Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.     

Purification, characterization, and solvent-induced thermal stabilization of ficin from Ficus carica

Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur in multiple forms. Although crude ficin is of considerable commercial importance, ficin as such has not been fully characterized. A major ficin from the commercial crude proteinase mixture preparation of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography and is a single polypeptide chain protein with a molecular mass of 23 100 +/- 300 Da as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core sequence of ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme contains three disulfide bonds and a single free cysteine residue at the active site. The effect of co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of ficin was determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent thermal denaturation temperature (T(m)) of ficin was significantly increased from the control value of 72 +/- 1 degrees C in the presence of all co-solvents. However, the maximum stabilization effect was observed in terms of thermal stabilization by the co-solvent trehalose.     

A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria

A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.     

Biochemical and spectroscopic characterization of morning glory peroxidase from an invasive and hallucinogenic plant weed Ipomoea carnea

A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.     

Anionic relationships in leaf petiole sap of tomato and capsicum plants growing in a glasshouse

The nitrate, chloride and sulphate content and their interaction effects in capsicum and tomato plants growing in glasshouse under fertigation systems was studied. Using leaf petiolesap concentrations as an index of the uptake rhythm, it was found that nitrate‐chloride and nitrate‐chloride plus sulphate relationships are regulated by potential or logarithmical laws. Nitrate‐sulphate interactions only appear clear in capsicum plants, but not for tomato.

The utilization of these interaction curves may permit the use of waters with a relatively high saline level for the irrigation of the both capsicum and tomato plants, by suitable planning of the nitrate supply in the fertigation program.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

Characterizing nicotianamine aminotransferase: Improving its assay system and details of the regulation of its activity by Fe nutrition status

Under iron deficient conditions, graminaceous plants secrete mugineic acid family phytosiderophores (MAs) from their roots to dissolve sparingly soluble iron compounds in the rhizosphere, and take up iron in the form of an Fe³⁺-MAs complex (Takagi 1976). A good correlation has been reported between the tolerance of Fe-deficiency and the amount of secreted MAs (Takagi 1993). Therefore, by using the genes involved in MAs biosynthesis, molecular breeding might produce transgenic plants tolerant to Fe-deficiency with a high level of MAs secretion. The biosynthetic pathway of MAs from L-methionine has been clarified (Fig. 1) and the enzymes participating in this process are now being investigated to isolate the genes responsible. Nicotianamine aminotransferase (NAAT) catalyzes the amino group transfer between nicotianamine (NA) and 2-oxoglutaric acid (Fig. 1). In order to purify NAAT, enzyme assay methods for NAAT have been developed and modified (Shojima et al. 1990; Ohata et al. 1993; Kanazawa et al. 1994). Some characteristics of NAAT have been reported using these enzyme assay methods (Kanazawa et al. 1994, 1995). Here, we further investigate some characteristics of this enzyme to improve the enzyme assay method, namely 1) the effect of K⁺ and Mg²⁺ on NAAT activity in vitro, and 2) the direct influence of MAs, Fe³⁺, and Fe²⁺ on NAAT activity. In addition, based on these results, the induction of enzyme activity by Fe-deficiency and suppression of the activity by Fe-resupply was investigated, by applying the new enzyme assay method.     

Kinetic study of oxalic acid inhibition on enzymatic browning

Oxalic acid has a strong antibrowning activity. The inhibitory pattern on catechol-PPO model system appeared to be competitive, with a K(i) value of 2.0 mM. When the PPO was incubated with oxalic acid, the activity was not recovered via dialysis, but the inactivated enzyme partially recovered its activity when cupric ion was added. Comparing the relative antibrowning effectiveness of oxalic acid with other common antibrowning agents, oxalic acid with I(50) value of 1.1 mM is as effective as kojic acid and more potent than cysteine and glutathione.     

The effect of peatland drainage and rewetting (ditch blocking) on extracellular enzyme activities and water chemistry

Extensive areas of European peatlands have been drained by digging ditches in an attempt to improve the land, resulting in increased carbon dioxide fluxes to the atmosphere and enhanced fluvial dissolved organic carbon (DOC) concentrations. Numerous peatland restoration projects have been initiated which aim to raise water tables by ditch blocking, thus reversing drainage‐induced carbon losses. It has been suggested that extracellular hydrolase and phenol oxidase enzymes are partly responsible for controlling peatland carbon dynamics and that these enzymes are affected by environmental change. The aim of this study was to investigate how drainage and ditch blocking affect enzyme activities and water chemistry in a Welsh blanket bog, and to study the relationship between enzyme activity and water chemistry. A comparison of a drained and undrained site showed that the drained site had higher phenol oxidase and hydrolase activities, and lower concentrations of phenolic compounds which inhibit hydrolase enzymes. Ditch blocking had little impact upon enzyme activities; although hydrolase activities were lowered 4–9 months after restoration, the only significant difference was for arylsulphatase. Finally, we noted a negative correlation between β‐glucosidase activity and DOC concentrations, and a positive correlation between arylsulphatase activity and sulphate concentration. Phenol oxidase activity was negatively correlated with DOC concentrations in pore water, but for ditch water phenol oxidase correlated negatively with the ratio of phenolics to DOC. Our results imply that drainage could exacerbate gaseous and fluvial carbon losses and that peatland restoration may not reverse the effects, at least in the short term.     

Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

影响原料乳品质的纤溶酶活性的因素分析

为了充分了解原料乳中纤溶酶活性,以便从源头上提高原料乳品质,该文针对中国原料乳的生产现状,分析了奶牛品种、胎次、养殖模式及挤奶时间对原料乳中纤溶酶活性的影响,并对纤溶酶的热稳定性进行了研究。结果表明,养殖模式、奶牛品种及胎次对原料乳中纤溶酶活性影响显著（P＜0.05）,牧场养殖、1、4胎和娟珊牛原料乳中纤溶酶活性显著低于养殖小区、2、3胎及荷斯坦牛乳中的纤溶酶活性;挤奶时间对原料乳中纤溶酶活性影响不显著（P＞0.05）;原料乳中纤溶酶活性随热处理温度的升高而逐渐下降,巴氏杀菌（75℃、15 s）仅可使原料乳中纤溶酶活性下降25%;半胱氨酸对乳中纤溶酶活性具有一定的抑制作用。研究结果对根据纤溶酶活性对原料乳进行品质评价及分级具有一定借鉴作用。     

Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix)

Cathepsin L in silver carp musle was purified to 48.4-fold by acid-heat treatment and ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 30 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64 and insensitive to PMSF and pepstatin A, suggesting that the purified enzyme belongs to a family of cysteine proteinase. Consistent with this conclusion, Zn2+, Cu2+, Co2+, Ni2+, and Fe2+ could strongly inhibit the activity of this enzyme. The optimal pH and temperature were 5.0 and 55 degrees C, respectively. The enzyme catalyzed the hydrolysis of Z-Phe-Arg-MCA with a parameter of K(m) (8.27 microM) and K(cat) (28.7 s(-1)) but hardly hydrolyzed Z-Arg-Arg-MCA, Arg-MCA, and Boc-Val-Leu-Lys-MCA. The microstructure analysis by scanning electron microscopy showed that this proteinase is capable of destroying the network structure of silver carp surimi gels. The enzyme exhibited a higher hydrolytic activity on surimi protein at 65 degrees C than at 40 degrees C.     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Effects of lime and phosphate additions on changes in enzyme activities,microbial biomass and levels of extractable nitrogen,sulphur and phosphorus in an acid soil

Summary The effects of adding lime and/or phosphate to an acid, phosphate-deficient soil on microbial activity, enzyme activities and levels of biomass and extractable N, S and P were studied under laboratory conditions. Following rewetting there was, as expected, an initial flush in microbial growth and activity, as shown by large increases in CO₂ evolution, in levels of biomass N, S and P and by accumulation of extractable mineral N and sulphate in the soil. Following rewetting, additions of lime and phosphate further stimulated mineralization of C, N and S. In the first 4 weeks of incubation, the mineralized N accumulated in the soil as ammonium N and there was a concomitant rise in soil pH. After this initial period, nitrification increased substantially and soil pH decreased again. Additions of lime generally increased protease and sulphatase activities but decreased phosphatase activity. Additions of phosphate decreased the activities of all three enzymes. The positive effect of liming on protease and sulphatase activities persisted for the duration of the experiment while accumulation of mineral N and sulphate effectively ceased after about 4 weeks. Furthermore, although phosphate additions decreased the activities of protease and sulphatase they increased the accumulation of mineral N and sulphate. Thus, protease and sulphatase activities were not reliable indicators of the relative amounts of mineral N and sulphate accumulated in the soil during incubation. Some uncertainty surrounded the validity of biomass S and P values estimated by the chloroform fumigation technique because differing proportions of the sulphate and phosphate released from the lysed cells may have been extracted from the different treatments.     

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

镁原卟啉Ⅸ甲基转移酶研究进展

镁原卟啉Ⅸ甲基转移酶(ChlM)是叶绿素合成过程中的关键酶,对光合自养生物中叶绿素合成、叶绿体的正常发育以及光合作用的顺利进行起到至关重要的作用。ChlM不仅影响着光系统Ⅰ(PSⅠ)和光系统Ⅱ(PSⅡ)的形成以及脱落酸(ABA)的信号转导,还可以通过调控镁螯合酶H亚基基因(ChlH)表达而影响镁螯合酶活性。ChlM的产物可能参与叶绿体到细胞核的反向信号转导,同时,ChlM受到光照、叶绿体内氧化还原状态和叶片中叶酸含量等因素的影响。本文主要综述镁原卟啉Ⅸ甲基转移酶的结构特点、作用机制、酶活性调控及其参与的代谢调节,并展望镁原卟啉Ⅸ甲基转移酶的研究前景,以期为进一步深入研究并提高植物光合效率提供参考。     

泥蚶能量代谢相关的同工酶的分析

利用聚丙烯酰胺凝胶电泳分析了泥蚶（Tegillarca granosa）能量代谢相关的10种同工酶。酯酶和α-淀粉酶是同能量吸收有关的酶，主要分布于消化腺里。苹果酸脱氢酶、苹果酸酶、异柠檬酸脱氢酶、琥珀酸脱氢酶、醇脱氢酶、乳酸脱氢酶、6-磷酸葡萄糖脱氢酶和三磷酸腺苷激酶是与能量代谢相关的酶。泥蚶的能量供应有3种途径：有氧呼吸、无氧酵解和磷酸戊糖途径。从泥蚶体内含有很高活性的6-磷酸葡萄糖脱氢酶可知，磷酸戊糖途径是泥蚶糖代谢的主要途径，NADP为供能体。同其它贝类相比，泥蚶的三磷酸腺苷激酶的活性很低，这与它们的埋栖生活不活跃运动的习性是一致的。     

Iron chlorosis and nitrate reductase activity in response to Fe stress and additional nitrate in sorghum

Effect of additional nitrate supply on chlorosis development at different levels of Fe stress was examined in iron efficient and inefficient sorghum cultivars. Nitrate reductase, the enzyme primarily responsible for nitrate reduction was estimated in roots and leaves as affected by Fe stress and additional nitrate.

Young leaves showed differences in chlorosis progression at Fe stress levels. Nitrate reductase activity was depressed in roots and young leaves when iron supply was reduced beyond certain level (<0.0015 gm FeSO₄/1). Addition of extra nitrate was not able to enhance NR activity when iron supply was lower than ½ level. It was inferred that nitrate utilisation and iron nutrition influence each other and some minimu level of iron supply is necessary for efficient enzyme activity.     

Effect of nitrogen and sulphur fertilization on the yield and composition of winter oilseed rape

Abstract

A field experiment was conducted to assess the effects of varying levels of sulphur (S) and nitrogen (N) supply on oilseed rape (Brassica napus L. var. Rafal) grown in a soil assessed to be in the low category for plant available sulphate (SO₄). There were no significant effects on crop yield as a result of applied S for any of the treatments. This was probably due to a significant input of atmospheric S as a result of the wetter than average year. However, there were significant compositional effects on total S, total N and sulphate‐sulphur (SO₄‐S) which has implications for the supply of S from soil which can often display a range of S availabilities. Effects on composition were most marked as the crop reached maturity.