摘要
采用溶菌酶处理、超声破碎、硫酸铵分级、3次DEAE SepharoseCL 6B层析、SephacrylS 200凝胶过滤等手段,从乳酸菌细胞中分离纯化得到谷氨酸脱羧酶(GAD;EC4.1.1.15).纯化酶的比活力为14.4U/mg,纯化倍数31,回收率3.8%,SDS PAGE得到亚基的相对分子质量为65000.L 谷氨酸是测试的18种氨基酸中的惟一作用底物,表明乳酸菌GAD具有高度的底物专一性.酶在pH值3.6～5.4时具有活性,pH值4.7时活性最高,pH值6.0以上时没有活力.耐热性实验表明,pH值4.7条件下处理5h后,60℃时该酶仍能保持80%以上的活性,80℃以上迅速失活,由Lineweaver Burk作图得到的GAD的Km值为1.9mmol/L.
Glutamate Decarboxylase (GAD; EC4.1.1.15) was purified from Lactococcus lactis SYFS1.009 with lysozymic treatment, sonication, ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme, concentrated about 31-fold, is 14.4 U/mL with a yield of 3.8%. Single band obtained by SDS-PAGE showed subunit Mw is 65 000. L-glutamate is the unique decarboxylation substrate among 18 amino acids tested, suggesting the enzyme be highly specific. The enzyme shows is activity at acidic pH (3.6～5.4) range and the optimal pH is (4.7). No activity was detected above pH6.0. The enzyme keeps more than 80% of its activity below 60 ℃ during 5-hour storage and was inactivated rapidly at 80 ℃. The apparent K(m) of the enzyme obtained from Lineweaver-Burk plot was 1.9 mmol/L.
出处
《无锡轻工大学学报（食品与生物技术）》
CSCD
北大核心
2004年第3期79-84,共6页
Journal of Wuxi University of Light Industry
