黄孢原毛平革菌产漆酶优化培养及其对刚果红的脱色降解

以白腐真菌模式菌株黄孢原毛平革菌Phanerochaete chrysosporium为研究对象,探讨培养条件、重金属和芳香族化合物对产漆酶的影响,并进一步研究漆酶对刚果红的降解效果。结果表明,P. chrysosporium产漆酶最适培养条件：葡萄糖为碳源,蛋白胨为氮源,碳氮比为90。培养8d后,漆酶酶活为911.1U/L;Mn²⁺严格地控制着P.chrysosporium产漆酶,而Cu²⁺对其影响不大,在添加4.0mmol/L Mn²⁺时,漆酶酶活为2 001.7U/L;芳香族化合物中藜芦醇（veratryl alcohol,VA）、4-香豆酸、香草醛和香草酸对菌体产漆酶能力均有促进作用,最高可提升至1 459.1U/L,而咖啡酸对菌体产漆酶稍有抑制。100U/L漆酶粗酶液可降解40mg/L刚果红,降解率为24.0%;而当介体物质VA存在时,该降解效率可提升至87.7%。     

毛栓孔菌XYG422菌株产漆酶发酵条件优化及对玉米秸秆生物降解的研究

利用基础产酶培养基从保藏的9株白腐真菌中筛选得到一株高产漆酶菌株毛栓孔菌XYG422,并通过单因素试验对该菌株发酵培养基及培养条件进行优化筛选,获得较高产漆酶能力,同时研究了该菌株对玉米秸秆的生物降解。研究结果表明:在液体发酵条件下,XYG422产漆酶最适宜碳氮源成分为玉米粉和酒石酸铵,菌株XYG422发酵条件优化后产漆酶酶活显著提升,该菌株最佳发酵培养条件为:玉米粉40g/L、酒石酸铵3g/L、温度30℃、pH 8.0、接种量5个直径1cm的菌饼、转速180r/min,诱导剂吐温-80和2,5-二甲基苯胺在低浓度时对菌株产漆酶有明显的促进作用,菌株产漆酶活性最高可达到41.6U/m L。该菌株表现出了对玉米秸秆较好的生物降解效率,培养60d后,玉米秸秆中木质素、纤维素和半纤维素的降解率依次为83.54%、50.65%和19.53%。     

不同固体发酵培养基下三种白腐真菌分泌的木质纤维素酶活性

本研究选取众所周知的典型白腐真菌树舌灵芝Ganoderma applanatum、毛栓孔菌Trametes hirsuta和木蹄层孔菌Fomes fomentarius作为研究对象,对其利用木质纤维生物质进行发酵及添加有机营养、无机盐、金属离子、表面活性剂等进行了探索,期间以测定漆酶、滤纸纤维素酶、木聚糖酶活性表征3种菌株对木质纤维生物质的预处理能力,为确定白腐真菌菌株及单环境因子而达到高效预处理木质纤维生物质提高生物转化效率的目的奠定了重要的理论基础。结果显示,3种菌株分泌的木质纤维素酶在10周内基本都呈现先上升后下降的趋势,且酶活都较高,均可作为木质纤维生物质预处理的备选菌株。相比于针叶树（落叶松）基质,阔叶树（白桦）基质更适宜于3种菌株生长及分泌木质纤维素酶。各环境因子中,Cu²⁺的添加可提高漆酶活性,表面活性剂对于3种酶活的诱导作用均十分显著。     

栓孔菌属漆酶高产菌株的初步筛选及其产酶条件的优化

利用显色反应对栓孔菌属(Trametes)进行了漆酶高产菌株的筛选,并对目标菌株的产酶条件进行了优化,在添加愈创木酚的固体培养基中,通过显色反应初步筛选出漆酶高产菌株东方栓孔菌Trametes orientalis Cui 6300;进一步通过单因子分析、正交试验和ABTS法确定了菌株Cui 6300的最适产酶条件:麦芽糖15 g/L,蛋白胨3 g/L,pH 4.8,Cu2+2.0 mmol/L,培养温度28°C,接种饼直径1.5 cm,此时酶活最高可达19.923 U/mL;同时探索了Cu2+浓度及添加时间对其菌丝生物量和漆酶活力的影响。研究表明,Cu2+最适添加浓度为2.0 mmol/L,添加时间为接种后第3天。     

Trametes sp. LS-10C固态发酵产漆酶培养基优化及其对双酚A的降解

漆酶是一种绿色高效的多酚氧化酶,在降解双酚A方面具有巨大潜力。为降低发酵产漆酶的成本及考察漆酶在双酚A降解中的能力,本研究以麸皮和柚皮为主要基质,优化了栓菌固态发酵产漆酶条件,对优化后获得的漆酶在双酚A降解中的应用进行了研究。结果表明,在培养基组分为：麸皮和柚皮粉比例为6:4（W/W）、固液比1:2.5（W/V）、铜离子2%（W/W）、蔗糖3%（W/W）、硝酸钾2%（W/W）、稻壳20%（W/W）的条件下,栓菌发酵产漆酶的酶活最高,发酵11d后,酶活可达到38.4U/gds。当双酚A初始浓度为10μg/mL时,在55℃条件下酶解140min后,双酚A基本降解完全。     

低能离子注入诱变选育漆酶高产菌株

利用低能N+束注入技术对漆酶产牛菌灵芝菌(Ganoderma lucidum)U60菌丝体进行辐照诱变.通过研究15 keV能量下不同注入剂量与灵芝菌存活率及突变率的生物学效应关系,确定了在2.6×1015-3.9×1015ions/cm2注入剂茸范围内可获得高比例正突变株.选择3.12×1015ions/cm2的注入剂量参数后,经多轮注入诱变,获得了遗传性稳定的漆酶高产突变株UIM-281;发酵产酶实验表明,UIM-281的产漆酶活力峰值分别是出发菌株U60的1.7倍及2.28倍,且产酶发酵周期相对缩短24 h,是工业发酵中更加经济高效的灵芝漆酶发酵菌株.     

粗毛栓菌(Trametes hirsuta) D2固态发酵山核桃蒲壳产漆酶的营养条件研究

【目的】为提高漆酶产量,降低生产成本,以山核桃蒲壳作为基质,对粗毛栓菌D2固态发酵产漆酶的营养条件进行研究。【方法】对不同碳源、氮源、碳氮比、蒲壳含量对漆酶产量的影响进行分析。【结果】山核桃蒲壳是粗毛栓菌生长的良好载体,能够促进漆酶的合成。粗毛栓菌D2漆酶固态发酵培养基干物质组成为:山核桃蒲壳40%(质量比),玉米粉24%(质量比),菜籽饼粉36%(质量比)。发酵6 d时,漆酶活性为126.8 U/g干基。【结论】粗毛栓菌固态发酵山核桃蒲壳产漆酶具有效率高,生产成本低的优点,具有潜在的工业化应用前景。     

茶树内生真菌CSN-4产漆酶培养基及培养条件研究

从茶树内生真菌筛选产漆酶的菌株,分析不同营养因素和培养条件对菌株漆酶酶活力的影响。采用6种显色底物的平板初筛和酶活测定的复筛方法,从15株茶树内生真菌菌株中筛选获得1株产漆酶酶活较高的菌株CSN 4。单因素分析结果显示,液态发酵条件下菌株CSN-4适宜的主要培养基成分是麸皮和蛋白胨;菌株CSN-4分别在麸皮30 g/L、蛋白胨2.5 g/L、CuSO₄·5H₂O 0.015 g/L和茶水6 g/L时发酵产漆酶酶活最高。发酵条件试验结果表明,菌株CSN-4分别在接种量为6个菌饼（直径6 mm）、装液量60 mL/250 mL、pH 4.8、摇床转速120 r/min,培养温度为28 ℃时产漆酶酶活较高。在培养基中添加麸皮和茶水对菌株CSN-4产漆酶有明显的促进作用。经过培养基成分及培养条件优化后,菌株CSN 4产漆酶酶活显著升高,达到2 417 U/L。     

稀土元素对短叶对齿藓生理生化的研究

通过测定分析3个主要生理指标过氧化物酶（POD）活性、丙二醛（MDA）含量和叶绿素含量的变化,研究了短叶对齿藓组培苗在6个不同浓度梯度的轻稀土La³⁺,Ce⁴⁺和重稀土Y³⁺单一元素胁迫下的生理响应和变化。结果如下：（1）短叶对齿藓体内的过氧化物酶（POD）活性和丙二醛（MDA）含量各处理组均低于对照组;Ce⁴⁺元素在3.55×10^-2 mmol·L^-1时显著提高了POD活性,在7.1×10^-2 mmol·L^-1时显著增加了膜脂过氧化产物丙二醛（MDA）含量。表明短叶对齿藓对Ce⁴⁺元素胁迫响应较强;而La³⁺元素各处理浓度间POD的变化较为平缓,胁迫响应较弱;Y³⁺处理居中。（2）较低浓度的La³⁺（1.8×10^-2 mmol·L^-1）和Ce⁴⁺（3.55×10^-2 mmol·L^-1）时显著提高叶绿素a、叶绿素b和总叶绿素含量;3种稀土元素在较高浓度时叶绿素的含量均有明显下降。本研究为进一步探明白云鄂博稀土矿区的苔藓植物生长发育受稀土元素的影响奠定了基础。     

一株木蹄层孔菌SO3高产漆酶发酵工艺及部分酶学特性研究

从采自新疆阿勒泰山的木蹄层孔菌(Fomes fomentarius)中筛选获得一株高产漆酶的SO3菌株,对其产酶发酵工艺进行优化,并对部分酶学特性进行了研究。结果表明:SO3菌株产漆酶的最佳碳、氮源分别为麦麸和酵母粉; Mn~(2+)、Ca~(2+)、Zn~(2+)和Mg~(2+)的添加对SO3产漆酶有一定的促进作用,而Cu~(2+)具有明显的抑制作用;没食子酸和单宁酸可使产酶高峰期提前2天;添加ABTS可使酶活由6 736 U/L提高至8 470 U/L;正交实验L_9(3~4)优化培养基最佳组分为:酵母粉0.5%、麦麸2.5%、葡萄糖0.5%,磷酸二氢铵0.5%,漆酶活性达到10 863 U/L。酶学特性结果表明:SO3菌漆酶最适反应温度为50℃,且在80℃仍具有漆酶活性,最适反应pH为3.0,在40℃,pH 4.0～5.0之间,酶活性最稳定;同时研究表明Mn~(2+)和Zn~(2+)对酶稳定性有一定的促进作用,Co~(2+)、Cd~(2+)、Cr~(2+)和Pb~(2+)对酶的稳定性具有明显的破坏作用;对菌体及发酵液进行了双向电泳检测,测定其等电点为4.1,获得了1个纯化酶蛋白质谱序列。结果表明,该蛋白为LaccaseE(Trametes sp.420)。SO3菌株具有潜在的应用价值。     

稀土镧对铁皮石斛不定芽诱导、植株生长及次生代谢产物合成的影响

采用培养基中添加30~150 μmol·L^-1 La（NO₃）₃的方式，研究La³⁺对铁皮石斛不定芽诱导、植株生长及次生代谢产物合成的影响。结果表明：70~90 μmol·L^-1的La³⁺对茎段不定芽诱导和促生效果最好；130 μmol·L^-1的La³⁺能显著提高叶绿素含量，植株在含110 μmol·L^-1 La³⁺培养基上生长最好，鲜重增加了15.22倍，显著高于对照，干鲜比为12.05∶100，比对照提高了64.39%；一定浓度的La³⁺显著提高干品中4种成分含量，110 μmol·L^-1 La³⁺处理下其多糖含量最高，为98.84 mg·g^-1，添加90 μmol·L^-1时，总黄酮、总酚和联苄含量最高，为4.31，7.56和21.01 mg·g^-1。联苄和总黄酮含量与其他5个指标相关性较大，而总酚和叶绿素含量与其他5个指标相关性小。本研究为进一步探究稀土对铁皮石斛促生及改善品质的作用机制奠定基础，为植物组培中如何科学合理使用稀土元素提供理论依据。     

Stabilities and rates in the laccase/TEMPO-catalyzed oxidation of alcohols

The influence of alcohol, 4-acetylamino,2,2,6,6'-tetramethylpiperidinyloxy (4-acetylamino-TEMPO) and laccase (from Trametes versicolor, TvL) concentration in the aerobic oxidation of furfuryl alcohol was investigated. Studies show that the K _m for 4-acetylamino-TEMPO is around 6.3 mM (V _max=0.18 mM min^-1) using 6.6 U mL^-1 of laccase and a furfuryl alcohol concentration of 140 mM. Under these optimized conditions, the reaction rate is still dependent on the concentration of enzyme in solution. Laccase can be reused, with a residual activity of around 25%. An important conclusion is that laccase is not stable in the presence of oxoammonium salts, presumably due to degradation via oxidation of essential amino acid residues or the glycosyl moieties on the periphery of the enzyme.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

Indispensability of Iron for the Growth of Cultured Chick Cells

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe²⁺, Fe³⁺, Cr³⁺, Cu²⁺, Mn²⁺, Co²⁺, Cd²⁺, Zn²⁺ and Ni²⁺), Tf complexes with these metals and metal-free apoTf.
The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe²⁺ or Fe³⁺ (10–100 μM) or iron-bound Tf (10–500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells.
The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.     

Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn2+ and Mn2+

Salts at high concentrations may cause oxidative damage to plant cells since many studies indicated the involvement of reactive oxygen species in salt-stress response. Recently, we have demonstrated that treatment of tobacco ( Nicotiana tabacum ) cell suspension culture with various salts result in an immediate burst of superoxide production via activation of NADPH oxidase by ions of alkali metals (Li⁺, Na⁺, K⁺), alkali earth metals (Mg²⁺, Ca²⁺) or lanthanides (La³⁺, Gd³⁺). In this study, we tested the effect of extracellular supplementation of Zn²⁺ and Mn²⁺ on the cation-induced oxidative burst in tobacco cell suspension culture, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent. Extracellular supplementation of Zn²⁺ and Mn²⁺ inhibited the generation of superoxide in response to addition of salts. Although both Zn²⁺ and Mn²⁺ inhibited the salt-induced generation of superoxide, the modes of inhibition by those ions seemed to be different since Mn²⁺ simply inhibited total production of superoxide while Zn²⁺ inhibited the early phase of superoxide production and induced the slow release of superoxide. Roles of Mn²⁺ and Zn²⁺ in protection of plant cells from salt stress, as an effective superoxide scavenger and an effective inhibitor of plasma membrane-bound NADPH oxidase, respectively, are discussed.     

Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha

The hard wood-colonizing ascomycete Xylaria polymorpha, that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l⁻¹). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a pI of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 10³ to 7 × 10⁴ M⁻¹ s⁻¹). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic β-O-4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin.     

Ca2+ acts synergistically with brassinosteroid and indole-3-acetic acid in stimulating ethylene production in etiolated mung bean hypocotyl segments

Brassinosteroid (BR) and indole-3-acetic acid (IAA) were used in combination with Ca²⁺ in order to determine if there was a synergistic effect in the stimulation of ethylene production in etiolated mung bean ( Vigna radiata L. Rwilez ev. Berken) hypocotyl segments. Ca²⁺ was found to act synergistically with BR. IAA or a combination of the two in promoting a stimulation in ethylene production. EDTA, which chelates Ca²⁺, greatly reduced the effectiveness of calcium salts in promoting ethylene production in the presene of either BR, IAA or a combination of the two. Neither K⁺, Mg²⁺ nor Mn²⁴ (chloride salts) acted synergistically with BR and IAA.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.