全 文 ：食用菌学报 2009.16(2):51～ 55
收稿日期:2009-05-06原稿;2009-06-06 修改稿
基金项目:教育部科学技术研究重点项目“ 香菇采后保鲜和加工过程中内生甲醛的形成和调控机理”(编号:
208054)的部分研究内容
作者简介:尹　洁(1983-), 女 ,浙江工商大学食品学院在读硕士研究生 ,主要从事食品生物技术方面的研究。
＊本文通讯作者
文章编号:1005-9873(2009)02-0051-05
香菇中γ-谷氨酰转肽酶的分离纯化及其酶学性质研究
尹　洁 , 朱军莉 , 傅玲琳 , 励建荣＊
(浙江工商大学食品与生物工程学院 ,浙江省食品安全重点实验室 ,浙江杭州 310035)
摘　要:通过(NH4)2SO4 沉淀 、Phenyl Sepharose FF 疏水层析分离纯化香菇中的 γ-谷氨酰转肽酶(γ-
Glutamyltr anspeptidase ,GGT),纯化后的 GGT 的比活力到达了 19.59 U/mg。SDS-PAGE 分析表明 ,GGT 由
分子量分别为 28 kDa 和60 kDa的两个亚基组成。酶学性质试验结果表明 , GGT 反应的最适温度为 37 ℃, 最
适 pH 值为 7.6;金属离子 Na+ 、K +和 Ca2+对酶有激活作用 , 而 Ag+ 、Cu2+、Zn2+和 Fe3+则有抑制作用;试验
范围内 , GGT 水解 γ-glutamyl-p-nitr oanilide 的米氏动力学参数 K m 值为 2.601 μmol/mL , V max 值为
0.0287 μmol/min;组成的氨基酸中 ,Glu和 Asp 含量较高 , Met和 Cys 含量较低。
关键词:香菇;γ-谷氨酰转肽酶;分离纯化;酶学性质
　　γ-谷氨酰转肽酶(γ-Glutamyltranspeptidase ,
GGT)是广泛存在于生物体内参与谷胱甘肽循环
的酶 , 是香菇中催化该反应产生甲醛的关键
酶[ 1 ～ 5] 。胡子豪等向香菇子实体中分别添加 Vc ,
NO以及谷胱甘肽代谢循环的各中间产物 ,能使
香菇中甲醛的含量发生显著地变化[ 6] 。我们从
香菇中提取 、分离纯化 GGT ,并对其相关的特性
进行研究 ,以期为阐述香菇内源性甲醛的形成机
理提供酶学背景资料。
1　材料与方法
1.1 材料
　　新鲜香菇采自浙江庆元 ,经真空冷冻干燥后
粉碎 ,保存于-70 ℃。
1.2 试剂
　 　γ-谷氨 酰-对硝 基苯 胺(γ-Glutamyl-p-
nit roanil ide)、β-巯基乙醇(β-mercaptoethanol)、
对硝基苯胺(p-ni troanilide)购于美国 Sigma 公
司;Phenyl Sephar ose FF 购于瑞典 Pharmacia
公司;其余试剂为国产 AR 级试剂。
1.3 方法
1.3.1 GGT 的粗提
　　25 g冻干的香菇粉 ,加入 300 mL 0.05 mol/L
的 Tris-HCl 缓 冲 液 (含 1 mmol/L EDTA 、
5 mmo l/L β-巯基乙醇 , pH 7.6),在 4 ℃下匀浆 4
次 ,每次 30 s(IKA T25 匀浆机 , 德国), 离心
(4 ℃, 8 000 g , 20 min), 取上清 ,加(NH4)2SO4
至 30%饱和度 , 4 ℃静置 2 h 后 , 离心(4 ℃,
8 000 g ,20 min),取上清 ,加(NH4)2SO4 至 70%
饱和度 , 4 ℃静置 2 h 后 , 离心(4 ℃, 8 000 g ,
20 min),取沉淀 ,溶解于 10 mL 的 0.05 mol/L
的 Tris-HCl缓冲液 ,得到粗酶液。
1.3.2 Phenyl Sephar ose FF 柱层析
　　将得到的粗酶液过 Phenyl Sephar ose FF 层
析柱(2.6 cm×20 cm)分离纯化。平衡缓冲液为
0.05 mol/ L Tris-HCl 、1.0 mol/L(NH4)2SO4
(pH 7.6),洗脱缓冲液为 0.05 mol/ L Tris-HCl
(pH 7.6),流速为 2 mL/min [ 7] 。
1.3.3 蛋白质含量的测定
　　采用考马斯亮蓝法测定 GGT 的蛋白质含
量 , 以牛血清白蛋白(bovine ser um albumin ,
BSA)作为标准蛋白[ 8] 。
1.3.4 电泳分析
　　纯化后的 GGT按文献[ 9]的方法进行 SDS-
聚丙烯酰胺凝胶电泳(SDS-PAGE)。
1.3.5 GGT活性的测定
　　GGT 催化 γ-glutamyl-p-nitr oanil ide 的产
物 p-nit roanil ide 在 410 nm 处有吸光值[ 10] , 酶
食　用　菌　学　报 第 16 卷
活性由生 成产物 的量反映 。反应 体系为
0.5 mol/LTris-HCl(pH 7.6)缓冲液 4 mL 、酶液
0.5 mL 、3.5μmol/mL底物 0.5 mL , 37 ℃反应
20 min ,加入 3 mL 1.5 mol/L乙酸终止反应 ,过
滤后在410 nm处测吸光值 。酶活单位(U)定义
为在上述反应条件下 ,每分钟生成 1 μmol的 p-
nit roanil ide 为 1个酶活单位。
1.3.6 酶的热稳定性试验
　　将 0.5 mL酶液分别置于 20 ℃, 25 ℃,30 ℃,
35 ℃,40 ℃,45 ℃,50 ℃,60 ℃,70 ℃和 80 ℃,保
温30 min ,再按 1.3.5中的方法测定酶活性。
1.3.7 最适反应温度试验
　　将反应温度分别设为 20 ℃, 25 ℃, 30 ℃,
35 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃和 70 ℃,按照
1.3.5中的方法测定酶活性。
1.3.8 最适 pH 值试验
　　将酶反应液的 pH 值分别调整为 3.0 、4.0 、
5.0 、5.5 、6.0 、6.5 、7.0 、7.5 、8.0 、8.5 、9.0 、10.0
(pH 4.0 ～ 6.5用 0.1 mol/L 乙酸缓冲液调整 ,
pH 7.5 ～ 10.0用 0.1 mol/L Tris 调整),再在最
适反应温度下按 1.3.5中的方法测定酶活性 。
1.3.9 金属离子对酶活性的影响
　　在 Tris-HCl 缓冲液中分别加入浓度为
10 mmol/ L 的 N a+ , Ag+ , K + , Fe3+ , Ca2+ ,
Mn2+ , Mg2+ , Zn2 +和 Cu2+ ,再在最适反应温度
下 ,按1.3.5中的方法测定酶活性 。
1.3.10 GGT 催化水解 γ-glutamyl-p-nit roanilide
的动力学参数测定
　　将底物浓度梯度分别设为 0 、50 、100 、150 、
200 和 250 mmol/ L , 再在最适反应温度下 ,按
1.3.5中的方法测定酶活性。以 1/v 对 1/ [ S]作
图 , 用 Linewear-Bur k 作图法求得 GGT 对 γ-
glutamyl-p-nitr oanilide 的 K m 和V max值。
1.3.11 氨基酸组成分析
　　采用 L-8800型氨基酸自动分析仪测定 GGT
的氨基酸组成及含量(Hitachi ,日本)[ 11] 。
2　结果与分析
2.1 γ-GGT的分离纯化
2.1.1 (NH 4)2SO 4 沉淀
　　(NH 4)2 SO 4 分步沉淀结果表明 ,在饱和度为
30%时 ,GGT 开始析出 ,饱和度为 70%时 ,可以
将>90%的GG T 沉淀出 ,即 70%(NH 4)2SO4 饱
和度即可有效沉淀目标蛋白 。
2.1.2 疏水性相互作用层析
　　(NH4)2SO4 沉淀后得到的粗酶液经 Phenyl-
Sepharose FF柱层析洗脱得到1个有 GGT 活性的
峰(图 1),将活性洗脱峰收集浓缩后 ,采用 SDS-
PAGE的方法检测其纯度。结果表明 ,该活性峰的
纯度较高 ,基本为单一蛋白质;含有两个亚基 ,分子
量分别为 28 kDa和 60 kDa左右(图 2)。
2.1.3 分离纯化效率
　　在整个 GGT 的分离纯化过程中 ,各步骤的分
离纯化效率见表 1。结果表明 ,GGT 经分离纯化
后其比活力增加了 178.1倍 ,回收率为23.7%。
2.2 γ-GGT的酶学性质
2.2.1 酶的热稳定性
　　热稳定性试验结果表明 ,GGT 在 20 ～ 40 ℃保
温30 min后基本不失活 ,但温度>45 ℃时 ,酶活力
迅速降低 ,在 70 ℃保温 30 min后酶活力完全丧失。
52
第 2 期 尹　洁 ,等:香菇中γ-谷氨酰转肽酶的分离纯化及其酶学性质研究
表 1　GGT的收率和纯度
Table 1　Recovery and purification of GGT from L.edodes fruit bodies
样品
Pur if ica tion step
体积
Volume
(mL)
总蛋白
Protein
(mg)
总活力
Total activity
(U)
比活力
Specific activity
(U/mg)
提纯倍数
Purif ication
fold
得率
Yie ld
(%)
匀浆液 Homogenate 200 4 617.0 515 0.11 1.0 100.0
离心后上清液 Crude enzyme 168 426.0 387 0.91 8.3 75.2
30%硫酸铵沉淀后的上清
Supe rnat ant af te r 30%(NH4)2SO4 satura tion 157 196.0 289 1.48 13.5 56.1
70%硫酸铵沉淀
Redissolved precipitate after 70%
(NH4)2SO4 satur ation
10 31.0 136 4.39 39.9 26.4
Phenyl-Sepha rose柱纯化后的酶液
Phenyl-Sepharose chromatography 125 6.3 122 19.59 178.1 23.7
2.2.2 酶反应的最适温度
　　酶的最适反应温度试验结果表明(图 3),
GGT的最适反应温度为 37 ℃。
数据为 3次重复的平均值 Values are the means of three replicates
图 3　温度对 GGT活力的影响
Fig.3　Effect of temperature on GGT activity
2.2.3 酶反应的最适 pH 值
　　GGT反应时最适 pH 为 7.5 ～ 8.0 ,该酶在
pH 高于 8.5的碱性条件或 pH 低于 6.5的酸性
条件时 ,酶活力明显下降(图 4)。
数据为 3次重复的平均值 Values are th e means of three replicates
图 4　pH对 GGT活力的影响
Fig.4　Effect of pH on GGT activity
2.2.4 金属离子对酶活性的影响
　　Na+ 、K+和 Ca2+对 GGT 活性有激活作用 ,而
Cu
2+、Ag+、Zn2+和Fe3+对酶活性有抑制作用(图 5)。
　　　　　　　数据为 3次重复的平均值 Values ar e the means of th ree replicates
图 5　金属离子对 GGT活性的影响
Fig.5　Effect of metal ions on GGT activity
53
食　用　菌　学　报 第 16 卷
2.2.5 酶催化水解 γ-glutamyl-p-nit roanil ide 的
动力学特性
　　图 6表明 ,在底物浓度为 50 ～ 250 mmol/L
范围内 , GGT 催化水解 γ-glutamyl-p-nit ro-
anilide的速度符合米-门二氏动力学模式 ,求得
的 K m 和 V max 值分别为 2.601 μmol/mL 和
0.0287 μmol/min 。
数据为 3次重复的平均值 Values are the means of three replicates
图 6　GGT的 Lineweaver-Burk图
Fig.6　Lineweaver-Burk plot for GGT
2.2.6 GGT的氨基酸组成及含量
　　GGT 的氨基酸组成中(表 2),碱性氨基酸
(Arg 、Lys 、His 、Pr o)与酸性氨基酸(Glu 、Asp)比
值为 1∶1.84 ,谷氨酸和天门冬氨酸含量很高 ,亮
氨酸 、苏氨酸的含量也较高 ,半胱氨酸 、蛋氨酸的
含量很低 。
表 2　GGT的氨基酸分析
Table 2　Amino acid composition of GGT
氨基酸
Amino acid
含量(%)
Concentration
氨基酸
Amino acid
含量(%)
Concentrat ion
天门冬氨酸(Asp)11.95 异亮氨酸(Ile) 6.03
苏氨酸(Thr) 6.86 亮氨酸(Leu) 9.25
丝氨酸(Ser) 5.82 苯丙氨酸(Phe) 4.78
谷氨酸(Glu) 12.58 赖氨酸(Lys) 4.26
甘氨酸(Gly) 5.61 组氨酸(His) 2.81
丙氨酸(Ala) 5.82 精氨酸(Arg) 6.24
半胱氨酸(Cys) 2.08 脯氨酸(Pro) 4.37
蛋氨酸(Met) 1.46 缬氨酸(Val) 4.99
酪氨酸(Typ) 5.09
数据为 3次重复的平均值 Values are the means of three replicates
3　讨论
　　本试验通 过(NH4)2SO4 沉淀 、 Pheny l
Sepharose FF 疏水层析分离纯化香菇中的
GG T ,所得回收液中 GGT 的纯度较高 ,比活力到
达了19.59 U/mg 。疏水层析由于采用水盐系
统 ,避免了使用有机溶剂 ,更有利于维持蛋白质
的活性[ 12] 。实验中盐析处理后的上清液中盐浓
度较高 ,可以直接作为疏水层析进料 ,经疏水层
析分离纯化 ,最终的纯化倍数为 178.1 。纯化后
的 GG T 经 SDS-PAGE 电泳检验为单一条带 ,两
个亚基分子量分别为28 kDa和 60 kDa左右。与
KEILLOR等报道的该酶结构是二聚体一致 ,但
是两个亚基的大小和来自于动物体内 GG T 的亚
基不同[ 13 , 14] 。本文获得的纯化酶与已报道的
GG T 粗酶酶学性质相似[ 15] 。金属离子 N a+ 、K +
和 Ca2+对酶有激活作用 ,而 Ag+ 、Cu2+ 、Zn2+和
Fe
3+则有抑制作用。
参考文献
[ 1] YASUMOTO K , IWAM I K , MITSUDA H .
Enzyme-cataly zed evolution of lenthionine fr om
lentinic acid [ J] .Agric Biol Chem , 1971 , 35:
2070-2080.
[ 2] YASUMOTO K , IWAMI K , MITS UDA H.A new
sulfur-containing peptide from Lentinus edode acting
a s a precurso r fo r lenthionine[ J] .Agric Bio l Chem ,
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[ 3] IWAMI K , YAS UMOTO K.Alliinase-like enzymes
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Chem , 1980 , 44(12):3003-3004.
[ 4] YASUMOTO K , IWAMI K , MITSUDA H .
Enzymatic cleavage of cy steine sulfoxide in Lentinus
edodes [ J] .Agric Biol Chem , 1975 , 39(10):
1947-1955.
[ 5] FUJIMOTO K.The mechanism of formaldehyde
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Sci , P roc 9 th Inte rna tional Scientific Cong ress on the
Cultivation of Edible Fungi , Tokyo , 1974.
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[ 7] 李冲峰 , 王仁伟 ,刘淑珍 , 等.盐析与疏水层析相结合
快速分离提纯猪胰激肽释放酶[ J] .过程工程学报 ,
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[ 8] BRADFORD MA.Rapid and sensitive method fo r the
quantitation of micro g ram quantities of pr otein
utilizing the principle of protein-dye binging [ J] .
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[ 9] AUSUBEL FM , BRENT R, KINGS TON RE , et al.
54
第 2 期 尹　洁 ,等:香菇中γ-谷氨酰转肽酶的分离纯化及其酶学性质研究
Cur rent pro tocols in molecular bio lo gy [ M] .USA:
John wileg &Sons Inc, 1997:1024-1068.
[ 10] KIMIKAZU I , KYODEN Y , KATSUMI N , et al.
P roper ties of γ-Glut amyltr ansfer ase f rom Lentinus
edodes [ J] . Agr boil Chem , 1975 , 39 (10):
1933-1940.
[ 11] KOZ LOWSKA HJ , NDWAK H , NOWAK J.
Characte risation of myrosinase in polish varieties
of r apeseed[ J] .Food Agric , 1983 , 34:1171-1178.
[ 12] 苏志国 ,李京京.一种复性蛋白质的方法[ P] .中国
专利:02156513.9 , 2002-12-16.
[ 13] KEILLOR JW CASTONGUAY R , LHERBET C ,
et al.Gamma-glutamyl transpeptidase substr ate
specificity and catalytic mechanism [ J] .Meth in
Enzymol , 2005 , 401:449-467.
[ 14] HANIGAN MH. g-Glutamyl transpeptidase , a
glutathionase: its expression and function in
car cinogenesis[ J] .Chem Biol Inter act , 1998 , 111-
112 :333-342.
[ 15] 张　烨.香菇中甲醛的影响因素及其存在状态研究
[ D] .重庆:西南大学 , 2007.
[本文编辑] 　朱丽娜
55
ACTA EDULIS FUNGI 2009.16(2):56 ～ 59
Received:May 6 , 2009;　Accepted:June 6 , 2009
Sponsored by the Foundation of Ministr y of Education(No.208054)
＊Corresponding author.Tel:+86-571-88056656　E-mail:lijianrong@zjgsu.edu.cn
Pur if ication and Propert ies of γ-Glutamyltranspeptidase
from Lent inula edodes
YIN Jie , ZHU Junli , FU Linglin , LI Jianr ong＊
(College of Food Science and Biotechnology , Zhejiang Gongshang University , Key Labora tory of Food
Safe ty for Zhejiang Province , Hangzhou , Zhejiang 310035 , China)
Abstract:γ-Glutamyltranspeptidase (GGT) was extr acted from Len tinula edodes by ammonium sulf ate
precipita tion and pur if ied by Phenyl Sepharose FF column chroma tography.The specific ity activity of the
pur if ied GGT was 19.59 U/mg , and SDS-PAGE revealed tha t the enzyme consisted of two subunits of 28 kDa
and 60 kDa , respectively.The optimal temperatur e and pH values for enzyme activity were 37 ℃ and 7.6 ,
respectively.Na+ , K + and Ca2+ exer ted a slight activating ef fec t on GGT , whereas Cu2+ , Ag+ , Z n2+ and
Fe3+ inhibited enzyme activity.The rate of γ-glutamyl-p-nitr oanilide hydrolysis by purified GGT followed
Mich aelis-Menten kinetics over the substra te concentr ation r ange 50 ～ 250 mmol/ L(K m =2.601 μmol/mL ,
V max =0.0287μmol/min).Pur if ied GGT contained highest amounts of glutamic and aspar tic acids , whereas
Cys and Met were pr esent in low amounts.
Key words:Lentinula edodes ;γ-glutamyltranspeptidase;enzyme pur if ica tion;enzyme proper ties
　　γ-Glutamyl tr anspeptidase (GGT)is widely
distr ibuted in living organisms and is a key
enzyme in the glutathione cycle and
formaldehyde product ion in Lent inula
edodes
[ 1 ～ 5] . Formaldehyde concentrat ions
changed significantly after adding various
compounds including nit ric oxide , vi tamin C
and intermediates in the gluta thione metabolic
pathway to L .edodes fruit bodies[ 6] .I n order
to bet ter understand the mechanism of
endogenous formaldehyde pr oduct ion , GGT
was purif ied from L .edodes f rui t bodies and
part ially characterized.
1　Materials and Methods
1.1 Fungal material
　 　Fresh f rui t bodies o f L .edodes f rom
Qingyuan , Zhejiang Province , we re f reeze-dried
unde r vacuum , g round to a powder , and stored
at -70 ℃.
1.2 Chemicals
　　γ-Glutamyl-p-nitr oanil ide , β-mercaptoe-
thanol and p-nitr oanilide were from Sigma
Chemical Company , Phenyl-Sepharose FF was
from Pharmacia , and other re agents were from
Chinese suppliers and of AR grade.
1.3 Methods
1.3.1 Extract ion of GGT
　　L .edodes powder (25 g)were suspended
in 300 mL 0.05 mol/ L Tris-HCl buf fer
(pH 7.6) containing 1 mmol/ L EDTA and
5 mmo l/L β-mer captoethanol , and homo-
genized (IKA T25 Homogenizer , Germany)
for four 30 s intervals a t 4 ℃.The homogenate
was centr ifuged at 8 000 g for 20 min a t 4 ℃,
solid (NH 4)2SO4 was added to the supernatant
fract ion with gentle st ir ring to a final
concentra tion of 30% (w/v), and the
suspension was allowed to stand at 4 ℃ for 2 h.
The precipitate was removed by centr if ugat ion
No.2 YIN Jie , ZHU Junli , FU Linglin , et al
as above and solid (NH 4)2SO4 added to the
supernatant to a final concentr ation of 70%.
After standing at 4 ℃ for 2 h , the precipitated
protein was collected by centr if ugat ion as above
and redissolved in 10 mL Tr is-HCl buffer
(0.05 mol/L).
1.3.2 Phenyl-Sephar ose FF chromatography
　　The par tially purified enzyme solut ion
from 1.3.1 was applied to a Phenyl-Sephar ose
FF chromatography column (2.6 cm×20 cm)
equilibrated in 0.05 mol/L Tris-HCl /1.0 mol/L
(NH4)2SO4 buffer , pH 7.6 , and eluted with
0.05 mol/L Tris-HCl buffer , pH 7.6 , at a f low
ra te of 2 mL/min[ 7] .
1.3.3 Protein determination
　　Protein was determined by the Coomassie
brilliant blue me thod using bovine serum
albumin as the standard
[ 8] .
1.3.4 Elect rophoresis
　　SDS-polyacrylamide gel elect rophoresis
(SDS-PAGE)was carr ied out according to the
procedure described in Ref erence[ 9] .
1.3.5 GGT assay
　　GGT act ivi ty was determined by measuring
the increase in absorbance at 410 nm due to the
release of p-nitr oanilide fr om γ-glutamyl-p-
nit roanil ide
[ 10] .React ion mixtures contained
4 mL Tris-HCl buffer (0.5 mol/L , pH 7.6),
0.5 mL enzyme solution and 0.5 mL substra te
(3.5μmol/mL).Af ter incubat ion at 37 ℃ for
20 min , 3 mL acet ic acid (1.5 mol/ L)was
added to terminate the react ion and the
absorbance at 410 nm was determined after
fil tra tion.One unit(U)of enzyme act ivity was
de fined as the amount of enzyme pr oducing
1 μmol p-ni troanilide per minute under the
assay conditions.
1.3.6 Heat stability of GGT
　　The heat stabil ity of GGT was determined
by incubating 0.5 mL of purif ied enzyme
solut ion for 30 min to temperatures over the
range 20 ～ 80 ℃ pr ior to assay of the residual
enzyme activity under the standard
condit ions.
1.3.7 Ef fect of temperature on GGT act ivity
　　The ef fect of temper atur e on GGT act ivity
was measured under standard condi tions over
the tempera tur e r ange 20 ～ 70 ℃.
1.3.8 Ef fect of pH on GGT act ivity
　　The effect of pH on GGT act ivi ty was
measured under standard condit ions at the
opt imum temperature in 0.1 mol/L acet ic acid
buffer ove r the range of pH 4.0 ～ 6.5 , and
in 0.1 mol/L Tris-HCl buffer over the pH
range 7.5 ～ 10.0.
1.3.9 Ef fect of metal ions on GGT act ivity
　　The eff ects of var ious me tal ions (Na+ ,
Ag
+ , K+ , Fe3+ , Ca2+ , Mn2+ , Mg2+ , Zn2+ and
Cu
2+) a t 10 mmol/L concentrat ion on GGT
act ivity in Tris-HCl buffer at 37 ℃ was
measured under standard condit ions at the
opt imum temperature.
1.3.10 Determinat ion of K m and V max values
　 　 The rate of γ-glutamyl-p-nitr oanil ide
hydrolysis by purif ied GGT was determined a t
37 ℃ over the subst ra te concentr ation range
50 ～ 250 mmol/L , and K m and V max values
were determined f rom double-reciprocal plots.
1.3.11 Amino acid analysis
　　The amino acid composition of GGT was
determined as descr ibed in Refer ence [ 11]
using a L-8800 automa tic amino acid analyzer
(Hitachi).
2　Results and Analysis
2.1 Isolation and purification of GGT
2.1.1 (NH 4)2SO 4 precipita tion
　 　 S tepw ise f ractionat ion w ith solid
(NH4)2 SO 4 revealed that >90% of the GG T
activity present in ex t racts of L .edodes f rui t
bodies w as precipita ted betw een 30%～ 70% sal t
sa turation.
2.1.2 Hydrophobic interaction chromatography
　　GG T eluted as a single discrete peak from a
Pheny l-Sepharose FF column (see Fig .1 in the
Chinese ver sion).SDS-PAGE revealed that the
purified enzyme consisted of tw o subuni ts of 28
kDa and 60 kDa , respectively[ 14] (see Fig.2 in
57
ACTA EDULIS FUNGI V ol.16
the Chinese version).
2.1.3 Summary o f GGT purification pro to col
　　The results o f a representat ive purification
of GG T are summarized in Table 1 (see the
Chinese version).After a four-step purification
protoco l , the specif icity activi ty o f puri fied GG T
was increased 178.1 fold wi th a 23.7% recovery
yield.
2.2 Characterisation of γ-GGT from L.edodes
2.2.1 Heat stabi li ty
　　GG T activi ty w as stable fo r 30 m in at 20 ～
40 ℃ but decreased rapidly following exposure
to temperatures above 45 ℃.The enzyme w as
comple tely inactivated af ter 30 min at 70 ℃.
2.2.2 Ef fect of temperature on GG T activi ty
　　GG T activi ty w as measured under standard
condi tions over the temperature range 20 ～ 70 ℃
and the opt imum temperature for enzyme
act ivity w as 37 ℃(see Fig.3 in the Chinese
version).
2.2.3 Ef fect of pH on GGT act ivity
　 　 GG T displayed a relativ ely sharp pH
optimum betw een 7.5 ～ 8.0 (see Fig .4 in the
Chinese version).
2.2.4 Ef fect of metal ions on GGT activi ty
　 　 The ef fects o f various metal ions
(10 mmol/ L concentrat ion)on GG T activity at
37 ℃ are show n in Fig.5 (see the Chinese
version).N a+ , K+ and Ca2+ had a small
act ivating ef fect on GG T , whereas Cu2+ , Ag +
and Fe3+ all inhibited enzyme activi ty by
>50%.
2.2.5 Kinetic parame ters
　　The rate of γ-glutamyl-p-nitr oanilide
hydrolysis at 37 ℃ by pur ified GGT over the
substr ate concentrat ion range 50 ～ 250 mmol/L
followed Michaelis-Menten kinet ics.Double-
reciprocal plots revealed an apparent K m
value of 2.601 μmol/mL and a V max value
of 0.0287 μmol/min.
2.2.6 GGT amino acid composit ion
　　The amino acid composi tion of GGT is
shown in Table 2 (see the Chinese version).
The r atio of basic amino acids(Arg , Lys , His ,
Pro) to acidic amino acids (Glu , Asp)was
1∶1.84.Glutamic and aspart ic acids were
present at the highest concentr at ions , whereas
Cys and Me t wer e present in low amounts.
3　Discussion
　 　γ-Glutamyl tr anspept idase (GGT) was
purif ied from Lent inus edodes fruit bodies by
ammonium sulf ate precipitat ion and Phenyl
Sepharose FF column chromatography , thereby
avoiding the use of organic solvents which can
adversely affe ct enzyme act ivity
[ 12] .The high
salt concentr at ion in the re-dissolved fract ion
following precipitation with (NH4)2SO4
allowed direct applicat ion to the hydrophobic
chromatography column.After a four-step
purif ication protocol , the specif ici ty act ivity of
purif ied GGT was increased 178.1-f old.SDS-
PAGE indicated that L .edodes GGT consisted
of two subunits.This is consistent with the
repor t of KEILLOR et al .(2005) that GGT
was dimer ic , although the molecular weights of
the subunits of the L .edodes enzyme are
different compared with GGTs from animal
sources
[ 13 , 14] .The proper ties of pur ified GGT
from L .edodes descr ibed here were similar to
those reported earl ier for a crude GGT
preparat ion
[ 15] .Na+ , K+ and Ca2+ had a small
act ivat ing ef fect on GGT , whereas Cu2+ , Ag+
and Fe
3+
all inhibited enzyme activity by
>50%.
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