平菇液体培养液全波长光谱特性与漆酶产量关系研究

漆酶是一种广泛分布在真菌中的酶,其底物有非专一性,在木质素的降解和环境保护方面有重要作用.为测定和估计平菇菌丝液体发酵期间的漆酶产生量,使用紫外-可见分光光度计对菌液进行了全波长扫描,结合常规化学测定方法和平板固体培养基培养时的漆酶圈直径来进行研究,探明粗酶液全波长光谱特性与漆酶产生量的关系.结果发现,发酵粗酶液全波长扫描图谱在300 nm处的波峰数目与漆酶的产量呈现相关关系;300 nm处吸光值大于1.5的波长范围逐渐增大,11天的培养期间其波长范围从5 nm变化到80 nm与漆酶的产量呈正相关关系;在320～400 nm出现有规律的光吸收,且在349,365,388 nm处各出现光吸收峰,其中在365 nm处出现最大吸收,且吸光值均与漆酶的产量呈正相关关系.据此可以应用于粗酶液中漆酶含量的估计或测定.与常规化学分析相比,全波长扫描方法有测定速度快、简单、节省溶剂、无化学污染等优点,为实际工业生产中的漆酶产量估计和测定提供了新方法.     

蒲а芯吭?杭州市农业科学研究院 杭州310024,杭州310024,杭州310024,杭州31

在发芽48d后,测定过氧化物酶、α-淀粉酶和SOD酶的活性,最后进行统计和分析。试验结果表明:低温抑制甜玉米幼苗的生长,但经50mg/LGA3、2mg/LKT和0.05mg/LBR处理后,对甜玉米种子发芽率的提高和幼苗生长具有促进作用。用2mg/LKT处理效果α-淀粉酶活性最好。低温情况下用0.05mg/LBR处理SOD酶活性明显提高。     

乳菇和红菇的红外光谱及其二维相关红外光谱的分析与鉴别

采用傅里叶变换红外光谱原始谱、二阶导数光谱以及二维相关谱的三级鉴定方法,对红菇属和乳菇属六种蘑菇进行了鉴别分析。六种样品的原始光谱吸收峰非常相似,主要为蛋白质和碳水化合物的吸收组成,但在各样品吸收峰强度、峰形和峰位上仍有微小差异。对其进行二阶导数分析,在二阶导数谱中,1 800～1 400和1 200～800 cm-1范围内的吸收峰强度、位置、峰形状显示出明显差异。二维相关红外光谱提高了光谱的分辨率;对六种蘑菇样品进行二维相关红外光谱分析,发现在1 690～1 420 cm-1范围内,三种乳菇出现了3个明显的自动峰,另外三种红菇出现了4个自动峰,而且自动峰和交叉峰位置、强度均不同;在1 110～920 cm-1二维光谱范围内,六种蘑菇的自动峰和交叉峰的数量、位置和强度也都不同;说明不同样品中蛋白质和糖类化合物的显著不同。结果表明：应用红外光谱、二阶导数谱和二维相关红外光谱三级鉴定的技术可以快速有效地分析和鉴别辣乳菇、绒白乳菇、香乳菇、稀褶红菇、变绿红菇和近似酒红菇。该多级鉴定的方法对于分类鉴别蘑菇是一种快速、准确、有效的方法。     

He—Ne激光在香菇速生高产菌株选育中的应用：Ⅰ菌株的诱变及选育

采用Ｈｅ－Ｎｅ激光对２株香菇生产菌株分别进行两次诱变处理，从中筛选出６株生长速率比亲本明显加快的变异株。所选变异株过氧化物酶活性与亲本相比有不同程度的提高，过氧化物酶活性提高程度与香菇菌丝生长速率呈相关性。所选变异株具有良好的遗传稳定性。     

光镊捕获金福菇孢子的拉曼光谱分析

使用光镊拉曼光谱系统俘获悬浮在生理盐水中的单个金福菇孢子,激发并收集其拉曼信号,结果显示单个金福菇孢子的拉曼光谱基本能呈现其内含物的组成和结构信息,脂类物质是其主要成分。同种栽培方式的3个不同菌株单个金福菇孢子的平均拉曼光谱信号基本一致,分别对其进行多元统计分析(PCA),无法区分其拉曼光谱信号;同一菌株不同栽培方式的单个孢子平均拉曼光谱的差异光谱表明其主要成分相同,都以脂类物质为主;而源自于脂类1743、1655、1442、1125、1080、1070、876、728cm-1等峰的信号强度基本一致,说明其脂类的含量基本相同。从单细胞拉曼光谱角度初步分析金福菇孢子,为光镊拉曼光谱技术研究食用菌孢子萌发机理提供有意义的参考。     

绿豆酶解过程中Se的形态分布分析

采用AFS-230E 型原子荧光光度计对绿豆中Se的含量及绿豆分步酶解过程中Se的形态分布进行了分析。结果表明：（1）绿豆中Se含量为54.79 μg·g-1（干重） ;（2）通过酶解,绿豆中90%以上的Se被提取出来,而水煮对Se的提取率只有19.26%,其中淀粉酶、蛋白酶和纤维素酶酶解过程对Se的提取率分别是36.64%,55.96%和5.189%,可推断,绿豆中大部分Se被蛋白结合或络合;（3）总酶解产物中Se的有机态分布系数为59.87%,远大于绿豆汤中Se的有机态分布系数（3.64%）,其中淀粉酶解产物和蛋白酶解产物中Se的有机态分布系数分别为15.51%和44.36%,纤维素酶解产物中未检测到有机态Se,可见,Se在绿豆纤维素中主要以络合形式存在。回收实验表明,方法回收率为97.8%,RSD为1.1%（n=11 ）。     

褪黑素对绿豆在增强UV-B辐射下的防护作用

研究了外源褪黑素对于绿豆幼苗在增强UV-B辐射下叶片内丙二醛含量、过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶酶活的影响.实验结果表明,增强UV-B辐射对绿豆产生了氧化胁迫,使其叶片内的丙二醛含量升高,引起抗氧化酶包括过氧化物酶、抗坏血酸过氧化物酶、超氧化物歧化酶和过氧化氢酶酶活提高.而外源褪黑素和UV-B辐射复合处理可以显著地降低绿豆叶片内的丙二醛含量、过氧化物酶、过氧化氢酶和抗坏血酸过氧化物酶酶活.褪黑素单独处理绿豆使各项指标与对照无显著差异.表明在植物体内,褪黑素是一种保护性抗氧化剂,加入一定浓度的外源褪黑素可以减缓UV-B辐射引起绿豆的氧化胁迫.     

高压处理种子对西瓜生长发育的影响

以8个汴梁西瓜品种(京引21、花皮88、圳板、郭牟、金玫黑、金小兰、日本瓜和蜜玫绿)为实验材料,采用样本率的显著性检验t、检验及单因素方差分析方法,研究了26~28 MPa的高氮气压及50~150 MPa的高静水压处理种子对西瓜出苗率、叶绿素含量、过氧化物酶活性、单瓜重、含糖量等生长特性的影响。结果表明:高压处理会显著(P0.05)或极显著(P0.01)地降低西瓜的出苗率和幼苗期叶片的类胡萝卜素含量,提高幼苗期叶片的叶绿素a含量和过氧化物酶的活性。高压处理种子对于含糖量和单瓜重这两个农艺性状的影响,品种间存在较大差异。其中,在50~150 MPa高压下处理种子20 min主要影响日本瓜(P0.001)和金小兰(P0.092)的单瓜重以及郭牟的含糖量(P0.064)。     

生物磁技术在食用菌栽培中的应用

用磁场处理水（简称磁水，下同）喷浇食用菌能获得高产，这是生物磁技术在食用菌栽培中应用的新成果，实验表明：用磁不拌料加菇期喷浇磁水的方法最好，它可使食用菌增产１０％－５０％，磁水能使纤维素酶，脂酶，淀粉酶等酶的活性增加，加速了纤维素，脂肪，淀粉等养分的分解，并提高这些养分的利用率，达到增产的效果，这是食用菌喷施磁水获得增产的重要原因。     

红外光谱分析木质素在漆酶酶法改性中的反应性

随着非水酶学的发展,木质素的酶法催化改性得到日益广泛的运用。文章主要考察了漆酶酶法改性反相微乳液体系中云杉碱木质素、乙醇水溶液中乙醇木质素、缓冲液体系中木质素磺酸盐、以及碱溶液中汽爆麦草碱木质素的反应。通过红外光谱对各反应体系中,漆酶催化改性后的木质素结构特点进行分析,结合凝胶色谱法考察木质素的分子量及其分布,结果表明经过YY-5漆酶处理后各种木质素的分子量分布均趋向于高分子区域,且分子量分散性降低,各种木质素的红外光谱结构也发生了明显的变化。初步确定了木质素在漆酶催化改性中存在的反应位点,主要是酚羟基、苯环侧链取代基、羰基等。在四种木质素中,漆酶对碱木质素的改性反应性要高于其他类型的木质素,这可能与碱性溶液体系中漆酶的活性较高有关。     

Microbial degradation of lignin: Role of lignin peroxidase,manganese peroxidase,and laccase

Lignin peroxidase (LiP), laccase (LA) and manganese peroxidase (MnP) of white-rot basidiomycetes such as Phanerochaete chrysosporium, Coliorus versicolor, Phlebia radiata and Pleurotus eryngii catalyze oxidative degradation of lignin substructure model compounds and synthetic lignins (DHPs). Side chain- and aromatic ring cleavage products of both phenolic and non-phenolic substrates oxidized by LiP were isolated and characterized by NMR and MS. The cleavage mechanism was elucidated by using ¹⁸O, ²H, and ¹³C labeled lignin substructure dimers with ¹⁸O₂ and H₂ ¹⁸O. Recent studies suggested that LiP is capable of oxidizing lignin directly at the protein surface via a long-range electron transfer process. LA and MnP, which oxidize phenolic but not non-phenolic moieties, generally degrade lignin stepwise from phenolic moieties. However, recent studies indicated that MnP and LA can degrade both phenolic and non-phenolic aromatic moieties of lignin with some special mediators.     

低氮胁迫下苦荞根际土壤纤维素酶活性的响应机制：荧光光谱法测定

土壤纤维素酶参与土壤中养分的循环过程,分解纤维素为小分子糖分微生物生命活动提供能源物质,并且对生态系统有重要的指示作用。如何快速、精确的测定纤维素酶活性对土壤生态系统诊断、恢复等有重要指示意义。自然界中不同的氮肥处理及种植不同的苦荞品种均会对土壤纤维素酶活性产生影响。利用德国进口全自动进样酶标仪(InfiniteR 200 PRO),以4-Methylumbelliferyl β-D-cellobiopyranoside (Cel) 纤维素荧光物质为底物,将不同耐瘠性苦荞(迪庆苦荞-耐低氮;黑丰1号-不耐低氮)根际土壤以悬液形式用排枪吸取微量和荧光底物混合加入到96孔微孔板进行充分反映、培养测定土壤中纤维素酶活性,其中将对照板和样品放在同一个板块上,减少培养空间占用和操作时间。结果表明,除了苦荞成熟期的常氮处理,苗期和开花期的低氮处理条件下,纤维素酶活性均表现为迪庆苦荞根际土壤显著高于黑丰1号,迪庆根际纤维素酶活性在苗期低氮处理下高于黑丰39.50%;纤维素酶活性在迪庆苦荞的开花期和成熟期氮处理间有显著性差异,表现为常氮高于其他处理,而黑丰1号仅在开花期时常氮处理和其他处理之间有显著差异;灭菌处理酶活性显著低于常氮处理的54.29%,说明迪庆苦荞根际纤维素酶一部分来源于微生物的贡献,养分充足时微生物的活性更高,贡献越大。荧光光谱法分析出的纤维素酶活性表明耐瘠性品种迪庆苦荞可以通过增加根际土壤酶活性的方法来抵抗外界环境的胁迫,面对黄土高原养分贫瘠的土壤,可以考虑选种耐贫瘠的品种来降低成本增加收益。试验过程中,荧光标记的酶标仪测定方法与传统的测定方法相比更加快捷、准确和节约,同样适合大批量样品测定,可以为未来精准农业生产,测土配方施肥提供及时的数据支持。     

ESR Study in Reactive Oxygen Species Free Radical Production of Pinus kesiya var. langbianensis Heartwood Treated with Laccase

Enzymatic oxidation of lignin phenolic hydroxyl groups can enhance the level of autoadhesion between wood fibers or particles depending upon the bonding mechanism of wood-based materials without synthetic adhesives such as urea and phenol formaldehyde. The adhesive effect is caused by an increased number of reactive oxygen groups at the fiber surface. The parameters of laccase-treated wood fibers play vital roles in generating reactive oxygen species (ROS) free radicals. Laccase I (white-rot fungi) and laccase II (Aspergillus sp.) are used to catalyze the oxidation of heartwood powder of Pinus kesiya var. langbianensis in suspension under different pH values, temperatures, treatment times and different laccase concentrations. Electron spin resonance spectroscopy and spin trapping technique were used to detect the ROS free radicals generated in the laccase-treated heartwood powder and it was found that in it the concentration of ROS free radicals was higher than that in the control. Analysis of variance indicates that there was a significant difference between the ROS concentration values of laccase-treated heartwood powder under different pH values, treatment temperatures and times, and laccase dosages for both laccase I and II. Furthermore, the concentration of ROS free radicals generated by laccase I is higher than that generated by laccase II. It was found that the optimum conditions for generation of ROS in laccase-treated heartwood powder by the two kinds of laccase are pH 3.0; treatment temperature, 50 °C; treatment time, 2 h; enzyme concentration, 20 units/g of wood powder. Authors' address: Xinfang Duan, Research Institute of Wood Industry, Chinese Academy of Forestry, Wan Shou Shan, Beijing 100091, People's Republic of China     

Orientation selective ESEEM studies on the blue oxidases laccase and ascorbate oxidase

The blue oxidases, laccase and ascorbate oxidase, contain three spectroscopically distinct copper binding sites, two of which are EPR detectable in the oxidized Cu(II) state, called type 1 (T1) and type 2 (T2). The three dimensional structure of ascorbate oxidase has recently been determined (Messerschmidt A.et al.: J. Mol. Biol.206, 513 (1989)) while that of laccase has not. We have therefore carried out comparative electron spin echo envelope modulation (ESEEM) measurements on ascorbate oxidase, laccase and laccase in which T1 Cu(II) was substituted with Hg(II) in order to obtain structural information about the copper sites in laccase. The ESEEM results clearly show that there are as many histidines in laccase as in ascorbate oxidase, i.e., at least two at each site. Orientation selective ESEEM experiments showed that in the T1 site in both enzymes the two remote (uncoordinated) nitrogens are magnetically inequivalent and have different hyperfine interactions. Furthermore, the isotropic hyperfine constants of both remote nitrogens in laccase T1 are larger than those in ascorbate oxidase T1. In laccase T2 two remote nitrogens show similar hyperfine couplings and the modulation depth is significantly deeper than in ascorbate oxidase. Finally, it is suggested that the difference between the NQR frequencies of the remote nitrogens in T1 and T2 in both oxidases can be attributed to the alkyl group of the side chain being adjacent to the bound imidazole in T1 and to the remote nitrogen in T2. This is in accordance with the known X-ray structure of ascorbate oxidase.     

H2/air autoignition: The nature and interaction of the developing explosive modes

Appropriate algorithmic tools are employed for the analysis of the explosive modes developing during the autoignition of homogeneous mixtures. The ability of these tools to provide significant physical understanding is demonstrated in the case of the homogeneous ignition of a stoichiometric H₂/air mixture, modelled by two different chemical kinetics mechanisms. It is shown that the ignition process evolves in two stages. The first stage is characterised by the development of two explosive timescales (one fast and one slow), that lead the system away from equilibrium. As the end of the first stage is approached, the two explosive timescales converge, they merge and then they disappear. In the second stage only dissipative timescales develop, which drive the system all the way to equilibrium. It is shown that throughout the first stage the fast explosive timescale is generated by chain reactions. The slow explosive timescale is initially generated by an initiation reaction that produces the radicals required for the start-up of the fast mode, while later on it is generated by reactions that are responsible for the heat released. These findings are validated with sensitivity analysis results for the ignition delay time and are employed in order to clarify the discrepancies in the solution provided by the two different chemical kinetics mechanisms considered.     

Tryptophan radicals as reaction intermediates in versatile peroxidases: Multifrequency EPR, ENDOR and density functional theory studies

Versatile peroxidases are a new class of ligninolytic enzymes secreted by fungi from the group of white-rot basidiomycetes. Versatile peroxidase (VP) is a structural hybrid between lignin and manganese peroxidase. This hybrid combines the catalytic properties of the two above peroxidases being able to catalyze Mn(II)-dependent and Mn(II)-independent reactions. A long-range electron transfer mechanism has been inferred for the oxidation of big substrate molecules starting from an exposed tryptophan to the heme cofactor. A neutral tryptophan radical has been identified in VP fromBjerkandera adusta andPleurotus eryngii, after H₂O₂ activation and assigned to a specific tryptophan residue using multifrequency electron paramagnetic resonance and electron-nuclear double resonance spectroscopy. Comparative density functional theory calculations were performed for tryptophan neutral and cation radical species, considering also the effect of the polar environment surrounding the radical. The functional role of the radicals is discussed in the context of mechanistic models for VP.     

高压对米曲霉理化性质影响及诱变的研究

 在100~400 MPa压力、保压20 min的条件下处理酱油酿造菌种——米曲霉,研究高压对米曲霉存活率、形态特征、生理性质、蛋白酶、淀粉酶活性等的影响,并诱变筛选优良菌株。结果表明：高压对米曲霉的存活率、形态特征有明显的影响;压力对蛋白酶及淀粉酶活性的影响也有特异的规律,即在一定压力范围内（0.1~200 MPa）蛋白酶的活性随着压力的增加而减小,但随着压力的进一步升高（200~400 MPa）蛋白酶的活性又逐渐增强,在300 MPa时超过对照组,400 MPa时蛋白酶的活性达到最高值;淀粉酶在0.1~100 MPa时活性下降,在200 MPa时其平均的糊化和糖化活性最强、活力最高,当压力升高活性又开始降低,400 MPa时几乎又回到对照值。另外,高压处理后获得一株理想的变异菌株HP300a：生长速度快、孢子数量多、蛋白酶活力高,且不易被杂菌污染。其成曲的几项主要指标均优于对照株,酿出酱油的几项主要指标也明显优于对照株。为利用高压诱变筛选米曲霉优良菌种、提高酿造酱油产品的产量及质量提供了理论依据,并发现了高压处理米曲霉引起其蛋白酶及淀粉酶活性改变的特殊规律。     

双模瑞利-泰勒不稳定性的预热烧蚀效应研究

针对双模扰动下的烧蚀瑞利-泰勒不稳定性增长问题,采用高精度的数值计算方法,研究了不同预热程度下模耦合产生的多个高次谐波幅值的发展和演化问题。研究表明,三种预热烧蚀条件下,当扰动基模满足长波与短波耦合方式时,谐波中的长波模态占主导,而短波模发展明显受到抑制;当满足短波与短波耦合时,耦合结果带来了许多新的增长较快的长波模态,此时短波模增长呈现小幅震荡形式。比较两种耦合方式可以发现,长波结构在烧蚀瑞利-泰勒不稳定性弱非线性阶段都占主导地位,尤其是短波与短波耦合中气泡与尖钉表现出不同于两个基模的长波模结构。进一步分析预热效应对模耦合增长的影响,发现预热程度越强就越能削弱耦合谐波的增长,这说明预热对烧蚀瑞利-泰勒不稳定性具有致稳作用,这对惯性约束聚变工程中控制烧蚀瑞利-泰勒不稳定性发展具有重要意义。     

Studies on the application of ultrasound in leather enzymatic unhairing

Enzymatic unhairing is a clean technology for leather industry. However, one problem hinders the utilization of enzymes in leather unhairing is the slow diffusion of enzymes through skins. As another clean technology, ultrasound can be applied to accelerate the diffusion of enzymes through skins. In this work, effects of ultrasound on the enzymatic unhairing were investigated. Effect of frequency of ultrasound on the enzymatic unhairing was compared, and 20 kHz was chosen in this work. Under our conditions, ultrasound has little effect on enzyme activities, and also has little influence on the physical and mechanical properties of leather. Ultrasound can enhance the diffusion of enzyme through skins, thus improving the unhairing capacities of enzymes. It is noted that amylase can interact with proteoglycans by attacking saccharide side chains, thus having some unhairing capacity. Ultrasound can improve the unhairing capacity of amylase more effectively. Furthermore, amylase does no harm on the collagen fibrous structure and can be used safely without the need for a high degree of control. So it is possible to use amylase in enzymatic unhairing in combination with protease. In short, using ultrasound in amylase and protease unhairing is a promising clean technology.