川西高山林线交错带凋落物纤维素分解酶活性研究

以川西高山林线交错带3种典型植被类型(针叶林、高山灌丛、高山草甸)下两个层次(LF层: 新鲜凋落物层和发酵层; H层: 腐殖质层)的凋落物为研究对象, 分别模拟凋落物分解的前期和后期阶段, 对凋落物分解过程中的纤维素酶活性及凋落物质量进行了研究。结果表明, 凋落物分解前期的纤维素酶活性和纤维素含量均显著高于分解后期, 但植被类型对LF和H层的纤维素含量的影响都不显著。双因素方差分析结果表明, 凋落物分解阶段对纤维素酶活性和纤维素含量的影响比植被类型对纤维素酶活性和纤维素含量的影响更大。不同种类的纤维素酶活性在分解前期和分解后期受到不同因子的限制。凋落物分解前期, 微晶纤维素酶和β-葡萄糖苷酶活性可能受N、P含量的限制, 而羧甲基纤维素酶主要受底物纤维素含量控制; 凋落物分解后期, 羧甲基纤维素酶和β-葡萄糖苷酶可能受C、N含量的限制。生态化学计量学的理论预测, 底物质量比C:N > 27或C:P > 186时会限制微生物生长, 因此判断高山林线交错带凋落物微生物生物量和纤维素酶活性同时受到底物N、P的限制, 尤其是高山草甸上微生物生物量在凋落物分解前期受到底物N、P的限制比分解后期更显著, 这充分说明了底物质量调控着凋落物分解过程中的纤维素酶活性和微生物生物量。     

Fibrolytic activities and cellulolytic bacterial community structure in the solid and liquid phases of rumen contents.

Four sheep were fed an alfalfa hay diet. Rumen content samples were collected three hours after feeding in order to total microorganism population (TP), solid attached population (SAP) and solid attached firmly population (SAFP). Fibrolytic specific activities (xylanase, CMCase and beta-glycosidases) were estimated by the amount of reducing sugars or p-nitrophenol released from the appropriate substrate. The distribution of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens) was quantified by dot-blot hybridisation using specific 16S-rRNA-targeting probes. Specific activities of polysaccharidase enzymes were higher in SAP than in TP, and in SAFP than in SAP. The sum of RNA of the three cellulolytic bacterial species represented on average 9% of the total bacterial RNA, and increased after filtration. In all samples, the relative population size of F. succinogenes was higher than that of R. albus and of R. flavefaciens. These results demonstrate that the most active enzymes are secreted by the particle-associated microorganisms. The differences in composition of the microflora between the solid and liquid phase suggest that bacteria are not equally distributed throughout the rumen content: the cellulolytic species are present in a higher proportion in the solid phase of rumen contents.     

发酵条件对纤维素分解菌酶活性的影响研究*

研究了4株可用作饲料添加剂的纤维素分解菌,在不同发酵时间、发酵温度和厌氧发酵条件下,菌体蛋白含量、纤维素酶和半纤维素酶活性等的变化。结果表明发酵20h酶活性最高;供试菌都适宜在28℃恒温发酵;在厌氧条件下,39℃士2℃发酵12h、24h、36h,供试菌均可在PDA平板上正常生长,发酵48h供试菌生长和酶活均受不同程度的影响。研究为菌株的生产、保存和应用提供了重要依据。     

Interactions among cellulolytic bacteria from an anaerobic digester

High cellulolytic activity of particular strains did not cause dominance of one, or a few, species of fiber-digesting bacteria in a cattlewaste anaerobic digester. The population contained a large number of species and varieties with different cellulolytic and fiber-digesting activities. Although mixed cultures of some of these bacteria showed no intereffects, with others, cellulolysis was less or in some cases greater than that shown by individual components of the cultures. The interactions were probably related to effects on growth of the bacteria rather than on activities of components of the cellulase enzyme complex, and culture filtrates of two of the more numerous cellulolytic species ofClostridium affected growth of other cellulolytic bacteria. The inhibitory factor(s) appeared to be of bacteriocin type, but the stimulatory factor(s) was unknown. It was suggested that these interactions are localized or short-lived in the digester, and so the population remains in a dynamic steady state.Some inhibitions of growth of rumen cellulolytic bacteria were caused by the digester bacteria, but it was suggested that factors other than these inhibitions are responsible for the absence of rumen bacteria from anaerobic digesters.     

Prospecting for cellulolytic activity in insect digestive fluids

Efficient cellulolytic enzymes are needed to degrade recalcitrant plant biomass during ethanol purification and make lignocellulosic biofuels a cost-effective alternative to fossil fuels. Despite the large number of insect species that feed on lignocellulosic material, limited availability of quantitative studies comparing cellulase activity among insect taxa constrains identification of candidate species for more targeted identification of effective cellulolytic systems. We describe quantitative determinations of the cellulolytic activity in gut or head-derived fluids from 68 phytophagous or xylophagous insect species belonging to eight different taxonomic orders. Enzymatic activity was determined for two different substrates, carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), approximating endo-β-1,4-glucanase and complete cellulolytic activity, respectively. Highest CMC gut fluid activities were found in Dictyoptera, Coleoptera, Isoptera, and Orthoptera, while highest MCC gut fluid activities were found in Coleoptera, Hymenoptera, Lepidoptera, and Orthoptera. In most cases, gut fluid activities were greater with CMC compared to MCC substrate, except in Diptera, Hymenoptera, and Lepidoptera. In contrast, cellulolytic activity levels in most head fluids were greater on the MCC substrate. Our data suggests that a phylogenetic relationship may exist for the origin of cellulolytic enzymes in insects, and that cellulase activity levels correlate with taxonomic classification, probably reflecting differences in plant host or feeding strategies.     

环境胁迫和抗坏血酸的氧化还原状态

为了解环境胁迫对植物体中抗坏血酸含量及氧化还原状态的影响,以不同强度的冰冻和干旱两种胁迫为例,研究了它们对沈阳几种针叶树离体叶抗坏血酸、脱氢抗坏血酸含量以及抗坏血酸-谷胱甘肽循环中4种酶活性的影响.结果表明,两种胁迫达到一定强度后,都能使还原态抗坏血酸含量下降而使脱氢抗坏血酸含量上升.冰冻使抗坏血酸过氧化酶和单脱氢抗坏血酸还原酶活性下降.轻度失水使这两种酶活性上升,失水加重后转而趋于下降.脱氢抗坏血酸还原酶和谷胱甘肽还原酶活性对两种胁迫反应均不如前两种酶敏感.结合以前的研究结果,认为这一H2O2清除系统在导致驯化(acclimation)的轻度胁迫作用下可以得到加强,而当胁迫强度过大时则其清除能力下降并使组织受到伤害.文中还报告了沈阳几种针叶树抗寒性和针叶中抗坏血酸含量及上述4种酶活性之间的相关关系.     

环境胁迫和抗坏血酸的氧化还原状态

为了解环境胁迫对植物体中抗坏血酸含量及氧化还原状态的影响，以不同强度的冰冻和干旱两种胁迫为例，研究了它们对沈阳几种针叶树离体叶抗坏血酸、脱氢抗坏血酸含量以及抗坏血酸-谷胱甘肽循环中4种酶活性的影响。结果表明，两种胁迫达到一定强度后，都能使还原态抗坏血酸含量下降而使脱氢抗坏血酸含量上升。冰冻使抗坏血酸过氧化酶和单脱氢抗坏血酸还原酶活性下降。轻度失水使这两种酶活性上升，失水加重后转而趋于下降。脱氢抗坏血酸还原酶和谷胱甘肽还原酶活性对两种胁迫反应均不如前两种酶敏感。结合以前的研究结果，认为这一H2O2清除系统在导致驯化(acclimation)的轻度胁迫作用下可以得到加强，而当胁迫强度过大时则其清除能力下降并使组织受到伤害。文中还报告了沈阳几种针叶树抗寒性和针叶中抗坏血酸含量及上述4种酶活性之间的相关关系。     

Changes in Sulfur Metabolism During Needle Development of Norway Spruce*

In a field-study with approx. 150-year-old spruce trees, seasonal changes in thiol composition and content, sulfate content, and oxtractable activities of enzymes of sulfate reduction and assimilation were analyzed in needles and buds and were related to developmental processes during flushing. GSH was the predominant thiol in spruce needles throughout the year, with maximum contents of more than 400nmol g FW^?1 during winter and minimum contents of less than 200 nmol g FW^?1 during summer. Negative correlation of changes in GSI1 contents during winter and spring with air temperature showed a low correlation coefficient (r =– 0.55), suggesting minor significance of GSH in frost protection. Drastic changes in thiol contents during flushing suggested that GSH accumulated in the previous year's needles during winter supports the growing needles with reduced sulfur. Sulfate and other substrates for sulfate assimilation appeared to be available in buds and the new needles. An external supply with reduced sulfur may still be required because of insufficient activities of enzymes of sulfate reduction and assimilation, esp. ATP-sulfurylase and APS-sulfotransferase.     

Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees

Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD-malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate.     

Comparison of the xylanolytic and cellulolytic activities of Cellulomonas

Summary A highly cellulolytic Cellulomonas mutant, CS1-17, has been shown to be improved over the original parent strain, CS1-1, with respect to xylanase and -xylosidase activities. From induction studies during growth on xylan, crystalline cellulose and carboxymethylcellulose it can be deduced that, although both activities have been similarly affected by the mutation, xylanolytic activity is distinct from cellulolytic activity; however, the possibility of some cross-specificity has not been eliminated.     

Extracellular cellulase production by tropical isolates of Aureobasidium pullulans

Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and alpha-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 micromol glucose.(mg protein)-1.h-1, while activities on alpha-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 micromol glucose.(mg protein)-1.day-1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.     

Cellulolytic complex exists in cellulolytic myxobacterium Sorangium

Cellulolytic myxobacterium Sorangium can efficiently degrade cellulose materials. The cellulolytic activities are linked to cellular surfaces and organized into a large complex, which is presumed to be the protuberant structures that were found on the growing cell surfaces. The separated cellulolytic complex was determined to be 1000–2000 kDa from gel chromatogram, and contained at least cellulase and xylanase activities. The separated complex was unstable and could release smaller fractions when they stored in solution at refrigerator. There were more than ten bands on SDS-PAGE after the complex was heat-treated with SDS. The HPLC chromatogram of the complex on DIOL-300 column also supports the result.     

华西雨屏区不同退耕模式细根(包括草根)分解过程中土壤酶动态

采用原状土芯(intact core)法, 探讨了四川洪雅柳江退耕模式——光皮桦(Betula luminifera)与扁穗牛鞭草(Hemarthria compressa)复合模式(HN)、扁穗牛鞭草草地模式(NC)、柳杉(Cryptameria fortunei)人工林模式(LS)、光皮桦人工林模式(H)细根(包括草根)分解过程中土壤酶动态。结果表明: 1) HN下的土壤脲酶、蔗糖酶、酸性磷酸酶活性较大, LS下的土壤脲酶、酸性磷酸酶活性最小, 显著低于其他模式(p < 0.05)。2) HN、NC和LS下的土壤脲酶与细根(包括草根)分解速率显著相关, HN的蔗糖酶、NC的酸性磷酸酶、LS的多酚氧化酶活性与细根(包括草根)分解速率也呈显著正相关关系(p < 0.05)。3) 除H外, 土壤脲酶活性与细根C/N、纤维素绝对含量呈显著负相关关系(p < 0.05); 除NC外, 多酚氧化酶活性与细根纤维素绝对含量呈显著负相关关系。4)土壤脲酶活性与需氧固氮细菌或与真菌数量显著相关, HN下的土壤蔗糖酶活性与细菌和纤维素分解菌数量呈正相关关系, H与NC下的土壤酸性磷酸酶还分别与细菌和纤维素分解菌数量呈正相关关系(p < 0.05)。以上结果显示: 由光皮桦与扁穗牛鞭草不同生活型植物构成的复合模式有利于土壤酶活性的提高; 土壤脲酶活性高低能够反映这几种退耕模式细根(包括草根)分解速率的快慢, 细根(包括草根)的C/N是影响土壤脲酶活性的一个重要因素; 土壤酶活性与土壤真菌、需氧固氮细菌、纤维分解菌及细菌数量有关。     

Molecular cloning of cellulolytic enzyme genes from Cellulomonas flavigena in E. coli

A genomic bank of Cellulomonas flavigena was constructed in E. coli using the pUC18 vector, and over 14000 clones screened for cellulolytic activity. Three different cellulolytic enzyme genes were cloned, one coding for an endo-β-glucanase (pJS10, CMC activity) and two coding for β-glucosidases, each with a distinct substrate specificity (pJS3, X-glu, and pJS4, X-glu and MUC activities). These three inserts have different restriction patterns to each other and the previously isolated cellulolytic enzyme genes from C. fimi and C. uda.     

Characterization of Cellulolytic Activities of Environmental Bacterial Consortia from an Argentinian Native Forest

Cellulolytic activities of three bacterial consortia derived from a forest soil sample from Chaco region, Argentina, were characterized. The phylogenetic analysis of consortia revealed two main highly supported groups including Achromobacter and Pseudomonas genera. All three consortia presented cellulolytic activity. The carboxymethylcellulase (CMCase) and total cellulase activities were studied both quantitatively and qualitatively and optimal enzymatic conditions were characterized and compared among the three consortia. Thermal and pH stability were analyzed. Based on its cellulolytic activity, one consortium was selected for further characterization by zymography. We detected a specific protein of 55 kDa with CMCase activity. In this study, we have shown that these consortia encode for cellulolytic enzymes. These enzymes could be useful for lignocellulosic biomass degradation into simple components and for different industrial applications.     

Shade effect alters leaf pigments and photosynthetic responses in Norway spruce (Picea abies L.) grown under field conditions

The contents of chlorophyll (Chl) and carotenoids (Car) per fresh mass were lower in shade needles than in sun needles. Ribulose-1,5-bisphosphate carboxylase (RuBPC) activity and contents of soluble proteins were also significantly lower in shade needles. In isolated thylakoids, a marked lower rate of whole chain and photosystem (PS) 2 activities were observed in shade needles. Smaller lower rate of PS1 activity was also observed in shade needles. The artificial exogenous electron donors, diphenyl carbazide (DPC) and NH₂OH, significantly restored the loss of PS2 activity in shade needles. Similar results were obtained when F_v/F_m was evaluated by Chl fluorescence measurements. The marked lower rate of PS2 activity in shade needles was due to the lower contents of 47, 33, 28–25, 23, and 17 kDa polypeptides. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the watersplitting complex was diminished significantly in shade needles.     

Strategies for the isolation of cellulolytic fungi for composting of wheat straw

It was shown that inoculation of straw with cellulolytic fungi offers potential for manipulating and improving the composting of cellulose waste, where the C:N ratio is not optimal for composting. In this paper we report on a screening strategy used to isolate novel cellulolytic fungi from field samples. The screen comprised of two phases. In phase I, 300 cellulolytic fungi were isolated to pure culture from field samples collected from Hawaii, China and the UK. Isolates were selected on the basis of high cellulolytic activities and growth rates on cellulose agar. A total of 137 lead isolates progressed to an unreplicated phase II screen to rapidly identify strains that improved quality of the resulting compost over and above that of the uninoculated control. Compost quality was assessed by measuring C:N ratio, water holding capacity, water content and potential and polysaccharide content of the resulting compost. Effect on the aggregate stability of soil and the growth of wheat seedlings was assessed when compost was added to a sandy loam soil. Performance of each isolate was quantified by allocating a utility score for each compost analysed. Utility scores were based on the sum of the logged ranked score in each assay. The 10 highest scoring isolates were subsequently processed through a replicated phase II screen and the best performing isolates identified by calculating utility scores as before. Significantly lower C:N ratios, higher water-holding capacities and improved aggregate stabilities were obtained with some inoculated treatments compared to the uninoculated control, whilst the results obtained for polysaccharide content and plant growth showed no significant differences. Isolate 304, isolated from decomposing vegetation obtained from Egham, Surrey, UK, and identifed as a Trichurus sp., appeared the most effective inoculant, significantly decreasing the C:N ratio by 36% and increasing the aggregate stability of soil by 54% compared to the uninoculated control. As a result of adopting this screening strategy, it has been possible to identify cellulolytic fungi that can, under non-sterile (laboratory) conditions, significantly improve the quality of compost. This screening approach therefore offers real possibilities for selecting microbial inoculants in low-tech agricultural practices.     

Effects of ozone on organic acids in needles of Norway spruce and Scots pine

Summary The effect of ozone, needle age, and season on the pH of homogenate and acid contents of Scots pine and Norway spruce needles is presented. In addition enzyme activities of cytochrome C-oxidase (cyt. C-ox), phosphoenolpyruvate-carboxylase (PEPC), shikimic acid-dehydrogenase (SHDH) and malate-dehydrogenase (MDH) were measured in Scots pine needles. In freshly sprouted spruce needles the level of quinic acid is high and the pH of the needle homogenate is low. Shikimic acid starts at low levels, increases with increasing needle age and becomes dominant, whereas the quinic acid content decreases. Malic acid has a marked seasonal trend; no trend was found in citric acid. Ozone (200 g/m³) decreased shikimic acid and quinic acid, whereas pH, malic acid and citric acid increased. Ozone (100 g/m³) had a similar effect, except in the current-year spruce needles. In Scots pine needles ozone led to increased enzymatic activities of cyt. C-ox, PEPC and SHDH, and a decrease in the activity of MDH. This effect was more pronounced in summer than in autumn, but the visible damage was greater in autumn. These effects can be found with other stresses and are not specific for ozone.     

Antioxidant systems in the leaf apoplast compartment of Pinus pinaster Ait. and Pinus radiata D. Don. Plants exposed to SO2

The activities of guaiacol peroxidase (GuPOD), ascorbate peroxidase (ASAp), superoxide dismutase (SOD) and ascorbate/glutathione cycle (AGC) enzymes, together with ascorbate (ASC) and glutathione contents, were determined in apoplastic-fluid and cell-wall fractions of needles of Pinus pinaster Ait. and Pinus radiata D. Don. exposed for up to 6 months to SO₂ (0.01 ppm or 0.30 ppm) in fumigation chambers. AGC enzyme activities (monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) were in all cases undetectable, as was glutathione content. In needles of P. pinaster plants exposed to SO₂, ascorbate content and all enzyme activities considered (except AGC enzymes) increased. The increases were most marked in response to the higher SO₂ concentration. In needles of P. radiata, similar but less marked responses were observed. These findings suggest a) that enzyme activities and ascorbate contents increase in order to deal with the reactive oxygen intermediates produced during long-term contamination with SO₂, and b) that P. pinaster has more effective defences against contamination of this type than P. radiata.     

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H₂SO₄ with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2³ central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.