双胸蚓纤溶酶的纯化及性质

 用硫酸铵分段盐析、超滤膜分级分离及DEAE-纤维素、Sephadex A-25和Sephadex G-50三种柱层析方法从双胸蚓组织的粗提取液中分离纯化出一种纤溶酶,分子量为29kD,由一条肽链组成。此晦具有强烈的溶解纤维蛋白的作用,对家兎实验性血凝块也具有明显的溶解作用。此酶的最适pH为8.0,在pH7.6～8.4之间活力相差不到2％;酶在PH4.7—11.0范围内稳定;酶作用的最适温度为57℃;此酶热稳定性较好,于25～50℃保温3小时,酶活力基本不变,60℃时,活力保留65％。金属离子Na~(+)、K~(+)、Mg~(2+)等可提高此酶的活力,而Hg~(2+)、Ca~(2+)等金属离子对此酶有不同程度的抑制作用。     

粘质赛氏菌胞外蛋白酶的提取、纯化及部分性质研究

经过硫酸铵３０％～５０％分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达５３％,并制备了酶的结晶,该酶以ＳｅｐｈａｄｅｘＧ１００柱层析及ＳＤＳ－ＰＡＧＥ测得分子量约为８１０００,该酶的最适ｐＨ为７．０,最适温度为４５℃,Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋、Ｃｕ２＋、Ｃｏ２＋等重金属离子不同程度地抑制酶活性。     

疣孢漆斑菌(Myrothecium Verrucaria)J-1胆红素氧化酶的纯化及性质初步研究

从中国土样中筛选到一株能产生胆红素氧化酶的微生物(Myrothecium Verrucaria)J-1,培养后,分离纯化,最后经QAE—Sephadex A50柱层析,得到胆红素氧化酶比活为207.65 U/A 280nm,总产率为22.3％。纯酶紫外吸收峰为278 nm,凝胶电泳为单一色带。分子量估计为52000。它能迅速、特异地氧化胆红素为胆绿素,并进一步氧化成目前还不清楚的紫色化合物。最佳作用pH为7.0,最佳作用温度为40℃。     

外来入侵植物空心莲子草几丁质酶特性研究

外来入侵植物空心莲子草因其强抗逆性而迅速蔓延,成为有害杂草.拟研究其病程相关蛋白--几丁质酶酶学性质,为探讨该植物的抗逆机理和入侵机制提供理论依据.空心莲子草经乙烯利处理后,其叶片蛋白质提取液在4℃经40～60%饱和硫酸铵溶液沉淀,SephadexG-75洗脱后得几丁质酶液.结果表明:空心莲子草几丁质酶最适反应pH为5.7左右,在pH4.0～8.0均表现稳定,当pH小于4.0和大于8.0时酶活力丧失50%以上,在pH2.0和10.0时没有活力;在30℃-80℃范围内,酶均具较高活性,最适反应温度为70℃左右,温度高于80℃后,酶稳定性下降;当底物浓度小于1.25%时,酶活力随底物浓度的增加而增加,在底物浓度大于1.25%浓度时,活力增加的趋势减缓.结果提示:也许空心莲子草的强抗逆性是因为其几丁质酶具较高的温度耐受性和较大的pH作用范围.     

Phosphoenolpyruvate Phosphatase Activity and Partial Properties in Rice Leaves

Phosphoenolpyruvate (PEP) specific phosphatase was isolated from rice (Oryza sativa) leaves. Its Km (PEP) was 0. 42 mmol/L. The partially purified PEP phosphatase showed a narrow pH profile and had an optimum pH of 8.7. It was relatively stable at the pH ranging from 6.2 to 9.5 and at temperature below 40℃. Pi showed no effect on the enzyme activity at low concentrations, but slight inhibitory effect at the concentrations above 5 mmol/L. The enzyme activity was activated by Mgz+ and inhibited by CaCl2, CoCl2, CuSO4, FeSO4 and ZnSO4.     

Ornithine transcarbamylase from Neurospora crassa: purification and properties

Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and Km's of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Km's and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

大凉疣螈碱性磷酸酶的分离纯化及部分性质

碱性磷酸酶 (alkaline phosphatase,AKP)在生物界的分布很广 ,动物、植物、微生物中均广泛存在 .提纯的 AKP常被应用于对核酸等的研究 ,是基因工程常用的工具酶 ,也是酶标免疫测定技术的常用工具酶之一 .人类血清中的 AKP在不同疾病状态下有显著变化 ,临床上将血清 AKP变化指标作为诊断某些疾病的依据 .对于细菌和高等动物的 AKP已有广泛的研究 ,但国内外对两栖爬行类动物 AKP的研究报导却很少 ,仅有蛇毒中 AKP的研究报导 [1,2 ] .本文对大凉疣螈皮肤的 AKP进行了分离纯化 ,并对其部分性质进行了初步研究 .1　材料和方法1 .1　材…     

黑曲霉(Aspergillus niger)β-D-葡萄糖苷酶的纯化及其性质

利用垂直板凝胶制备电泳从黑曲霉(Aspergillus niger,AS 3.316)中分离提纯了β-D-葡萄糖苷酶(EC3.2.1.21),经凝胶电泳鉴定为单一带。酶作用的最适pH为4.4,在pH4.0—6.2稳定;最适温度65℃,热稳定性较好,于60℃保温4小时,活力保留80％。此酶作用于纤维二糖的Km值为6.09mM。聚丙烯酰胺薄层等电聚焦测得其pI值为5.5;用SDS凝胶电泳测得其分子量为77000。此酶不仅能水解纤维二糖和对硝基苯-β-D-葡萄糖苷,还能微弱地水解对硝基苯β-D-半乳糖苷和β-D-木糖苷。金属离子Fe~(2+)、Hg~(2+)、Cu~(2+)、Al~(3+)、Hg~+和Ag~+等对此酶有不同程度的抑制作用,蛋白质侧链修饰剂N-溴代琥珀酰亚胺对此酶有较强的抑制作用,2-羟基-5-硝基溴苯对酶也有一定的抑制作用,推测色氨酸残基对β-D-葡萄糖苷酶的活力是非常必要的。     

β-葡萄糖苷酶的分离纯化和性质研究

β－葡萄糖苷酶是纤维素酶的重要组分之一,它不仅可水解纤维二糖和寡糖,更可解除纤维二糖对β－１,４－内切葡聚糖酶和外切葡聚糖酶的抑制,提高水解速率和程度．利用ＳｅｐｈａｄｅｘＧ－１５０和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０层析法从黑曲霉变异株Ｌ－２２中分离提纯了β－葡萄糖苷酶,该酶是由两个分子量相同的亚基组成的二聚体,每个亚基分子量为２０３ｋＤ．该酶最适ｐＨ为４．８,ｐＨ稳定范围在３．６～６．４;最适温度是６０℃,温度稳定范围为４～６０℃;酶分子含糖量为８．３５％．它是一个酸性β－葡萄糖苷水解酶,专一性地水解β－糖苷键．而不水解α－糖苷键,对短链底物表现了相对高的活力．用动力学分析和共价化学修饰方法探讨了与该酶活力有关的必需基团．由ｐＨ对ｌｇＶｍ和ｌｇＶｍ／Ｋｍ的影响,推测出酶活性部位至少有两个可解离基团为酶活性所必需,它们在酶－底物复合物中的ｐＫｅｓ１和ｐＫｅｓ２的值分别为４．０和５．６,在游离酶中的ｐＫ值分别为４．２和５．９．由此可初步判断这两个可解离基团可能为组氨酸和含羧基的氨基酸,它们与酶的催化和底物结合可能有关．     

树状多节孢酸性磷酸酶的分离纯化与性质研究

树状多节孢Nodulisporium sylviforme是从东北红豆杉Taxus cuspidata分离、可产生紫杉醇的内生真菌。研究以树状多节孢为材料,利用液体发酵手段获得菌丝体,通过CM-cellulose阴离子交换柱层析、Q-Sepharose阳离子交换柱层析和FPLC凝胶过滤层析（Superdex 75）,获得纯化的树状多节孢酸性磷酸酶蛋白（Nod-ACP）。结合FPLC和SDS-PAGE分析,判定该磷酸酶为分子量44kDa单亚基蛋白。酶学性质研究表明,其最适pH值为3.0,最适温度为58℃。6     

Low molecular weight DNA ligase of chick embryo.

A nuclear DNA ligase from chick embryos was isolated by the non-aqueous method and partially purified. Its activity is several fold lower than that of the enzyme found in the cytoplasmic fraction of the chick embryos. The pH dependance curve shows a single optimum for the nuclear enzyme activity, over a very narrow pH range. The molecular weight of the nuclear enzyme is 82000 and the activity is inhibited with a low K_Iby d-ATP.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

虎纹捕鸟蛛毒液中透明质酸酶的纯化和部分性质的研究

用分子筛（岛津ＤＩＯＬ－１５０柱）和阳离子交换（岛津ＷＣＸ－１柱）高效液相色谱从虎纹捕鸟蛛（Ｓｅｌｅｎｏｃｏｓｍｉａｈｕｗｅｎａ）毒液中分离提纯透明质酸酶（Ｈｙａｌｕｒｏｎｉｄａｓｅ,ＥＣ３．２．１．３５）．经等电聚焦电泳为一条带,ｐＩ＝７．２．经ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为４０ｋＤ,经凝胶过滤测得分子量为４０．７ｋＤ。以透明质酸为底物时在ｐＨ３．５─５．５范围内有较大活性,最适ｐＨ值为４．０;在ｐＨ４．５─６．０范围内稳定,在反应温度为３０─６０℃时有较大活性,最适温度为５０℃;对热稳定,０．１５ｍｏｌ／Ｌ的ＮａＣｌ对酶活性有一定的稳定作用．３％的肝素、５００μｍｏｌ／Ｌ的Ｈｇ~（２＋）、Ｆｅ~（２＋）、Ｃｕ~（２＋）对酶活性有明显的抑制作用。     

聚球藻PCC 7942氢酶的分离纯化及特性研究

报道了室温、空气环境下聚球藻Synechococcus sp.PCC7942氢酶的分离纯化.经过超声破碎、超速离心、离子交换层析、疏水层析及凝胶层析等步骤,氢酶被纯化了218倍,得率为6.5%,比活为1.46U·mg^-1蛋白.纯化氢酶的SDS-PAGE图显示五条蛋白带,分子量约为83kDa,60kDa,47kDa,30kDa和27kDa.该氢酶为可溶性的双向氢酶,其催化放氢的最佳电子供体为还原态的甲基紫精,最适温度50℃,最适pH8.0.     

米黑毛霉(M.miehei)天冬氨酸蛋白酶的研究

用硫酸铵分部盐析及离子交换层析技术从米黑毛霉半固体培养物的浸提液中提纯了天冬氨酸蛋白酶,酶活力收率为19.5％,,比活力达3080SU/mg蛋白,提纯8.5倍。用聚丙烯酰胺凝胶电泳、SDS-凝胶电泳、等电聚焦、双向免疫扩散及免疫电泳等方法鉴定该酶均一。本文还报道了关于该酶生化性质、动力学性质及化学修饰的研究结果。该酶以天冬氨酸为其活性的必需基团,系一典型的天冬氨酸蛋白酶。     

一种食源性溶栓酶的分离纯化与部分酶学性质的研究

目的对以豆渣为原料,接种纳豆菌的发酵物分离纯化和酶学性质研究。方法采用生理盐水浸提、(NH4)2SO4分级沉淀、Sephadex G-100凝胶层析等纯化步骤,得到层析纯的食源性溶栓酶-纳豆激酶,结果经聚丙烯酰胺凝胶电泳显示为二个组分。酶学性质研究表明,以酪蛋白为底物时,最适反应温度为60℃,最适反应pH为8.0,在pH7~9溶液中,37℃以下基本稳定。体外溶栓作用表明,纳豆激酶溶解纤维蛋白的方式主要是直接溶解,而不是纤溶酶原激活剂。     

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。     

Purification and properties of an acid deoxyribonuclease from rat small intestinal mucosa

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.     

类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。