Ganodermin, an antifungal protein from fruiting bodies of the medicinal mushroom Ganoderma lucidum

A 15-kDa antifungal protein, designated ganodermin, was isolated from the medical mushroom Ganoderma lucidum. The isolation procedure utilized chromatography on DEAE-cellulose, Affi-gel blue gel, CM-Sepharose and Superdex 75. Ganodermin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. Ganodermin inhibited the mycelial growth of Botrytis cinerea, Fusarium oxysporum and Physalospora piricola with an IC50 value of 15.2 microM, 12.4 microM and 18.1 microM, respectively. It was devoid of hemagglutinating, deoxyribonuclease, ribonuclease and protease inhibitory activities.     

Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908

A plant pathogenic fungus, Colletotrichum lagenarium, causing watermelon anthracnose, was isolated from naturally infected leaves, stems, and fruits of watermelon. A bacterial strain, MET0908, showing a potent antifungal activity against C. lagenarium, was isolated from soil. An antifungal protein was purified by 30% ammonium sulfate saturation and concentrated using Centricon 10, DEAE-Sepharose(TM) Fast Flow column and Sephacryl S-100 gel filtration chromatography. The molecular weight of the purified protein was estimated as 40 kDa by SDS-PAGE. The purified protein was stable at 80 degrees C for 20 min and exhibited a broad spectrum of antifungal activity against various plant pathogenic fungi. Confocal microscopy image analysis and scanning electron microscopy showed that the protein acted on the cell wall of C. lagenarium. The purified antifungal protein exhibited beta-1,3-glucanase activity. The N-terminal amino acid sequence of the purified protein was determined as Ser-Lys-Ile-x-Ile-Asn-Ile-Asn-Ile-x-Gln-Ala-Pro-Ala-Pro-x-Ala. A search of the sequence with NCBI BLAST showed no significant homology with any known proteins, suggesting that the purified protein may be novel.     

A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems

A novel small antifungal peptide produced by a Bacillus strain B-TL2 isolated from tobacco stems was purified. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on CM-Sepharose Fast Flow column and reverse-phase HPLC on SOURCE 5RPC column. After the final isolation step, one peptide with antifungal activity, designated as BTL, was obtained. The molecular mass of the purified BTL was determined as 2500 Da and 2237.7 Da by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry, respectively. The N-amino acid sequence of BTL was determined to be NH(2)-KQQLATEAESAGPIL, which shows relatively low identity to other antimicrobial peptides from bacteria. The peptide exhibited strong inhibitory activity against mycelial growth of Bipolaris maydis, Alternaria brassicae, Aspergillus niger, Cercospora personata. The purified BTL displayed thermostability, almost retaining 100% activity at 100 degrees C for 15 min.     

Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10

Bacillus licheniformis HS10 is a good biocontrol agent against Pseudoperonospora cubensis which caused cucumber downy disease. To identify and characterize the antifungal proteins produced by B.licheniformis HS10, the proteins from HS10 were isolated by using 30–60% ammonium sulfate precipitation, and purified with column chromatography on DEAE Sepharose Fast Flow, RESOURCE Q and Sephadex G-75. And the SDS–PAGE and MALDI-TOF/TOF-MS analysis results demonstrated that the antifungal protein was a monomer with molecular weight of about 55 kDa, identified as carboxypeptidase. Our experiments also showed that the antifungal protein from B. licheniformis HS10 had significantly inhibition on eight different kinds of plant pathogenic fungi, and it was stable with good biological activity at as high as 100 °C for 30 min and in pH value ranged from 6 to 10. The biological activity was negatively affected by protease K and 10 mM metal cations except Ca²⁺.     

一种新的蛇毒凝血酶原激活物的分离纯化及特征

为获得矛头蝮蛇（Bothrops atrox）蛇毒凝血酶原激活物并研究其基本性质,采用SP Sepharose Fast Flow, DEAE Sepharose Fast Flow 和SP Sepharose High Performance等层析方法从巴西矛头蝮蛇蛇毒中分离纯化得到1种单一组分的凝血酶原激活物（prothrombin activator,FⅡA）.还原性SDS-PAGE结果显示,其分子质量约为72 kD,等电点为6.67. HPSEC显示纯度大于95%.该酶是1种N连接的糖蛋白,N末端氨基酸序列为ALVLIAFAQYLQQCP,获得登录号为：B3A0N1其活性可被EDTA-Na₂ 抑制,PMSF对其活性无影响,对凝血酶原的激活过程无需Ca2+、FⅤa、磷脂的参与,为P-Ⅰ金属蛋白酶,对凝血酶原的激活方式与FⅩa相似.本研究纯化与鉴定的新凝血酶原激活物为其药学研究及临床应用提供参考.     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

小麦叶片β-1,3-葡聚糖酶的诱导、纯化与抗菌活性

三个小麦品种331、抗倒680和鲁麦23经氯化汞、水杨酸或核黄素处理后,叶片中的β-1,3-葡聚糖酶活性均有不同程度的升高.氯化汞处理24h对品种331该酶活性的诱导作用最强.因此取用氯化汞处理24 h的331小麦叶片研磨得到粗酶液.将粗酶液经硫酸铵分级沉淀、Phenyl-Sepharose Fast Flow疏水层析、DEAE-Sepharose Fast Flow阴离子交换层析和Sephacryl S-100分子筛层析,得到了SDS-PAGE凝胶电泳谱带单一的β-1,3-葡聚糖酶样品.经SDS-PAGE(12%)和凝胶过滤层析,测得其分子量约为52.0～53.6 kD.抗菌试验测定显示,纯化的β-1,3-葡聚糖酶对供试的4种病原真菌的生长、孢子萌发或芽管伸长都有一定程度的抑制作用.     

Purification of a protein C activator from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix)

A protease has been purified by ion-exchange chromatography from the venom of Agkistrodon contortrix contortrix (Southern copperhead snake) that can activate the vitamin K dependent protein, protein C. The apparent molecular weight of this protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 20,000 under nonreducing conditions. Incubation of this protease with plasma resulted in a prolongation of the clotting time and a time-dependent increase in amidolytic activity. Incubation of the protease with purified protein C resulted in an increase in both amidolytic and anticoagulant activity. The protease had no inhibitory effect on thrombin, factor V, fibrinogen, or factor X. It had slight clotting activity toward fibrinogen. The apparent Km of the protease for protein C was 0.28 microM. Calcium ions were observed to inhibit protein C activation with an apparent Ki of 0.2 mM. Ethylenediaminetetraacetic acid, diisopropyl fluorophosphate, and soybean trypsin inhibitor were observed to inhibit the venom protease. These results suggest that the venom of the Southern copperhead snake contains a protease that is a specific activator of protein C.     

Isolation of alliumin, a novel protein with antimicrobial and antiproliferative activities from multiple-cloved garlic bulbs

A protein designated alliumin, with a molecular mass of 13 kDa and an N-terminal sequence similar to a partial sequence of glucanase, and demonstrating antifungal activity against Mycosphaerella arachidicola, but not against Fusarium oxysporum, was isolated from multiple-cloved garlic (Allium sativum) bulbs. The protein, designated as alliumin, was purified using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Mono S, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75. Alliumin was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel, CM-cellulose and Mono S. Its antifungal activity was retained after boiling for 1 h and also after treatment with trypsin or chymotrypsin (1:1, w/w) for 30 min at room temperature. Alliumin was inhibitory to the bacterium Pseudomonas fluorescens and exerted antiproliferative activity toward leukemia L1210 cells. However, it was devoid of ribonuclease activity, protease activity, mitogenic activity toward mouse splenocytes, and antiproliferative activity toward hepatoma Hep G2 cells.     

Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds

An antifungal protein designated as Psc-AFP, with an apparent molecular mass of 18kDa, was isolated from a traditional Chinese herb, malaytea scurfpea (Psoralea corylifolia L.). The isolation procedure entailed extraction, cation exchange chromatography on CM FF, gel filtration chromatography on Superdex 75 and reversed-phase high performance liquid chromatography on SOURCE 5RPC column. Automated Edman degradation determined the partial N-terminal sequence of Psc-AFP to be NH2-EWEPVQNGGSSYYMVPRIWA, which displayed homology with plant trypsin inhibitors. The protease inhibitor activity of Psc-AFP was then confirmed by the inhibition on trypsin. Psc-AFP at 10 microM inhibited the mycelial growth of Alternari brassicae, Aspergillus niger, Fusarium oxysporum and Rhizoctonia cerealis, suggesting that Psc-AFP has a role in the defense against pathogens.     

胸腺素a1与复合a干扰素融合蛋白的表达及其生物学活性

实验旨在获得具有双重生物学活性的重组胸腺素a1(Thymosin alpha1, TM-a1)与复合a干扰素(IFNa-con)融合蛋白。选择大肠杆菌偏爱的密码子, 将合成的TM-a1与IFNa-con编码序列构成的融和基因克隆至大肠杆菌表达载体pET-22b(+)、在宿主菌BL21(DE3)-Codon plus-RP-X中成功表达了可溶性融合蛋白(TM-a1-IFN-con)。表达量占总蛋白的20%以上。通过硫酸铵沉淀、疏水层析、阴离子交换层析、阳离子交换层析、分子筛层析后, 产品纯度达到96%以上。采用细胞病变抑制法测定融合蛋白的抗病毒活性, 采用细胞增殖实验检测融合蛋白对小鼠脾淋巴细胞增殖的影响。结果表明, 融合蛋白的抗病毒活性优于市售的IFNa1b和IFNa2a。对小鼠脾淋巴细胞增殖的影响与市售的合成胸腺素a1相同。已有研究证实, 该融合蛋白具有良好的体外抗HBV作用, 其体外抗HBV活性比联合应用TM-a1和干扰素a强, 且细胞毒性明显低于联合应用TM-a1和干扰素a。以上结果表明, 通过大肠杆菌表达的可溶性融合蛋白(TM-a1- IFN-con), 既具有良好的干扰素a抗病毒作用, 也具有胸腺素a1促淋巴细胞增殖作用。