Gene Cloning of Cellobiohydrolase II from the White Rot Fungus Irpex lacteus MC-2 and Its Expression in Pichia pastoris

A gene (cel4) coding for a cellobiohydrolase II (Ex-4) was isolated from the white rot basidiomycete, Irpex lacteus strain MC-2. The cel4 ORF was composed of 452 amino acid residues and was interrupted by eight introns. Its deduced amino acid sequence revealed a multi domain structure composed of a cellulose-binding domain, a linker, and a catalytic domain belonging to family 6 of glycosyl hydrolases, from the N-terminus. cel4 cDNA was successfully expressed in the yeast Pichia pastoris. Recombinant Ex-4 showed endo-processive degrading activity towards cellulosic substrates, and a synergistic effect in the degradation of Avicel was observed when the enzyme acted together with either cellobiohydrolase I (Ex-1) or endoglucanase (En-1) produced by I. lacteus MC-2.     

Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.     

Production of Higher-quality Plastein from a Crude Single-cell Protein

From defatted n-paraffin-assimilating yeast cells, a crude protein was obtained by alkaliextraction followed by acid-precipitation. Then the protein was treated with ether until extractable substances were removed exhaustively at this stage. However, at the next stage where the ether-treated protein had been partially hydrolyzed with pepsin, when the hydrolysate was retreated with ether, it was found that ether-extractable substances totalling 270 mg/100 g were obtainable additionally. Chromatographic investigations demonstrated that the substances included significant amounts of aliphatic and aromatic hydrocarbons, some indoles, and a ubiquinone (n = 8).

From the protein hydrolysate (substrate) after the above ether-treatment, a plastein was synthesized with Bioprase under the specific conditions. The plastein was obtained as a precipitate when the whole reaction mixture was treated with aqueous ethanol or acetone. The quantity and quality (nitrogen content) of the plastein depended on the ethanol or acetone concentration. Roughly speaking, the higher the concentration, the more the plastein quantity. The converse relation held for the quality; a plastein precipitated by treatment solely with water showed a higher quality than any other case.     

耙齿菌糖蛋白的提取分离、理化性质及抗炎活性

耙齿菌发酵物经水提醇沉得醇沉Ⅰ、醇溶Ⅱ两部分;Ⅰ透析得内液Ⅰ1、外液Ⅰ2。苯酚-硫酸法、Lowry法、间羟基联苯法测Ⅰ的总糖、蛋白质、糖醛酸的含量分别为43．22、21．11、4．32％;除蛋白和氨基酸分析证明Ⅰ为糖蛋白。气相色谱测定Ⅰ的组成糖及摩尔比为Ara：Xyl:Man：Gal：Glu=1：0．5：4．2：1．6：6．1;HPLC测定分子量为60kD;小鼠耳肿胀实验证实Ⅰ具有抗炎活性。     

一株产木质素降解酶真菌的分离与鉴定

从自然界中分离到一株产木质素降解酶真菌.平板显色反应显示,该菌株具有产多种木质素降解酶的能力,并通过酶活力测定得到证实.为确定该菌株的分类地位,对其ITS序列进行了扩增并测序,并利用MEGA4.0生物学软件计算其与同属其它菌株的遗传距离并构建系统发育树,在分子水平上确定它们之间的亲缘关系.试验结果表明,该菌株的ITS序列与Irpex lacteus乳白耙菌序列相似度达99%,且与Irpex lacteus XSD-2亲缘关系最近.     

乳白耙齿菌F17对单一和复合多环芳烃的降解差异解析

[目的] 针对菲、蒽、荧蒽多环芳烃（PAHs）污染物,利用乳白耙齿菌F17,研究单一和复合PAHs污染物的生物降解规律。[方法] 采用气相色谱-质谱法（GC-MS）分析降解过程中PAHs的浓度,并采用准一级反应动力学模型对降解结果进行拟合。[结果] 对于单一PAHs,第15天时菲、蒽、荧蒽的降解率由高到低依次为菲（97.8%） > 蒽（89.3%） > 荧蒽（81.5%）。菲、蒽和荧蒽的降解过程具有准一级反应动力学特征,菲的生物降解速率最快,其次是蒽,荧蒽的降解速率最慢。与单一PAHs的降解相比,在复合PAHs的降解过程中,乳白耙齿菌F17的生长和锰过氧化物酶的合成均表现出不同的特征。此外,水溶性极可能是复合污染物降解的重要控制因子,三者水溶性为：菲 > 荧蒽 > 蒽。因此,在菲或荧蒽加入条件下,微生物能优先降解这些污染物,抑制了污染物蒽的降解;同时,蒽或菲的存在对荧蒽的降解也有抑制作用;然而外源加入水溶性较差的蒽和荧蒽,则对菲的生物降解无显著影响。[结论] 复合PAHs的生物降解主要表现为相互竞争的特点,通过GC-MS分析了PAHs的生物降解途径。     

Crystal structure of aspartic proteinase from Irpex lacteus in complex with inhibitor pepstatin

The crystal structure of Irpex lacteus aspartic proteinase (ILAP) in complex with pepstatin (a six amino acid residue peptide-like inhibitor) was determined at 1.3A resolution. ILAP is a pepsin-like enzyme, widely distributed in nature, with high milk-clotting activity relative to proteolytic activity. The overall structure was in good topological agreement with pepsin and other aspartic proteases. The structure and interaction pattern around the catalytic site were conserved, in agreement with the other aspartic proteinase/inhibitor complex structures reported previously. The high-resolution data also supported the transition state model, as proposed previously for the catalytic mechanism of aspartic proteinase. Unlike the other aspartic proteinases, ILAP was found to require hydrophobic residues either in the P(1) or P(1') site, and also in the P(4) and/or P(3) site(s) for secondary interactions. The inhibitor complex structure also revealed the substrate binding mechanism of ILAP at the P(3) and P(4) site of the substrate, where the inserted loop built up the unique hydrophobic pocket at the P(4) site.     

Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The effect of manganese and selected synthetic dyes on the production of manganese-dependent peroxidase (MnP) by Irpex lacteus immobilized on polyurethane foam was studied. In the cultures grown in a medium containing 65 μM Mn (II), up to three various isoenzymes of MnP were resolved by isolectrofocusing, with pI values within the range of 3.50–6.04. In the cultures grown in a medium containing 2.9 mM Mn (II), two new MnP isoforms (pI 3.28, 3.75) were produced. The addition of structurally different synthetic dyes, an azo dye Reactive Orange 16 (RO16), an anthraquinonic dye Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye Bromophenol Blue (BPB), to the fungal cultures grown in the presence of high manganese inhibited the production of low pI MnP isoforms. However, in the presence of BPB a new MnP isoform with pI 5.67 was detected. BPB was found to induce MnP isoforms which are more effective in RBBR decolorization in vitro than the low pI isoforms present in the control cultures.