产木质纤维素降解酶真菌的筛选及产酶特性

【背景】利用微生物处理秸秆引起研究者的广泛关注。【目的】筛选生长速度快、木质纤维素降解酶活性强的真菌菌株,用于植物秸秆降解和高效利用。【方法】从自然界采集的样品中分离纯化真菌菌株,利用PDA-愈创木酚和PDA-羧甲基纤维素钠平板初筛,再经过液体发酵检测漆酶酶活、羧甲基纤维素酶酶活及菌丝生长速率复筛目的菌株,通过内转录间隔区(internal transcribed spacer,ITS)测序法对目的菌株进行鉴定,对目的菌株产漆酶和羧甲基纤维素酶活力进行测定及酶学性质研究。【结果】从样品中分离纯化到18株真菌,通过初筛筛选出9株产木质纤维素降解酶真菌菌株,再经过复筛,筛选出一株产漆酶、羧甲基纤维素酶活力高、菌丝生长快的菌株M1,经过分子生物学鉴定M1为糙皮侧耳(Pleurotus ostreatus),其漆酶酶活为(243.59±1.11)U/mL,羧甲基纤维素酶酶活为(36.03±0.63) U/mL。在5 d的培养期内,菌丝生长速率为(9.43±0.32) mm/d。对菌株M1的发酵粗酶液的酶学性质进行了检测分析,结果表明,所产的漆酶在pH5.0-6.5相对酶活为90%以上,在pH ...     

木质纤维素酶高产菌株的筛选及其对玉米秸秆降解效果的影响

为了提高玉米秸秆中木质素的降解率,从腐烂的树枝和土壤中筛选木质纤维素酶高产菌株,以秸秆为唯一C源富集培养后,采用PDA-愈创木酚法进行初筛,筛选出产木质素酶真菌5株,然后以玉米秸秆为主要C源进行固态发酵和复筛。结果表明:第5号菌株在发酵玉米秸秆5 d后,使木质素的降解率达到最高(34.95%),粗纤维的降解率达到20.00%,显著高于其他4种菌株(P<0.05),其羧甲基纤维素酶比酶活达到116.35 U/g;10 d后,其木质素酶比酶活达到最高(45.64 U/g)。     

白灵侧耳栽培过程中胞外酶和呼吸酶活及离子流速的研究

研究了白灵侧耳Pleurotus nebrodensis栽培过程中生长发育各阶段的漆酶、羧甲基纤维素酶（CMC酶）、半纤维素酶和淀粉酶等4种胞外酶及苹果酸脱氢酶、葡萄糖-6-脱氢酶和6-磷酸葡萄糖酸脱氢酶等3种呼吸酶的活性变化,同时测定了菌丝表面H+、K+、Ca2+离子流速。结果表明,整个生育期的胞外酶活性变化具有明显的阶段性。漆酶酶活在菌丝生长阶段第28天时达到最大值,纤维素酶和半纤维素酶酶活在子实体生长阶段第107天时达到最大值,子实体收获后活性下降,而淀粉酶活性在各时期均比较低,说明白灵侧耳对木质素类物质利用最早。苹果酸脱氢酶、葡萄糖-6-脱氢酶和6-磷酸葡萄糖酸脱氢酶酶活在后熟期、温差刺激及子实体生长期出现3个峰值。整个栽培周期的菌丝表面H+、K+、Ca2+离子流速各个阶段有不同的变化规律,Ca2+在菌丝生长阶段内流,从后熟期开始外排,K+和H+在整个周期外排;C/N和温度对离子的流速有一定的影响。     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T.reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50℃,在Fe³⁺(≤4 mmol/L)和Cu²⁺(0–10 mmol/L)存在条件下酶活稳定。     

筛选两株稻杆降解放线菌

【目的】筛选能够同时降解纤维素、半纤维素、木质素的微生物菌株,并研究其对稻杆的降解效果。【方法】采用羧甲基纤维素钠、半纤维素平板水解圈法、苯胺蓝平板脱色法进行初筛;利用DNS法测定胞外酶活性;在含有2%稻秆的液体发酵培养基中摇瓶培养10天后,洗去菌体测定稻杆失重率、木质纤维素类物质降解率,同时测定稻杆断裂拉力进行复筛。【结果】筛选出两株能够同时高效降解木质纤维素的放线菌A3和A6,其纤维素和半纤维素酶活较高,最高纤维素全酶活、β-葡萄糖苷酶活、外切酶活和内切酶活分别为:12.84和12.85、6.23和6.53、24.56和17.80、14.00和18.80 U/mL;最高半纤维素酶活分别为:83.05和52.98 U/mL;木质素酶活较低,菌株A3和A6的木素过氧化物酶最大值为:12.72和14.67 U/mL,锰过氧化物酶最大值分别为:22.48和24.67 U/mL,漆酶最大值分别为:28.40和33.04 U/mL。经形态学、培养特征和分子生物学分析,两株菌株鉴定为链霉菌,对稻杆均有较好的降解效果,在第10 d后稻杆断裂拉力测定值分别比初始时降低62.67%和66.67%;稻杆失重率在31.50%和35.83%;菌株A3对纤维素、半纤维素、木质素降解率为38.73%、33.16%和20.68%,菌株A6为47.69%、28.64和22.59%。【结论】放线菌A3和A6对纤维素、半纤维素、木质素均具有降解作用,且酶活较高,具有较好的应用前景。     

秸秆降解放线菌GC的筛选及其应用基础研究

运用循环流化技术,从土壤样品中筛选出1株具有单独降解秸秆能力的菌株GC,考察了该菌的生长特性及产纤维素酶和木质素酶能力,验证了该菌对小麦秸秆的处理效果。结果表明,该菌为放线菌的左式链霉菌(Streptomyces drozdowiczii);可在LB等基础培养基中快速繁殖;纤维素内切酶活和滤纸酶活分别可达67.57 U/mL和19.69 U/mL,并且具备木质素降解能力;该菌单独处理小麦秸秆20 d的秸秆失重率为11.52%;处理产物含多种石油烃、有机醇和植物甾醇等,表明该菌在秸秆等农业面源污染物的资源化利用方面具有良好的开发应用前景。     

三株高效秸秆纤维素降解真菌的筛选及其降解效果

【目的】利用多种筛选方法,获得高效秸秆纤维素降解真菌,并研究其秸秆纤维素的降解能力。【方法】采用滤纸片孔洞法、滤纸条降解法、羧甲基纤维素钠(CMC-Na)水解圈测定法、秸秆失重法、纤维素分解率测定法、胞外酶活测定法等常规秸秆纤维素降解菌的筛选方法。【结果】筛选到3株具有较强纤维素降解能力的真菌菌株,经初步鉴定菌株98MJ为草酸青霉(Penicillium oxalicum)、菌株W3为木霉(Trichoderma sp.)、菌株W4为扩张青霉(Penicillium expansum)。菌株W4具有非常强的秸秆纤维素降解能力,10d内对秸秆的降解率可达56.3%,对纤维素、半纤维素和木质素的分解率分别为59.06%、78.75%和33.79%。菌株W4的胞外纤维素酶活力在14.25-49.75U/mL之间。【结论】筛选获得3株高效秸秆纤维素降解真菌菌株,其中菌株W4的纤维素酶活高于已报道的菌株,是一株十分具有研究开发潜力的纤维素酶生产菌株。     

高效木质素降解菌的筛选及其对玉米秸秆的降解效果

本研究利用愈创木酚和苯胺蓝固体培养基对菌株进行初筛,利用形态学和分子生物学对筛选出的菌株进行鉴定,以黄孢原毛平革菌Phanerochaete chrysosporium CGMCC 5.0776为对照,利用其对玉米秸秆进行预处理并测定木质素和纤维素的降解率,测定筛选菌株在预处理玉米秸秆过程中漆酶、锰过氧化物酶（manganese peroxidase,MnP）和木质素过氧化物酶（lignin peroxidase,LiP）活性。结果表明:利用愈创木酚和苯胺蓝固体培养基,从16株白腐真菌菌株中筛选出2株具有较高漆酶或MnP活性的菌株,鉴定其为桦栓孔菌Trametes betulina (L.) Pilát（ZT-153）和亚黑管孔菌Bjerkandera fumosa (Pers.) P. Karst.（ZT-307）,测定T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆酸不溶木质素降解效率分别为13.60%和21.87%,较对照P. chrysosporium CGMCC 5.0776高1.58%和9.85%,对纤维素的降解率较低,分别为4.10%和4.50%。2株菌株在预处理玉米秸秆过程中,T. betulina ZT-153表现出漆酶和MnP活性,B. fumosa ZT-307只表现出LiP活性。其中B. fumosa ZT-307对玉米秸秆酸不溶木质素的降解效率最高,在秸秆资源的综合利用方面具有较好的潜力和应用前景。     

2株瘤胃纤维降解细菌的分离鉴定

从日粮精粗比为3:7的小尾寒羊×蒙古羊杂交一代绵羊的瘤胃内容物中分离到2株严格厌氧细菌,一株球菌WQ-1,1株弧菌WH-2,二者对滤纸有很好的降解能力。通过酶活力试验测得WQ-1的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.66,7.0 U/mL和15.3 U/mL;WH-2的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.52,6.9 U/mL和17.2 U/mL。经形态学、生理生化反应、生态特性和遗传型的鉴定,WQ-1归类为瘤胃球菌属(Ruminococcus)的黄色瘤胃球菌(Ruminococcus flavefaciens)。WH-2归类为丁酸弧菌属(Butyrivibrio)的溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)。     

白腐真菌对稻草秸秆的降解及其有关酶活性的变化*

以稻草秸秆加20%棉籽壳为培养基质,接种侧耳Z17、921、1024菌株,在不同生长阶段,测定培养物的主要化学成分和有关酶活的变化。结果表明,从接种到子实体形成,所试菌株培养物的纤维素、木质素等呈持续不断的下降趋势,水份、粗蛋白含量却逐渐升高。基质中漆酶酶活性在菌丝生长初期呈迅速上升趋势,后稍降低,而愈创木酚酶活性在菌丝生长初期及子实体形成时达到高峰,后有所降低或消失。     

Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT2

Chaetomium thermophilum CT2 produced endocellulases at 50 °C, when grown on 2% microcrystalline cellulose, 1% soluble starch, and 0.4% yeast extract medium. A major endocellulase component was purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography and gel filtration on Sephacryl S-100. The molecular weight of the enzyme was estimated to be 67.8 kDa and the enzyme was found to be a glycoprotein containing 18.9% carbohydrate. The K_m of the purified enzyme for carboxymethyl cellulose, sodium salt (CMC), was 4.6 mg ml⁻¹. The enzyme displayed highest activity towards CMC and significantly lower activities towards phosphoric acid swollen cellulose and filter paper. The activity was enhanced in the presence of Na⁺, K⁺ and Ca²⁺ but inhibited by Hg²⁺, Zn²⁺, Ag⁺, Mn²⁺, Ba²⁺, Fe²⁺, Cu²⁺, Mg²⁺ and NH₄⁺. Optimum activity was at 60 °C and pH 4.0. The enzyme was stable over 60 min incubation at 60 °C and half-life at 70, 80 and 90 °C was approximately 45, 24 and 7 min, respectively.     

Cloning of sucrase genes from Streptococcus mutans in bacteriophage lambda

Abstract An extracellular peroxidase was purified by chromatofocusing column chromatography from the growth medium of ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium Burds BKM-1767. The enzyme was electrophoretically pure with an M _r of 45 000–47 000. It contained an easily dissociable heme, and required Mn²⁺ ions for activity. In the presence of hydrogen peroxide and Mn²⁺ it oxidized compounds such as vanillylacetone, 2,6-dimethyloxyphenol, curcumin, syringic acid, guaiacol, syringaldazine, divanillylacetone, and coniferyl alcohol. It did not oxidize veratryl alcohol. In reactions requiring Mn²⁺ and O₂, but not hydrogen peroxide, the enzyme oxidized glutathione, dithiothreitol, and NADPH with production of hydrogen peroxide. The hydrogen peroxide produced could be used as a co-substrate by ligninases such as those that oxidize veratryl alcohol, or by the peroxidase itself to oxidize lignin model compounds.     

Cu2+对拟南芥根的局部毒性及诱导DNA损伤和细胞死亡

该文探讨了不同浓度的Cu²⁺胁迫对拟南芥(Arabidopsis thaliana)根生长、活性氧(ROS)积累、抗氧化酶活性、质膜完整性和细胞活性的影响, 通过分根实验初步分析了Cu²⁺毒性效应的影响范围。结果表明, Cu²⁺胁迫可显著抑制拟南芥主根伸长, 诱导ROS积累及DNA损伤, 促发抗氧化酶活性升高, 破坏质膜完整性, 且Cu²⁺浓度越高, 毒性效应越明显, 在高浓度Cu²⁺胁迫下细胞活性显著降低。分析各参数之间的关系, 表明ROS的积累与超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)及抗坏血酸过氧化物酶(APX)的活性呈显著正相关; ROS积累与DNA损伤、质膜完整性、细胞活性之间具有显著的近线性关系。分根实验结果表明, 只有在添加重金属Cu²⁺(75 μmol·L^–1)一侧培养基中的根生长受抑制, 并出现ROS积累、细胞死亡, 暗示Cu²⁺对拟南芥根系的局部毒性效应可能是由于ROS的局部性积累导致受胁迫根系一侧的细胞死亡所引起的。     

硅对铜胁迫下小麦幼根细胞超微结构的影响

为探索硅对重金属铜胁迫下小麦的缓解作用,以矮抗58为供试材料,采用室内溶液培养法,研究不同浓度重金属铜(0、15、30 mg·L^-1)胁迫及铜胁迫下添加外源硅(50 mg·L^-1)对小麦幼苗生长、根系活力及根尖超微结构的影响.结果表明: 铜处理下,小麦幼苗根长、株高和根系活力均极显著低于对照(P＜0.01),加硅处理后小麦根长、株高和根系活力与单一铜处理相比均有所提高.铜胁迫下,小麦根尖细胞壁和细胞膜均遭到不同程度的破坏,细胞间空隙加大、细胞器消失;加硅处理后,虽然与对照相比,部分细胞和细胞器仍有变形,但细胞结构基本保持完好.综上,外源硅能在一定程度上缓解重金属铜对小麦幼苗生长及细胞组分的胁迫.
     

亚热带不同树种凋落叶分解对氮添加的响应

为探究不同质量凋落物对氮(N)沉降的响应, 该研究采用尼龙网袋分解法, 在亚热带福建三明格氏栲(Castanopsis kawakamii)自然保护区的米槠(Castanopsis carlesii)天然林, 选取4种本区常见的具有不同初始化学性质的树种凋落叶进行模拟N沉降(N添加)分解实验(施N水平为对照0和50 kg·hm ^-2·a ^-1)。研究结果表明: 在2年的分解期内, 对照处理的各树种凋落叶的分解速率依次为观光木(Michelia odora, 0.557 a ^-1)、米槠(0.440 a ^-1)、台湾相思(Acacia confusa, 0.357 a ^-1)、杉木(Cunninghamia lanceolata, 0.354 a ^-1); N添加处理凋落叶分解速率依次为观光木(0.447 a ^-1)、米槠(0.354 a ^-1)、杉木(0.291 a ^-1)、台湾相思(0.230 a ^-1), 除杉木凋落叶外, N添加显著降低了其他3种凋落叶分解速率。N添加不仅使4种树木凋落叶分解过程中的N释放减慢, 同时还抑制凋落叶化学组成中木质素和纤维素的降解; N添加在凋落叶分解过程中总体上提高β-葡萄糖苷酶(βG)和酸性磷酸酶活性, 对纤维素水解酶的活性影响不一致, 而降低β-N-乙酰氨基葡萄糖苷酶活性和酚氧化酶活性。凋落叶分解速率与凋落叶中的碳获取酶(βG)活性以及其化学组分中的可萃取物含量极显著正相关, 与初始碳浓度、纤维素和木质素含量极显著负相关, 与初始N含量没有显著相关性。凋落物类型和N添加的交互作用虽未影响干质量损失速率, 但对木质素和纤维素的降解具有显著效应。综上所述, 化学组分比初始N含量能更好地预测凋落叶分解速率, 而N添加主要通过抑制分解木质素的氧化酶(如PHO)来降低凋落叶分解速率。     

Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng

Heterogeneity of ginsenosides is an interesting and important issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. In this work, a new enzyme UDP-glucose:ginsenoside Rd glucosyltransferase (UGRdGT), which catalyzes the formation of ginsenoside Rb₁ from ginsenoside Rd [Biotechnol. Bioeng. 89: 444–52, 2005], was purified approximately 145-fold from suspended cells of Panax notoginseng with an overall yield of 0.2%. Purification to apparent homogeneity, as judged by SDS-PAGE, was successfully achieved by using sequential ammonium sulphate precipitation, anion-exchange chromatography and native PAGE. The enzyme had a molecular mass of 36 kDa, and its activity was optimal at pH 8.5 and 35 °C. The enzyme activity was enhanced by Mn²⁺, Ca²⁺ and Mg²⁺, but strongly inhibited by Zn²⁺, Hg²⁺, Co²⁺, Fe²⁺ and Cu²⁺. The apparent K_m value for UDP-glucose and ginsenoside Rd was 0.32 and 0.14 mM, respectively. The biotransformation yield from ginsenoside Rd to Rb₁ by UGRdGT in 50 mM Tris–HCl buffer at pH 8.5 and 35 °C was over 80%. This work provides a basis for further molecular study on the ginsenoside Rb₁ biosynthesis by P. notoginseng cells and it is also useful for potential application to in vitro biotransformation from ginsenoside Rd to Rb₁.     

铜离子调控木质纤维素降解和糙皮侧耳形态发育的研究

漆酶是一种含铜的多酚氧化酶,在木质素的降解中起重要作用。铜离子对漆酶的产生和活性有重要影响。通过向秸秆固体培养基中添加铜离子,研究铜离子对糙皮侧耳木质纤维素降解酶、木质纤维素降解和形态发育的影响。结果表明,添加铜离子能在一定程度上提高漆酶的活性,添加3mmol/L铜离子在第7天漆酶活性比对照高出71.2%,28d后添加铜离子的样品木质素降解率稍高出对照,添加铜离子能促进糙皮侧耳原基的分化和子实体的发育。     

Cu2+-catalyzed oxidative degradation of thyroglobulin

Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H₂O₂) alone on the Tg degradation, the inclusion of Cu²⁺ (30 μM), in combination with 2 mM H₂O₂, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu²⁺ was not mimicked by Fe²⁺, suggesting that Tg may interact selectively with Cu²⁺. A similar degradation of Tg was also observed with Cu²⁺corbate system, and the concentration of Cu²⁺ (5-10 μM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu²⁺ (10-30 μM) in combination with H₂O₂. In support of involvement of H₂O₂ in the Cu²⁺ corbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu²⁺, diminished the oxidative degradation, presumably consistent with the mechanism for Cu²⁺-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3-100 μM) of Cu²⁺ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu²⁺ up to 200 μM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu²⁺-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu²⁺-catalyzed oxidation.     

Purification and characterization of a streptomycete collagenase

A soil streptomycete designated as Streptomyces sp. A₈ produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase'as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as α-α-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetracetate completely inhibited the enzyme activity. Among the cations tested only Ca^{2 +} and Mg^{2 +} enhanced the collagenase activity. Heavy metal ions like Pb^{2 +}, Ag⁺, Cu^{2 +} and Zn^{2 +} strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca^{2 +}. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.