玉米尿卟啉原Ⅲ脱羧酶同功酶生物信息学分析、基因克隆和原核表达

尿卟啉原Ⅲ脱羧酶是生物卟啉类化合物分支合成的关键酶。从GenBank中搜寻到4种玉米尿卟啉原Ⅲ脱羧酶:Les22、UROD1、UROD2和截短UROD,氨基酸序列比对显示N端的同源性较差;玉米les22基因比urod1基因在开放阅读框的上游少3个碱基,导致阅读框移码。植物除玉米外存在高度同源的UROD1和UROD2,具有结构完整性。本文以玉米幼叶总RNA为模板,通过RT-PCR克隆了玉米les22和urod2基因,并对突变位点进行校正,同时通过定点突变获得urod1基因,它们都编码去除叶绿体导肽的尿卟啉原Ⅲ脱羧酶。再分别将les22、urod2和urod1基因插入大肠杆菌的不同表达载体,转化表达菌株BL21(DE3),16℃诱导表达18h,SDS-PAGE分析显示,Les22的N端含有组氨酸标签或SUMO蛋白,重组蛋白表达为包涵体,UROD2的N端含有组氨酸标签或SUMO、TRX、GST或MBP蛋白,未检测到目的蛋白在上清液的特异表达,而UROD1和MBP融合为可溶性表达,表明玉米UROD的N端氨基酸残基可能参与蛋白在大肠杆菌的折叠。这为研究玉米UROD的种类和功能奠定基础。     

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．     

荚膜红细菌尿卟啉原Ⅲ转甲基酶的纯化及其酶学性质

S-腺苷-_L-甲硫氨酸依赖型尿卟啉原Ⅲ转甲基酶(S-adenosy-_L-methionine uroprophyrinogenⅢmethyltransferase,SUMT)催化尿卟啉原Ⅲ(UroprophyrinogenⅢ,urogenⅢ)的中心碳原子C-2和C-7位上甲基化生成前咕啉-2,是维生素B12生物合成途径中的一步关键酶,但大部分SUMT受其底物urogenⅢ和副产物S-腺苷同型半胱氨酸(S-adenosy-_L-homocysteine,SAH)的抑制作用。为了挖掘能耐受高浓度urogenⅢ的转甲基酶,文中从荚膜红细菌Rhodobacter capsulatus SB1003中克隆2个SUMT基因(RCcobA1,RCcobA2),经表达与纯化后,检测发现RCcobA1和RCcobA2的酶活分别为27.3 U/mg和68.9 U/mg,后者比VB_(12)工业生产菌株脱氮假单胞菌Pseudomonas denitrificans中内源的SUMT(PDcob A,27.9 U/mg)高2.4倍,并且当urogenⅢ浓度高达70μmol/L时都几乎不受抑制作用。因此,RCcobA2的发现可以为解除VB_(12)合成途径的瓶颈以及提高VB_(12)产量提供理论支持和方向指导。     

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,利用硫酸铵分段盐析,丙酮沉淀,ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦＦ离子交换柱层析,ＳｅｐｈａｃｒｙｌＳ３００凝胶过滤柱层析及ＦＰＬ－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,分离纯化了谷氨酰羧酶,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．ｍｇ＾－１,纯化保数３８．２倍,经ＳＤＳ－ＰＡＧＥ测定,其亚基分子量为７０ｋＤ,经工ＰＡＧＥ确定     

毛栓菌胞外漆酶的纯化及部分性质研究

毛栓菌胞外漆酶经盐析、透析、sephadexG75和G2 5四步纯化 ,粗酶液被纯化了 39 1倍 ,比活力 12 152 ,回收率 4 5 3%。漆酶最适pH值为 4 0 ,最适反应温度为 30℃。K Cu 2 、Zn2 离子可激活漆酶 ,而Ag 、Fe3 离子可抑制漆酶的活性。漆酶的Km值为 1 81× 10 3mol/L。     

对生玉米和互生玉米染色体核型比较研究

本文利用对生性状转育获得的六组对生和互生姊妹系,通过风油精法染色体制片,首次研究了对生玉米与互生玉米的染色体核型差异。结果表明：对生玉米的染色体含有2对随体,而互生玉米却只有1对。对生玉米和互生玉米共有的一对随体分布在第6对染色体短臂上,对生玉米额外一对随体主要位于第10对染色体短臂上。     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

青稞中β-1,3葡聚糖酶的纯化及部分性质研究

β-1,3-葡聚糖酶[EC．3．2．1．39]存在于大多数的高等植物中,它们由一个小的基因家族编码,在植物不同的生理活动中起着重要的作用。采用NaCl抽提、硫酸铵分部沉淀和2步离子交换法和分子筛从青稞的胚芽中提纯得到了一种β-1,3-葡聚糖酶。在非变性电泳中纯化的β-1,3-葡聚糖酶用银染只有一条蛋白带,运用底物进行的活性染色时在相同位置也只显示一条酶活性带。此活性染色可以直接在电泳胶板上快速鉴定和检验β-1,3-葡聚糖酶。纯化的β-1,3-葡聚糖酶在还原及非还原条件下的SDS-PAGE变性电泳中,呈现一条分子量为32kD的主要蛋白带及2条低分子量的弱带,表明此酶无链内二硫键存在。等电聚焦分析显示其等电点为8．1。上述结果表明纯化得到的是一种碱性β-1,3-葡聚糖酶。     

红曲霉丙酮酸脱羧酶基因克隆及酶学性质分析

目的:研究生物乙醇发酵关键酶丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)的性质和功能.方法:从SMART技术构建的红曲霉(Monascus anka CICC 5031 )cDNA文库中筛选pdc基因,亚克隆到表达载体pET-21b(+),原核表达、亲和层析纯化重组PDC.分析重组PDC和野生型PDC的酶学性质.结果:pdc基因的开放阅读框长1 713bp,编码一个570个氨基酸残基的蛋白质.重组PDC在E.coli BL21( DE3)中的表达量为菌体总蛋白的32.7％.重组PDC与野生型PDC比活分别为20.2U/mg和30.1U/mg.二者的最适反应条件均为pH6.0、30℃.重组PDC和野生型PDC的Km值分别为2.6mmol/L和0.56mmol/L.结论:红曲霉pdc基因可在大肠杆菌中高效表达,重组PDC稳定性较好,可用作生物乙醇生产的候选资源.     

赤霉菌超氧化物歧化酶的纯化及部分理化性质

采用加热、Sephadex G—100凝胶过滤及DEAE-Sephadex A-50柱层析的方法,提纯了赤霉菌的超氧化物歧化酶(SOD),纯酶比活力为2640U/mg蛋白,最大紫外吸收峰为276nm,为Mn-SOD,由二个亚基组成,亚基分子量为14.5kD。此外还报道了该酶的氨基酸组成。     

玉米花粉32kD蛋白的纯化及性质

利用丙酮粉、硫酸铵分段盐析及离子交换层析技术,从玉米花粉细胞质中分离纯化了一种３２ｋＤ的可溶性蛋白,其ＧＴＰａｓｅ活性大于ＡＴＰａｓｅ。ＳＤＳ－ＰＡＧＥ表明达银染电泳纯。等电聚焦电泳测得等电点为５．２５。免疫印迹鉴定表明该蛋白与抗牛脑动蛋白或动力蛋白的抗体无免疫交叉反应。最大紫外吸收波长为２８２ｎｍ,ＣＤ谱分析说明具有球蛋白特征。     

小麦谷氨酸脱羧酶的纯化及部分性质研究

谷氨酸脱羧酶（ｇｌｕｔａｍａｔｅｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）催化谷氨酸脱羧生成γ－氨基丁酸（γ－ａｍｉｎｏｂｕｔｙｒａｔｅ,ＢＡ）,植物中已从南瓜［１］、马铃薯和林生山黧豆［２］纯化了ＧＡＤ．ＧＡＤ活性在禾本科作物中作为...     

Interaction of the Molecular Chaperone HtpG with Uroporphyrinogen Decarboxylase in the Cyanobacterium Synechococcus elongatus PCC 7942

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.     

Subcellular Localization of Oxaloacetate Decarboxylase and Its Isolation from Maize Leaves

The activity of oxaloacetate decarboxylase was revealed in leaves of a C₄ plant, maize (Zea mays L.). This activity was unrelated to decarboxylase activities of other enzymes, e.g., NAD-malate dehydrogenase (EC 1.1.1.38) or NADP-malate dehydrogenase (EC 1.1.1.40), and was located in chloroplasts (83.1%). Using a four-step purification procedure, an electrophoretically pure enzyme preparation of oxaloacetate decarboxylase was obtained from maize leaves. The specific activity of the enzyme was 3.150 EU/mg protein, the factor of purification was 40.4, and the yield was 11.0%. The enzyme exhibited Michaelis–Menten kinetics with K _m for oxaloacetate 30 ± 5 M and pH optimum 7.1 ± 0.5. The metabolite-mediated regulation of oxaloacetate decarboxylase activity has been investigated. It is found that sodium chloride (1.0 mM) activates the enzyme, whereas ATP inhibits the enzyme activity.     

Purification and Some Properties of Thiosulfate Dehydrogenase from Acidithiobacillus ferrooxidans

Abstract

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion‐exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine‐selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.     

露花叶片PEP羧化酶的纯化及某些分子特性

经硫酸铵分部沉淀,DEAE-纤维素(DE52),DEAE-Sephadex A-50,SephacrylS-200和二次羟基磷灰石等柱层析,从露花叶片中分离得到纯化63.9倍、电泳均一的磷酸烯醇式丙酮酸羧化酶。此酶的天然分子量经聚丙烯酰胺梯度凝胶电泳测定为260kD,经SephadexG-200凝胶过滤法测定为240kD。用SDS-聚丙烯酰胺梯度凝胶电泳测得酶的亚基分子量为115kD,表明此酶是个二同聚体。此酶的等电点为PI=5.6。免疫双扩散的结果表明此酶与高梁PEPG的抗原决定簇呈部分同一性。     

A Rapid Purification of L-Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria

L-Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chromatofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 +/- 4,000 and 93,000 +/- 5,000, respectively. When analysed by sodium dodecyl sulphate-PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 +/- 1,000 and 44,000 +/- 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0-7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl-trapping reagent aminooxyacetic acid with an I50 value of 0.2 microM.     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.