同步分泌高效纤维素酶和木聚糖酶菌株YB的鉴定及其酶学性质研究

筛选和鉴定可降解木质纤维素的真菌,并研究其产酶特征。采用刚果红平板涂布法,从荔枝腐叶中筛选具有木质纤维素降解能力的真菌,结合ITS-rDNA序列分析进行鉴定,初步测定其产酶条件,然后采用DEAE Sepharose Fast Flow阴离子交换层析与Sephadex G-100凝胶层析对硫酸铵沉淀的粗酶液进行分离纯化,对其开展酶学性质研究。结果显示,筛选出一株可降解木质纤维素降解的菌株YB,鉴定为绿木霉(Trichoderma virens)。在发酵过程中,纤维素酶和木聚糖酶的最大活力分别为313.53±26.78 U/mL和18 120.87±500.37 U/mL。分离纯化得到纤维素酶(CMC酶)Ⅰb、Ⅳ和木聚糖酶Ⅰa;通过SDS-PAGE检测,其分子量分别为58.5 kD、22.8 kD和44.5 kD。3种酶的最适酶促反应条件均为:50℃,pH 5.0。其中,木聚糖酶能有效降解玉米芯木聚糖为木糖和多种木寡糖。菌株Trichoderma virens YB可分泌高效木质纤维素降解酶,具有应用于木聚糖酶和木寡糖生产的潜力。     

毛木耳降解木质纤维素的研究

测定了毛木耳“沪毛一号”菌株瓶栽0—60天期间,其木屑基质中木素纤维素的降解和木质纤维分解酶活性的变化。结果表明,该菌能够同时降解木素、半纤维素和纤维素,尤以分解木素的能力为强(以各自相对百分含量减少计算),故毛木耳为白腐真菌。该菌的多酚氧化酶、羧甲基纤维素酶、滤纸纤维素酶和木聚糖酶的活性高峰均出现于菌丝体发育阶段,在子实体原基发生时(22天),羧甲基纤维素酶、滤纸纤维素酶、木聚糖酶的活性明显下降,在子实体发生后,该三种酶活性又复上升,直到头批耳成熟酶活仍处于较高水平。     

产纤维素酶黏细菌在生物转化的作用

目的:预处理对木质纤维素降解的影响.方法:从土壤中分离筛选到高纤维素酶活的黏细菌菌株So ce sh1008.该菌具有CMC酶活(CMCase)及微晶纤维素酶活性.研究NaOH联合黏细菌降解盐蒿、稻草、棉花秸秆和甘蔗渣四种木质纤维素的情况.结果:碱(2％ NaOH) -黏细菌处理的方法优于黏细菌-碱的方法,其中降解棉花秸秆降解效果最明显,以5.0g木质纤维素为原料,其最终干重损失达2.1g,溶液中总糖含量和还原糖含量均值分别为12.8 mg/mL和0.93 mg/mL.酵母菌发酵产乙醇的研究结果表明,最佳发酵时间为47h,碱-黏细菌甘蔗渣降解液发酵效果最好,乙醇产出达6.0％.结论:黏细菌联合2％ NaOH能有效降解甘蔗渣,提高乙醇产量.     

嗜热厌氧细菌Caldicellulosiruptor bescii降解木质纤维素研究进展

嗜热厌氧菌Caldicellulosiruptor bescii具有强大的木质纤维素降解能力,能以多种模式植物细胞壁多糖如微晶纤维素Avicel和木聚糖,甚至未经预处理的木质纤维素如柳枝稷作为唯一碳源快速生长,该菌还具有少见的厌氧降解木质素的能力。对基因组注释发现,该菌所编码的蛋白大多为多结构域双功能酶,即在多肽链的N端和C端分别是不同家族的糖苷水解酶,间隔以2-3个碳水化合物结合结构域。该菌降解纤维素相关的酶基因多集中于一个植物细胞壁多糖降解利用的基因簇,例如纤维素酶/木聚糖酶、纤维素酶/甘露聚糖酶和纤维素酶/木葡聚糖酶等。C.bescii的木聚糖酶主要属于GH10家族,该家族的酶底物特异性较为宽泛,氨基酸序列的同源性在18.7%-59.5%间。Caldicellulosiruptor属细菌进化出了一系列的机制使得糖苷水解酶和底物、细菌和木质纤维素能更好的吸附在一起,从而有利于木质纤维素的酶解。C.bescii有12个含SLH结构域的蛋白,以及新发现的黏附蛋白Tāpirin,可能参与了木质纤维素的吸附与利用。综述了近年来对C.bescii降解植物细胞壁的糖苷水解酶的基因资源挖掘方面和降解分子机制方面的研究进展,对高效、多功能高效木质纤维素降解酶的设计和优化具有积极的意义。     

利用毛壳霉固体发酵木聚糖酶

毛壳属真菌大多数具有较强的纤维素降解能力地,其中球毛菌所产生的木聚糖酶活性较强.木聚糖酶通过水解木糖分子间的β-1,4-糖苷键,将木聚糖水解成低聚木糖及少量木糖和阿拉伯糖.     

β-木糖苷酶及其在纤维素乙醇生产中的应用研究进展

将木质纤维素类生物质生物转化生产液体燃料,如纤维素乙醇和大宗化学品,对缓解当前人类社会面临的能源和资源危机以及保护环境具有重要意义。半纤维素是木质纤维素类生物质的主要组成成分之一,它的生物降解转化对实现木质纤维素生物炼制意义重大。由于半纤维素糖种类的多样性和半纤维素结构的复杂性,需要一个复杂的半纤维素酶系才能完成对半纤维素的有效降解。除了木聚糖酶等以外,β-木糖苷酶也是半纤维素酶系的主要组分。在半纤维素降解过程中,β-木糖苷酶将木聚糖酶的水解产物木寡糖和木二糖水解为木糖,不仅在木聚糖的彻底降解过程中起着重要作用,而且可以缓解木寡糖对木聚糖酶和纤维素酶的抑制作用。该文综述了目前在β-木糖苷酶方面的研究进展,包括β-木糖苷酶的分类、酶学性质、酶结构及其催化机制、基因的克隆与表达等,并对β-木糖苷酶在纤维素乙醇生产中的应用情况进行了简述。     

白蚁及其共生菌来源的4种木质纤维素酶的共表达

天然的木质纤维素材料含有纤维素、半纤维素和木质素等成分。降解天然木质纤维素底物时,需要木质纤维素酶共同作用。近年在木质纤维素酶的相互协同作用方面的研究引起人们的关注,成为一个新的研究热点,文中使用两个不同的共表达载体pETDuet-1和pRSFDuet-1,在大肠杆菌中共表达了白蚁及其肠道微生物来源的β-葡萄糖苷酶、内切β-1,4-葡聚糖酶、漆酶和木聚糖酶这4种木质纤维素酶,经过SDS-PAGE分析得到了与理论值一致的蛋白条带,同时经过酶活验证,这4种蛋白都具有酶活性。以磷酸处理的微晶纤维素(PASC)为底物,测定了共表达酶粗酶液与单独表达酶混合液的协同作用因子,从还原糖的产量上经计算共表达的粗酶液比单独表达酶的混合液对PASC的降解协同作用提高44%;以滤纸和磷酸处理的玉米芯为底物,测定降解协同作用,分别提高了34%和20%。结果表明,共表达酶的降解效率要高于混合的单组分酶液降解效率的总和。     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株Bacilluspumilus WL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为26.0kD ,pI值9.5 ,以燕麦木聚糖为底物时的表观Km 值为16.6mg mL ,Vmax值为12.63μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7.2至8.0之间,是耐碱性木聚糖酶;最适作用温度为45℃～55℃,在37℃、45℃以下时该酶热稳定性均较好;50℃保温时,该酶活力的半衰期大约为2h ,在超过50℃的环境下,该酶的热稳定较差,55℃和60℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL-11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL-11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL-11主要合成内切型木聚糖酶A,同时发酵液中不含木糖苷酶,适合用来酶法制备低聚木糖。     

纤维素降解的菌株筛选及其运用

目的:筛选降解稻草纤维素菌株,为纤维素的高效降解提供理论依据.方法:采用羧甲基纤维素钠刚果红培养基与滤纸条培养基从采集的腐木、腐土和腐叶等样品中筛选出纤维素降解菌.然后经液态发酵后测定其羧甲基纤维素酶活力与降解稻草的天然纤维素酶活力,综合考虑这两种酶活力,对其进行单独与混合发酵培养.筛选分解稻草能力较强的菌株组合.结果:初筛到5株真菌和5株细菌纤维素降解力较优的菌株.经酶活力测定后,得到分解纤维素能力较强的两株真菌F3和F5与两株细菌B1和B5,其中F3和B1的羧甲基纤维素酶活分别为705.6U、214.6U;F5和B5天然纤维素酶活分别为466.5U、204.8 U.混合培养在一定程度上能提高纤维素酶活,F3/F5具有稳定而较高的酶活力,某时间段酶活高达646.8U,且后续酶活力也保持在较高水平.而F3/B5在某时间段的酶活高达788.6U.结论:菌株的混合培养可以提高纤维素酶活.     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

秸秆纤维素分解菌的酶活力测定

目的:测定秸秆纤维素分解菌的酶活力。方法:从土壤中分离出具有分解纤维素能力的菌株,采用刚果红染色法进行粗选,得到7株透明圈较大的菌株。将这7株菌株液体发酵培养6d,再分别用滤纸分解度观察、羧甲基纤维素酶活法(CMC)、滤纸酶活法(FPA)和天然纤维素酶活法测定其酶活力。结果:在7株菌株中,F-1、F-2、F-3、F-5的酶活力测定结果与其溶解圈的测定结果、滤纸分解结果基本相同。且天然纤维素酶活力高的菌株,其CMC酶活、FPA酶活也高,滤纸分解效果也比较明显。结论:CMC法、FPA法和天然纤维素酶活法适于测定秸秆纤维素分解菌的酶活力。     

Effects of fire on soil respiration,ATP content and enzyme activities in Mediterranean maquis

Question: Do low or high intensity fires affect micro‐organism activity in the upper soil layer of Mediterranean maquis? Location: 600 m from the sea in the Nature Reserve of Castel Volturno (Campania, southern Italy, 40°57’N; 13°55’E). Methods: Soil respiration was measured in situ on intact soil; enzyme activity (cellulase, xylanase, invertase, trehalase and protease) and ATP content were measured on soil samples collected under three species of maquis vegetation: Phillyrea angustifolia L., Myrtus communis L. and Cistus incanus L. Results: Soil microbial respiration showed no significant differences in CO₂ flux in treated and untreated plots, but the ATP content in the soil under C. incanus and M. communis was lower in the treated plots for most of the study period. In the soil under Ph. angustifolia, ATP content was low only for one week after fire. The reduction was more marked in the samples from ‘high fire intensity’ than from ‘low fire intensity’ plots. Soil respiration and ATP content exhibited seasonal variations linked to soil water content. Among the enzyme activity measured in the soil under the three plant covers, only invertase declined in burned plots throughout the study period, particularly in the ‘high fire intensity’ plots. Activity of the enzymes cellulase, xylanase, trehalase and protease had a different sensitivity depending on the respective shrub cover. Conclusions: Impact of fire on soil microbial activity is largely dependent on vegetation mosaic and species identity.     

Isolation of lipid- and polysaccharide-degrading micro-organisms from subtropical forest soil, and analysis of lipolytic strain Bacillus sp. CR-179

AIMS: To isolate the micro-organisms from three soil samples obtained from a subtropical forest of Puerto Iguazu (Argentina), to analyse them for detection of the biotechnologically interesting enzymatic activities lipase, esterase, cellulase, xylanase and pectinase, and to identify the most active strain. METHODS AND RESULTS: A total of 724 strains were isolated using different culture media and temperatures, and 449 of them showed at least one of the hydrolytic activities pursued. Lipolytic activity of the lipid-degrading strains was further determined using MUF-butyrate and MUF-oleate as substrates. The alkalophilic strain CR-179, one of the most active for all the enzymatic activities assayed, was characterized and preliminarily identified by morphological, physiological and 16S rDNA tests, as a Bacillus sp. closely related to Bacillus subtilis. CONCLUSIONS: Highly hydrolytic strains were isolated from all soil samples, suggesting the existence of a microbial community well-adapted to nutrient recycling. Strain CR-179, one of the most active, has been preliminarily identified as a Bacillus sp. SIGNIFICANCE AND IMPACT OF THE STUDY: A collection of hydrolytic strains with high biotechnological potential was obtained. Presence of sequences codifying for a lipolytic system related to the B. subtilis group lipases was revealed by PCR for the best lipolytic strain.     

Evaluation of a novel bifunctional xylanase–cellulase constructed by gene fusion

An artificial bifunctional enzyme, xylanase–cellulase, has been prepared by gene fusion. Three chimeric genes were constructed that encoded fusion proteins of different lengths. The fusion proteins exhibited both xylanase (XynX) and cellulase (Cel5Z::Ω) activity when cel5Z::Ω was fused downstream of xynX, but not when xynX was fused downstream of cel5Z::Ω. Activities of bifunctional enzymes decreased when a shorter xylanase peptide was fused. Three fusion enzymes were purified, and the molecular weights of the enzymes were estimated by CMC-SDS-PAGE and XYN-SDS-PAGE to be 149, 129, and 87 kDa, respectively. The fusion enzymes displayed optimum cellulase activity at pH 8.0 and 50 °C and optimum xylanase activity at pH 8.0 and 70 °C.     

Xylanase active at high pH from an alkalotolerant Cephalosporium species

Summary An alkalotolerant Cephalosporium (NCL 87.11.9) strain capable of rapid growth and xylanase secretion over a wide pH range (pH 4–10) has been isolated from soil samples. When grown in shake flasks on a wheat bran, yeast extract medium for 96 h it produced 15 to 18 IU/ml. The novel feature of this study is that it is the first report of an extracellular fungal xylanase which is active and stable at high alkaline pH (8–9.5). The culture filtrate did not show any significant cellulase activity. Gel filtration studies indicated two peaks of xylanase activities corresponding to molecular weights of 70,000 and 30,000 in the proportion of 10:90.     

Cellulolytic microbes in the Yanbaru, a subtropical rainforest with an endemic biota on Okinawa Island, Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.     

Cellulolytic Microbes in the Yanbaru,a Subtropical Rainforest with an Endemic Biota on Okinawa Island,Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.     

重金属污染区土壤酶活性变化

从福建龙岩新罗区特钢厂污灌区农田采集土壤,测定土壤基本理化性质及脲酶、纤维素酶、碱性磷酸酶、多酚氧化酶、过氧化氢酶活性和Cu、Cd、Pb、Zn含量,探讨重金属污染和土壤性质对土壤酶活性的影响.结果表明： 4种全量或有效态重金属与土壤脲酶、纤维素酶、碱性磷酸酶和多酚氧化酶活性呈显著正相关,与过氧化氢酶活性呈显著或极显著负相关;土壤pH与碱性磷酸酶活性呈极显著正相关,粉粒含量与过氧化氢酶活性呈显著负相关.经通径分析,重金属污染刺激了脲酶、多酚氧化酶和纤维素酶活性,但对碱性磷酸酶活性的影响较小.有效态Cu、Cd、Pb、Zn对过氧化氢酶活性的直接影响并不大,但通过间接途径抑制了过氧化氢酶活性.土壤理化性质对5种土壤酶活性的影响较大,碱解氮直接抑制了脲酶活性;全磷直接刺激了碱性磷酸酶和过氧化氢酶活性,并通过有效磷刺激了纤维素酶活性;有效磷直接刺激了纤维素酶活性,直接抑制了碱性磷酸酶和过氧化氢酶活性;全钾直接抑制了碱性磷酸酶和多酚氧化酶活性;速效钾通过有效磷刺激了纤维素酶活性;土壤颗粒组成明显影响多酚氧化酶和过氧化氢酶活性.5种酶活性与土壤Cu、Cd、Pb、Zn含量之间的关系不明确,因此其活性不是指示土壤Cu、Cd、Pb、Zn污染的良好指标.     

不同水位时期东洞庭湖湿地土壤微生物生物量碳氮和酶活性变化

为从土壤微生物的角度分析东洞庭湖不同植被类型湿地土壤质量状况,本研究选取了苔草、芦苇和柳树3种典型植被类型为对象,在平水期、丰水期和枯水期对其土壤微生物生物量碳(MBC)、氮(MBN)和酶活性进行监测,并分析其主要影响因子。结果表明: 1)3个水位时期,各植被类型湿地土壤MBC、MBN、蔗糖酶和纤维素酶活性(枯水期纤维素酶除外)均表现为0～10 cm高于10～20 cm,而土壤过氧化氢酶活性则相反。2)各植被类型湿地0～20 cm土层土壤MBC、MBN和MBC/TOC(总有机碳)、MBN/TN(总氮)皆以丰水期最低。3)各植被类型湿地0～20 cm土层土壤蔗糖酶活性峰值均出现在枯水期,而纤维素酶活性峰值出现在平水期,过氧化氢酶活性季节性波动较小,以丰水期稍高。4)不同植被类型间比较:平水期和丰水期,芦苇湿地土壤蔗糖酶活性显著高于其他植被类型,而其土壤纤维素酶活性最低,枯水期不同湿地间两种酶活性差异不显著。土壤过氧化氢酶活性在平水期以苔草湿地最高,枯水期以柳树湿地最高,丰水期以芦苇湿地最低。5)相关性分析表明,土壤MBC、MBN和蔗糖酶与TOC、TN、总磷(TP)呈显著正相关,而与pH值呈显著负相关。土壤纤维素酶和过氧化氢酶与TOC、TN、TP呈显著负相关,与pH值呈显著正相关。表明季节性水位波动影响土壤C、N、P和pH值,并对土壤微生物生物量碳、氮和酶活性产生显著影响,使其呈现明显的季节性变化特征。     

重金属污染区土壤酶活性变化——以福建龙岩新罗区特钢厂污水灌溉区为例

从福建龙岩新罗区特钢厂污灌区农田采集土壤,测定土壤基本理化性质及脲酶、纤维素酶、碱性磷酸酶、多酚氧化酶、过氧化氢酶活性和Cu、Cd、Pb、Zn含量,探讨重金属污染和土壤性质对土壤酶活性的影响.结果表明:4种全量或有效态重金属与土壤脲酶、纤维素酶、碱性磷酸酶和多酚氧化酶活性呈显著正相关,与过氧化氢酶活性呈显著或极显著负相关;土壤pH与碱性磷酸酶活性呈极显著正相关,粉粒含量与过氧化氢酶活性呈显著负相关.经通径分析,重金属污染刺激了脲酶、多酚氧化酶和纤维素酶活性,但对碱性磷酸酶活性的影响较小.有效态Cu、Cd、Pb、zn对过氧化氢酶活性的直接影响并不大,但通过间接途径抑制了过氧化氢酶活性.土壤理化性质对5种土壤酶活性的影响较大,碱解氮直接抑制了脲酶活性;全磷直接刺激了碱性磷酸酶和过氧化氢酶活性,并通过有效磷刺激了纤维素酶活性;有效磷直接刺激了纤维素酶活性,直接抑制了碱性磷酸酶和过氧化氢酶活性;全钾直接抑制了碱性磷酸酶和多酚氧化酶活性;速效钾通过有效磷刺激了纤维素酶活性;土壤颗粒组成明显影响多酚氧化酶和过氧化氢酶活性.5种酶活性与土壤Cu、Cd、Pb、Zn含量之间的关系不明确,因此其活性不是指示土壤Cu、Cd、Pb、Zn污染的良好指标.