真菌产纤维素酶的诱导物及其调控机理研究进展

木质纤维素原料作为一种储量丰富、价格低廉的可再生资源,在生物燃料以及相关高附加值产品领域的应用已成为一个研究热点.纤维素酶是木质纤维素原料资源化利用过程中的关键酶,但从自然中筛选的纤维素降解菌株酶活较低,因而制约了木质纤维素原料的资源化利用.本文综述了真菌产纤维素酶的诱导物及纤维素酶表达调控机理研究的主要进展,着重讨论了纤维素、纤维二糖、槐糖、龙胆二糖等诱导物对真菌产纤维素酶的诱导作用以及葡萄糖等代谢产物的抑制作用,并阐述了真菌纤维素酶诱导表达调控的机理以及纤维素酶基因表达激活子(ACE)、木聚糖酶转录激活因子(Xyr1)、内切葡聚糖苷酶激活元件(CAE)、分解代谢物抑制蛋白(CRE)等调控因子的研究进展.提出通过对纤维素酶合成代谢进行调控来高效合成纤维素酶,为提高纤维素酶的活性和纤维素酶工业化应用提供参考.     

基于木质素生物降解的堆肥化接种剂开发

选取由农林废物堆肥中筛选出的木质素降解优势土著微生物枯草芽孢杆菌(Bacillus subtilis)、铜绿假单孢菌(Pseudomonas aeruginosa)、黑曲霉(Aspergillus niger)、简青霉(Penicillium simplicissim)、栗褐链霉菌(Streptomyces badius),依据PLFA-PLS定量分析所得堆肥化2次发酵期有效的木质素降解微生物群落组成比例混合接种至稻草基质发酵瓶中,做1组L9(34)正交试验以优化混合比例,期望开发1种基于木质素降解的高效堆肥化接种剂.试验结果表明:混合菌剂具有较强的木质素降解能力,其对木质素的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;当按照个数比细菌∶放线菌∶真菌为85∶5∶ 10,枯草芽孢杆菌∶铜绿假单孢菌为55∶25,黑曲霉∶简青霉为2∶1配比时,木质素、纤维素、半纤维素降解率最高,分别达到22.13%,48.97%和55.93%;在不灭菌前提下,按此配比接入菌剂,其木质素、纤维素、半纤维素降解率分别比不接菌剂发酵稻草提高19.16,38.25和46.30百分点.     

细胞表面展示木聚糖酶对纤维素酶水解底物的协同促进作用

为了解木质纤维素水解过程中木聚糖酶作为辅助酶对纤维素酶的协同促进作用,采用实验室保存的单展示3种纤维素酶(内切葡聚糖酶EGII、外切葡聚糖酶CBHII和β-葡糖苷酶BGLI)和2种木聚糖酶(β-D-1,4内切木聚糖酶Xyn II和β-D-1,4外切木聚糖酶Xyl A)的酿酒酵母功能菌群,以蒸汽爆破玉米秸秆为底物进行乙醇发酵实验(SSF).结果显示,以蒸汽爆破玉米秸秆为底物时,加入纤维素酶和木聚糖酶共发酵96 h的最高乙醇浓度达到0.695 g/L,乙醇产率为0.254 g/g,相当于理论值的49.8%,纤维素酶与木聚糖酶之间的协同因子(DS)最高达到1.16(始终大于1).本研究表明在细胞表面展示体系中适量添加木聚糖酶对纤维素酶水解底物具有较明显的促进作用,为直接以木质纤维素为原料制取纤维素乙醇提供了一定的可行性依据,可通过调节单展示酵母细胞在菌群间的动态比例实现对酶协同作用的优化调控.     

低温纤维素降解菌的分离与鉴定

对内蒙古部分地区土壤中低温降解纤维素的微生物进行研究,以期获得一些高酶活的低温纤维素酶产生菌.采用纯培养的方法,在10℃下培养获得纯培养物.以细菌16S rDNA通用引物PCR扩增后进行序列同源性比对确定种属.以DNS法测定纤维素酶活性,并对酶活较高的菌株进行产酶条件的优化.结果共分离得到55株可低温降解纤维素的菌株,16S rDNA序列分析表明它们分别属于γ-变形菌纲(γ-Proteobacteria)、硬壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)以及β-变形菌纲(β-Proteobacteria).该55株菌的纤维素酶活性均在22℃下最高.其中菌株CF11在10℃下的酶活在分离得到的55株细菌中最高.通过优化,菌株CF11产纤维素酶的最佳条件初步确定为pH值为6.5,培养时间为10 d,并且是以酵母提取物作为氮源,其纤维素酶活为58.091 IU.因此菌株CF11是一株极具开发潜力的低温纤维素酶产生菌.     

高效纤维素降解菌分离筛选、复合菌系构建及秸秆降解效果分析

从川西高原贡嘎山区杜鹃林下土壤中分离纤维素降解菌,构建具有高效降解纤维素能力的复合菌系,并对秸秆降解效果进行分析,为农业废弃物的循环利用提供菌种资源和理论依据.样品及经风干、高温等预处理后,采用平板涂布法进行分离,共获得79株菌株;通过刚果红实验对分离获得的菌株进行初步筛选,运用DNS法测定各菌株的羧甲基纤维素酶活(Carboxymethyl cellulase,CMCase),复筛得到15株具有CMCase活能力的菌株.经滤纸条崩解实验、秸秆崩解实验及降解率测定,最终确定了各菌株的纤维素降解能力,进一步经拮抗实验,选取相互无拮抗的菌株构建5个复合菌系:A(112、146、156、171),B(145、147、150、153),C(110、116、174),D(147、154、171),E(145、146、150、152、153).复合菌系的滤纸酶活(Fpase)与秸秆降解率测定结果显示,组合C对秸秆的降解率较单菌株116提高了50.71%,组合D对秸秆的降解率较单菌株154提高了41.54%.经形态学和分子生物学鉴定,纤维素降解能力比较好的两个组合中的菌株分别被鉴定为类芽孢杆菌属(Paenibacillus sp.)、芽孢杆菌属(Bacillus sp.)、不动杆菌属(Acinetobacter sp.)以及链霉菌属(Streptomyces sp.).本研究表明,复合菌系纤维素降解能力优于单一菌株,C、D两组复合菌系表现出较高的纤维素降解能力,具有进一步开发的价值.     

一株产耐热纤维素酶菌株的筛选及酶学性质

采用纤维素刚果红平板法从大田蘑菇种植场建堆的稻草样中分离得到了1株能产具有较好温度耐受性和pH稳定性的纤维素酶的放线菌DY3.综合形态、生理生化特征以及16S rDNA序列分析,将其初步鉴定为嗜热裂孢菌(Thermobifida fusca).对该菌所产纤维素酶的性质研究表明:最适催化温度为65℃,在70℃保温60 min后仍有75%以上的活力;最适催化pH为7.5,在pH 5.5～10.0之间该酶的稳定性较好,pH 10.0的条件下仍有80%的活力;最适作用底物是羧甲基纤维素钠.研究可为木质纤维素的生物预处理提供一定参考价值.     

蚁巢伞对木质纤维素的降解作用

白蚁-细菌-真菌共生系统对自然界生物质降解具有重要作用,研究蚁巢伞(Termitomyces)对木质纤维素的降解能力有助于揭示其共生关系,为蚁巢伞应用于生物质能源开发提供理论依据.利用黑翅土白蚁(Odontotermes formosanus)菌圃(OFC)及其固体培养蚁巢伞(TA)、液体培养蚁巢伞(TL)分别对未经预处理和经密褐褶孔菌(Gloeophyllum trabeum)预处理材料进行降解试验,探究蚁巢伞的木质纤维素降解能力.结果显示,未经预处理时,处理90 d后,OFC对纤维素、半纤维素和木质素的降解率分别为15.22%、29.34%和6.01%;处理120 d后,TA对纤维素、半纤维素和木质素的降解率分别为20.98%、31.89%和11.68%,TL对纤维素、半纤维素和木质素的降解率分别为14.39%、24.62%和5.05%.经过预处理时,处理120d后,OFC对纤维素、半纤维素和木质素的降解率分别为37.09%、42.20%和24.95%,TA对纤维素、半纤维素和木质素的降解率分别为34.77%、38.29%和29.74%,TL对纤维素、半纤维素和木质素的降解率分别为30.57%、30.47%和24.36%. 3种状态蚁巢伞对经预处理材料木质纤维素降解率均高于未经预处理材料.本研究表明,蚁巢伞对木质纤维素具有一定的降解能力,尤其是蚁巢伞对木质素的降解说明其可以打破木质素屏障,证实了利用蚁巢伞的木质纤维素降解能力来实现生物质能源化的潜力,结果可为蚁巢伞的人工栽培提供数据和参考.(图4表2参39)     

不同配方培养料生产双孢蘑菇过程中主要木质纤维素降解酶及物料组分的变化

为探讨双孢蘑菇生产过程木质纤维素的利用情况,以麦草秸秆、玉米秸秆和杂草秸秆为主料的3种不同配方的培养料为研究材料,分别测定各配方培养料不同时期主要胞外木质纤维素降解酶活性和木质纤维素组分(纤维素、半纤维素和木质素)的相对含量,并统计各配方产量.结果显示,麦草配方(FWS)的纤维素酶总活性和木聚糖酶活性不断升高,出菇期达到稳定;玉米秸秆配方和杂草配方堆肥期的纤维素酶总活性保持在1 U/g左右,发菌期大幅升高,出菇期先稳定后下降,木聚糖酶活性始终保持在7.97-23.85 U/g之间;3个配方的漆酶活性在堆肥期未检测到,发菌期升至最高,出菇期快速降低.3个配方堆肥期纤维素和半纤维素的相对含量明显下降,木质素相对含量则几乎不变.发菌及出菇期木质素与半纤维素的相对含量较纤维素的下降明显.3个配方双孢蘑菇的产量关系为麦草配方(30.00 kg/m~2)玉米秸秆配方(17.21 kg/m~2)杂草配方(16.67 kg/m~2).本研究表明堆肥期主要为堆肥微生物及物理化学作用降解纤维素和半纤维素,发菌期则主要是双孢蘑菇菌丝利用木质素;从播种至三潮菇清床,双孢蘑菇菌丝主要利用木质素和半纤维素;本研究中麦草配方是栽培双孢蘑菇的最佳培养料,而玉米秸秆配方和杂草配方有待进一步优化.     

黑胸散白蚁纤维素酶的体外酶学特性

采用DNS法研究了我国广泛分布的一种低等木食性白蚁——黑胸散白蚁纤维素酶的体外酶活特性以了解其纤维素降解机制.结果表明,内切β-1,4-葡聚糖酶(EG)、纤维二糖水解酶(CBH)和β-葡萄糖苷酶(BG)这3种酶的最佳反应时间均为15 min,最佳底物浓度为1%,最适反应pH为5.6,最适反应温度为35℃.在最适反应条件下,EG、CBH和BG的活性分别达到71.3(±13.9)U/mg、5.8(±0.8)U/mg和4.1(±0.7)U/mg.EG在体外的热稳定性较差,在50℃及更高温度酶活很低或完全失活,但该酶对pH稳定性较好,在pH 3.2～8.0范围内酶活力变化不大.Native-PAGE电泳检测到该白蚁体内至少有8种不同的EG活性条带,肠道不同部位纤维素酶活性条带种类不同.这些研究表明,木食性白蚁降解纤维素是一个复杂的过程,需要多种纤维素酶的共同作用.     

厌氧纤维素降解产氢复合菌系L-3研究

从厌氧发酵污泥中筛选到一组高效、传代稳定的厌氧纤维素降解复合菌系L-3.该复合菌系的内切葡聚糖酶活(Cx)、滤纸酶活(FPA)、外切葡聚糖酶活(C1)、β-葡聚糖苷酶活(β-glucodase)分别为0.216、0.101、0.132、0.002U/mL;该复合菌系可使滤纸在42h内溃烂,并能在降解纤维素的同时产生氢气,气体中氢气含量最高可达70.2%,d13时滤纸失重率为70.6%.DGGE结果表明,该复合菌系主要由14种菌组成.在所选实验条件内,该复合菌系产纤维素降解酶的最适条件为:最佳碳源为滤纸,最佳氮源为硫酸铵,温度36℃,pH6.5～7.0,接种量5%.     

添加剂对绿色木霉JQF-04纤维素酶热稳定性的影响

如何提高酶蛋白的热稳定性是分子生物学、微生物学、生化工程学等学科长期所关注的重要研究课题之一.本文研究了多种添加剂对绿色木霉纤维素酶热稳定性的影响.在60℃的溶液中,多元醇(乙二醇、甘油、赤藓糖醇、木糖醇和山梨糖醇)能提高该酶的热稳定性,随着浓度的增加,赤藓糖醇、木糖醇和山梨糖醇促进酶的热稳定性呈线性增高;适当的多元醇分子长度对该酶的热稳定性有最优的保护效应;不同浓度和不同分子量的聚乙二醇对该酶的热稳定性具有明显的影响;在无机盐中,单价金属阳离子比二价金属阳离子更能显著地提高该酶的热稳定性;酶液溶剂的改变直接影响着该酶的热稳定性,该酶在D2O溶液中比在水溶液中稳定,其酶活半衰期延长了2.6倍.研究表明,热环境使酶蛋白分子的螺旋结构发生变化而失活,但某些溶质和溶剂的存在可能通过作用于蛋白质分子的三维结构而影响该酶的热稳定性.图7参15     

Effect of phosphogypsum amendment on soil physico-chemical properties, microbial load and enzyme activities

Phosphogypsum (PG) is produced as a solid waste from phosphatic fertilizer plants. The waste slurry is disposed off in settling ponds or in heaps. This solid waste is now increasingly being used as a calcium supplement in agriculture. This study reports the effectof PG amendmenton soil physico chemical properties, bacterial and fungal count and activities of soil enzymes such as invertase, cellulase and amylase over an incubation period of 28 days. The highest mean percent carbon loss (55.98%) was recorded in 15% PG amended soil followed by (55.28%) in 10% PG amended soil and the minimum (1.68%) in control soil. The highest number of bacterial colonies (47.4 CFU g(-1) soil), fungal count (17.8 CFU g(-1) soil), highest amylase activity (38.4 microg g(-1) soil hr(-1)) and cellulase activity (38.37 microg g(-1) soil hr(-1)) were recorded in 10% amended soil. Statistically significant difference (p<0.05) has been recorded in the activities of amylase and cellulase over the period of incubation irrespective of amendments. Considering the bacterial and fungal growth and the activities of the three soil enzymes in the control and amended sets, it appears that 10% PG amendment is optimal for microbial growth and soil enzyme activities.     

湿地根际土壤碳矿化及相关酶活性分异特征

研究了中国三江平原小叶章湿地根际土壤基础呼吸速率及相关酶活性，以了解碳矿化及其相关酶活性空间分异特征。结果表明，β-葡萄糖苷酶、淀粉酶、纤维素酶均为碳循环的良好指示酶，它们均存在着显著的空间分异。从表层土向下，由根表土向外，碳矿化速率及其相关的各种酶活性均呈下降趋势。当从田间取出土壤样品时，土壤样品在取出后的最初阶段碳矿化速率较高，2．5h以后达到一个较为平稳的水平，然后呈平缓降低的趋势。β-葡萄糖苷酶、淀粉酶、纤维素酶是碳循环的真正催化剂，而土壤有机碳则是此反应的低物。     

Effects of pesticides on soil enzymes: a review

The use of pesticides in agriculture has highly increased during the last 40 years to increase crop yields. However, today most pesticides are polluting water, soil, atmosphere and food. Pesticides are also impact soil enzymes, which are essential catalysts ruling the quality of soil life. In particular, the activity of soil enzymes control nutrient cycles, and, in turn, fertilization. Here, we review the effects of pesticides on the activity of soil enzymes in terrestrial ecosystems. Enzymes include dehydrogenase, fluorescein diacetate hydrolase, acid phosphatase, alkaline phosphatase, phosphatase, β-glucosidase, cellulase, urease and aryl-sulfatase. Those enzymes are involved in the cycles of carbon, nitrogen, sulfur and phosphorus. The main points of our analysis are (1) the common inhibition of dehydrogenase in 61 % of studies, stimulation of cellulase in 56 % of studies and no response of aryl-sulfatase in 67 % of studies. (2) Fungicides have mainly negative effects on enzymatic activities. (3) Insecticides can be classified into two groups, the first group represented by endosulfan having an overall positive impact while the second group having a negative effect. (4) Herbicides can be classified into two groups, one group with few positive effect and another group with negative effect.     

崇明岛典型土地利用方式对土壤有机碳和酶活性的影响

为了研究上海崇明岛不同土地利用方式对土壤有机碳以及酶活性的影响,对冬小麦地、蔬菜地、柑桔地和冬绿肥地4种土地利用方式的土壤进行了调查、样品测定和分析。结果表明,土地利用方式对有机碳和4种酶活性的影响极显著。冬小麦地总有机碳和活性有机碳质量分数均最高,分别为19.23g·kg^-1和2.26mg·g^-1,柑桔地土壤脲酶、碱性磷酸酶、纤维素酶和蛋白酶等4种酶活性均高于其他3种土地利用方式,表明采用秸秆还田式的冬小麦地,可以增加土壤总有机碳和活性有机碳质量分数;常年处于免耕状态的柑桔地可以使土壤酶活性增强。     

Agricultural waste from the tequila industry as substrate for the production of commercially important enzymes

Approximately 1 million tons of Agave tequilana plants are processed annually by the Mexican Tequila industry generating vast amounts of agricultural waste. The aim of this study was to investigate the potential use of Agave tequilana waste as substrate for the production of commercially important enzymes. Two strains of Aspergillus niger (CH-A-2010 and CH-A-2016), isolated from agave fields, were found to grow and propagate in submerged cultures using Agave tequilana waste as substrate. Isolates showed simultaneous extracellular inulinase, xylanase, pectinase, and cellulase activities. Aspergillus CH-A-2010 showed the highest production of inulinase activity (1.48 U/ml), whereas Aspergillus niger CH-A-2016 produced the highest xylanase (1.52 U/ml) and endo-pectinase (2.7U/ml) activities. In both cases production of enzyme activities was significantly higher on Agave tequilana waste than that observed on lemon peel and specific polymeric carbohydrates. Enzymatic hydrolysis of raw A. tequilana stems and leaves, by enzymes secreted by the isolates yielded maximum concentrations of reducing sugars of 28.2 g/l, and 9.9 g/l respectively. In conclusion, Agave tequilana waste can be utilized as substrate for the production of important biotechnological enzymes.     

木薯皮堆肥过程中酶活性的变化

以木薯皮为原料进行高温堆肥发酵,研究了堆肥化过程中理化性质和酶活性的变化情况.结果表明:在堆肥发酵过程中.温度变化呈先上升后下降趋势,到后期已趋近于初始温度;而含水率在堆肥处理过程中变化不大.各处理的pH值在发酵结束时为7.3～7.5.过氧化氢酶活性初期较高,随后迅速降低,并维持在较低水平.纤维素酶和脲酶活性在堆肥初期增加,之后逐渐降低.由于堆肥原料不同,不同处理的酶活性变化趋势表现出一定的差异.考虑到堆肥腐熟度受多方面因素的影响,判断堆肥腐熟度时应根据多种指标(包括生物学、化学、物理学指标)综合判断.     

Time-course of digestive-enzyme acclimation in the cockle Cerastoderma edule

We determined the temporal evolution of amylase, cellulase, laminarinase and protease in the digestive gland and crystalline style of cockles Cerastoderma edule held over 9 to 12 d in the presence and absence of food. Cockles were fed a constant diet of 1.5 mm³ l⁻¹ of Tetraselmis suecica for 9 to 12 d and were then starved for 6 to 8 d in late summer (September 1992) and in winter (January 1993). Feeding increased the dry weight and total cellulase, laminarinase and protease activities of the digestive gland irrespective of season, whereas amylase activity remained unchanged. In winter (i.e. when cockles are metabolically weak) the response was faster and stronger, especially for protease. An additional experiment in September starved cockles for 20 d before resuming feeding. In agreement with the seasonal differences, the presence of food after prolonged starvation induced a rapid and marked increase in protease in the digestive gland of the cockles. In winter, the possible effects of the biochemical composition of food on their enzymatic response were tested by feeding two groups of cockles with the same ration of T. suecica but harvested at different growth phases. A compensatory induction of cellulases occurred in cockles fed on T. suecica with a lower carbohydrate content. In the crystalline style, the protein level and carbohydrase fell during the first day of feeding and increased during the first day of subsequent starvation. These results indicate that the release of enzymes from the style prevails over the incorporation of enzymes during the early stages of feeding, whereas the opposite occurs during starvation. Received: 15 February 1998 / Accepted: 22 February 1999     

Research progress in microbial cytochrome P450 and xenobiotic metabolism北大核心CSCD

Cytochrome P450 (CYP450s) is a type of heme-mercaptide protein superfamily, which is distributed widely in animals, plants, and microorganisms. CYP450s can oxidize and degrade many exogenous compounds such as drugs, herbicides, pesticides, some persistent organic pollutants, and so on. Based on recent researches, this paper reviews the nomenclature, classification, structure, and catalytic mechanism of P450 enzymes, and summarizes the research progresses in the metabolism and biodegradation of xenobiotics using P450 enzymes from microorganisms. The nomenclature and classification of the P450 gene superfamily mainly rely on the similarities of amino acid sequences. Although the structures of P450 are conserved, their recognition sites towards to the substrates are variable. This is also the structural basis for the catalytic diversities of P450 enzymes. Few P450 enzymes from bacteria and fungi can metabolize and degrade xenobiotics such as polycyclic aromatic hydrocarbons, herbicides, and so on. However, these P450 enzymes are less likely be used in practical applications because of their low catalytic activities. In the future, more P450 enzymes with high degradable efficiencies towards xenobiotics are needed to be obtained using multiple omics tools or modifying the existing P450 enzymes, to achieve the bioremediation of the environment. © 2018 Science Press. All rights reserved.