石油污染土壤的生物修复与土壤酶活性关系

研究了不同油浓度污染土壤经过两个为期125d的生物修复后的土壤中过氧化氢酶、多酚氧化酶和脂肪酶的酶活变化,分析了土壤中3种酶活性的变化特征与规律。结果表明,随着油浓度的增加,土壤中过氧化氢酶和多酚氧化酶的活性下降,脂肪酶活性增加;经过生物修复后,土壤中的过氧化氢酶和多酚氧化酶的活性在第二周期要比第一周期提高,而脂肪酶活性下降;这3种土壤酶活性变化受污染物浓度影响不显著,但不同浓度油污染土壤的修复对过氧化氢酶的影响要大于对多酚氧化酶和脂肪酶的影响。     

双加氧酶活力对细菌降解菲的指示作用

在液体培养基中选用两种石油降解细菌进行菲降解实验,研究了菲降解率和双加氧酶活力的变化。结果表明,菲降解率受其浓度的影响,当菲浓度为100mg·L-1时,其降解率为最高。而菲浓度高于100mg·L-1时,其降解率下降。实验发现在菲浓度为50～250mg·L-1条件下,细菌的双加氧酶活力与其菲的降解率存在较好的相关性,对细菌降解菲具有指示作用,可将双加氧酶活力作为菲降解率变化的评价指标。     

出芽短梗霉胞外酸性漆酶

通过愈创木酚法平板检测10株出芽短梗霉,发现5株菌能够分泌胞外多酚氧化酶,反应最适pH在2.0左右,均属于酸性多酚氧化酶。菌株NG的酶活最高,达110 U/mL。添加H2O2、EDTA以及过氧化氢酶不显著影响菌株NG胞外酶活,表明NG分泌的多酚氧化酶中不含有锰过氧化物酶（MnP）和不依赖Mn2＋的过氧化物酶（MiP）,属于漆酶（Lac）。     

低温条件下O3对红桃采后几种生理指标的影响

低温条件下,8 mg·m-3O3可延缓采后红桃果肉硬度下降以及呼吸速率和乙烯释放峰值出现时间,抑制丙二醛(MDA)含量和相对细胞膜透性的升高,过氧化氢酶(CAT)的活性保持相对较高水平,淀粉酶、过氧化物酶(POD)和多酚氧化酶(PPO)活性均明显受抑制,腐烂率下降.     

铜污染对三叶草幼苗生长及活性氧代谢影响的研究

通过水培实验研究了重金属铜(Cu)污染对三叶草(Trifolium pratense)幼苗生长及活性氧代谢系统的影响。结果表明,低浓度Cu污染(<10mg·L^-1)对三叶草幼苗生长无明显抑制现象,甚至促进幼苗生长,植株干重、鲜重和叶片可溶性蛋白及叶绿素含量均略微升高,丙二醛(MDA)水平降低,活性氧清除系统内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性均略微升高,保护酶系统仍保持平衡.但随Cu浓度(10～100mg·L^-1)增加则显示出一定的负效应,三叶草幼苗与对照组相比,植株矮小,须根短且数目少,植株干重、鲜重和可溶性蛋白含量均明显减少,叶片发黄,叶片色素含量下降,并随Cu浓度的增加而变化更显著。同时,随Cu浓度增加,叶片细胞膜透性增大,电导率显著升高,MDA水平上升,且活性氧清除系统遭到破坏,保护酶系统失衡,SOD和CAT活性显著下降,分别降低了26．7％和71．3％,而POD活性却明显上升,比对照升高了10．6倍。     

气体NO处理对蒜苗生长和抗氧化酶活性的影响

以改良的蒜为材料,用不同浓度NO气体（0．1、0．5、1．0μmol&#183;L^-1）在无氧环境中熏蒸大蒜3h后,检测蒜苗生长、光合色素和可溶性蛋白质含量以及抗氧化酶活性的结果表明：0．1和0．5μmol&#183;L^-1NO气体熏蒸的蒜种长成的幼苗,叶中光合色素和可溶性蛋白含量、假茎长、株高和假茎粗均大于未经NO熏蒸的植株,叶中超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性均提高;而1．0μmol&#183;L^-1的NO则抑制蒜苗生长,且抗氧化酶活性亦下降。气态NO处理的鳞茎幼苗生长也得到促进,以0．5μmol&#183;L^-1NO处理的效果最佳。     

壳寡糖诱导黄瓜抗黄瓜黑星病的初步研究

本文研究了壳寡糖诱导黄瓜对黑星病的抗性作用。利用6 mg/mL壳寡糖溶液对苗期黄瓜诱导,进行病情调查统计及测定处理前后黄瓜叶片的主要防御酶系———苯丙氨酸解氨酶,过氧化物酶,多酚氧化酶,超氧化物歧化酶,过氧化氢酶的活性变化。结果显示,壳寡糖对黄瓜黑星病在10 d和17 d的诱抗效果分别为60.25%和47.59%,且作为诱导因子可显著提高黄瓜叶片内苯丙氨酸解氨酶(PAL)活性,过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)活性也有所提高,但叶片内过氧化氢酶(CAT)活性无较明显变化。研究结果表明壳寡糖对黄瓜抗黑星病产生诱导作用,为研究壳寡糖作为新型生物农药提供了依据。     

黄河三角洲滩地不同造林模式的土壤酶活性

该文对东营市垦利县柽柳(Tamarix chinensis)林、枣(Ziziphus jujuba)园、旱柳(Salix matsudana)林、刺槐(Robinia pseudoacacia)林、白蜡(Fraxinus chinensis)林、桑(Morus alba)园土壤酶活性及其与土壤化学性质和土壤微生物的关系进行了分析。结果表明:在土壤空间上,脲酶和过氧化物酶活性随土层的增加而减小,多酚氧化酶和过氧化氢酶的活性在土层之间变化不明显。过氧化氢酶与脲酶和多酚氧化酶显著相关,脲酶与多酚氧化酶呈负相关。不同造林模式对土壤过氧化氢酶和过氧化物酶影响不大,对脲酶和多酚氧化活性有显著的影响。各种造林模式增加了脲酶的活性,均高于柽柳林;减小了多酚氧化酶的活性,除刺槐林外,均显著低于柽柳林;而造林模式对过氧化物酶和过氧化氢酶的影响较小。土壤酶活性与土壤养分有一定的相关,其中脲酶与全氮、有机质显著正相关,与速效氮正相关;多酚氧化酶与速效钾正相关,而过氧化氢酶与全氮、速效磷和有机质呈负相关,与土壤pH值相关性不显著;与微生物的相关系数不大,其中脲酶与固氮菌、纤维素分解菌显著正相关,与细菌正相关;多酚氧化酶与固氮菌和细菌负相关;过氧化氢酶与固氮菌显著负相关,与纤维素分解菌负相关。脲酶和多酚氧化酶可以作为滩地土壤质量评价的指标。     

环境条件对芽孢杆菌B-9987菌株防御酶诱导效应的影响

研究室内外条件下,海洋芽孢杆菌不同处理物对番茄防御酶系的诱导作用。分别使用不同浓度芽孢杆菌B-9987发酵液和无细胞滤液喷施番茄植株,于室内外两种环境条件下养殖,测定了处理11d内番茄5种防御酶的变化趋势。两种处理诱导抗性结果显示,在两种环境条件下处理植株苯丙氨酸解氨酶（PAL）、多酚氧化酶（ppo）、过氧化物酶（POD）、过氧化氢酶（CAT）较空白对照植株酶活性均有不同程度增加。除超氧化物歧化酶（SOD）外,室内环境较室外更利于B-9987菌株处理液对番茄防御酶系的诱导作用发挥;相同环境条件下,发酵液的处理诱导活性较强。B-9987菌株对番茄防御酶系有较好的诱导作用,但诱导作用的发挥受环境条件影响明显。     

中度嗜盐菌Martelella sp. AD-3 降解菲过程中水杨酸-5-羟化酶的酶学性质

[目的]研究中度嗜盐菌Martelella sp.AD-3在降解菲过程中水杨酸-5-羟化酶的活性与菲降解效率的关系及其酶学性质.[方法]通过HPLC分析菲的降解效率和AD-3菌粗酶液催化水杨酸的产物,根据NADH在340 nm处的吸光度变化计算水杨酸-5-羟化酶的活性.[结果]水杨酸-5-羟化酶是一种诱导酶,在AD-3菌的对数生长期和稳定初期时活性较高,酶活力大小与该菌对菲的降解速率基本一致.在菲浓度为200 mg/L、生长盐度为3％、pH为9.0的培养条件下,AD-3菌株表达的水杨酸-5-羟化酶的活力最高,为132.8 nmol/(min·mg).水杨酸-5-羟化酶催化水杨酸降解时的最适温度、pH和盐度分别为30℃、7.5和3％,酶的最大反应速率为200 nmol/(min· mg)、米氏常数Km为8.7μmol/L.[结论]AD-3菌在降解菲的过程中表达水杨酸-5-羟化酶,该酶的活性与菲降解速率具有相关性.     

Proteolytic activity of Staphylococcus xylosus strains on pork myofibrillar and sarcoplasmic proteins and use of selected strains in the production of "Naples type" salami

AIMS: The aim of this study was to determine the proteolytic activities of Staphylococcus xylosus strains on sarcoplasmic and myofibrillar proteins in order to evaluate the suitability of selected strains as starter cultures in the processing of a dry fermented pork sausage. METHODS AND RESULTS: The proteolytic activity of 27 strains of Staphylococcus xylosus on sarcoplasmic and myofibrillar proteins was determined by agar plate method, o-phtaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four strains were selected for the formulation of six starter cultures to use in the production of "Naples type" salami. The proteolytic contribution of starters was determined by SDS-PAGE, comparing the protein profile of inoculated sausages with that of uninoculated sausages after 0, 15 and 33 days of ripening. The results showed that the proteolytic activity of some strains, determined by the agar plate method, were not confirmed by electrophoretic and spectrophotometric assays. In fact, of 24 strains of Staphylococcus xylosus able to hydrolyse muscle protein extracts on agar plate, only 12 strains were shown to change SDS-PAGE profile of pork proteins. The SDS-PAGE profile of sarcoplasmic proteins extracted from all sausages showed that the major changes were produced with starters S3, S4 and S5 after 15 days of ripening. Also myofibrillar proteins undergo major changes after 15 days of ripening and the protein profiles showed the same pattern in all samples, except for the sausages produced with starter S4. CONCLUSIONS: The results of this work showed that the muscle protein extracts hydrolysis test is suitable for preliminary screening of Staphylococcus xylosus strains on the basis of their proteolytic activity. However, evaluation of muscle protein hydrolysis in a food model system could then be more appropriate for selecting micro-organisms for use as starter cultures for fermented sausages. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the formulation of starter cultures for the dry fermented sausages production.     

Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

芽胞形成相关基因缺失对解淀粉芽胞杆菌生物量及胞外酶表达的影响

解淀粉芽胞杆菌作为公认的安全生产宿主菌(GRAS),在高效表达异源蛋白方面具有巨大的发展潜力和应用前景.本研究以解淀粉芽胞杆菌TCCC111018为出发菌株,通过敲除芽胞形成相关基因(spo0A,sigF和sigE),构建了一系列突变菌株(BA△spo0A,BA△sigF,BA△sigE),并对突变株生物量和胞外酶表达...     

Biosurfactant Production by a Soil Pseudomonas Strain Growing on Polycyclic Aromatic Hydrocarbons

The capacity of polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria to produce biosurfactants was investigated. Twenty-three bacteria isolated from a soil contaminated with petroleum wastes were able to form clearing zones on mineral salt agar plates sprayed with solutions of PAHs. Naphthalene and phenanthrene were utilized as sole substrates. Biosurfactant production was detected by surface tension lowering and emulsifying activities from 10 of these strains grown in an iron-limited salt medium supplemented with high concentrations of dextrose or mannitol, as well as with naphthalene or phenanthrene. Glycolipid determinations showed that in cultures of Pseudomonas aeruginosa 19SJ on naphthalene, the maximal productivity of biosurfactants was delayed compared with that in cultures grown on mannitol. However, when small amounts of biosurfactants and naphthalene degradation intermediates were present at the onset of the cultivation, the delay was markedly shortened. Production of biosurfactants was accompanied by an increase in the aqueous concentration of naphthalene, indicating that the microorganism was promoting the solubility of its substrate. Detectable amounts of glycolipids were also produced on phenanthrene. This is the first report of biosurfactant production resulting from PAH metabolism.     

蚓粪和益生菌互作对土壤性状及番茄产量和品质的影响

本试验研究了两株益生细菌(巨大芽孢杆菌BM和解淀粉芽孢杆菌BA)与化肥和蚓粪配施对土壤性状、番茄产量和品质的影响．结果表明:与化肥相比,在等养分条件下蚓粪能够提高番茄产量、果实可溶性糖和蛋白质含量,并提高土壤pH和速效磷含量．与单施蚓粪相比,益生菌与蚓粪配施不仅能提高番茄产量、果实可溶性糖、蛋白质、维生素C含量和糖酸比,降低有机酸和硝态氮含量,而且增加了土壤pH和硝态氮含量,降低了土壤电导率;益生菌与化肥配施的效果不如益生菌与蚓粪配施．BA和BM与化肥或蚓粪配施时,番茄品质无显著差异,但BA配施蚓粪处理的番茄产量显著高于BM配施蚓粪处理;BM与化肥配施处理显著提高了土壤速效磷含量,而BA与蚓粪配施处理则显著提高了土壤速效钾含量．本研究表明,益生菌和蚓粪可替代化肥用于番茄生产和土壤肥力改良.     

Identification of inducible proteins in the phenanthrene degrader <Emphasis Type="Italic">Sphingobium chungbukense</Emphasis> DJ77 by 2-dimentional electrophoresis and liquid chromatography/tandem mass spectrometry

Sphingomonas chungbukensis DJ77 is a novel aromatic hydrocarbon-degrading bacterium capable of growing on phenanthrene as its sole source of carbon and energy. In this study, the protein expression profiles of S. chungbukensis DJ77 grown in the presence of phenanthrene were investigated by using two-dimensional gel electrophoresis (2-DE). Among 1000 protein spots visualized by 2-DE, the four proteins (i.e. 4-oxalocrotonate decarboxylase, 2-hydroxy-6-oxo-phenylhexa-2,4-dienoate hydrolase, glutathione S-transferase, and 2,3-dihydroxybiphenyl 1,2-dioxygenase) showing the significant upregulation by phenanthrene were identified by reversed-phase liquid chromatography–tandem mass spectrometry. Evidently, these proteins were involved in the metabolism of aromatic hydrocarbons. This can explain why S. chungbukensis DJ77 shows a significantly higher rate of phenanthrene consumption during the degradation process. The present analysis of proteomic responses and the detailed analysis results will be quite helpful to better understand the global physiology of S. chungbukensis DJ77, as proteome databases for various aromatic hydrocarbon-degrading strains have already been established.     

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.     

A New Collection of Thermosensitive Endocytosis Mutants in the Cellular Slime Mold Dictyostelium discoideum

ABSTRACT. We used a photoactivatable fluid-phase marker to isolate a new collection of thermosensitive endocytosis mutants in the cellular slime mold Dicfyostelium discoideum. All the strains were thermosensitive for growth on bacteria or axenic medium at 27° C. Initial rates of endocytosis rapidly decreased upon incubation at the restrictive temperature, but surprisingly most of the strains showed a transient recovery of activity with prolonged exposure to 27° C. Endocytosis and exocytosis activities were uncoupled for some of the cell lines at 27° C whereas the others had to be shifted to 29° C. Further molecular analysis of these mutants could lead to the discovery of new proteins involved in endocytosis and its regulation.