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1.
目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。 相似文献
2.
地衣二元性及其概念的双重理解与思考 总被引:1,自引:0,他引:1
本文简短回顾人类对地衣的认识过程之后,认为从地衣二元性(地衣的菌藻共生联合)和从地衣一元性(地衣的真菌本质)不同角度理解地衣都是正确的,被你之为“地衣概念的双重理解”。但作为地衣的定义似乎只能主要从地衣的二元性概念去表达,并分析其原因。对现在使用的地衣定义提出了某些看法与修改意见。 相似文献
3.
Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase.
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The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions. 相似文献
4.
Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branches 总被引:4,自引:0,他引:4
Xiang JQ Moody ML Soltis DE Fan C Soltis PS Fan Cz 《Molecular phylogenetics and evolution》2002,24(1):35-57
Phylogenetic relationships in Cornales were assessed using sequences rbcL and matK. Various combinations of outgroups were assessed for their suitability and the effects of long branches and outgroups on tree topology were examined using RASA 2.4 prior to conducting phylogenetic analyses. RASA identified several potentially problematic taxa having long branches in individual data sets that may have obscured phylogenetic signal, but when data sets were combined RASA no longer detected long branch problems. t(RASA) provides a more conservative measurement for phylogenetic signal than the PTP and skewness tests. The separate matK and rbcL sequence data sets were measured as not containing phylogenetic signal by RASA, but PTP and skewness tests suggested the reverse [corrected]. Nonetheless, the matK and rbcL sequence data sets suggested relationships within Cornales largely congruent with those suggested by the combined matK-rbcL sequence data set that contains significant phylogenetic signal as measured by t(RASA), PTP, and skewness tests. Our analyses also showed that a taxon having a long branch on the tree may not be identified as a "long-branched" taxon by RASA. The long branches identified by RASA had little effect on the arrangement of other taxa in the tree, but the placements of the long-branched taxa themselves were often problematic. Removing the long-branched taxa from analyses generally increased bootstrap support, often substantially. Use of non-optimal outgroups (as identified by RASA) decreased phylogenetic resolution in parsimony analyses and suggested different relationships in maximum likelihood analyses, although usually weakly supported clades (less than 50% support) were impacted. Our results do not recommend using t(RASA) as a sole criterion to discard data or taxa in phylogenetic analyses, but t(RASA) and the taxon variance ratio obtained from RASA may be useful as a guide for improved phylogenetic analyses. Results of parsimony and ML analyses of the sequence data using optimal outgroups suggested by RASA revealed four major clades within Cornales: (1) Curtisia-Grubbia, (2) Cornus-Alangium, (3) Nyssa-Camptotheca-Davidia-Mastixia-Diplopanax, and (4) Hydrangeaceae-Loasaceae, with clades (2) and (3) forming a monophyletic group sister to clade (4) and clade (1) sister to the remainder of Cornales. However, there was not strong bootstrap support for relationships among the major clades. The placement of Hydrostachys could not be reliably determined, although most analyses place the genus within Hydrangeaceae; ML analyses, for example, placed the genus as the sister of Hydrangeeae. Our results supported a Cornales including the systematically problematic Hydrostachys, a Cornaceae consisting of Cornus and Alangium, a Nyssaceae consisting of Nyssa and Camptotheca, a monogeneric Davidiaceae, a Mastixiaceae consisting of Mastixia and Diplopanax, and an expanded Grubbiaceae consisting of Grubbia and Curtisia, and two larger families, Hydrangeaceae and Loasaceae. 相似文献
5.
Anu Jose Mattam James M. Clomburg Ramon Gonzalez Syed Shams Yazdani 《Biotechnology letters》2013,35(6):831-842
Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms. 相似文献
6.
Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation 总被引:1,自引:0,他引:1
The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor. 相似文献
7.
Chromic nitrate and aluminon (ATA) react to form highly colored lakes. Examination of these lakes by the Vosburgh and Cooper method has led to the identification of a pigment [(ATA)Cr(H2O)x] which is insoluble in water and absolute alcohol, a deep red anionic component [(ATA)BCr(H2O)x]-y, and a purplish-red cationic component [(ATA)Cr4(H2O)x]+y. These components of the lake have been isolated and separated in the dry form. The anionic compound is apparently a simple coordination compound and can be used as a cytoplasmic stain while the cationic component is a chelate and can be used as a rather selective nuclear stain. In addition to these components, a number of other components were also found. Not only the ratio between chromium and aluminon but also the concentration of the reactants influences the formation of these different components. The amount of pigment formed is maximal with a molar ratio of 1 and a concentration of 10-2 M or 10-1 M. The anionic component is maximal with a molar ratio of 1 Cr to 6 aluminon at the 10-1 M concentration, the cationic component is maximal with a molar ratio of 4 chromium to 1 aluminon at the 10-1 M concentration. None of these are formed at the 10-4 M concentration but only a deep redpurple soluble compound at the 1 : 1 ratio, which was not further investigated. 相似文献
8.
JOHN R. WIBLE DESUI MIAO JAMES A. HOPSON 《Zoological Journal of the Linnean Society》1990,98(2):203-228
The septomaxilla is a paired intramembranous ossification in the external nares that occurs in Lepidosauria among Recent Sauropsida and is purported to be present in Monotremata and Dasypodidae (armadillos) among Recent Mammalia. A review of neontological and palaeontological evidence regarding this element in mammals supports the following conclusions: (1) monotremes have a true septomaxilla resembling that known for non-mammalian therapsids and some Mesozoic mammals; (2) the element in dasypodids is a neomorph; it neither resembles the septomaxilla of other synapsids nor does it exhibit the same relationship to the developing nasal-floor cartilage as the septomaxilla of lepidosaurs and monotremes; (3) a septomaxilla is lacking in all Recent therians, and there is no evidence that this bone is fused to the premaxilla in Recent therians, as has been suggested by previous authors. 相似文献
9.
JOHN R. WIBLE DESUI MIAO JAMES A. HOPSON 《Zoological Journal of the Linnean Society》1990,98(3):203-228
The septomaxilla is a paired intramembranous ossification in the external nares that occurs in Lepidosauria among Recent Sauropsida and is purported to be present in Monotremata and Dasypodidae (armadillos) among Recent Mammalia. A review of neontological and palaeontological evidence regarding this element in mammals supports the following conclusions: (1) monotremes have a true septomaxilla resembling that known for non-mammalian therapsids and some Mesozoic mammals; (2) the element in dasypodids is a neomorph; it neither resembles the septomaxilla of other synapsids nor does it exhibit the same relationship to the developing nasal-floor cartilage as the septomaxilla of lepidosaurs and monotremes; (3) a septomaxilla is lacking in all Recent therians, and there is no evidence that this bone is fused to the premaxilla in Recent therians, as has been suggested by previous authors. 相似文献
10.
Column chromatographic separation of acidic and neutral and basic metabolites of tyrosine and dihydroxyphenylalanine 总被引:1,自引:0,他引:1
Liquid column chromatographic techniques for separation of tyrosine; dihydroxyphenylalanine; and acidic, neutral, and basic metabolites of tyrosine and catecholamines are described. These techniques permitted simultaneous separation of a wide spectrum of acidic and neutral metabolites of tyrosine and dihydroxyphenylalanine on one column. Rapid and complete separation of basic metabolites was carried out on a second column. 相似文献
11.
The paper brings data set of seed reproduction of about 500 wild herbaceous species of Central Europe and presents a number
of produced seeds in a new way. Number of seeds — reproductive capacity of a population (RCP) was defined as a number of seeds
produced by one species per 1 m2 at its one-hundred-per-cent cover per one season. About 23% of seed weight variability and about 26% of variability in RCP
were explainable on a family level. The trade-off between RCP and seed weight was confirmed both within families and also
among families. Both characteristics had higher variability on a family level and on lower taxonomic levels than on subclass
and class levels. Species with an annual life cycle have a larger number of seeds than perennials. Geophytes and species without
lateral spread have a tendency to produce a large amount of relatively weighty seeds in comparison to other life forms, and
to species with vegetative lateral spread. Species with seeds dispersed by wind usually have a large number of lightweight
seeds. Multivariate analyses confirmed some tendencies of reproductive traits which correspond to the definition of C-, S-,
R- strategies by Grime — “verification” of the RCP as ecological trait. The R- strategy was well distinguished from the C-
and S- strategies by reproductive traits, whereas C- and S- strategies are very similar to each other. Species with insect
pollination agent often correspond to C- strategy, wind agent to S- strategy, and species with self-pollination to R- strategy. 相似文献
12.
Matthew A. Campbell Bruno Chanet Jhen‐Nien Chen Mao‐Ying Lee Wei‐Jen Chen 《Zoologica scripta》2019,48(5):640-656
Flatfishes (Pleuronectiformes) are a species‐rich and distinct group of fishes characterized by cranial asymmetry. Flatfishes occupy a wide diversity of habitats, including the tropical deep‐sea and freshwaters, and often are small‐bodied fishes. Most scientific effort, however, has been focused on large‐bodied temperate marine species important in fisheries. Phylogenetic study of flatfishes has also long been limited in scope and focused on the placement and monophyly of flatfishes. As a result, several questions in systematic biology have persisted that molecular phylogenetic study can answer. We examine the Pleuronectoidei, the largest suborder of Pleuronectiformes with >99% of species diversity of the order, in detail with a multilocus nuclear and mitochondrial data set of 57 pleuronectoids from 13 families covering a wide range of habitats. We combine the molecular data with a morphological matrix to construct a total evidence phylogeny that places fossil flatfishes among extant lineages. Utilizing a time‐calibrated phylogeny, we examine the timing of diversification, area of origin and ancestral temperature preference of Pleuronectoidei. We find polyphyly or paraphyly of two flatfish families, the Paralichthyidae and the Rhombosoleidae, and support the creation of two additional families—Cyclopsettidae and Oncopteridae—to resolve their non‐monophyletic status. Our findings also support the distinctiveness of Paralichthodidae and refine the placement of that lineage. Despite a core fossil record in Europe, the observed recent diversity of pleuronectoids in the Indo‐West Pacific is most likely a result of the Indo‐West Pacific being the area of origin for pleuronectoids and the ancestral temperature preference of flatfishes is most likely tropical. 相似文献
13.
Mervyn W. Thenabadu 《Plant and Soil》1972,36(1-3):15-29
Summary Results of a greenhouse sand-culture experiment conducted to study the effect of increasing or decreasing nitrogen supply
at various stages of growth on the growth, yield, and yield components of rice are reported.
Adequate nitrogen during the vegetative phase of growth encouraged tillering which had a bearing on the number of panicles
per plant. Excess nitrogen during the reproductive phase of growth produced panicles with unfilled or partly filled grains.
Similar effects were observed when the nitrogen status of the growth medium was changed from a low to a high level at the
stages of reduction division and anthesis.
Decrease in yield was observed when plants growing at a low level of nitrogen were transferred to a high level at the end
of the vegetative phase.
High levels of nitrogen in the growth medium at time of heading and after increased the concentration of nitrogen in the grain.
There was an inverse relationship between yield of filled grain and nitrogen in the grain. 相似文献
14.
15.
Lapcík O Stursa J Kleinová T Vítková M Dvoráková H Klejdus B Moravcová J 《Steroids》2003,68(14):1147-1155
3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples. 相似文献
16.
Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission 总被引:4,自引:0,他引:4
The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer. 相似文献
17.
Summary A mesophilic bacterium,Proteus mirabilis, which was known to be able to accumulate monomer silicate ions, a thermophilic bacterium,Bacillus caldolyticus, originating from a habitat with high silica concentrations, and a silicautilizing plant,Equisetum arvense, were all found to produce monomer silica from its polymer. The monomer silica, resulting from the mineralysis of either
experimentally polymerized silica, or from quartz, is taken up byP. mirabilis cells, and also byEquisetum, which then deposits the silica again as a polymer in its stem and leaves. WithB. caldolyticus, which does not utilize the depolymerized product under the given conditions, we found that the intensity of the mineralysis
depends on the growth rate of the organism. 相似文献
18.
Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin
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The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke. 相似文献
19.
Taurocyamine kinase (TK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase. TK is found only in certain marine annelids. In this study we used PCR to amplify two cDNAs coding for TKs from the polychaete Arenicola brasiliensis, cloned these cDNAs into the pMAL plasmid and expressed the TKs as fusion proteins with the maltose-binding protein. These are the first TK cDNA and deduced amino acid sequences to be reported. One of the two cDNA-derived amino acid sequences of TKs shows a high amino acid identity to lombricine kinase, another phosphagen kinase unique to annelids, and appears to be a cytoplasmic isoform. The other sequence appears to be a mitochondrial isoform; it has a long N-terminal extension that was judged to be a mitochondrial targeting peptide by several on-line programs and shows a higher similarity in amino acid sequence to mitochondrial creatine kinases from both vertebrates and invertebrates. The recombinant cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% of that of taurocyamine). However, the mitochondrial TK showed activity for taurocyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Neither TK catalyzed the phosphorylation of creatine. Comparison of the deduced amino acid sequences of mitochondrial CK and TK indicated that several key residues required for CK activity are lacking in the mitochondrial TK sequence. Homology models for both cytoplasmic and mitochondrial TK, constructed using CK templates, provided some insight into the structural correlation of differences in substrate specificity between the two TKs. A phylogenetic analysis using amino acid sequences from a broad spectrum of phosphagen kinases showed that annelid-specific phosphagen kinases (lombricine kinase, glycocyamine kinase and cytoplasmic and mitochondrial TKs) are grouped in one cluster, and form a sister-group with CK sequences from vertebrate and invertebrate groups. It appears that the annelid-specific phosphagen kinases, including cytoplasmic and mitochondrial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplasmic and mitochondrial isoforms of TK evolved independently. 相似文献
20.
Administration of the methionine analogue methoxinine (O-methyl-DL-homoserine) to sheep substantially changed the composition of wool; in addition wool fibres were weakened and the staple crimp frequency was reduced for a prolonged period. The proportions of high-tyrosine proteins were reduced by 40-45% whereas the high-sulfur proteins were usually slightly increased. The content of high-tyrosine proteins in wool was still depressed in most sheep 70 days after dosing with methoxinine. These experiments supported a previous finding that the cystine content of wool and its crimp frequency are not causally related. Ethionine, another methionine analogue, did not consistently change the composition of wool. In some sheep there was no change in the proportions of high-tyrosine proteins following administration of ethionine, even though weak wool was produced. This result, together with the lack of association between the content of high-tyrosine proteins and the strength of wool fibres in a sheep given methoxinine plus methionine, indicates that a reduction of the high-tyrosine proteins is not a prerequisite for the production of weak wool. Neither a threefold increase in the phenylalanine intake by mice nor the administration of three analogues of phenylalanine (4-fluoro-DL-phenylalanine, 4-chloro-DL-phenylalanine and beta-(2-thienyl)-DL-alanine) to sheep altered the composition of hair or wool. Fluorophenylalanine was incorporated into all the constituent proteins of wool to the extent of c. 2% of phenylalanine residues. The other analogues studied could not be detected in wool. 相似文献