巴西蘑菇对木质纤维素的降解与转化*

巴西蘑菇能够降解棉籽壳和麦草两种培养基中木质纤维素复合体中的全部组分,属于白腐真菌;巴西蘑菇降解的有机物质的绝大部分被菌体的呼吸过程消耗掉,其绝对生物学效率较低,仅为4.41%~5.25%;在栽培前期木质素的降解速率大于纤维素和半纤维素,这对纤维素和半纤维素的降解十分有利;非木质纤维素组分主要在菌丝生长阶段被利用,而木质纤维素是子实体生长发育阶段的主要碳源;就整个栽培过程而言,巴西蘑菇生长发育所需要的82.39%~84.50%的碳源来自木质纤维素。     

滑菇营养生理研究

本文研究了滑菇在木屑-麦麸基质上生长期间,菌体对基质的转化效率和基质中主要组分的降解规律。实验结果表明:子实体绝对生物学效率为12.43%,产量系数为16.94%。子实体阶段的木素、半纤维素和纤维素的降解量以及非木质纤维素组分的减少量占子实体阶段基物中干物质减少量的百分比值分别为:5.85、17.54、49.13和27.48%。由此可见,纤维素是子实体生长阶段的主要碳源。滑菇在菌丝生长和子实体生长阶段分别有纤维素酶和半纤维素酶的活性高峰出现。进一步证明了:子实体阶段这两种酶的活性增加与子实体形成有密切关系     

巴西蘑菇对木质纤维素的降解与转化

巴西蘑菇能够降解棉籽壳和麦草两种培养基中木质纤维素复合体中的全部组分,属于白腐真菌,巴西蘑菇降解的有机物质的绝大部分被菌体的呼吸过程消耗掉,其绝对生物学效率较低,仅为4．41％－5．25％,在载培前期木质素的降解速率大于纤维素和半纤维素,这对纤维素和半纤维素的降解十分有利;非木质纤维素组分主要在菌丝生长阶段被利用,而木质纤维素是子实体生长发育阶段的主要碳源;就整个栽培过程而言,巴西蘑菇生长发育所需要的82．39％－84．50％的碳源来自木质纤维素。     

不同木质纤维素基质上白腐菌降解特性的研究

通过测定木质素、纤维素、半纤维素和漆酶分泌的变化,研究白腐菌在稻草、木屑、粗纤维素、滤纸、黑液木素基质上的降解特性。结果表明,除黑液木素上白腐菌不能生长外,在前25d,各基质中纤维素、半纤维素和木质素含量呈持续下降趋势,之后,降解速率减少,其中木质素的降解速率大于纤维素和半纤维素的降解速率。漆酶分泌在生长初期呈快速上升趋势,第10d酶活达到最大,第10～20d快速下降,其后基本不变,基质中酶活大小顺序为稻草基质、木屑基质、粗纤维和滤纸基质,显示了木质素存在对漆酶分泌的诱导作用。     

解淀粉芽孢杆菌MN-8对玉米秸秆木质纤维素的降解

微生物降解木质纤维素既是生物质资源化利用中的关键问题,也是亟需解决的难点问题.本文在前期获得木质素降解菌——解淀粉芽孢杆菌MN-8菌株的基础上,进一步研究该菌株对玉米秸秆木质纤维素的降解作用.研究利用玉米秸秆粉-MSM培养基对MN-8菌株进行固态发酵,监测发酵过程中木质纤维素酶活力和木质纤维素含量变化情况,并通过傅立叶红外光谱(FTIR)和气质联用色谱(GC/MS)对木质纤维素的降解情况及产物进行分析.结果表明:解淀粉芽孢杆菌MN-8菌株可产生木质素过氧化物酶、锰过氧化物酶、纤维素酶和半纤维素酶等木质纤维素降解酶,在发酵10～16 d陆续达到酶活力峰值,最高酶活力分别为55.0、16.7、45.4和60.5 U·g-1.发酵24 d后,玉米秸秆中木质素、纤维素和半纤维素的降解率可分别达到42.9％、40.6％和27.1％.FTIR光谱数据表明,玉米秸秆发酵后木质素、纤维素和半纤维素的特征吸收峰强度均有一定程度的下降,表明木质纤维素被部分降解.GC/MS分析结果也证实,解淀粉芽孢杆菌MN-8能有效降解秸秆木质纤维素.MN-8菌株可断裂玉米秸秆木质素单体之间的连接键β-O-4,将秸秆木质素解聚为苯丙胺、苯丙酮和苯丙酸等保留木质素苯丙烷结构的单体化合物,并将部分单体化合物进一步氧化为Cα羰基化合物,如2-氨基-1-苯丙酮和紫丁香基苯乙酮等.在对纤维素和半纤维素降解产物的GC/MS分析中发现,降解产物包含葡萄糖、甘露糖和半乳糖等多种单糖化合物以及甲酸、乙酸、丙酸、1,1-乙二醇和3-羟基丁酸等代谢产物.表明解淀粉芽孢杆菌MN-8对秸秆木质纤维素表现出强降解作用,且该作用依赖于菌株产木质纤维素降解酶的能力.     

香菇HMG-box转录因子lelcrp1基因的功能

【目的】研究香菇(Lentinula edodes) HMG-box转录因子LELCRP1 (Lentinula edodes lignocellulase genes regulation protein 1)在木质纤维素降解相关酶基因表达中的功能与作用。【方法】通过double-joint及同源重组方法构建lelcrp1基因RNAi载体,采用根癌农杆菌介导转化的方法转入香菇异核菌株W1菌丝中,筛选得到RNAi转化子,通过Southern杂交检测插入片段在菌株W1基因组中的拷贝数量。采用荧光定量PCR检测RNAi转化子木质纤维素降解酶基因表达水平变化,并在含有3.5μg/mL潮霉素的MYG平板上测定RNAi转化子的菌丝生长速度。【结果】获得了4个lelcrp1基因表达水平与出发菌株W1相比显著下调6–7倍的RNAi转化子。Southern杂交结果显示,lelcrp1基因RNAi片段已成功整合至香菇菌株W1基因组内,并以单拷贝形式存在。对其中2个RNAi转化子的26个木质纤维素降解酶基因表达水平进行分析,发现其中9个纤维素酶基因、1个半纤维素酶基因、2个辅助酶AA9基因和1个锰过氧化物酶基因的表达水平均表现出明显的下调。平板生长试验表明,RNAi转化子菌丝生长速度均显著慢于出发菌株W1。【结论】通过RNAi技术成功抑制了香菇异核菌株中lelcrp1基因表达水平,并导致部分纤维素及木质素酶基因表达水平相应下调,首次发现HMG-box结构域的转录因子能调控木质纤维素降解相关酶基因表达。     

木质素酶及其生产菌的筛选育种

木质素酶降解木质纤维素材料中的木质素,使木质素-半纤维素-纤维素结构解体,纤维素得以暴露出来供后续步骤处理.它广泛应用于生物制浆、生物漂白、废水处理等工业过程中.由于近年利用可再生木质纤维素材料用酶法水解生产酒精成了研究热点,因而作为纤维素材料生物转化工艺预处理过程中的关键角色,木质素酶也极大地唤起人们的研究兴趣.本文介绍了木质素与白腐真菌(Phanerochaete chrysosporium)木质素降解酶系的特征以及锰过氧化物酶、木质素过氧化物酶、漆酶等3种木质素酶的催化作用机理,归纳了目前流行的木质素酶产生菌的筛选方法及近年来从自然界筛选木质素酶高产菌的种类,并对产木质素酶野生菌株的诱变育种与基因工程改造的进展进行了阐述.     

玉蕈降解木质纤维素的生理生化基础

本文研究了玉蕈在纤维废弃物上生长期间,培养基中主要成分的降解规律及有关的酶学分析。实验结果表明:1.玉蕈分解纤维素和半纤维素的能力较强,分解木素的能力很弱。因此,玉蕈是褐腐型木腐菌。2.纤维素是玉蕈子实体生长阶段的主要碳源。3.玉蕈生长期间可向培养基中释放羧甲基纤维素酶、滤纸纤维素酶、半纤维素酶、淀粉酶和蛋白酶。酶活性在子实体生长阶段显著增加。进一步证明了子实体阶段酶活性增加与培养温度和子实体形成有密切关系。     

枯草芽胞杆菌降解木质纤维素能力及产酶研究

从农林废物堆肥中分离得到1株细菌经鉴定为枯草芽胞杆菌,将该细菌用于木质素类化合物利用.固态培养条件下考察其对木质纤维素的降解能力及产酶特性,另外对发酵前后的稻草结构进行了红外光谱分析.结果表明,枯草芽胞杆菌具有木质素降解能力,兼具低分子量木质素酚型、非酚型类物质的降解能力.其对木质素降解是木质素过氧化物酶、锰过氧化物酶、漆酶、纤维素酶和半纤维素酶共同作用的结果.在实验条件下,培养30 d使木质素降解率达9.47%,同时对纤维素、半纤维素也有较高程度的降解;降解率分别为38.8%、41.84%.红外光谱分析结果表明,稻草木质素结构被破坏,枯草芽胞杆菌对木质素各官能团的降解作用有所不同.     

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。     

废次烟叶提取液源木质素降解菌Bacillus subtilis SM降解特性

【目的】目前造纸法再造烟叶工艺已经成为我国重要的废烟叶处理和利用方式,该工艺中烟梗中高木质素的降解是个挑战性的需解决问题。从废次烟叶提取液(Tobacco waste extract,TWE)中筛选木质素的降解微生物用来直接处理烟梗或烟末提取液,可实现对木质素含量的调控。【方法】将废次烟叶提取液(TWE)浓缩液中分离出的Bacillus subtilis SM接种到以Kraft木质素为唯一碳源的无机盐培养基中,在pH 7.0、30°C培养基中培养4 d来检测菌株对木质素的降解效果。通过HPLC、TOC、GPC和色度来表征SM对木质素的降解,并采用烟梗无机盐培养基在pH 7.0、30°C培养4 d检测SM对烟梗木质素的降解。【结果】HPLC结果显示SM在以木质素磺酸钠为唯一碳源的无机盐培养基中可全部降解分子质量为534.5的木质素磺酸钠,而对Kraft木质素降解不明显,仅观察到组分的变化。脱色结果显示脱色率达到40.7%,但在对Kraft木质素矿化方面矿化率只能达到5.4%。SM在烟梗无机盐培养基中可使烟梗失重率分别达到50%以上(对照组为18.9%),烟梗中木质素含量减少了70%左右。【结论】来源于废次烟叶提取液(TWE)的Bacillus subtilis SM能够以Kraft木质素为唯一碳源生长,也能够有效降解烟梗中的木质素,可应用于烟草废弃物原料中木质素的降解。     

Phenotypic classes of phenoloxidase-negative mutants of the lignin-degrading fungus Phanerochaete chrysosporium

This paper reports the isolation of phenoloxidase-negative mutants of the white-rot fungus Phanerochaete chrysosporium and the results of a survey of idiophasic functions among these mutants. The mutant strains were isolated from a medium containing o-anisidine after gamma irradiation of wild-type spores and fell into four classes, divided by the manner in which they mineralized 14C-lignin wheat lignocellulose. Examples are strain LMT7, which degraded lignin at a rate similar to that of the wild type; strain LMT26, in which degradation was enhanced; strain LMT16, whose degradation rate was apparently unaffected, although the onset of lignin attack was delayed compared with that in the wild type; and strain LMT24, which was unable to evolve significant amounts of 14CO2 from the radiolabeled substrate. The mutants were not necessarily defective in other functions associated with idiophasic activities (intracellular cyclic AMP levels, sporulation, extracellular glucan production, veratryl alcohol synthesis). We conclude that phenoloxidase activity as detected by the o-anisidine plate test is not necessary for lignin degradation. In addition, mutations resulting in the loss of lignin-degrading ability were not necessarily pleiotropic with other idiophasic functions.     

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens.

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.     

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens.

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.     

Lignin-Degrading Enzymes of the Commercial Button Mushroom, Agaricus bisporus

Agaricus bisporus, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by A. bisporus. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in A. bisporus suggests significant roles for these two enzymes in lignin degradation by this fungus.     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

Influence of cultivation conditions on production of lignocellulolytic enzymes by Ceriporiopsis subvermispora

The aim of this work was to make a survey describing factors that influence the production of extracellular enzymes by white-rot fungus Ceriporiopsis subvermispora responsible for the degradation of lignocellulolytic materials. These factors were: carbon sources (glucose, cellulose, hemicellulose, lignin, maltose and starch), nitrogen sources (ammonium sulphate, potassium nitrate, urea, albumin and peptone), pH, temperature and addition of three different concentrations of Cu²⁺ and Mn²⁺. The cellulase and xylanase activities were similar in medium with different carbon sources and the highest cellulase and xylanase activities were measured in medium with urea and potassium nitrate as nitrogen sources, respectively. The highest laccase activity was observed in medium with lignin and peptone as carbon and nitrogen sources. In other experiments, time course of production of lignocellulolytic enzymes by white-rot fungus C. subvermispora in medium with lignin or glucose as carbon sources was observed.     

白腐菌混合菌降解木质素最佳条件的优化

通过正交试验对3种白腐菌混合菌降解竹材木质素的条件进行优化,结果表明,在温度为32℃、pH3.0、固体发酵时间20 d、培养液与竹材基质质量百分比110%时降解木质素的效率最高.在此基础上,研究了两种诱导剂对白腐菌混合菌降解木质素的影响.结果表明,两种诱导剂均能促进木质素的降解,其中H_2O_2在浓度1%时,木质素降解率高达62.9%,苯甲酸在浓度0.1%时,木质素降解率最高,为67.8%.     

The effects of various nutrients on extracellular peroxidases and acid-precipitable polymeric lignin production byStreptomyces chromofuscus A2 andS. viridosporus T7A

Summary The relationships between growth, medium pH, assimilation of glucose and amino acids, presence or absence of lignocellulose in the medium, lignin solubilization, and the appearance of extracellular peroxidase activity were compared for two lignin-solubilizing actinomycetes, Streptomyces chromofuscus A2 and S. viridosporus T7A. In a mineral salt medium containing yeast extract and three amino acids S. chromofuscus A2 grew faster than S. viridosporus T7A. When d-glucose was added to this medium, it was used in preference to the amino acids, the assimilation of which was delayed. Extracellular peroxidase activity peaked during the stationary phase, and glucose supplementation delayed peroxidase production. The eventual peak in peroxidase activity was higher in glucose-containing medium than in medium without glucose. Supplementation of the medium with lignocellulose did not affect either the level or time of appearance of extracellular peroxidase. However, lignin solubilization in lignocellulose-supplemented medium correlated positively with peroxidase activity: both increased after the cells entered the stationary phase. Supplementation of lignocellulose-containing medium with glucose delayed peroxidase production and lignin solubilization until the glucose had been assimilated. With S. viridosporus T7A, addition of d-glucose to the standard medium affected amino acid assimilation differently from S. chromofuscus A2. Glucose was consumed concomitantly with the amino acids. In the medium supplemented with lignocellulose, peroxidase activity and lignin solubilization correlated as they did for S. chromofuscus A2. A correlation of unknown significance was observed between the peroxidase activities of both strains and increasing medium pH. S. chromofuscus A2 produced more peroxidase and solubilized more lignin from lignocellulose than did S. viridosporus T7A. Overall, these findings show that extracellular peroxidases of both Streptomyces ssp. appear extracellularly primarily after cells cease growing and nutrients have been depleted from the medium. Also, increasing extracellular peroxidase activity and rates of lignin solubilization in both organisms are correlated and subject to glucose repression. These results point to the involvement of stationary-phase active peroxidases in the Streptomyces-catalyzed solubilization of lignin.Paper No. 90518 of the Idaho Agricultural Experiment Station Offprint requests to: D. L. Crawford     

木质素含量对四倍体刺槐嫩枝插穗扦插生根的影响

以四倍体刺槐1年生嫩枝插条为试验材料,分析插穗木质素含量与其横截面剪切强度、相关酶活性和激素关系,并探究不同木质素含量的插穗扦插生根性状的效应,为四倍体刺槐扦插选择合适插穗提供理论参考。结果表明:(1)插穗的木质素含量与其横截面剪切强度呈极显著正相关关系(相关系数为0.99),根据剪切强度可以间接地估算其木质素含量。(2)插穗的木质素含量与其POD和PPO活性呈极显著正相关关系(POD相关系数为0.98,PPO相关系数为0.92),也与激素ABA含量呈极显著的正相关关系(相关系数为0.97),而同IAA、IBA呈不显著的相关性,根据POD和PPO活性以及激素ABA含量对生根的影响可以推测木质素含量对生根有一定的影响。(3)不同木质素含量的插穗生根性状差异显著,木质素含量为19.47%时插穗生根能力最强,其插穗生根率为60.39%,平均每株生根量为9.70个,根长为4.85cm;木质素含量为10.60%时插穗生根能力最差。(4)生根性状最佳时的木质素含量为19.47%,其对应的剪切强度范围为40～50kg。