Purification and characterization of a beta-glucosidase with beta-xylosidase activity from Aspergillus sojae

A beta-glucosidase (EC 3.2.1.21) was purified as an electrophoretically homogeneous protein from a solid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 250 kDa by gel filtration chromatography and 118 kDa by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point of the enzyme was 3.80. The maximum velocity of rho-nitrophenyl beta-d-glucopyranoside degradation by the beta-glucosidase was attained at 60 degrees C and at pH 5.0. The purified enzyme was stable from pH 6.0 to 8.0, and up to 50 degrees C. The activity of the enzyme was significantly inhibited by Hg2+ and Cu2+, and stimulated by Mn2+ and Fe3+. The purified enzyme hydrolyzed beta-D-xylopyranosides as well as beta-D-glucopyranosides; the Km and Vmax values on rho-nitrophenyl beta-D-glucopyranoside were 0.14 mM and 16.7 micromol/min/mg protein, and on rho-nitrophenyl beta-D-xylopyranoside 0.51 mM and 12.2 micromol/min/mg protein, respectively.     

法尔皮有孢汉生酵母(Hanseniaspora valbyensis)醇乙酰基转移酶的分离纯化及特性

乙酸苯乙酯是苹果酒里的重要香气物质之一,是在醇乙酰基转移酶(AATase)的催化下由乙酰辅酶A和苯乙醇缩合而成。从法尔皮有孢汉生酵母(Hanseniasporavalbyensis)中收集细胞膜片段,然后经过DEAESepharose、SephadexG-75、OctylSepharose三步层析分离得到纯酶。纯化后的酶经SDS-PAGE电泳,得到一条明显的条带,分子量在37kD左右。此酶的最适反应条件是在30℃、pH7.0,在10℃以下酶比较稳定,pH稳定在7.0～8.0。重金属离子Hg2+、Zn2+、Pb2+对酶活有明显的抑制,EDTA、Mn2+对酶活没有太大的影响,Mg2+能激活此酶。此酶也能催化其它重要乙酸酯的合成,但对乙酸苯乙酯的合成具有特异性。     

Purification and characterization of N-Acetylglucosamine 6-phosphate deacetylase from Thermus caldophilus

N-Acetylglucosamine 6-phosphate deacetylase [EC 3.5.1.25] was purified and biochemically characterized from an extreme thermophile, Thermus caldophilus GK24. The optimum temperature and pH of the enzyme were 80 degrees C and 7.5, respectively. The enzyme is a tetramer composed of identical 45 kDa subunits. The N-terminal amino acid sequence of the purified enzyme was determined to be MSVDLKTLHRRHVLTP. It hydrolyzed GlcNAc-6-P, but not GlcNAc-1-P or chitin oligosaccharides. The deacetylase activity was completely inhibited by the addition of 1 mM Cu2+, but moderately activated by that of 1 mM Mn2+ and Co2+. Within 2 h of reaction, 2 mM GlcNAc-6-P was completely hydrolyzed to GlcN-6-P and acetate by the action of the deacetylase.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.     

Purification and comparison of triosephosphate isomerases from ammonia-oxidizing bacteria isolated from terrestrial and marine environments

Triosephosphate isomerases [TIMs, EC 5.3.1.1] were purified from two ammonia-oxidizing bacteria: Nitrosomonas sp. K1 (K1), Nitrosomonas sp. TNO632 (TNO). The molecular masses of the native enzymes were estimated to be about 53.6 (K1-TIM) and 51.9 kDa (TNO-TIM) by gel filtration, whereas SDS-PAGE produced one band for each enzyme with M(r) values of 27.1 (K1-TIM) and 26.4 kDa (TNO-TIM), respectively, suggesting that the enzymes consist of identical subunits. The apparent K(m) for d-glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) were about 1.19 and 4.78 mM (K1-TIM), and 0.41 and 6.01 mM (TNO-TIM), respectively. The two TIMs had different pH-activity curves with an optimum pH range of 6.5 (K1-TIM) and 8.0 (TNO-TIM). The temperature optima of K1-TIM and TNO-TIM were 50-60 and 60-65 degrees C, respectively. Both enzymes were strongly inhibited by 5,5'-ditiobis at 1.0 mM. The N-terminal amino acid sequences of K1-TIM and TNO-TIM were MRAGFVAGNWKMHG (K1-TIM) and MVRTGLVAGNWKMNG (TNO-TIM). A homology of 74.1% was observed between K1-TIM and TNO-TIM.     

黏质沙雷氏菌胞外几丁质酶的纯化及特性

黏质沙雷氏菌几丁质酶发酵上清液经硫酸铵沉淀、透析、DEAE-琼脂糖凝胶阴离子交换层析和苯基-琼脂糖凝胶疏水层析,得到电泳纯的几丁质酶和几丁质结合蛋白CBP21。该几丁质酶和CBP21分子质量分别约为58 ku和21 ku,CBP21对该几丁质酶水解几丁质增效明显。几丁质酶反应最适温度为50℃,最适pH约为6.5～7.0。该酶在55℃以下、pH 4.5～8.0范围内稳定。酶的Km值为0.22 mg/mL,Vm为1.26μmol/(min.mg)。金属离子K+、Sn2+、Mn2+对酶有一定激活作用,而Pb2+、Hg2+和Cu2+则强烈抑制其活性。该几丁质酶的糖基含量约为3.3%。EDTA和2-ME可分别提高酶活力65%和105%。H2O2强烈抑制酶活力,提示其活性中心可能存在硫氢基。     

金龟子绿僵菌(Metarhizium anisopliae Ma83)几丁质酶的纯化及性质

由金龟子绿僵茵Ma83菌株产生的几丁质酶经硫酸铵盐盐析,Sephadex G-100柱层析,DEAE-纤维素柱层析分离纯化后,得到SDS-PAGE均一样品.酶的最适反应温度为50℃,半失活温度为65℃;酶的最适反应pH值为5.0,酶在pH4.0～7.0范围内较稳定.Ag+、Co2+、K+、Mg2+对Ma83几丁质酶有激活作用,而Hg2+、Zn2+、Pb2+对几丁质酶活力有抑制作用.经计算Ma83菌株几丁质酶对胶体几丁质的Km值为1.05 mg/mL.     

环糊精葡萄糖基转移酶分离纯化及酶学性质研究

通过筛选和诱变获得一株高产环糊精葡萄糖基转移酶芽孢杆菌菌株,研究该菌所产环糊精葡萄糖基转移酶的分离纯化及酶学性质,结果显示：发酵液经离心处理后,采用硫酸铵溶液分步盐析、DEAE-cellulose 52 离子交换层析、Sephadex G-200 凝胶过滤层析方法得到电泳级环糊精葡萄糖基转移酶,SDS-PAGE 电泳显示该酶分子量为33kD,纯化倍数为10,得率为14.4%。该酶反应的最适温度为50℃,在40～60℃基本稳定,最适pH 值为8.0,在pH6.0～10.0 范围内基本稳定。Fe2+、Cu2+、Mg2+ 对该酶活力有明显的抑制作用。     

重组1,3-丙二醇氧化还原酶酶学性质和稳定性的研究

1,3-丙二醇氧化还原酶(PDOR)是以甘油为底物生产1,3-PD途径中的关键酶之一。将dhaT在E.coliBL21(DE3)pLysS进行了表达,对含有6×His标记的PDOR进行了纯化,同时考察了重组PDOR的酶学性质和稳定性。重组PDOR反应的最适pH和温度分别是10.0和55°C;在pH7.0～8.0,酶保持了较高的稳定性,酶在30°C保温表现出较高的稳定性;Ca2+,Mg2+和Cu2+对酶活性有抑制作用,而Fe2+,Na+,NH4+和Mn2+对酶活有促进作用;冷冻干燥处理后,PDOR酶活有一定的损失;添加适当浓度的保护剂——海藻糖、葡萄糖、蔗糖、聚乙二醇,对酶在冷冻干燥时有保护作用;添加5%蔗糖的固体酶制剂在保存过程中表现出较好的稳定性。     

杨桃多酚氧化酶的部分纯化及其特性研究

杨桃多酚氧化酶粗酶液经过DEAE-Toyopearl650M离子交换柱层析,Butyl-Toyopearl650M疏水柱层析后,被纯化了21.6倍,回收率为45.2%。该酶能迅速地催化焦性没食子酸的酶促氧化反应,而对邻苯二酚、间苯二酚、对苯二酚和绿原酸则完全无催化活性。该酶对焦性没食子酸的Km值为7.92mmol/L,其最适pH为8.0,pH稳定性范围在pH4.0-11.0,最适温度为60℃,热稳定性相对较高,在≥90℃加热30min后仍残留约9%的酶活性。该酶的最佳抑制剂是抗坏血酸,其次是NaHSO3和盐酸-L-半胱氨酸,Cu2+、Zn2+、Ca2+等金属离子对该酶也有一定的抑制作用。     

Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719

An alkaline protease was purified to apparent homogeneity from culture supernatants of Bacillus sp. PS719, a novel alkaliphilic, thermophilic bacterium isolated from a thermal spring soil sample, by ammonium sulfate precipitation followed by DEAE-cellulose and alpha-casein agarose column chromatographies. The purified enzyme migrated as a single protein band of 42 kDa during both denaturing and nondenaturing gel electrophoresis, suggesting that it consists of a single polypeptide chain. Its isoelectric point was approximately 4.8. The protease exhibited maximum activity towards azocasein at pH 9.0 and at 75 degrees C. The enzyme activity was stimulated by Ca2+, but was inhibited in the presence of Fe2+ or Cu2+. The enzyme was stable in the pH range 8.0 to 10.0 and up to 80 degrees C in the absence of Ca2+. Since phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin (DCI) in addition to N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) completely inhibited the activity, this enzyme appears to be a trypsin-like serine protease. Among the various oligopeptidyl-p-nitroanilides tested, the protease showed a preference for cleavage at arginine residues on the carboxylic side of the scissile bond of the substrate, liberating p-nitroaniline from N-carbobenzoxy (CBZ)-L-arginine-p-nitroanilide with the K(m) and V(max) values of 0.6 mM and 1.0 micromol.min(-1).mg protein(-1), respectively.     

鸭肝谷氨酸脱氢酶的纯化与酶学性质研究

目的：获得鸭肝谷氨酸脱氢酶纯品并对其酶学性质进行研究。方法：采用丙酮脱脂、重金属离子沉淀、硫酸铵分级沉淀、DEAE-Sepharose 离子交换层析和Sephacryl S-200 凝胶层析方法,分离纯化鸭肝谷氨酸脱氢酶,用SDS- 聚丙烯酰胺凝胶电泳法进行纯度鉴定和酶相对分子质量测定。结果：从鸭肝中分离纯化获得电泳纯的谷氨酸脱氢酶,纯化倍数为60.93 倍,酶活力回收率为11.02%,比活力达24.37U/mg。酶相对分子质量为371.41,亚基相对分子质量为61.60。推测该酶由6 个相同亚基构成。该酶对NADH 的Km 为53.19μmol/L,最适pH 值为10.0,最适反应温度为35℃。该酶在pH8.0 左右较稳定;在40℃以下酶活力保持稳定。Zn2+、Li+ 和Cu2+ 对该酶具有显著的抑制作用。结论：分离纯化获得谷氨酸脱氢酶,该酶具有较高应用价值。     

Purification of isocitrate lyase from Saccharomyces cerevisiae

Isocitrate lyase purified to homogeneity from Saccharomyces cerevisiae was composed of four identical subunits with a molecular mass of 75 kDa. The enzyme was most active at pH 7.0 in the presence of 5 mM-Mg2+. The Km value for threo-Ds-isocitrate was 1.4 mM. Isocitrate lyase was shown to be thermostable at 50 degrees C for 60 min at a high salt concentration, but rapidly lost activity at -20 degrees C or by dialysis.     

Bacillus sp.CBB272脱枝酶的酶学性质

脱枝酶是淀粉加工过程中水解其1,6-糖苷键的水解酶,为淀粉彻底糖化所必需。作者从高温菌Bacillus sp.CBB272的培养液中,经过盐析、凝胶过滤层析、弱阴离子交换层析以及强阴离子交换层析联用的方法,纯化出一种新型脱枝酶。该酶在SDS-PAGE上的相对分子质量约为70 000,最适作用温度为70℃,最适作用pH为6.0。该酶在30~70℃、pH 4.5~9.0之间具有优良的稳定性。该酶在50℃和pH 6.0下水解支链淀粉的Km、Vmax分别为4.0324 mg/mL和0.1841 mg/(min.mL)。研究了不同金属离子对该酶活性和热稳定性的影响,发现Ca2+、Mg2+、Mn2+等金属离子对于该酶具有显著的激活效果,并且Ca2+对该酶的热稳定性具有很好的提升作用。     

Purification and characterization of fenitrothion hydrolase from Burkholderia sp. NF100

The organophosphorus pesticide hydrolase was purified to homogeneity from Burkholderia sp. NF100 by detergent extraction of the cell membrane fraction, anion-exchange, chromatofocusing, and gel filtration chromatographies. The purified enzyme had a molecular mass of 55 kDa and a pI 5.8, and the hydrolase activity was strongly inhibited by EDTA, dithiothreitol (DTT), Hg2+ and 1,10-phenanthroline. The optimum pH and temperature for the enzyme activity were 8.0 and 40 degrees C, respectively. The enzyme hydrolyzed five organophosphorus pesticides.     

杂优-2平菇漆酶的分离纯化及酶学性质

将杂优-2平菇菌丝进行液体培养,发酵液经硫酸铵分级沉淀、DEAE（diethylaminoethyl）-Sepharose fastflow层析和Superdex-200 prep grade层析等方法纯化,获得了电泳纯的杂优-2平菇漆酶,并对纯化的漆酶进行了部分酶学性质研究。结果显示,杂优-2平菇漆酶比活力为115 U/mg,分子质量约为244.0 kD,亚基分子质量约为85.6 kD。最适反应pH值和最适反应温度分别为5.0和55 ℃,在pH 6.0～8.0及40～55 ℃范围内稳定性较好;最适条件下,以2,2’-联氮-二（3-乙基苯并噻唑-6-磺酸）为底物的Km值为2.1 mmol/L,最大反应速率（vmax）为0.117 μmol/（min·L）。Fe2＋、抗坏血酸对该酶活性具有完全抑制作用,乙二胺四乙酸、Ag＋、Mg2＋、Li＋对该酶活性影响较小;草酸、甲醇、正丁醇、K＋、Ca2＋、Ba2＋、Zn2＋、Cd2＋、Pb2＋、Mn2＋、Co2＋对该酶活性有不同程度的抑制作用;Cu2＋激活作用不明显;尿素、乙醇、异丙醇对该酶活性具有激活作用。     

Purification and characterization of three extracellular proteinases produced by Pseudomonas fluorescens INIA 745, an isolate from ewe's milk.

Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45 degrees C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded beta-casein, with preference to alphas1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.     

坎皮纳斯类芽孢杆菌木聚糖酶的纯化以及酶学性质

采用硫酸铵盐析、Sephadex G-50凝胶过滤、DEAE Sepharose Fast Flow离子交换层析对坎皮纳斯类芽孢杆菌（Paenibacillus campinasensis）xy-7发酵液进行分离纯化,获得纯化的木聚糖酶,纯化倍数为26.36,酶活回收率为5.13%。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果为单一条带,分子质量为24.5 ku。酶学性质研究结果表明,该酶的最适反应温度为60 ℃,最适pH值为8.0。K+、Fe2+对酶有激活作用,Zn2+、Cu2+对酶有强烈的抑制作用。以榉木木聚糖为底物时,米氏常数Km=4.733 mg/mL,最大酶反应速率Vm=315.85 μmol/（min·mg）。     

醋酸菌中乙醛脱氢酶的分离纯化及酶学性质

以实验室筛选得到的醋酸菌(Acetobacter pomorum)为实验菌株发酵产酶,通过细胞破壁,采用(NH₄)₂SO₄沉淀、透析、DEAE-Sepharose 离子交换层析及 Superdex G-75凝胶过滤层析分离纯化得到乙醛脱氢酶的酶液,并考察其酶学性质。该酶分子质量为221.60 kDa,单个亚基分子质量约为54.41 kDa,为四聚体结构;纯酶液比活力20.25 U/mg,纯化倍数为10.16倍,乙醛脱氢酶(aldehyde dehydrogenase,ALDH)的回收率为6.53%。酶学性质研究表明,ALDH促进乙醛分解的最适温度为50 ℃,40~50 ℃相对酶活力稳定性好;该酶的最适pH为7.0,当pH在5.5~7.5内酶活力表现稳定;金属离子对酶活性的影响实验表明,Na+、K+、Zn2+、Ba2+对该酶酶有不同程度抑制作用,而Mg2+、Ca2+、Al3+、Li+、Cu2+具有促进作用;ALDH的最适底物为乙醛,相对偏好直链醛类。ALDH活性较大,为后期表达和深入研究其生物学功能提供理论和数据支持。     

Leucine aminopeptidase from Streptomyces hygroscopicus is controlled by a low molecular weight inhibitor

In culture filtrate of Streptomyces hygroscopicus a producer of polyketide antibiotics, a leucine aminopeptidase and its autogenous inhibitor were detected. The leucine aminopeptidase was purified 4573-fold with yield of 82% by combination of ion exchange and hydrophobic chromatography. The enzyme is monomeric with a molecular mass of 51 kDa determined by gel chromatography and 67 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal activity was at pH 8.0 and 40 degrees C. The pI of leucine aminopeptidase is 8.2. The enzyme is strongly inhibited by 1,10-phenantroline, amastatin and dithiothreitol. Atomic absorption spectrometry indicated 2 mols of ion zinc per mol of enzyme. The enzyme is stable at up to 70 degrees C. Leucine aminopeptidase prefers leucine and methionine as N-terminal amino acids. Activity of leucine aminopeptidase is strongly modulated by an autogenous low-molecular weight inhibitor during fermentation, especially during periods of intensive antibiotic production.