Lipid changes in tobacco cell suspensions following treatment with cellulase elicitor

Tobacco ( Nicotians tabacum ) KY14 cell cultures have previously been reported to produce capsidiol and other stress metabolites when treated with fungal elicitor or cellulase. Using a new high performance liquid chromatographic technique, we have measured the changes in sesquiterpene phytoalexins and membrane lipid classes thai occur upon elicitation of tobacco cell cultures with cellulase. Measurable levels of capsidiol and debneyol were found in the tobacco cells and in the culture medium after 8 h of elicitor treatment, with levels continuing to increase for up to 24 h. For the duration of the experiments, the levels of most of the galactolipids and phospholipids were found to decrease in elicited cells and increase in control cells. The most striking change was a rapid decrease in the level of digalactosyldiacylglycerol in elicited cells, to less than 10% of the level in control cells. Among the sterol lipid classes, the most notable changes occurred in the levels of sterol esters and acylated sterol glycosides, which increased significantly in elicited cells within 2 to 4 h after addition of cellulase, but remained unchanged in control cells. Free sterols and sterol glycosides declined slightly, while free fatty acids dropped to low levels 24 h after treatment of cells with cellulase. The present results and those of previous studies indicate that esterification of phytosterols may be a widespread response to environmental or chemical stress.     

木聚糖酶基因研究进展

半纤维素分解微生物在自然界碳素循环中起着重要作用,半纤维素是植物多糖的重要成分之一。木聚糖则是半纤维素的主要成分。木聚糖酶(EC3.2.1.8)可催化木聚糖的水解,在各种各样的生物体里都发现有木聚糖酶,如细菌、放线菌、真菌。在过去几十年里,有超过100个木聚糖酶基因被克隆进同源或异源宿主中,其目的是为了超表达木聚糖酶和改变它们的特性以适应商业应用。木聚糖酶的应用极其广泛,可用于生物转化、造纸、食品、饲料、能源、纺织等行业。尤其是迫切的环境问题将进一步促进木聚糖酶研究的开展。     

微生物产生的木聚糖酶的功能和应用

术聚糖是一种异质多糖,主要由木糖和阿拉伯糖组成。微生物产生的木聚糖酶来源广泛,能将木聚糖水解为木寡糖和D-木糖。该酶具有极大的应用价值,如可用于纸浆的漂白以减少环境污染,也可将造纸工业及农业废料中的木聚糖转化为D-木糖。     

工业分枝杆菌植物甾醇转化途径及菌种改造研究进展

相比于传统的化学转化法,微生物转化法在甾体药物的生产中显示出了明显的优势.利用分枝杆菌降解植物甾醇可以生成一系列甾体药物的中间体,这极大地方便了甾体药物的生产.通过基因工程、分子生物学和结构生物学等学科的技术手段,人们对甾醇转化菌株进行了深入的探索和改造.本文对工业分枝杆菌植物甾醇转化途径及菌种改造的研究进展进行了综述...     

地衣芽孢杆菌H-1的鉴定及其产木聚糖酶性质的研究

本实验室分离到一株木聚糖酶高产菌,经形态、生理以及生化鉴定,确认为地衣穿孢杆菌,定名为H-1。对H-1的胞外木聚糖酶进行初步的研究。从碳源利用方面,认为 H-1的木聚糖酶为组成型合成。木聚糖和纤维二糖对它有诱导作用,而木糖和葡萄糖对酶的产生有阻抑作用。对酶解产物分析表明,有小分子糖产生、但并非都是单糖。H-1胞外木聚糖酶经60％硫酸铵沉淀,过DEAE-50柱,以及超过滤,得到了部分纯化酶。该酶作用的最适pH为7.6,在pH4～5范围较为稳定;最适温度为50℃; 70℃25min则完全失活。米氏常数为10.42×10~(-3)g/ml,最大反应速度为0.345μmol/min。2mM Ag~+使酶活增加了1.5倍,2mM EDTA抑制了 22％的酶活性,而2mM Cu~(++)则使酶完全失活。     

Rapid quantification of free and esterified phytosterols in human serum using APPI-LC-MS/MS

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free and esterified beta-sitosterol, campesterol, brassicasterol, and stigmasterol. The total time for sample pretreatment and analysis could be reduced from approximately 3 h [gas chromatography-mass spectrometry (GC-MS)] to 15 min. The detection limits of the different phytosterols ranged between 0.25 and 0.68 microg/l. Linear ranges were between 1 and 1,000 microg/l. The within-run and between-run variabilities ranged between 1.4% and 9.9%. The analytical sensitivity was at least 150-fold higher compared with GC-MS. Our new method allows a rapid and simultaneous determination of free and esterified phytosterols in serum.     

斜卧青霉纤维素酶和木聚糖酶高产菌株的选育

以纤维素酶高产菌株斜卧青霉A50为出发菌株,通过紫外诱变原生质体获得1株木聚糖酶活力提高80%而纤维素酶活力没有改变的6号菌。蛋白质电泳和酶谱检测结果显示,纤维素酶谱基本无差别,而木聚糖酶谱显示6号菌比A50多了一条带。6号菌优化后的产酶培养基组成为:麸皮7%、葡萄糖0.1%,该条件下,纤维素酶活为19.7IU/mL,木聚糖酶活力为215.4IU/mL。     

Constitutive and elicitation induced metabolism of isoflavones and pterocarpans in chickpea (Cicer arietinum) cell suspension cultures

Constitutive phenolics of chickpea cell suspension cultures are the isoflavones formononetin and biochanin A, the isoflavanones homoferreirin and cicerin and the pterocarpans medicarpin and maackiain. They accumulate as vacuolar malonylglucosides. The biosynthetic pathways to isoflavones, pterocarpans and malonylglucoside conjugates together with their enzymes are explained. Elicitation of cell cultures leads to pronounced increases in the activities of biosynthetic enzymes with differential effects on the enzymes involved in conjugate metabolism. Low elicitor doses favour pterocarpan conjugate formation whereas high doses lead to pterocarpan aglycone accumulation accompanied by vacuolar efflux of formononetin and pterocarpan malonylglucosides. Elicitor-induced changes in enzyme activities and vacuolar efflux of conjugates are prevented by application of 10^-3M concentrations of cinnamic acid. Cinnamate is alternatively metabolized to a glucose ester, a S-glutathionyl conjugate and to cell wall bounds forms; these reactions are intensified by elicitation. Isoflavone and pterocarpan biosynthesis and conjugate metabolism as regulated by elicitation and cinnamate is depicted in a metabolic grid to explain the complex regulatory pattern of phenolic accumulation in chickpea cell cultures.Abbreviations AOPP L--aminooxy--phenylpropionic acid - BGM biochanin A 7-0-glucoside-6-0-malonate - FGM formononetin 7-0-glucoside-6-0-malonate - HPLC high performance liquid chromatography - MaGM maackianin 3-0-glucoside-6-0-malonate - MeGM medicarpin 3-0-glucoside-6-0-malonate     

Sterol methyltransferase from Uromyces phaseoli: An investigation of the first and the second transmethylation reactions

The two-carbon unit at C-24 of many plant, algal and fungal sterols is known to be synthesized by two successive transmethylations with S-adenosylm     

p185蛋白及其抗体的研究进展

描述了p185在信号转导过程中的功能,讨论了抗p185抗体的蛋白质工程及它们在肿瘤治疗中的应用.     

Purification,Characterization, and Molecular Cloning of Acidophilic Xylanase from Penicillium sp.40

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family11 xylanases from fungi.     

Molecular Cloning,Overexpression, and Purification of a Major Xylanase from Aspergillus oryzae

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58°C. It had a Km of 5.1 mg/ml and a Vmax of 123 μmol/min/mg when birch wood xylan was used as a substrate.     

绿色糖单孢菌产木聚糖酶规律及其耐碱耐热性的初步研究

采用绿色糖单孢菌为实验材料,在不同诱导产酶培养基上经过192h的振荡培养,探索其产酶时程规律．结果表明,不同的诱导底物诱导产生的木聚糖酶活性差异不显著,但诱导产纤维素酶活性差异显著．其中松木粉加棉纱培养基诱导产纤维素酶活性为0．08IU／ml,与空白对照(5．40IU／ml)相比显著下降(P≤0．05)．为了适应纸浆漂白实际应用中纤维素酶越少越好的要求,选择该培养基为最佳诱导产酶培养基．绿色糖单孢菌在上述培养基中培养156h后达到木聚糖酶产酶高峰。粗酶液酶活可达到9．03IU／ml．通过对该酶进行高温及碱性处理。实验结果表明绿色糖单孢菌分泌的木聚糖酶在pH7．0下反应表现最高活性,同时在90℃下保温3h后酶活为原来的63．55％,具有较好的耐碱耐热性．     

Correlative Analysis of Cochliobolus sativus Pathogenicity and in vitro Xylanase Activity

Spot blotch (SB) caused by Cochliobolus sativus has been the major yield‐reducing factor for barley production during the last decade. In this study, the correlation between aggressiveness and in vitro xylanase production of 29 isolates of C. sativus was investigated. Isolate aggressiveness was evaluated in term of lesion form in barley leaves. Additionally, the isolates were compared for their ability to produce in vitro significant levels of xylanase activities when grown in a liquid medium. Aggressive isolates released more xylanase of weakly aggressive isolates. Correlation tests analysis revealed a significant relationship (r = 0.84, r = 0.50; P < 0.01) between the xylanase (per unit fungal mass) and aggressiveness on the two barley cultivars Arabi Abiad and Bowman, respectively. Correlation between the production of this enzyme and the origin of the isolates was not found. The results indicate that the production of xylanase influences the aggressiveness of the isolates of C. sativus towards barley seedlings.     

Increased N-acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells

Previous studies with tobacco (Nicotiana tabacum L.) cell suspensions indicated that elicitation of defense response (production of phytoalexins) with xylanase (1,4-β-D-xylanxylanohydrolase: EC 3.2.1.8) resulted in a dramatic acylation of phytosterols (Moreau et al. 1994). N-acylphosphatidylethanolamine (NAPE), an acylated derivative of phosphatidylethanolamine (PE), was recently demonstrated to be synthesized in vivo in plant tissues (Chapman and Moore 1993a). Here we report that acylation of PE was increased in elicitor-treated cells. NAPE levels increased 3-fold (from 1.6 to 4.8 mol% of total phospholipids) after a 2-h treatment of cell suspensions with xylanase (1 δg ml^?1). Specific activity of NAPE synthase increased in parallel with NAPE levels. Levels of NAPE and NAPE synthase activity declined during the period of 2–4 h after elicitation while levels of acylated sterolglycosides (ASG) continued to increase. Radiolabeling studies with [2^?14C]-ethanolamine confirmed that three times as much NAPE was synthesized in elicitor-treated cells compared to that in unelicited cells. Patterns of incorporation of [1-¹⁴C]-palmitic acid into membrane phospholipids in elicitor-treated cells suggested that increased acylation of lipids may be a result of changes in the acyl-coenzyme A pool. Treatment of cells with purified ethylene biosynthesis-inducing xylanase (EIX; 1 δg ml^?1 cells) resulted in increased levels of NAPE synthase activity comparable to those observed with the commercial preparations of xylanase. Boiled xylanase did not elicit an increase in the specific activity of NAPE synthase. Collectively our results demonstrate that the accumulation of NAPE in tobacco cells is attributable to increased activity of NAPE synthase. This suggests that NAPE may be specifically synthesized to play a protective role in membranes of plant cells as has been suggested for membranes of damaged animal cells.     

一株产木聚糖酶菌株的分离、鉴定及其酶学特性研究

以木聚糖为唯一碳源,采用平板水解圈筛选和摇瓶发酵相结合的方法,从土壤中分离、筛选到一株产木聚糖酶的细菌xy-7,根据其形态和生理、生化特性,并结合16S rDNA序列分析,初步鉴定为坎皮纳斯类芽孢杆菌(Paenibacillus campinasensis)。经测定,xy-7所产木聚糖酶的最适作用温度为60℃,最适作用pH为7.0。该酶热稳定性较好,60℃时保温2h酶活保持为原来的73%。此外,该酶的pH作用范围较广,pH 9.0时酶活仍能保持68%,属于耐碱性木聚糖酶。这些性质表明,该酶在制浆造纸等行业具有较好的应用前景。     

Acute ethanol treatment induces a bimodal response of phospholipid acylation rates in rat red blood cells

A single intraperitoneal injection of ethanol (4 g/kg) in rats elicited a bimodal response of acylation rates in phosphatidylcholine and phosphatidylethanolamine of intact red blood cells. Within an initial period, ethanol inhibited acylation rates. The inhibition then reversed, leading to increased values which persisted as long as ethanol was present in plasma. Acylation rates were not correlated to ethanol concentrations in plasma. We suggest that red cells first desensitize to, then overcompensate for the inhibitory effect of ethanol on acylation reactions. These adaptive changes may be one of the events mediating membrane tolerance to ethanol.     

Open Biology: overview for special issue on dynamics of protein fatty acylation

Fatty acylation is a widespread form of protein modification that occurs on specific intracellular and secreted proteins. Beyond increasing hydrophobicity and the affinity of the modified protein for lipid bilayers, covalent attachment of a fatty acid exerts effects on protein localization, inter- and intramolecular interactions and signal transduction. As such, research into protein fatty acylation has been embraced by an extensive community of biologists. This special issue highlights advances at the forefront of the field, by focusing on two families of enzymes that catalyse post-translational protein fatty acylation, zDHHC palmitoyl acyltransferases and membrane-bound O-acyl transferases, and signalling pathways regulated by their fatty acylated protein substrates. The collected contributions catalogue the tremendous progress that has been made in enzyme and substrate identification. In addition, articles in this special issue provide insights into the pivotal functions of fatty acylated proteins in immune cell, insulin and EGF receptor-mediated signalling pathways. As selective inhibitors of protein fatty acyltransferases are generated, the future holds great promise for therapeutic targeting of fatty acyltransferases that play key roles in human disease.     

同步分泌高效纤维素酶和木聚糖酶菌株YB的鉴定及其酶学性质研究

筛选和鉴定可降解木质纤维素的真菌,并研究其产酶特征。采用刚果红平板涂布法,从荔枝腐叶中筛选具有木质纤维素降解能力的真菌,结合ITS-rDNA序列分析进行鉴定,初步测定其产酶条件,然后采用DEAE Sepharose Fast Flow阴离子交换层析与Sephadex G-100凝胶层析对硫酸铵沉淀的粗酶液进行分离纯化,对其开展酶学性质研究。结果显示,筛选出一株可降解木质纤维素降解的菌株YB,鉴定为绿木霉(Trichoderma virens)。在发酵过程中,纤维素酶和木聚糖酶的最大活力分别为313.53±26.78 U/mL和18 120.87±500.37 U/mL。分离纯化得到纤维素酶(CMC酶)Ⅰb、Ⅳ和木聚糖酶Ⅰa;通过SDS-PAGE检测,其分子量分别为58.5 kD、22.8 kD和44.5 kD。3种酶的最适酶促反应条件均为:50℃,pH 5.0。其中,木聚糖酶能有效降解玉米芯木聚糖为木糖和多种木寡糖。菌株Trichoderma virens YB可分泌高效木质纤维素降解酶,具有应用于木聚糖酶和木寡糖生产的潜力。