壳聚糖固定化AS1．398中性蛋白酶稳定性的研究

对以壳聚糖为载体,由戊二醛作交联剂。制备的固定化ＡＳ１．３９８中性蛋白酶的稳定性进行了研究。实验结果表明,固定化ＡＳ１．３９８中性蛋白酶对热、乙醇、尿素以及ｐＨ值的稳定性均有明显的提高。     

Is glycine a surrogate for a d‐amino acid in the collagen triple helix?

Collagen is the most abundant protein in animals. Every third residue in a collagen strand is a glycine with phi, psi = -70 degrees, 175 degrees. A recent computational study suggested that replacing these glycine residues with D-alanine or D-serine would stabilize the collagen triple helix. This hypothesis is of substantial importance, as the glycine residues in collagen constitute nearly 10% of the amino acid residues in humans. To test this hypothesis, we synthesized a series of collagen mimic peptides that contain one or more D-alanine or D-serine residues replacing the canonical glycine residues. Circular dichroism spectroscopy and thermal denaturation experiments indicated clearly that the substitution of glycine with D-alanine or D-serine greatly disfavors the formation of a triple helix. Host-guest studies also revealed that replacing a single glycine residue with D-alanine is more destabilizing than is its replacement with L-alanine, a substitution that results from a common mutation in patients with collagen-related diseases. These data indicate that the glycine residues in collagen are not a surrogate for a D-amino acid and support the notion that the main-chain torsion angles of a glycine residue in the native structure (especially, phi > 0 degrees ) are critical determinants for its beneficial substitution with a D-amino acid in a protein.     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model.

The helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl-YSEEEEKAKKAXAEEAEKKKK-NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu-Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu- prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine-rich peptide modela, but not the previous Glu-Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared.     

Biodiversity and ecosystem stability across scales in metacommunities

Although diversity–stability relationships have been extensively studied in local ecosystems, the global biodiversity crisis calls for an improved understanding of these relationships in a spatial context. Here, we use a dynamical model of competitive metacommunities to study the relationships between species diversity and ecosystem variability across scales. We derive analytic relationships under a limiting case; these results are extended to more general cases with numerical simulations. Our model shows that, while alpha diversity decreases local ecosystem variability, beta diversity generally contributes to increasing spatial asynchrony among local ecosystems. Consequently, both alpha and beta diversity provide stabilising effects for regional ecosystems, through local and spatial insurance effects respectively. We further show that at the regional scale, the stabilising effect of biodiversity increases as spatial environmental correlation increases. Our findings have important implications for understanding the interactive effects of global environmental changes (e.g. environmental homogenisation) and biodiversity loss on ecosystem sustainability at large scales.     

Quantitative analysis of CryIA(b) expression in Bt maize plants,tissues, and silage and stability of expression over successive generations

The range and stability of expression of the transgenic CryIA(b) protein was examined in Ciba Seeds Bt maize plants derived from Event 176. Specifically, CryIA(b) levels were determined for: (1) various plant tissues and developmental stages in three maize lines from 1993 field tests; (2) pollen and leaves from plants representing four backcross generations of two genotypes; (3) leaves of 6 precommercial hybrids; and (4) silage from one Bt maize hybrid. Significant levels were found only in pollen and leaves. Genetic background did not greatly impact the level seen in either tissue. CryIA(b) expression in maize plants derived from transformation Event 176 was stable over at least four successive generations. On a per acre basis, the highest amount of CryIA(b) protein (estimated to be 2-4 g CryIA(b) protein/acre) was found to occur at anthesis, consistent with the stage at which maximum plant vegetative biomass is reached. CryIA(b) was not detected in silage prepared from CryIA(b)-expression plants. The maize-expressed CryIA(b) protein was found to have the expected size and to be immunoreactive with antibodies prepared against crystals from Bacillus thuringiensis subsp. kurstaki.     

Thermal stability enhancement of Candida rugosa lipase using ionic liquids

The thermal stability of Candida rugosa (C. rugosa) lipase was investigated and compared in n-hexane, benzene, dibutyl-ether as well as [bmim]PF₆ and [omim]PF₆ ionic liquids and the effect of solvent polarity and water activity were evaluated. Deactivation of the enzyme followed a series-type kinetic model. First order deactivation rate constants and the ratios of specific activities were determined and the kinetics of deactivation were studied. Among the organic solvents, the best stability was observed in n-hexane with a half-life of 6.5?h at water activity of 0.51. In ionic liquids, however, even longer half lives were obtained, and the enzyme was stable in these solvents at 50°C. The highest half-life times were obtained in [bmim]PF₆ (12.3?h) and [omim]PF₆ (10.6?h). A direct correlation was found between solvent polarity and thermal stability since the higher the polarity of the solvent, the lower was the stability decrease at 50°C comparing to that at 30°C.     

Spontaneous amplification of yeast CEN ARS plasmids

Summary Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-4 allele (as well as the URA3 and TRP1 markers) yielded His⁺ transformants at 0.1%–50% the frequency of Ura⁺ Trp⁺ transformants. Additional His⁺ derivatives arose on continuous growth of transformants originally scored as His^- Ura⁺ Trp⁺. In all cases, the His⁺ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His⁺ phenotype was lost more readily than the Ura⁺ Trp⁺ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura⁺ Trp⁺ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.     

A quantitative genetic method for estimating developmental instability

The concept of developmental instability (DI) is frequently used in evolutionary biology, and a range of definitions has been proposed. Moreover, numerous different statistical methods have been used for estimation of DI. The common basis for all methods is that measures need to be obtained from repeated structures within organisms. In the case of fluctuating asymmetry, mirror images could be interpreted as the repeats of each other. All repeats of a trait on one organism should, from a quantitative perspective, have the same genetic foundation. Most previous methods have not accounted for the genetics of the underlying trait. It is here shown how a statistical method from quantitative genetics (the repeated records animal model) can be used for assessment of DI, based on estimation of the variance due to the permanent environment. Moreover, Gibbs sampling is used for inference of the parameters, which provides a Bayesian framework where posterior distributions easily can be calculated from any functions of the variance components. The method is applied to a real dataset from two populations of the plant Scabiosa canescens, and results shows that it works well under realistic situations.     

Producing and scrounging can have stabilizing effects at multiple levels of organization

This study shows, for the first time, that the evolution of a simple behavior, scrounging, at the individual level can have effects on populations, food chains, and community structure. In particular, the addition of scrounging in consumer populations can allow multiple consumers to coexist while exploiting a single prey. Also, scrounging in the top predator of a tritrophic food chain can stabilize interactions between the top predator, its prey, and its prey's prey. This occurs because the payoffs to scrounging for food in a population are negative frequency dependent, allowing scroungers to invade a population and to coexist with producers at a frequency which is density‐dependent. The presence of scroungers, who do not search for resources but simply use those found by others (producers) reduces the total amount of resource acquired by the group. As scrounging increases with group size, this leads to less resource acquired per individual as the group grows. Ultimately, this limits the size of the group, its impact on its prey, and its ability to outcompete other species. These effects can promote stability and thus increase species diversity. I will further suggest that prey may alter their spatial distribution such that scrounging will be profitable among their predators thus reducing predation rate on the prey.