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1.
Background
In recent years, it has been gradually realized that bacterial inclusion bodies (IBs) could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D). As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. 相似文献2.
Søren Bastholm Lasse Wahlstrøm Louise Appel Bjergbæk Peter Roslev 《World journal of microbiology & biotechnology》2008,24(10):2323-2330
Traditional cultivation-dependent tests for coliform bacteria in food and drinking water take 18–24 h to complete. Bioluminescence-based
enzyme assays can potentially reduce analysis time for indicator bacteria such as coliforms. In the present study, we developed
a simple presence/absence (P/A) bioluminescence procedure for rapid detection of coliform bacteria in groundwater-based drinking
water. The bioluminescence procedure targeting β-d-galactosidase activity in coliform bacteria was based on hydrolysis of 6-O-β-galactopyranosyl-luciferin. Bacteria immobilized on membrane filters were enriched for 6–8 h in selective media containing
isopropyl-β-d-thiogalactopyranoside (IPTG) to induce β-d-galactosidase activity in coliform bacteria. The equivalent of approximately 300 E. coli cells was required for bioluminescence detection of β-d-galactosidase activity. In comparison, PCR based detection of E. coli in drinking water required approximately 30 target cells. Analysis of contaminated drinking water samples showed comparable
results for coliform bacteria using traditional multiple-tube fermentation, Colilert-18, and the bioluminescence procedure.
Aeromonas hydrophila or indigenous groundwater bacteria did not interfere with the procedure. The bioluminescence procedure can be combined with
commercial substrates such as Fluorocult or Colilert-18, and will allow the detection of one coliform in 100 ml drinking water
within one working day. The results suggest the bioluminescence assays targeting β-d-galactosidase activity may be used for or for early warning screening of drinking water and/or rapid identification of contaminated
drinking water wells. 相似文献
3.
Iordanka Georgieva Aglika Edreva Rossitza Rodeva Violeta Sotirova Elisaveta Stoimenova 《Acta Physiologiae Plantarum》2000,22(3):281-284
The metabolic changes in tomato fruits and seeds separately infected with cucumber mosaic virus, Pseudomonas syringae pv. tomato or Botrytis cinerea were investigated cytochemically. The changes of peroxidase (E.C. 1.11.1.7) and β-glucosidase (E.C. 3.2.1.21) were investigated
biochemically as well. Tomato fruits were involved in the study because of their high economic value. Tomato seeds were investigated
since they have been used most extensively as a model system for studying the physiology and biochemistry of seed development.
The diseases caused by the pathogens under study are of special importance for yield reduction in tomato. The three pathogens
provoked local changes in the activities of enzymes under study that affect the infected pericarp tissues and neighboring
seeds. It was established non-specific and specific responses. The non-specific responses of invaded tissues were expressed
as a local enhancement of peroxidase activity in both pericarp tissues and seeds as well as a decrease in activities of: i. enzymes taking part in aerobic and anaerobic respiration, ii. hydrolases esterase and acid phosphatase involved in the basic metabolism as well as an enhancement of their activities in
neighboring tissues. Furthermore, it was observed an enhancement of α-galactosidase activity in infected area was observed.
The specific responses depending on the type of the pathogen consisted in an enhancement of glucose-6-phosphate dehydrogenase
activity by virus infection and an increase of β-glucosidase activity by fungal invasion. 相似文献
4.
Salam A. Ibrahim Awfa Y. Alazzeh Saddam S. Awaisheh Danfeng Song Abolghasem Shahbazi Amer A. AbuGhazaleh 《Biological trace element research》2010,136(1):106-116
The hydrolysis of oligosaccharides and lactose is of great importance to the food industry. Normally, oligosaccharides like
raffinose, stachyose, and verbascose which are rich in different plants like soy bean are considered indigestible by the human
gut. Moreover, many humans suffer from lactose intolerance due to the absence of effective enzyme that can digest lactose.
α-Galactosidase can digest oligosaccharides like raffinose, while β-galactosidases can hydrolyze lactose. Therefore, selection
of microorganisms safe for human use and capable of producing high levels of enzymes becomes an attractive task. The objective
of this study was to investigate the enhancement of α- and β-galactosidase activity in Lactobacillus reuteri by different metal ions. Ten millimolar of Na+, K+, Fe2+, and Mg2+ and 1 mM of Mn2+ were added separately to the growth culture of six strains of L. reuteri (CF2-7F, DSM20016, MF14-C, MM2-3, MM7, and SD2112). Results showed that L. reuteri CF2-7F had the highest α- and β-galactosidase activity when grown in the medium with added Mn2+ ions (22.7 and 19.3 Gal U/ml, respectively). 0.0274% of Mn2+ ions lead to 27, 18% enhancement of α- and β-galactosidase activity over the control group, and therefore, it could be added
to the growth culture of CF2-7F to produce enhanced levels of α- and β-galactosidase activity. The addition of Fe2+ led to a significant (P < 0.01) decrease in the activity of both enzymes for most strains. This study shows that modified culture medium with that
0.0274% Mn2+ can be used to promote the production for α- and β-galactosidase in L. reuteri CF2-7F, which may lead to enhancement of α- and β-galactosidase activity and have a good potential to be used in the food
industry. 相似文献
5.
Glucose-6-phosphate dehydrogenase and mouse Kupffer cell activation: an ultrastructural dual staining enzyme-cytochemical study 总被引:1,自引:1,他引:0
Matsubara S 《Histochemistry and cell biology》2002,118(4):345-350
Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in Kupffer cell function, especially in phagocytosis activity. Although it was suggested that Kupffer G6PD may be upregulated in Kupffer phagocytosis/activation, direct morphological evidence has been lacking. Acid phosphatase (ACP), a representative lysosomal enzyme, can be used as a cytochemical marker for phagocyte activation. Using an ultrastructural enzyme-cytochemical dual staining method, I simultaneously localized G6PD and ACP activity in mouse Kupffer cells on a cell-by-cell basis, and examined whether or not cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Glucose-6-phosphate dehydrogenase labelings were observed in the cytoplasm and on the cytosolic side of the endoplasmic reticulum, and ACP labelings were seen in the lysosomes. In phagocytosing Kupffer cells, in which ACP deposits were observed not only in the lysosomes but also on the phagosomal membranes and phagosomal contents, G6PD labelings were denser than dormant Kupffer cells. Enzyme-cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Kupffer cell G6PD, activated in phagocytosing Kupffer cells, may play an important role not only in liver defense but also in liver disease pathogenesis/pathophysiology. 相似文献
6.
A. K. Singh 《The Histochemical journal》1993,25(8):562-568
Summary The question posed in the present study was: are there hydrolytic enzymes, including proteases, present in the extracellular
matrix of the glomerular basement membrane? If these enzymes are present they may play a role in the catabolism of the glomerular
basement membrane (GBM) and removal of macromolecular debris resulting from ultrafiltration. Enzymes, acid phosphatase - the
marker for lysosomal enzymes - β-galactosidase, β-glucuronidase and acid protease (using albumin as substrate) were biochemically
assayed in purified basement membrane preparations. It was found that all enzymes were present in significant amounts in the
basement membrane. Compared to other enzymes, acid protease activity was present in much higher amounts. The pH optima of
these enzymes were variable but all had significant activity at neutral pH. A method was developed to localize the marker
enzyme, acid phosphatase, ultrastructurally in the basement membrane in order to substantiate the biochemical findings. Activity
was shown by the presence of dense deposits of lead phosphate. Staining for acid phosphatase could also be shown on isolated,
purified basement membrane. The demonstration of acid hydrolases in the GBM matrix argues for their role in (i) the extracellular
turnover of basement membrane macromolecules, and (ii) clearance of debris of ultrafiltration which tend to clog the membrane
pores. 相似文献
7.
I. D. Georgieva 《Biologia Plantarum》1991,33(5):337-344
Changes in the activity and localization of nonspecific esterase, acid phosphatase, α-galactosidase and β-glucosidase inL. regale pistils after pollination with μ-irradiated pollen were studied. In the embryo sac and in the ovule reduction of AS-esterase
and α-galactosidase and, at the same time, enhancement of α-esterase, acid phosphatase and β-glucosidase activities were observed.
The changes in hydrolytic enzyme activities are discussed as manifestations of lethal factors resulting from structural disturbances
of DNA in the generative nucleus and in sperms caused by irradiation. 相似文献
8.
9.
10.
The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated,
as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex
medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high
levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase
activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric
β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose
feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented.
Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset
of stationary phase, glucose depletion and biomass concentration.
Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996 相似文献
11.
VALENTI VINCENZO; SCAGLIARINI SANDRA; PUPILLO PAOLO 《Journal of experimental botany》1986,37(5):606-614
Mesophyll protoplasts of one-month-old maize leaves were separatedenzymatically from bundle sheath strands, and purified by centrifugationthrough a Percoll layer. The protoplasts and BS strands wereessentially pure as judged by microscopy, chl a/b ratios, andlevels of enzyme markers (PEP carboxylase and NADP-malic enzyme).Chioroplasts were obtained from the protoplasts and from homogenates,and purified through Percoll. The distribution of four NAD P-linked dehydrogenases in tissuesand organdies was examined. NADP-triose phosphate dehydrogenasc,used as a chloroplast marker, shows high and comparable specificactivities in both main tissues. Glucose 6-phosphate dehydrogenaseis located mainly in the mesophyll (at a specific activity of15.1 µmol h1 mg1chl in protoplasts) andis exclusively cytosolic. 6-Phosphogluconate dehydrogenase,also present in both tissue types, has a higher activity inthe BS (12.6 in purified strands versus 7.3 µmol h1mg1 chl in protoplasts). It is a cytosolic enzyme, althoughplastids may contain a low activity. Glyceraldehyde 3-phosphate:NADP reductasc is entirely in the mcsophyll cytoplasm (11.2µmol h1 mg1 chl). It is suggested that thecytoplasm of mcsophyll cells is a site of diversion of sugarphosphates for production of NADPH, at rates, however, compatiblewith the operation of the triose phosphate shuttle to bundlesheath cells for the synthesis of starch. Key words: Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dchydrogenase, glyceralde-hyde-3-phosphate : NADP reductase, Zea mays 相似文献
12.
A quantitative study of the validity of the histochemical demonstration for pyridine nucleotide-linked dehydrogenases 总被引:1,自引:0,他引:1
Summary A quantitative histochemical and biochemical study has been made of the loss of pyridine nucleotide-linked dehydrogenases from frozen histological sections of rat liver. Glucose-6-phosphate, 6-phosphogluconate and lactate dehydrogenases were lost rapidly from the sections during incubation in the histochemical medium, but -OH-butyrate dehydrogenase was lost at a much slower rate. It was shown that a dehydrogenase reaction can occur in a section lacking that particular dehydrogenase if the section is incubated in the presence of another containing the dehydrogenase. The validity of the tetrazolium reaction for demonstrating pyridine-nucleotide-linked dehydrogenases is considered in the light of these results. 相似文献
13.
Domingues L Onnela ML Teixeira JA Lima N Penttilä M 《Applied microbiology and biotechnology》2000,54(1):97-103
One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being
the flocculating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production
processes. The construction of flocculent and non-flocculent brewer's yeast strains secreting β-galactosidase and growing
on lactose is presented. A plasmid was constructed coding for an extracellular β-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work
represents an important step towards the study of heterologous protein secretion by flocculent cells.
Received: 13 January 2000 / Accepted: 23 January 2000 相似文献
14.
Espinosa Yeison Trebotich Jovanka Sepúlveda Francisco Cadena Jeisson Vargas-Straube María-José Vaca Inmaculada Bull Paulina Levicán Gloria Chávez Renato 《World journal of microbiology & biotechnology》2011,27(12):3019-3023
The biotechnological applications of cheese-ripening fungi have been limited by a lack of genetics tools, in particular the
identification and characterization of suitable promoters for protein expression. In this study, the suitability of the glyceraldehyde-3-phosphate
dehydrogenase (gpdP) promoter from Penicillium camemberti to drive the production of a recombinant protein was evaluated. The gpdP gene and its promoter were isolated using PCR and Genome Walker. The promoter of gpdP has two regions with high identity to the regulatory elements gpd-box and ct-box previously described in Aspergillus
nidulans. Two fragments of the promoter containing the gpd- and ct-box or the ct-box alone were used to drive the in vivo production of recombinant β-galactosidase using A. nidulans as host. Our results indicate that larger fragment containing gpd-box enhances the production of β-galactosidase activity levels respect to ct-box alone, and that both boxes are necessary to obtain maximal enzymatic activity production. The smaller fragment (187 nt)
containing the ct-box alone was able to trigger up to 27% of β-galactosidase activity, and to our knowledge this is the smallest fragment from
a gpd gene used to produce a recombinant protein. Differences were not observed when glycerol, galactose or glucose were used as
carbon sources, suggesting that the promoter activity is carbohydrate-independent. This is the first report in which a Penicillium gpd promoter is used for recombinant protein production. Our results open the way for the future development of a system for
recombinant proteins expression in the biotechnologically important cheese-ripening fungus P. camemberti. 相似文献
15.
The β-Galactosidase Activity in <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> CBS6556 Decreases by High Concentrations of Galactose 总被引:1,自引:0,他引:1
In this paper we report on the effect of different concentrations of lactose and galactose in the production of β-galactosidase
by Kluyveromyces marxianus CBS6556. The results clearly demonstrate a decrease in enzyme specific activity during cultivation at high concentrations
of L-lactose or D-galactose, despite the fact that these carbohydrates are normally used for induction of the β-galactosidase
activity. Therefore, maximum induction of β-galactosidase in K. marxianus batch cultures was obtained at low concentrations of the inducer carbohydrates, in the range between 0.5 to 15 mM. Those
informations can help to design low cost medium with higher β-galactosidase productivity by K. marxianus cells.
Received: 8 August 2001 / Accepted: 15 October 2001 相似文献
16.
The work presented in this paper describes the purification and properties of a β-galactosidase from the protozoan Tritrichomonas foetus. An inexpensive and straightforward method for extraction of the enzyme involving ammonium sulphate precipitation, ion exchange
and affinity chromatography resulted in a high level of purification. After purification β-N-acetylglucosaminidase was the only enzyme present as a contaminant at a significant level. The β-galactosidase isolated had a pH optimum of 5.8. The Km determined at pH 5.8 was found to be 2.2 mM. Interesting results were obtained when studies were carried out to determine
the effect of various metal ions on enzyme activity. Of the metal ions used in this study only manganese ions were found to
activate the enzyme. This seems to be a characteristic of trichomonad enzymes, as N-acetyl-β-glucosaminidase, a-galactosidase and N-acetyl-a-galactosaminidase are also activated by manganese ions. The strongest inhibition was recorded with lead and to a lesser extent
by zinc. The result with lead is not unexpected as the heavy metal is known to cause irreversible inhibition by binding to
the amino-acid backbone of the enzyme. The result with zinc is interesting as high levels of zinc are present and trichomonads
are known to be apathogenic in semen. The purified β-galactosidase was found to have the capacity to hydrolyse lactose (Gal β1-4 Glc), lacto-N-biose 1 (Gal β1-3 GlcNAc) and N-acetyllactosamine (Gal β1-4 GlcNAc). When the enzyme was applied to a non-denaturing polyacrylamide gel a single band was observed when stained with
Coomassie brilliant blue. This band coincided with that obtained when the gel was stained with p-nitrophenyl β-galactopyranoside. When the same gel was incubated with p-nitrophenyl N-acetyl β-glucopyranoside a band was detected which did not coincide with that of β-galactosidase. Since the β-N-acetylglucosaminidase enzyme does not move to the same position on a non-denaturing gel as the
β-galactosidase, we will use this technique to isolate the latter enzyme and determine the N-terminal sequence as a prelude
to cloning and further study of the gene.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
17.
P. P. Tonini C. G. S. Lisboa C. O. Silva S. C. Mazzoni-Viveiros M. S. Buckeridge 《Trees - Structure and Function》2007,21(1):13-21
Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) contain galactomannan as a cell wall storage polysaccharide in the endosperm. After germination,
it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25).
This work aimed at studying the role of the testa (seed coat) on galactomannan degradation during and after germination. Seeds
were imbibed in water, with and without the testa, and used to evaluate the effect of this tissue on storage mobilisation,
as well as its possible role in the galactomannan hydrolases activities. Immunocytochemistry and immunodotblots were used
to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. Endo-β-mannanase
and α-galactosidase activities were found in the testa and latter in the endosperm during galactomannan degradation. The former
enzyme was immunologically detected in the testa, mainly during germination. The absence of the testa during imbibition led
to the anticipation of protein mobilisation and increased of the α-galactosidase activity and galactomannan degradation. Thus,
the testa appears to play a role during storage mobilisation in the legume seed of S. virgata probably by participating in the control of the production, modification and/or storage of the hydrolases. 相似文献
18.
Cytochemical localization of glucose-6-phosphatase activity in cerebral endothelial cells 总被引:2,自引:0,他引:2
R D Broadwell A M Cataldo M Salcman 《The journal of histochemistry and cytochemistry》1983,31(6):818-822
Glucose-6-phosphatase (G6Pase) activity, with glucose-6-phosphate and mannose-6-phosphate as substrates, was examined by cytochemistry in capillary and arteriole endothelial cells of the mouse brain. G6Pase activity was observed ultrastructurally in the lumen of the nuclear envelope and endoplasmic reticulum (ER) of these cells. The reactive ER and nuclear membrane appeared to be in continuity. Nucleoside diphosphatase activity, also a marker for the ER in some cell types, was not seen within the ER of the cerebral microvasculature. The ER of arterioles and capillaries did not bind lead nonspecifically when incubated in a substrate-free medium. Speculation is raised concerning the involvement of G6Pase in glucose metabolism of cerebral endothelial cells and in making blood-borne glucose available to brain parenchyma. 相似文献
19.
Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory
processes to the site of N2 fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed
relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities
of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction
reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following
sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H2 to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase,
NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD
and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing
photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme
iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate
dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein.
The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons
from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and
bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without
involvement of azotoflavin and bacteroid non-heme iron protein.
Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach
ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from
dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such
as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin. 相似文献
20.
Glucose-6-phosphate dehydrogenase, together with the other enzymesof pentose phosphate pathway, was found in the cytosol as wellas in the plastid from developing castor bean (Ricinus communisL.) seeds. The plastid enzyme was found in both the matrix andthe membrane. The plastid enzyme has a sharp pH profile withthe optimum at 8.5, while the cytosolic enzyme has a broad pHprofile, optimum at 7.5. The plastid enzyme was inactivatedby storage at 0°C and by detergents such as Triton X-100,Brij and Nonidet, but the cytosolic enzyme was not. Slab geldisc electrophoresis indicated that three isoenzymes of glucose-6-phosphatedehydrogenase were found in the plastid but one enzyme in thecytosol of developing castor bean seed. From the presence ofglucose-6-phosphate dehydrogenase in the plastid, the operationof whole pentose phosphate pathway in this organelle of developingcastor bean seeds is suggested. (Received September 21, 1982; Accepted January 17, 1983) 相似文献