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     

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...     

对生玉米幼叶尿卟啉原脱羧酶的纯化及部分性质研究

尿卟啉原Ⅲ脱羧酶是植物血红素和叶绿素合成的一个关键调控酶。对生玉米叶片中叶绿素含量较比互生玉米高。对生玉米幼苗经硫酸铵分级沉淀,DEAE Sepharose CL-6B、Sephacryl S-200、羟基磷灰石和B lueSepharose CL-6B层析,纯化了尿卟啉原脱羧酶。纯化倍数为1 060倍,得率约8%,比活约880 U/mg蛋白。纯化的UROD在SDS/PAGE显示一条带,亚基分子量约为40 kD,Sephacryl S-300测得全酶分子量约为55 kD。IEF-PAGE显示UROD为一条带,等电点约为6.0,酶的最适pH值约7.0,在55℃下保温12 m in,酶活力丧失90%,在100 mm ol/L的巯基乙醇下,UROD的酶活力提高7倍。体外5 mm ol/L的磷酸吡哆醛修饰显示UROD活力下降约30%。     

菠菜叶片蔗糖磷酸合成酶的纯化

经硫酸铵分部沉淀,DEAE-纤维素(DE 52),Sepharose 6B和 AH—4B连续三次柱层析,得到纯化88倍电泳均一的菠菜叶片蔗糖磷酸合成酶。电泳分析该酶分子量为490 kD,是由八个分子量为60 kD的相同亚基组成的寡聚体,等电点为PI=4.l,其最适pH值为6.9。     

GL-7-ACA酰化酶的分离纯化及性质研究

CU334是高表达GL-7-ACA酰化酶工程菌,其菌悬液用超声波处理后,经硫酸铵分级沉淀、DEAE-Sephadex A-50离子交换柱层析、DEAE—纤维素DE-52柱层析、Sephadex G-200凝胶过滤及羟基磷灰石吸附柱层析等步骤,得到了凝胶电泳均一的GL-7-ACA酰化酶蛋白,纯化了22倍,得率4.0％,比活力为13.8U/mg。用浓度梯度PAGE测得GL-7-ACA酰化酶的分子量为134kD,用SDS-PAGE测得两个亚基分子量分别为15.5kD和58.4kD。用PI法测得等电点为3.5。GL-7-ACA酰化酶反应最适pH为7.0。反应最适温度为37℃,GL-7-ACA酰化酶对底物GL-7-ACA的K_m值为0.50mmol/L,V_(max)为13.10U·mg^(-1)。Ca^(2+)、EDTA和巯基乙醇对该酶有激活作用,Cu^(2+)、Fe^(2+)和Mg^(2+)等有一定程度的抑制作用。产物7-ACA、戊二酸均为GL-7-ACA酰化酶的反竞争性抑制剂,其K_1值分别为16.58mmol·L^(-1)和9.88mmol·L^(-1)。     

谷氨酸生产菌S9114中的谷氨酸脱氢酶的研究

谷氨酸脱氢酶 (GDH)是谷氨酸生物合成的关键酶 ,谷氨酸棒杆菌S91 1 4是目前我国味精工业应用最广泛的生产菌种 ,其谷氨酸脱氢酶的研究尚未见报道。分离纯化该菌中的谷氨酸脱氢酶 ,研究其辅酶组成 ,对揭示谷氨酸脱氢酶的分子结构和性质 ,提高谷氨酸产率很有必要。将培养至对数期中期的细胞离心收集并用含适量DTT、ED TA的Tris_HCl缓冲液 (pH 7 5 )洗涤 ,用Frenchpressurecellpress破碎 ,离心去除菌体碎片得无细胞抽提液。然后使用 KTA_10 0快速纯化系统经DEAE_纤维素柱、疏水柱 (HIC)、G_2 0 0凝胶过滤柱层析得到纯化大约 70倍的以NAD PH为辅酶的GDH和部分纯化的以NADH辅酶的GDH。这两个酶分别对NADPH、NADH高度专一 ,不能相互代替。经HPLC和SDS_PAGE测得前一种酶的分子量和亚基分子量分别为 188kD和 32kD ,表明该酶为具有相同亚基的六聚体。酶活性测定使用HITACHIU_30 0 0分光光度计利用NAD(P)H在 340nm氧化的初速度进行。蛋白质含量测定利用Bradford方法进行 ,并以牛血清白蛋白为标准蛋白。纯化结果表明S91 1 4中确实存在两种GDH ,其中以NADH为辅酶的GDH尚未见报道。和某些具有两种GDH的微生物一样 ,S91 1 4可能也是以NADPH为辅酶的GDH参与谷氨酸的合成代谢 ,以NADH为辅酶的GDH参与谷氨酸的分解代谢。     

番茄果实中焦磷酸:D-果糖-6-磷酸 1-磷酸转移酶的两种酶型的分离纯化及其特性

用快速蛋白液相层析仪(FPLC)Mono Q柱(HR5/5)分离纯化成熟绿番茄果实中PFP的两种分子酶型及其特性。一种酶型为Q_1,是含两个β-亚基(60kD)的二聚体,比活为5μmol min~(-1) mg~(-1);另一种为Q_2,由四个α-亚基(66kD)和四个β-亚基(60kD)组成八聚体,比活为70.5μmol/min~(-1)·mg~(-1)。Q_1的分子量是120kD,Q_2的分子量介于500kD和530kD之间。用纯化的Q_2制备的抗血清专一地与Q_2起沉淀反应。PFP酶液贮存后,其Q_1/Q_2蛋白量比值增加明显,表明部分Q_2转化为Q_1。Q_1具有催化活力表明PFP的活性中心位于β-亚基。α-亚基可能借增强PFP酶对F2,6P_2的亲和力以提高酶的比活而起调节功能,但是Q_1的活力依赖于F2,6P_2的激活,表明β-亚基处也可能存在F_2,_6P_2的调节位点。Q_2含紧密结合的F2,6P_2分子,并表现出对F2,6P2_的不敏感性,基于此种现象,有必要重新认识PFP对F2,6P_2敏感性的内在实质。     

假单胞杆菌D-海因酶的纯化及酶学性质

D 海因酶是工业上生产D 型氨基酸的关键酶 ,用热变性 ,硫酸铵沉淀及SepharoseQfastflow ,Phenyl Sepharosefastflow ,Superose 1 2等柱层析步骤从Pseudomonas 2 2 62菌体中分离纯化了该酶 ,纯化倍数约为 60 ,活力回收约为 1 6%。该酶为同源二聚体 ,分子量约为 1 0 9kD ,亚基分子量约为 53 7kD ,反应最适pH为 8 0 ,最适温度为 70℃ ,在pH6.0～ 1 0 0和温度 60℃以下稳定 ,该酶对巯基试剂敏感 ,大多数二价金属离子如镁、锰离子等能促使酶活提高 ,但高浓度锌离子能抑制酶活 ,以二氢尿嘧啶为底物的米氏常数Km =2 .5× 1 0 - 2 mol L。该酶的N末端1 0个氨基酸残基依次为MDKLIKNGTI     

豇豆几丁质酶部分酶学特性的研究

该文测定了纯化的豇豆几丁质酶部分酶学特性。结果表明,该酶在pH5-8,温度低于60℃的范围内稳定性较好,酶活力最适pH为65,最适温度为50℃。10mmol/L浓度的Hg2 、Mn2 、Mg2 、Co2 等金属离子对酶活力有一定抑制作用,其中Hg2 离子抑制率最高(6883%)。Km(胶状几丁质)值为1662mg/ml;以SDS-PAGE电泳和SephadexG-100柱层析两种方法分别测得分子量为34kD、325kD;IEF电泳测得等电点为83。     

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。     

人胃腺苷脱氨酶的分离纯化及性质研究

用硫酸铵分级沉淀、ＤＥＡＥ－纤维素离子交换层析、免疫亲和层析、ＳｅｐｈａｄｅｘＧ１００凝胶柱层析从人胃组织中提取出腺苷脱氨酶,酶纯化１９３２４倍,比活力为５７９７Ｕ／ｍｇ蛋白．提取酶液经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和等电聚焦只呈现一条区带。测得该酶的分子量为４１．２ｋＤ,等电点为ｐＨ４．８．氨基酸组成分析表明该酶由３８８个氨基酸残基组成,Ｎ端氨基酸为精氨酸。酶的最适ｐＨ为６．５,ｐＨ小于５．０或大于９．０时不稳定;最适温度为３７℃,对热不太稳定,以腺苷及２－脱氧腺苷作为底物,其Ｋｍ分别为８７μｍｏｌ／Ｌ和４１μｍｏｌ／Ｌ。     

Apoplastic β-1, 3-Glucanases in Leaves of Tomato Systematically Infected by TMV

Apoplastic β-1, 3-glucanase was purified from leaves of tomato (Lycopersicon esculentum Mill. ) which were systematically infected by TMV (tobacco mosaic virus). The enzyme obtained through -20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, DEAE-Sephadex A-25 ion exchange chromatography and Sephadex G-75 gel filtration, showed homogeneity in PAGE, and SDS-PAGE which had two isoenzymes of 27 kD and 36 kD. The enzyme hydrolysed laminarin at an optimum pH of 4.8--5.2 and was stable between pH 4--8 and at an optimum temperature between 30--40℃, and stable at 40℃ after 1 hour of incubation, It had a Km of 9. 2 mg/mL. SDS-PAGE profiles of the proteins in the tomato leaf intercellular fluid had the bands of 22 kD, 27 kD and 36 kD that were β-1, 3-glucanases.     

Purification and biochemical characterization of ionically unbound polyphenol oxidase from Musa paradisiaca leaf

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77?min. The inactivation energy (Ea) value of PPO was estimated to be 91.3?kJ mol(-1). It showed higher specificity with catechol (K(m)?=?8?mM) as compared to 4-methylcatechol (K(m)?=?10?mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Purification and Characterization of Polygalacturonase-3 from Jamaica cherry (Muntingia calabura Linn)

Endo-polygalacturonase-3 (PG-3), the key enzyme of fruit ripening was purified to near homogeneity as judged by native PAGE from the fruit tissues of Jamaica cherry (Muntingia calabura) using ammonium sulphate fractionation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the PG-3 enzyme was determined as 85 kD, by size exclusion chromatography. SDS-PAGE of PG-3 revealed two dissimilar bands of 62 and 21 kD as heterogenous subunits. The optimum pH of PG-3 was found to be 4.0. The enzyme had an optimum temperature of 40°C and was relatively stable at 50°C and 60°C. K_m for the substrate polygalacturonic acid was found to be 0.27%. The purified enzyme was a glycoprotein with 6.6 % carbohydrate content.     

Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays).

A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.     

Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.     

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.     

Purification and properties of peroxidase from Pinus pinaster needles

Peroxidase (Ec 1.11.1.7) was purified from needles of Pinus pinaster to apparent homogeneity by DE-52 cellulose chromatography with a final recovery of enzyme activity of about 85%. The purified enzyme (A402/A275 = 1.05) had a specific activity of about 948 U/mg of protein and ran as a single protein band both on SDS-PAGE and native PAGE with Mr of 37,000 and 151,000, respectively. Both native PAGE and isoelectric focusing gels of the purified enzyme were stained for activity which coincided with the protein band. The pI of the purified enzyme was found to be 3.2 by isoelectric focusing on an ultrathin polyacrylamide gel. The enzyme has an optimum pH of activity of 5.0 and temperature optimum of 30 degrees C. Stability studies of the enzyme as a function of pH and temperature suggest that it is most stable at pH 5.0 and 0-40 degrees C, respectively.     

A Review of: “HPLC of Biological Macromolecules (Methods and Applications) K.M. Goodring F.E. Regnier,Eds Marcel Dekker,New York, 1990; hardbound, 676 pages, $150”

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0–9.0) and temperature (30–90°C). From the thermal inactivation studies in the range 60–75°C, the half-life (t_1/2) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol^?1. It showed higher specificity with catechol (K_m = 8 mM) as compared to 4-methylcatechol (K_m = 10 mM). Among metal ions and reagents tested, Cu²⁺, Fe²⁺, Hg²⁺, Mn²⁺, Ni²⁺, protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K⁺, Na⁺, Co²⁺, kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Purification and Identification of Antifreeze Proteins in Ammopiptanthus mongolicus

The conventional protein chromatography technique was adopted to purify the antifreeze proteins (AFPs) from the leaves of Ammopiptanthus mongolicus ( Maxim. ) Cheng f. Two bands on native PAGE gel showed thermal hysteresis activity, one was band Bi, whose thermal hysteresis was 0.46 cE at 8 g/L, which showed two bands (67 kD, 21 kD) on SDS-PAGE gel; the other was B3, whose thermal hysteresis was 0.45 cE at 10 g/L, and it contained only a single protein (39.8 kD). Both B1 and B3 are not glycoproteins, because neither do they interact with Shift-reagent, nor show ultraviolet characteristics of a typical glycoprotein.