Summary The stigmatic dimorphisms in the distylous speciesPrimula obconica, which has been dealt with in an earlier paper, has been described further. An ultrastructural localization of various enzymes in the exudates is hereby revealed, and evidence is given for intermorph differences. Esterases are confined to the lipid phase of the exudates, including the lipid globuli in the papillae walls of both morphs, but they are not found in the pellicle of the dry thrum stigma. However, this pellicle exhibits acid phosphatase activity, as does the lumen of the blisters in the viscous exudate on pin stigmas. The blisters are presumed to hold the watery phase of the pin secretion. From the present findings and from previous results with LM cytochemical methods it is suggested that the sites of peroxidase activity resemble those of acid phophatases. 相似文献
Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity. 相似文献
For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p < 0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p < 0.05), and significant differences were found between isolates (p < 0.001) and the amounts of enzymes produced (p < 0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors. 相似文献
Abstract: Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with ~40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05–0.25 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550–1,300%) than of the housekeeping enzyme (by 120–150%). Magnesium chloride (1–2 mM) inhibited the nonactivated housekeeping GDH (by 45–64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue-specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05–0.25 mM). Heat-inactivation experiments revealed that the half-life of the housekeeping and nerve tissue-specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue-specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands. 相似文献
Epoxide hydrolase from Aspergillus niger (E.C. 3.3.2.3) was immobilized by covalent linking to epoxide-activated silica gel under mild conditions. A very easy procedure allowed to prepare an immobilized biocatalyst with more than 90% retention of the initial enzymatic activity. Immobilized and free enzyme showed very similar behaviour with respect to the effect of pH on activity and stability. One benefit of immobilizing epoxide hydrolase from A. niger on silica gel was the enhanced enzyme stability in the presence of 20% DMSO. The kinetic resolution of racemic para-nitrostyrene oxide was investigated by using this new immobilized biocatalyst. The enantioselectivity of the enzyme was not altered by the immobilization reaction: both unreacted epoxide and formed diol were obtained with very high ee (99 and 92%, respectively). In addition, the biocatalyst could be easily separated from the reaction mixture and re-used for over nine cycles without any noticeable loss of enzymatic activity or change in the enantioselectivity extent. The activity of immobilized AnEH was retained for several months. 相似文献
The stability of β-galactosidase entrapped in Ca-alginate–K-κ-carrageenan gels under operation conditions was studied. The thermal deactivation of the immobilised enzyme and the biocatalyst protein loss due to gel swelling were taken into account in the mass balance of the enzymatic reaction rate expression.
Time-temperature effect was the most important factor in the biocatalyst deactivation reaction. However, results showed that the enzyme entrapped in gels was partially lost by gel swelling, which was a source of error in predicting results in continuous processes. The enzyme loss determined in this work showed a non-linear behaviour and it depended on mixing conditions of the reactor.
Values of protein loss were used in the modelling of a fixed-bed reactor with similar flow conditions to reduce the error in predicting the operation conditions to maintain a constant conversion.
For reaction conditions similar to those analysed in this work, the β-galactosidase was well entrapped in alginate-carrageenan matrices. 相似文献
The effect of ions on enzyme activity and stability usually follows the Hofmeister series (or the kosmotropicity order): kosmotropic anions and chaotropic cations stabilize enzymes while chaotropic anions and kosmotropic cations destabilize them. The effect of ionic liquids (ILs) on the enzyme activity/stability/enantioselectivity is complicated especially when there is no or little water presence in the IL media. However, when aqueous solutions of hydrophilic ILs are employed as reaction media, the enzyme seems to follow the Hofmeister series since ILs dissociate into individual ions in water. 相似文献
Two isoforms of adenosine kinase (AdK) have been identified in mammalian organisms with the long isoform (AdK-long) containing extra 20-21 amino acids at the N-terminus (NTS). The subcellular localizations of these isoforms are not known and they contain no identifiable targeting sequence. Immunofluorescence labeling of mammalian cells expressing either only AdK-long or both isoforms with AdK-specific antibody showed only nuclear labeling or both nucleus and cytoplasmic labeling, respectively. The AdK-long and -short isoforms fused at the C-terminus with c-myc epitope also localized in the nucleus and cytoplasm, respectively. Fusion of the AdK-long NTS to green fluorescent protein also resulted in its nuclear localization. AdK-long NTS contains a cluster of conserved amino acids (PKPKKLKVE). Replacement of KK in this sequence with either AA or AD abolished its nuclear localization capability, indicating that this cluster likely serves as a nuclear localization signal. AdK in nucleus is likely required for sustaining methylation reactions. 相似文献
Subcellular fractionation of Leishmania donovani promastigotes yielded plasma membranes, which were enriched in acid phosphatase (E.C.3.1.3.2.) activity. Cytochemically, the enzyme displayed a uniform distribution over the surface of intact protozoa. The enzyme was also visualized on the external face of the isolated plasma membranes, as indicated by the distribution of subpellicular microtubules. Various parameters of the membrane-bound enzyme were also determined including pH and temperature optima and substrate specificity. The results suggest that these organisms are adapted for existence in a hydrolytic environment. 相似文献