Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.     

Broccoli processing wastes as a source of peroxidase

A peroxidase isozyme (BP) was purified to homogeneity from broccoli stems ( Brassica oleraceae var. maraton) discarded from industrial processing wastes. BP specific activity was 1216 ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] units/mg, representing 466-fold that of crude extract. BP is a monomeric glycoprotein containing 16% carbohydrates, with a molecular mass of 49 kDa and an isoelectric point close to 4.2. From kinetic data it showed a two-substrate ping-pong mechanism, and the catalytic efficiency measured as the rate-limiting step of free BP regeneration was 3.4 x 10(6) M(-1) s(-1). The ABTS K m value was 0.2 mM, which was about 20 times lower than that reported for acidic commercial horseradish peroxidase (HRP). Assessment of BP secondary structure showed 30% helical character, similar to HRP and cytochrome c peroxidase. BP lost only 25% activity after 10 min of heating at 55 degrees C and pH 6; it was stable in the pH range from 4 to 9 and showed an optimum pH of 4.6 using ABTS as substrate. BP was active on substrates normally involved in lignin biosynthesis, such as caffeic and ferulic acids, and also displayed good catechol oxidation activity in the presence of hydrogen peroxide. Reverse micellar extraction was successfully used as potential large-scale prepurification of broccoli peroxidase, achieving a purification factor of 7, with 60% activity yield. Stems from the broccoli processing industry have a high potential as an alternative for peroxidase purification.     

Purification and partial characterization of broccoli (Brassica oleracea Var. Italica) peroxidases

Three peroxidase (POD) isoenzymes were purified from a soluble extract of broccoli stems. The acidic and neutral PODs were purified to homogeneity by using ion exchange and hydrophobic chromatography. The basic POD was purified by cation exchange and gel filtration chromatography. The neutral and basic PODs had molecular masses of approximately 43 kDa, and the acidic POD had a molecular mass of 48 kDa by SDS-PAGE. pI was approximately 4, 5, and 8 for acidic, neutral, and basic PODs, respectively. Optimum activity using guaiacol as the H donor was obtained at pH approximately 6 for both neutral and basic PODs and at pH approximately 4 for acidic POD. All three of the purified isoenzymes are glycosylated. Reaction rates with various substrates including guaiacol, guaiacol/MBTH, DMAB/MBTH, and ferulic acid/MBTH were different among the isoenzymes. K(m) and amino acid composition were also determined.     

Novel oxidations of (+)-catechin by horseradish peroxidase and laccase

Horseradish peroxidase (HRP; EC 1.11.1.7) catalyzed the H(2)O(2)-dependent oxidative coupling of (+)-catechin 1 to form three different biphenyl C-C dimers 2-4, whereas Rhus vernicifera laccase catalyzed the formation of two new catechin-hydroquinone adducts 5 and 6. Spectroscopic evidence showed that HRP dimers were linked through position 8 of the A-ring of one catechin moiety to C-5' of ring B in 2 and 4 and to C-2 of ring C in 3. The unusual catechin dicarboxylic acid dimer 4 was obtained by ortho cleavage of the E-ring. Hydroquinone served as both a shuttle oxidant and a reactant by coupling at C-2' and C-5' of the catechin B-ring during laccase oxidations. HRP and laccase oxidation products were compared to D,L-alpha-tocopherol and (+)-catechin for their abilities to inhibit iron-induced lipid peroxidation in rat brain homogenates and Fe(3+)-ADP/NADPH in rat liver microsomes, as measured by the intensity of thiobarbituric acid reactive substance. All metabolites exhibited anti-lipid peroxidation with IC(50) values approximately 2-8 times higher than those of standard compounds. Characteristic reaction products may prove to be novel markers for (+)-catechin antioxidant reactions in living systems.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

仿刺参体腔液补体类似物化学发光免疫检测

首次应用酶联化学发光免疫检测(Chemiluminesent Immunoassay,CLIA)技术测定仿刺参体腔液补体类似物AjC3和AjC4。羊抗人C3、C4抗体吸附到经过紫外线处理的聚苯乙烯管内,采用辣根过氧化物酶(HRP)标记抗体。过氧化氢和鲁米诺为辣根过氧化物酶的底物。捕获抗体包被最适浓度为1μg/ml,免疫反应20℃孵育2h达到平衡。HRP-IgC3、IgC4抗体复合物适宜稀释度为1:2000,HRP-IgC3I、gC4抗体复合物4℃下保存8d性能稳定,室温下5d内性能稳定。标准品浓度在0.1～10ng/ml范围内时与化学发光值之间具有良好的线性相关性,检测灵敏度为0.1ng/ml。结果表明应用酶联化学发光免疫检测技术能够检测到仿刺参体腔液中含有补体类似物,AjC3含量为6.58±1.4μg/ml,AjC4含量为0.67±0.3μg/ml。     

Characterization of novel β-glucosidases with transglycosylation properties from Trichosporon asahii

Two novel β-glucosidases from Trichosporon asahii, named BG1 and BG2, were purified to electrophoretic homogeneity using ammonium sulfate precipitation, hydrophobic interaction, ion exchange, and gelfiltration chromatography. The molecular weight of BG1 and BG2 were estimated as 160 kDa and 30 kDa, respectively. The K(m), V(max), K(cat), and K(cat)/K(m) values of the two β-glucosidases for p-nitrophenyl-β-D-glucopyranoside were determined. Both enzymes showed relatively high affinity to p-nitrophenyl-β-D-glucopyranoside in 4-nitrophenol glycosides and gentiobiose in saccharide substrates. The enzymes exhibited optimum activity at pH 6.0 and pH 5.5, respectively. Their respective optimum temperatures were 70 and 50 °C. Metal ions and inhibitors had different effects on the enzymes activities. Circular dichroism (CD) spectroscopy demonstrated that the purified BG1 exhibited a β-sheet-rich structure and that BG2 displayed a high random coil conformation. HPLC analysis of transglycosylation and reverse hydrolysis assays revealed that only BG1 possessed transglycosylation activity and synthesized cello-oligosaccharides by the addition of glucose. This suggested that BG1 could be used to produce complex bioactive glycosides and could be considered as a potential enzyme for industrial application.     

Phytoextraction of Cd and Pb and physiological effects in potato plants (Solanum tuberosum var. Spunta): importance of root temperature.

Three consecutive years of field experiments were carried out to investigate the effect of different root-zone temperatures, induced by the application of mulches, on the concentration and accumulation of Cd and Pb and on bioindicators (chlorophylls, catalase, peroxidase and cell wall fractions) in different organs of potato plants (roots, tubers, stems, and leaflets). Four different plastic covers were employed (T1, transparent polyethylene; T2, white polyethylene; T3, white and black coextruded polyethylene, and T4, black polyethylene), using uncovered plants as the control (T0). The different treatments had a significant effect on the mean root-zone temperatures (T0 = 16 degrees C, T1 = 20 degrees C, T2 = 23 degrees C, T3 = 27 degrees C, and T4 = 30 degrees C) and induced significantly different responses in the Cd and Pb concentrations and phytoaccumulation, with T2 (23 degrees C) and T3 (27 degrees C) giving high concentrations of Cd in the roots and low concentrations in other organs. In relation to Pb, T2 and T3 reached higher levels in the tubers and lower levels in the roots, stems, and leaves. In terms of phytoaccumulation, the roots and tubers were the most effective organs for Cd and Pb. On the other hand, the highest values of peroxidase and catalase activities were obtained for T3. In addition, most of the carbohydrate fractions in both the roots and the tubers were highest for T3. Meanwhile, the lowest pigment values were registered for T1 (20 degrees C). For phytoremediation, it is necessary to ascertain the relevance and control of the thermal regime of the soil to optimize the phytoextraction of pollutant elements (Cd and Pb).     

Screening of postharvest agricultural wastes as alternative sources of peroxidases: characterization and kinetics of a novel peroxidase from lentil ( Lens culinaris L.) stubble

Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL(-1)) did not vary for at least 15 months when stored at 4 °C (k(inact) = 0.146 year(-1), t(1/2 inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k(inact) = 2.2 × 10(-3) day(-1), t(1/2 inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurified by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H(2)O(2)-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K(m)(appH(2)O(2)) = 1.87 mM; V(max)(appH(2)O(2)) = 6.4 mM min(-1); K(m)(app guaicol) = 32 mM; V(max)(app guaicol) = 9.1 mM min(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases. Finally, after the effectiveness of the crude extracts of LSP in oxidizing and removing from solution a series of last-generation dyes present in effluents from textile industries (1) had been checked, a steady-state kinetic study of the H(2)O(2)-mediated oxidation and decolorization of Green Domalan BL by the catalytic action of the lentil stubble extract was carried out, with the observation of the same apparent Michaelian kinetic behavior (K(m)(appGD) = 471 μM; V(max)(appGD)= 23 μM min(-1)). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.     

Heat inactivation and reactivation of broccoli peroxidase

Heat inactivation characteristics differed for acidic (A), neutral (N), and basic (B) broccoli peroxidase. At 65 degrees C, A was the most heat stable followed by N and B. The activation energies for denaturation were 388, 189, and 269 kJ/mol for A, N, and B, respectively. Reactivation of N occurred rapidly, within 10 min after the heated enzyme was cooled and incubated at room temperature. The extent of reactivation varied from 0 to 50% depending on the isoenzyme and heating conditions (temperature and time). The denaturation temperature allowing the maximum reactivation was 90 degrees C for A and horseradish peroxidase (HRP) and 70 and 80 degrees C for B and N, respectively. In all cases, heat treatment at low temperatures for long times prevented reactivation of the heated enzymes. Calcium (5 mM) increased the thermal stability of N and B but had no effect on reactivation. The presence of 0.05% bovine serum albumin decreased thermal stability but increased the extent of reactivation of A..     

Beta-glucosidase from Chalara paradoxa CH32: purification and properties

The hyphomycete Chalara paradoxa CH32 produced an extracellular beta-glucosidase during the trophophase. The enzyme was purified to homogeneity by ion-exchange and size-exclusion chromatography. The purified enzyme had an estimated molecular mass of 170 kDa by size-exclusion chromatography and 167 kDa by SDS-PAGE. The enzyme had maximum activity at pH 4.0-5.0 and 45 degrees C. The enzyme was inactivated at 60 degrees C. At room temperature, it was unstable at acidic pH, but it was stable to alkaline pH. The purified enzyme was inhibited markedly by Hg(2+) and Ag(2+) and also to some extent by the detergents SDS, Tween 80, and Triton X-100 at 0.1%. Enzyme activity increased by 3-fold in the presence of 20% ethanol and to a lesser extent by other organic solvents. Purified beta-glucosidase was active against cellobiose and p-nitrophenyl-beta-D-glucopyranoside but did not hydrolyze lactose, maltose, sucrose, cellulosic substrates, or galactopyranoside, mannopyranoside, or xyloside derivatives of p-nitrophenol. The V(max) of the enzyme for p-NPG (K(m) = 0.52 mM) and cellobiose (K(m) = 0.58 mM) were 294 and 288.7 units/mg, respectively. Hydrolysis of pNPG was inhibited competitively by glucose (K(i) = 11.02 mM). Release of reducing sugars from carboxymethylcellulose by a purified endoglucanase produced by the same organism increased markedly in the presence of beta-glucosidase.     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

Purification and characterization of vanilla bean (Vanilla planifolia Andrews) beta-D-glucosidase

Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 microkat.mg(-1). The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.     

南岭山地土壤有机碳及组分海拔梯度变化特征

  目的  为了解我国亚热带山地土壤有机碳及组分海拔梯度变化规律及影响因素。  方法  以南岭国家级自然保护区不同海拔（400? ~ 1650?m）山地土壤为研究对象,调查了土壤有机碳及组分在不同土层深度的分布及密度特征,分析了土壤理化因子的影响。  结果  （1）总有机碳、易氧化碳、颗粒有机碳、惰性有机碳含量在相对较高海拔土壤中的含量整体更高,并在针阔混交林土壤中出现最大值,而水溶性有机碳含量则在低海拔的沟谷常绿阔叶林土壤中最高。（2）有机碳及组分含量随土层深度的增加呈明显下降趋势,随海拔变化幅度最大的组分为水溶性有机碳,随深度变化幅度最大的为颗粒有机碳,不同组分占总有机碳的比例在不同海拔和深度上的变化规律有所差异。（3）南岭山地土壤有机碳密度范围为8.81 ~ 26.59?kg m?2,整体略高于与其位置相近的山地土壤,有机碳及组分密度随海拔变化趋势与各自在土壤中的含量分布规律较为类似。（4）pH、黏粒含量、全氮与有机碳及组分含量的相关性较好,RDA分析结果表明全氮、全磷与土壤含水率对有机碳及组分变化的解释量占比较高。  结论  南岭山地土壤有机碳及组分具有明显的海拔梯度变化特征,土壤理化性质是影响有机碳及组分分布的重要因素。     

Both dioscorin,the tuber storage protein of yam (Dioscorea alata cv. Tainong No. 1), and its peptic hydrolysates exhibited angiotensin converting enzyme inhibitory activities

Dioscorin, the tuber storage protein of yam (Dioscorea alata cv. Tainong No. 1), was purified to homogeneity by DE-52 ion-exchange chromatography. This purified dioscorin was shown by spectrophotometric methods to inhibit angiotensin converting enzyme (ACE) in a dose-dependent manner (12.5-750 microg, respectively, 20.83-62.5% inhibitions) using N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly (FAPGG) as substrates. The 50% inhibition (IC(50)) of ACE activity was 6.404 microM dioscorin (250 microg corresponding to 7.81 nmol) compared to that of 0.00781 microM (0.0095 nmol) for captopril. The commercial bovine serum albumin and casein (bovine milk) showed less ACE inhibitory activity. The use of qualitative TLC also showed dioscorin as ACE inhibitors. Dioscorin showed mixed noncompetitive inhibitions against ACE; when 31.25 microg of dioscorin (0.8 microM) was added, the apparent inhibition constant (K(i)) was 2.738 microM. Pepsin was used for dioscorin hydrolysis at 37 degrees C for different times. It was found that the ACE inhibitory activity was increased from 51.32% to about 75% during 32 h hydrolysis. The smaller peptides were increased with increasing pepsin hydrolytic times. Dioscorin and its hydrolysates might be a potential for hypertension control when people consume yam tuber.     

Purification and characterization of polyphenol oxidase from garland chrysanthemum (Chrysanthemum coronarium L.)

Polyphenol oxidase (PPO) of garland chrysanthemum (Chrysanthemum coronarium L.) was purified approximately 32-fold with a recovery rate of 16% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as a single band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be about 47000 and 45000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (-)-epicatechin. The K(m) value (Michaelis constant) of the enzyme was 2.0 mM for chlorogenic acid (pH 4.0, 30 degrees C) and 10.0 mM for (-)-epicatechin (pH 8.0, 40 degrees C). The optimum pH was 4.0 for chlorogenic acid oxidase (ChO) and 8.0 for (-)-epicatechin oxidase (EpO). In the pH range from 5 to 11, their activities were quite stable at 5 degrees C for 22 h. The optimum temperatures of ChO and EpO activities were 30 and 40 degrees C, respectively. Both activities were stable at up to 50 degrees C after heat treatment for 30 min. The purified enzyme was strongly inhibited by l-ascorbic acid and l-cysteine at 1 mM.     

我国商品有机肥和有机废弃物中重金属、养分和盐分状况

【目的】 分析我国主要菜区商品有机肥和有机废弃物的重金属、养分和盐分状况,为蔬菜高效安全施肥和菜田土壤质量改善提供一定的理论依据。【方法】 针对全国主要菜区包括北方6省（市）和南方12省（市）典型商品有机肥和有机废弃物展开调查,取样时间是2014年1月上旬,共采取商品有机肥样品126个和有机废弃物样品255个,对3类商品有机肥和5类有机废弃物中重金属、养分和盐分状况进行了研究。【结果】 1）按照我国现有的NY525-2012有机肥料中重金属限量标准,商品鸡粪中Cd、Pb和Cr超标率分别为10.3%、17.2%和17.2%,商品猪粪中Cd和As超标率分别为20.0%和6.7%,其它商品有机肥中Cr、Cd、As和Hg超标率分别为13.4%、2.4%、2.4%和2.4%;鸡粪中Cd、As和Hg超标率分别为2.4%、7.1%和2.4%,猪粪中Cd、Pb、As和Hg超标率分别为1.5%、3.1%、15.4%和1.5%,牛粪中Cd和Hg超标率分别为1.7%和1.7%,羊粪中Cd超标率为12.5%,其它有机废弃物中Cr和As超标率分别为4.5%和13.6%。现在的畜禽粪便与传统有机肥已经大不相同,尤其是鸡粪和猪粪中Cu、Zn含量远高于上个世纪90年代初,分别增加1.5~16.2倍和1.3~4.7倍。2）商品有机肥中全氮含量、全钾含量和氮磷钾总含量较有机废弃物平均分别高出51.2%、32.0%和15.0%,有机废弃物中全磷含量、有机质含量、C/N比值和C/P比值较商品有机肥平均分别高出23.7%、41.5%、22.7%和15.2%。商品有机肥中,商品猪粪的全磷含量、有机质含量和C/N比值均相对较高,其它商品有机肥的全氮含量、全钾含量、氮磷钾总含量和C/P比值均相对较高;有机废弃物中,鸡粪和猪粪的全氮含量、全磷含量和氮磷钾总含量均相对较高,鸡粪、羊粪和其它有机废弃物的全钾含量相对较高,猪粪、牛粪和羊粪的有机质含量相对较高,牛粪的C/N比值相对较高,牛粪和羊粪的C/P比值相对较高。3）商品有机肥的EC值（平均23.5 mS/cm,n=126）较有机废弃物（平均7.7 mS/cm,n=255）高出2.1倍。商品有机肥中其它商品有机肥的EC值很高,其次是商品猪粪,商品鸡粪的EC值相对较低;有机废弃物中鸡粪和其它有机废弃物的EC值相对较高,其次是羊粪,猪粪和牛粪的EC值相对较低。【结论】 商品鸡粪以Cd、Pb和Cr超标为主,商品猪粪以Cd和As超标为主,其它商品有机肥以Cr超标为主;猪粪、鸡粪和其它有机废弃物以As超标为主,羊粪以Cd超标为主。商品有机肥中全氮含量、全钾含量和氮磷钾总含量均高于有机废弃物,有机废弃物中全磷含量、有机质含量、C/N比值和C/P比值均高于商品有机肥。商品有机肥的EC值远高于有机废弃物。     

Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas)

Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.     

Isolation and thermal characterization of an acidic isoperoxidase from turnip roots

An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.     

Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.