黑木耳原种胞外酶活性的研究

目的：研究黑木耳菌株原种胞外酶活分泌特性,为进行大规模生产用种早期判定提供检测手段。方法：以9个黑木耳栽培菌株的原种为实验材料,分别测定了其胞外漆酶、多酚氧化酶、羧甲基纤维素酶、滤纸维素酶的酶活性变化。结果：胞外漆酶、多酚氧化酶、纤维素酶活性的变化趋势大致相似,酶活随培养时间呈规律性的升高、降低,除个别菌株（3、6）外,不同菌株同种酶活性差别不大,酶活相对较高的菌株有2、3、6、9号。不同菌株酶活高峰出现的时间有差异,较早的出现在培养的第10d,较晚的出现在培养的第70d。结论：被测菌株胞外酶活存在一定的规律性,利用该规律性可检测菌种退化、老化和不利变异。     

碳素和氮素对Coriolus versicolor胞外酶分泌的影响

采用单因子相互比较法研究了不同碳素和氮素对彩绒革盖菌胞外漆酶,愈创木酚氧化酶,多酚氧化酶,锰过氧化物酶等木素降解酶分泌的影响,结果淀粉作碳源,干酪素作氮源有利于漆酶的分泌,麦芽粉作碳源,酵母膏作氮源有利于愈创木酚氧化酶和多酚氧化酶的分泌,淀粉作碳源,玉米粉作氮源有利于锰过氧化物酶的分泌。     

培养条件对毛栓菌菌丝体生长和多酚氧化酶分泌的影响

研究了碳源、氮源、培养温度、初始pH值、接种量、通气量等条件对毛栓菌（Trametes trogii)菌丝体生长及多酚氧化酶分泌的影响;结果表明,不同碳源和氮源对菌丝体生长影响较大,对多酚氧化酶分沁也有较大影响,麦草粉和麸皮为碳源,玉米粉、硫酸铵为氮源有利于多酚氧化酶的分泌;初始pH值对酶活影响较小,培养温度、通气量等对酶活影响较大。     

黄瓜植株性别表现与3种氧化酶同工酶的关系

采用同工酶电泳技术分析了二叶期纯雌株和雌雄株黄瓜（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ Ｌ．）子叶和真叶过氧化物酶、多酚氧化酶和超氧化物歧化酶同工酶,结果发现：给株比雌雄株酶活性强、酶带数量多,这种差异酶带大多与雌性或雌雄性别紧密相关,经检验可以作为黄瓜雌性株早期鉴定的生化标记,尤其以真叶中多酚氧化酶同工酶Ｒｆ０．２８７表现稳定,鉴定成功率高。等电聚焦电泳垂直平板聚丙烯酰胺凝胶电泳分辨效果好。     

柠檬酸处理对芒果采后生理活动的影响

有机酸的代谢对植物组织的成熟衰老关系密切 ,芒果采后用适当浓度的柠檬酸液处理 ,可明显减缓其采后的生理活动 ,呼吸高峰推迟出现 ,与芒果成熟密切相关的过氧化物酶、淀汾酶、多酚氧化酶等酶活性推迟上升 ,酶活高峰值也有所下降。但酸液浓度达 0 .5 %后过氧化物酶、淀粉酶的酶活高峰开始前移 ;而多酚氧化酶始终维持较低的酶活水平 ,几乎无酶活高峰出现     

一种烟粉虱成虫唾液酶鉴定与活性分析的方法

以B型烟粉虱Bemisia tabaci(Gennadius)成虫为材料,介绍了一种微型刺吸式昆虫唾液酶鉴定和分析的方法,主要包括人工饲养、唾液收集、唾液多酚氧化酶(PPO)和过氧化物酶(POD)的鉴定与活性分析。结果显示,B型烟粉虱在特异性嗜好寄主甘蓝上分泌的多酚氧化酶与过氧化物酶的比活力分别为嗜好寄主番茄上的1.54和1.65倍。该方法操作简捷,鉴定结果直观清晰,酶活测定灵敏,适合于其他微型刺吸式昆虫如蚜虫、木虱等的唾液酶研究。     

黄孢原毛平革菌木素过氧化物酶类在天然木素降解中作用的研究

通过诱变得到十一株木素过氧化物酶酶活降低的黄孢原毛平革菌（Ｐｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍ）突变株,用灰色理论分析了其木素过氧化物酶类的产生与木素降解能力间的相关性,并从中筛选到一株木素过氧化物酶缺陷、锰过氧化物酶酶活明显降低的突变株,其木素降解能力为原始菌株的８０％左右。该菌粗酶液作用于纤维素酶酶解杉木木素和天然褐腐木素,可产生小分子的木素降解产物,此反应不需Ｈ２Ｏ２参与。红外光谱分析表明粗酶液对木素的作用主要为氧化作用,因此推测此突变株粗酶液中含有不同于木素过氧化物酶和锰过氧化物酶的与木素氧化降解有关的酶类     

自腐菌漆酶的研究进展

自然界中术素的降解主要是通过丝状真菌,其中主要由白腐担子菌的分解作用来完成。白腐菌降解木质素,是通过其分泌的酶的作用来实现,白腐菌所分泌的木素降解酶主要有三种,即本素过氧化物酶（LigninPeroxidase,简称LIP）,依赖锰的过氧化物酶办hganes于depeMentoroxM用已简称MnP和漆酶（Ulccase。漆酶是一种含铜的多酚氧化酶中Aiphenoloxidase,ECI10．3．2）,最早是从漆树的分泌物中发现（Yoshi他1883）,随后人们发现一些高等真菌也能分泌这种酶（sertranct,lsoe;totrircte,is00）。现在人们知道,漆酶广泛地存在于担子菌、…     

一株产胆固醇氧化酶的不动杆菌的筛选鉴定及特性研究

目的：筛选鉴定产胆固醇氧化酶的菌株并对其酶的性质及发酵条件进行初步的研究。方法：利用唯一碳源的胆固醇平板筛选,酶活测定比较得酶活力最高的菌株;生理生化试验结合16S rDNA序列分析鉴定其种属,单因素及正交实验优化培养基及发酵条件。结果：所得菌株H4与产不动杆菌（Acinetobacter）有最近的亲缘关系,其胆固醇氧化酶作用的最适温度和pH分别为37℃和8.0,金属离子Mg2＋、Zn2＋、Fe2＋对该酶具有一定激活作用,菌株产酶的最适培养基为（g/L）：胆固醇1.5,蔗糖5,蛋白胨7,硝酸铵3,吐温1.0,pH7.5;最适培养条件为33℃,15mL培养基/100mL三角瓶,摇床培养（200r/min）48h,优化后发酵液酶活达135.8U/L。结论：获得了1株产胆固醇氧化酶的菌株H4,并初步鉴定为不动杆菌（Acinetobacter）。     

金针菇酶促褐变的区域分布及调控

金针菇的多酚氧化酶（ＰＰＯ）、过氧化物酶（ＰＯＤ）和过氧化氢酶（ＣＡＴ）活性在全株中呈现区域化分布。菌盖的酶活性最低,菌柄上部酶活性稍高,菌柄中部较高,菌柄下部活必同。用硫脲、亚硫酸盐、ＣａＣｌ２、高ＣＯ２、低Ｏ２、低温等方法处理金针菇,对以上三种酶的活性均有抑制作用。高Ｏ２、Ｈ２Ｏ２和温度的提高无益一种酶生。高Ｎ２（减压后充Ｎ２）不能抑制三种酶活性。     

7株放线菌在辣椒根部定殖及对辣椒叶片PAL与PPO活性的影响

采用盆栽接种试验、平皿涂抹法测数及常规酶活测定法研究了7株拮抗性放线菌在辣椒根部的定殖能力及接种24d对辣椒叶片苯丙氨酸解氨酶（PAl。）与多酚氧化酶活性（PPO）的诱导效应。结果表明：（1）供试7株放线菌单独接种均不能在辣椒根内定殖,但与辣椒疫霉P3混合接种时有5株可定殖;供试放线菌在辣椒根部的定殖能力与其体外平皿试验中产生的的拈抗圈大小基本无关;可定殖放线菌的定殖密度随时间延长而降低,至40d时均无活菌检出。（2）在放线菌单接处理中,5株菌接种后可诱导辣椒叶片PAL,活性提高,全部供试菌均能诱导PPO活性提高,其中可使两种酶同步提高的有5株菌;在放线菌＋P3混接处理中,有6株接种后可诱导PAL,活性提高,5株菌能诱导PPO活性提高,其中可使两种酶同步提高的有4株菌;在接入放线菌时同时混接辣椒疫霉,能增强2株供试放线菌对辣椒叶片PAL活性及6株供试放线菌对辣椒叶片PPO活性的诱导作用;供试放线菌的定殖能力与辣椒叶片PAL及PPO活性变化无明显规律性关系。     

Sucrose-dependent cell adherence and cariogenicity of serotype c Streptococcus mutans

Four strains of serotype c Streptococcus mutans differing in glucosyltransferase (GTase) and fructosyltransferase (FTase) activities were examined. These strains had been made resistant to streptomycin. FTase activity of an S. mutans clinical variant, MT6801R, which forms large mucoid colonies on sucrose-containing agar, was considerably higher than that of a typical serotype c strain, MT8148R, which forms small, rough colonies on the same agar. Two mutants, NG14 and NG7183, were induced from strain MT6801R by N-methyl-N'-nitro-N-nitrosoguanidine, and were found to be streptomycin-resistant. GTase and FTase activities of mutant NG14 were similar to those of the typical serotype c strain, while in mutant NG7183 the two enzyme activities were very low. Growing cells of these strains (except NG7183) adhered firmly to a glass surface in sucrose broth. Resting cells of all strains attached in small numbers to saliva-coated hydroxyapatite in the absence of sucrose. On the other hand, the presence of sucrose markedly enhanced the attachment of cells of strains MT8148R, MT6801R and NG14, but not NG7183. Cell-surface hydrophobicity and acid production of all strains were similar. Both strain MT8148R and NG14 colonized tooth surfaces and produced significant dental caries in specific-pathogen-free rats. Strain MT6801R had lower colonization ability and cariogenicity when compared with strains MT8148R and NG14. Furthermore, mutant NG7183 was able to colonize the tooth surfaces in small numbers, but failed to cause dental caries. These results indicate that sucrose-dependent cell adherence mediated by de novo glucan synthesis is necessary for the accumulation of serotype c S. mutans cells on the tooth surface and the induction of dental caries.     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

Production of extracellular emulsifying agent byPseudomonas aeruginosa UG1

Summary Twenty-three bacterial strains were isolated from oil-contaminated soil samples. Of these, 20 displayed some ability to effect oil dispersion and they were screened quantitatively for the ability to emulsify 0.5% (v/v) reference oil. One strain, identified asPseudomonas aeruginosa UG1, produced extracellular material that emulsified reference oil, hexadecane and 2-methylnaphthalene at concentrations as high as 6% (v/v) in nutrient broth. Emulsification activity increased during a 10 day incubation period at 30°C. The activity was not influenced by pH over the range 5 to 9. The emulsifying agent was precipitated by cold ethanol. The highest emulsifying activity was detected in the extracellular fraction precipitated between 30 and 50% (v/v) ethanol. A linear relationship was observed between emulsifier concentration (mg/ml) and emulsifying activity. Genetic analysis showed that thePseudomonas aeruginosa UG1 strain did not carry extrachromosomal plasmids, suggesting that the gene(s) coding for emulsifying activity was carried on the chromosome.     

Isolation and Characterization of Strain MMB-1 (CECT 4803), a Novel Melanogenic Marine Bacterium

A novel marine melanogenic bacterium, strain MMB-1, was isolated from the Mediterranean Sea. The taxonomic characterization of this strain indicated that it belongs to the genus Alteromonas. Under in vivo conditions, L-tyrosine was the specific monophenolic precursor for melanin synthesis. This bacterium contained all types of activities associated with polyphenol oxidases (PPOs), cresolase (EC 1.18.14.1), catecholase (EC 1.10.3.1), and laccase (EC 1.10.3.2). These activities were due to the presence of two different PPOs. The first one showed all the enzymatic activities, but it was not involved in melanogenesis in vivo, since amelanogenic mutant strains obtained by nitrosoguanidine treatment contained levels of this PPO similar to that of the wild-type MMB-1 strain. The second PPO showed cresolase and catecholase activities but no laccase, and it was involved in melanogenesis, since this enzyme was lost in amelanogenic mutant strains. This PPO was strongly activated by sodium dodecyl sulfate below the critical micelle concentration, and it is a tyrosinase-like enzyme showing a lag period in its tyrosine hydroxylase activity that could be avoided by small amounts of L-dopa. This is the first report of a bacterium that contains two PPOs and also the first report of a pluripotent PPO showing all types of oxidase activities. The bacterium and the pluripotent PPO may be useful models for exploring the roles of PPOs in cellular physiology, aside from melanin formation. On the other hand, the high oxidizing capacity of the PPO for a wide range of substrates could make possible its application in phenolic biotransformations, food processing, or the cosmetic industry, where fungal and plant PPOs are being used.     

A novel function of Goalpha: mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells

Go is the most abundant G protein expressed in brain but its function is less known. Here we show a novel function of Goalpha as a mediator of opioid receptor-induced extracellular signal-regulated kinase activation in neural cells. The current study found that, in neuroblastoma x glioma NG108-15 hybrid cells, activation of extracellular signal-regulated kinase through delta opioid receptors was mediated by pertussis toxin-sensitive G protein and independent of Gbetagamma subunits, PI3 kinase and receptor internalization. Overexpression of a dominant negative form of Goalpha1, but not Gialpha2, completely blocked delta opioid receptor-induced extracellular signal-regulated kinase activity. Decreasing Goalpha expression by RNA interference greatly reduced delta opioid receptor-induced extracellular signal-regulated kinase activity and extracellular signal-regulated kinase-dependent gene expression, while knocking down Gialpha2 did not. By taking advantage of differences between human and mouse Goalpha gene sequences, we simultaneously knocked down endogenous Goalpha expression and expressed exogenous human Goalpha subunits. We found that both human Goalpha1 and Goalpha2 could mediate delta opioid receptor-induced extracellular signal-regulated kinase activation. This study suggests that one of the functions of Goalpha in the brain is to mediate extracellular signal-regulated kinase activation by G protein-coupled receptors.     

Key enzymes enabling the growth of Arthrobacter sp. strain JBH1 with nitroglycerin as the sole source of carbon and nitrogen

Flavoprotein reductases that catalyze the transformation of nitroglycerin (NG) to dinitro- or mononitroglycerols enable bacteria containing such enzymes to use NG as the nitrogen source. The inability to use the resulting mononitroglycerols limits most strains to incomplete denitration of NG. Recently, Arthrobacter strain JBH1 was isolated for the ability to grow on NG as the sole source of carbon and nitrogen, but the enzymes and mechanisms involved were not established. Here, the enzymes that enable the Arthrobacter strain to incorporate NG into a productive pathway were identified. Enzyme assays indicated that the transformation of nitroglycerin to mononitroglycerol is NADPH dependent and that the subsequent transformation of mononitroglycerol is ATP dependent. Cloning and heterologous expression revealed that a flavoprotein catalyzes selective denitration of NG to 1-mononitroglycerol (1-MNG) and that 1-MNG is transformed to 1-nitro-3-phosphoglycerol by a glycerol kinase homolog. Phosphorylation of the nitroester intermediate enables the subsequent denitration of 1-MNG in a productive pathway that supports the growth of the isolate and mineralization of NG.     

chiA基因在稻根联合固氮菌E26和NG13中的表达

将带有粘质沙雷氏菌几丁质酶基因 (chiA)的 1 8kbHinfⅠ片段分别克隆到表达载体pKK2 2 3 3和质粒pMC71A上 ,构建成几丁质酶表达质粒pKChiA和pMChiA。将这 2种质粒转化稻根联合固氮菌阴沟肠杆菌E2 6 (EnterobactercloacaeE2 6 )和催娩克氏菌NG1 3 (Klebsiellaoxy tocaNG1 3 ) ,chiA基因在这 2菌株中获得高效表达。对表达产物的细胞定位测定表明 ,几丁质酶不仅存在于细胞周间质和胞内 ,而且还分泌到培养物上清液中。在对数生长后期 ,胞外、胞间质和胞内的几丁质酶活性分布分别为 2 3 %～ 2 8%、45 %～ 5 1 %和 2 1 %～ 3 2 %。经SDS 聚丙烯酰胺凝胶电泳检测表明 ,表达的几丁质酶蛋白分子量为 5 8kD。在受体细胞内 ,质粒pMChiA的稳定性要比质粒pKChiA高。