(Lathyrus sylvestris L. )was homogenous as evaluated by SDS-PAGE and native gradient PAGE on which single silver staining protein band was showed. It appeared that the enzyme was a dimmer of identical subunits because that there was one 140kD band on native gradient PAGE and one 70kD band on SDS-PAGE. The purified GAD from flat Pea was specific for L-glutamic acid and could not decarboxylate other amino acids tested, such as aspartic acid. The optimum pH value of the enzymre was 5. 4. The optimum temperature was 40℃. Km value of the enzyme for L-glutamic acid was 1. 62 × 10-3mol·L-1.