Purification and Chemical Properties of an Inhibitor of Plant Virus Infection from Fruiting Bodies of Lentinus edodes

A new inhibitor of plant virus infection from fruiting bodies of Lentinus edodes was purified by a method using fractionation with DEAE-Cellulose, gel filtration on Sephadex G-75, and column chromatography on CM-Toyopearl 650M. The purified inhibitor thus obtained was homogeneous on disc and SDS disc electrophoreses, and the molecular weight of the inhibitor was 23,000. The inhibitor consisted of 17.4% nitrogen, which was found to be a basic simple protein, but contained no neutral sugar, hexosamine nor sialic acid. The inhibitor was estimated to be composed of about 199 amino acid residues. This inhibitor was named “fruiting body protein (FBP).”     

Substrate Preference of γ-Glutamyltransferase from Caps of Fruiting Bodies of Lentinus edodes

Three kinds of proteins (BA-1, BA-2 and BA-3) allergenic to the IgE antibody of allergenic individuals were isolated from buckwheat seeds. These three proteins were essentially homogeneous as judged by both polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The amino acid composition of BA-1 and BA-2 was very similar, and the molecular weight of each allergenic protein was between 8000–9000 by SDS-polyacrylamide gel electrophoresis. One of them was a trypsin inhibitor, and their immunoreactivity was quite stable to heating at 100°C for 60 min.     

Inhibition of Protein Synthesis by Antiviral Protein from Yucca recurvifolia Leaves

We analyzed 18S and 26S rRNA partial base sequences [positions 1451–1618 (168 bases) of 18S rRNA and positions 1611–1835 (225 bases) and 493–622 (130 bases) of 26S rRNA] of a total of three strains of Pichia jadinii and Candida utilis. The three strains had identical base sequences with the type strain of P. jadinii (IFO 0987) in the 18S rRNA partial base sequencings. In the 26S rRNA partial base sequencings, there were partial base sequences similar to each other (1–0 base difference and 87–95 percent similarities). The sequence data obtained are discussed taxonomically and phylogenetically, especially in connection with Williopsis saturnus, the type species of the genus Williopsis ZENDER.     

Alliinase-like Enzymes in Fruiting Bodies of Lentinus edodes: Their Purification and Substrate Specificity

3-Chloro-d-alanine chloride-lyase, which occurs in the cells of Pseudomonas putida CR 1-1, catalyzes not only the α,β-elimination reaction of 3-chloro-d-alanine to form pyruvate, but also its β-replacement reaction in the presence of a high concentration of sodium hydrosulfide to form d-cysteine. Using the β-replacement reaction, the enzymatic synthesis of d-cysteine by resting cells was investigated. The culture conditions for cell production of the bacterium with high d-cysteine-producing activity and the reaction conditions for d-cysteine production were optimized. Under these optimal reaction conditions, 100% of the added 3-chloro-d-alanine could be converted to d-cysteine and, as the highest yield, 20.6 mg of d-cysteine per 1.0 ml of reaction mixture could be synthesized.     

Purification and Properties of Tyrosinase Isozymes from the Gill of Lentinus edodes Fruiting Body

Six tyrosinase isozymes were purified from the browned gill of the fruiting body of Lentinus edodes by ammonium sulfate fractionation, DEAE-Sephacel and Q-Sepharose column chromatography, and partially denaturing SDS–PAGE. At the step of Q-Sepharose column chromatography, two active fractions (A and B) were obtained. Each fraction was separated to three further fractions, A1, A2, and A3, and B1, B2, and B3, respectively, by partially denaturing SDS–PAGE. All these isozymes consisted of two types of polypeptides: a polypeptide (Aα or Bα) and either β (Aβ or Bβ) or γ polypeptide (Aγ or Bγ). The α polypeptide contained the consensus amino acid sequence of the active site of known tyrosinases, which is considered to act as a catalytic subunit. From the results of peptide mapping and the amino acid composition, Aα and Bα polypeptides were considered to be different proteins. The kinetic properties of the purified tyrosinase isozymes differed greatly according to whether they contained β or γ polypeptide, indicating these polypeptides to be a possible regulatory subunit.     

Purification and Some Properties of Metal Proteinases from Lentinus edodes

To investigate the function of proteinases in the fruiting of Basidiomycetes, we purified the neutral proteinase in vegetative mycelium of Lentinus edodes. About 1.6 mg of purified enzyme was obtained from 1.5 kg of mycelium. The purified enzyme was confirmed to be monodispersive on disc electrophoresis.

The neutral proteinase was most active around pH 7.5 toward hemoglobin and 7.0 toward casein and was extremely labile with temperature. The enzyme was strongly inhibited by EDTA or Talopeptin (MK-I). The molecular weight and isoelectric point of the enzyme were 45,000 and pH 5.3, respectively. The enzyme contained no methionine residues. The enzyme hydrolyzed the bonds involving hydrophobic or bulky amino acid residues of oxidized insulin B-chain such as His-Leu (10–11 and 5–6), Leu (17)-Val (18) and Ala (14)-Leu (15).

These characteristics are compared with those of the metal proteinase in the fruit-body of the same fungus, which was purified and characterized at the same time as in vegetative mycelium. We also compare it with proteinases from other microbes.     

Purification and Some Properties of Nucleases from Shii-take,Lentinus edodes

Three kinds of nuclease preparations, each of which having both endonuclease activity that formed 5′-mononucleotides and 3′-nucleotidase activity, were separated and partially purified from Shii-take, Lentinus edodes. Both enzyme activities of each preparation showed a similar thermostability and electrophoretic mobility on Polyacrylamide gel, and a competitive relationship was observed between RNA and 3′-AMP in their enzyme reactions. From these results, it is concluded that both enzyme activities of these three preparations reside in a single protein, respectively. They resemble one another in substrate specificity, cleavage pattern of RNA and thermostability, but are distinguishable from one another by molecular weight, electrophoretic mobility and optimum pH for degradation of RNA.     

一种含有单链RNA的香菇球状病毒

从生长不正常的香菇(Lentinus edodes(Berk.)Sing)菌株中分离到一种等轴对称含单链RNA的病毒颗粒。病毒颗粒在电镜下直径为33～34nm,在SDS-聚丙烯酰胺凝胶电泳中病毒外壳蛋白分子量为22000道尔顿。病毒核酸径DNase1和SI酶解试验及热变性紫外吸收曲线试验证明为单链RNA,在1.5％的琼脂糖凝胶电泳中,病毒核酸呈现一条带,分子量为2.38×10~6道尔顿。     

Trypsin-Like Proteinases and Trypsin Inhibitors in Fruiting Bodies of Higher Fungi

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).     

香菇谷氨酰胺转氨酶的分离和动力学研究

采用超滤,DEAE-纤维素和SephadexG-100层析,研究了香菇谷氨酰胺转氨酶的制备方法,对酶反应动力学的最适温度、最适pH、酶的稳定性和酶催化激活剂等进行了研究。结果表明,获得了香菇谷氨酰胺转氨酶,经SDS-PAGE电泳检测,为一均一性蛋白;香菇谷氨酰胺转氨酶酶动力学研究结果显示,最适温度为40℃,酶促反应的Vmax为0.020 4 mg/(mL.min),米氏常数Km为1.520 mg/mL。Na 、Ca2 、Pb2 、K 、Mg2 、Cu2 等离子对酶活影响甚微,为非Ca2 依赖性酶。该酶由2个亚基组成,分子量分别为53 ku和27 ku,pI为5.33。     

香菇蛋白的分级纯化和结构分析

香菇粉经10℃pH 10的水提取制备香菇蛋白,得率13.1%,其蛋白含量47.5%,多糖含量24.2%.香菇蛋白经DEAE Sepharose CL-6B柱层析分级得5个级分,收集级分F1、F2、F3、F4,它们都是由蛋白和多糖构成的复合物.Sepharose CL-6B凝胶色谱显示,F2和F4的分子量分布较为均匀,且以蛋白为主,多糖含量很低;F3主要由两个分子量不同的蛋白级分构成,含有一定的多糖;F1中多糖含量较高,蛋白含量较少,且多糖分子量分布均匀.香菇蛋白的分子量主要集中在20 kDa～40 kDa之间.F1、F3、F4都属于酸性蛋白质,含有除色氨酸之外的7种必需氨基酸,除蛋氨酸含量较低外,其余必需氮基酸含量接近,且赖氨酸含量较高.红外光谱分析表明,香菇蛋白的二级结构主要为α-螺旋和无规卷曲.     

6-BA对平菇和香菇菌丝体两种同工酶的影响

通过在平菇、香菇的马铃薯液体培养基中添加不同浓度的 6 BA(6 苄基腺嘌呤 ) ,应用聚丙烯酰胺凝胶垂直平板电泳技术 ,探讨了 6 BA对平菇、香菇菌丝体酯酶 (EST)和过氧化物酶 (PER) 2种同工酶的影响。结果显示 ,6 BA浓度在 5 g/L培养液和 15 g/L培养液时分别诱导出平菇、香菇菌丝体中各 1条新的酯酶同工酶带产生 ,不同的 6 BA浓度对平菇、香菇菌丝体其余的酯酶同工酶带强度也有影响 ;6 BA不能诱导平菇和香菇菌丝体中新的过氧化物酶同工酶产生 ,但在浓度为 15 g/L培养液时可使PER同工酶带增强 ;6 BA对平菇和香菇菌丝体中EST ,PER2种同工酶的Rf值没有影响。     

一种新香菇病毒基因组部分cDNA序列及病毒RT-PCR检测

本文报道从香菇菌丝体和子实体中分离到一种大小约20nm×(100-200)nm的杆形病毒颗粒,病毒基因组是大小约8.0kb的dsRNA。对病毒基因组部分cDNA序列进行克隆,完成1457bp的核酸序列测定(Accession No:GQ372842),该序列含1个不完整ORF,编码314个氨基酸残基,推测为病毒RNA聚合酶部分序列。病毒基因组部分cDNA序列与GenBank中的已知核酸序列无明显同源性,表明它可能是新发现食用真菌病毒。为了对实验室和野外的香菇病毒进行快速检测,我们根据得到的病毒基因组部分cDNA序列设计特异性引物,建立了一种方便、有效检测香菇病毒的RT-PCR方法,对感染病毒异常菌丝体中的病毒成功地进行了检测。     

Primary Structure of a Base Non-specific and Adenylic Acid Preferential Ribonuclease from the Fruit Bodies of Lentinus edodes

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase Le₂) from the fruit bodies of Lentinus edodes was analyzed. The sequence was mostly determined by analysis of the peptides generated by V₈ protease digestion and BrCN cleavage (including α-chymotryptic, and V₈ protease digest of BrCN fragments). It consists of 239 amino acid residues. The molecular weight is 25831. The location of 10 half cystine residues were almost superimposable on those of known fungal RNases of the RNase T₂ family. The sequence homologies between RNase Le₂ and four known fungal RNases of the RNase T₂ family, RNase T₂, RNase M, RNase Trv, and RNase Rh, are 102, 103, 109, and 74, respectively. The homologous sequences are concentrated around the three histidines, which are supposed to form the active site of RNase T₂ family RNases.     

Purification and Some Properties of Carboxyl Proteinase in Mycelium of Lentinus edodes

The effect of Streptomyces-pepsin inhibitor (S-PI) on fruit-body formation of Lentinus edodes (Berk.) Sing, was studied. The addition of S-PI to the culture medium (5 ~ 10 µg/ml) shortened the time required for mature fruit-bodies, and increased the fruiting-percentage and the overall yield 3.4 times compared to the control.

The intracellular proteinase in the mycelium was investigated. Proteinases having an optimal pH of 2.7 and 7.0 were found in the vegetative mycelial extract. When S-PI was added to the culture medium, their activities were strikingly changed; the carboxyl proteinase activity was remarkably decreased, and, in the contrary, the metal proteinase activity was increased to 1.5 times that of the control.

The carboxyl proteinase was purified. This enzyme was strongly inhibited by S-PI and synthetic pepsin inhibitors such as DAN and EPNP. The molecular weight and isoelectric point were 43,000 and pH 3.4, respectively.     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。