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Sae-Jin Kim Nag-Jong Kim Chang-Hun Shin Chan-Wha Kim 《Biotechnology and Bioprocess Engineering》2008,13(2):144-149
The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in
a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate
and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose,
which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50%
until specific stage of bacterial growth (OD600 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until
the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD600 81, respectively. By reproducing these optimized conditions in a 12-m3 fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD600 80. 相似文献
3.
A method for isolation of d-amino acid oxidase (DAAO) from disrupted Trigonopsis variabilis cells has been developed. In an aqueous two-phase system consisting of PEG6000 (220 g l–1), potassium phosphate (110 g l–1, K2HPO4 + KH2PO4 = 10.1:1, mol mol–1) and dl-methionine (11 g l–1), the major portion of cellular proteins (87%) was partitioned into the salt phase. By sequential extraction, 48% of DAAO was recovered in PEG phase, giving a yield of 211 U mg protein–1. 相似文献
4.
Xian-Feng Ma Hui-Min Yu Cheng Wen Hui Luo Qiang Li Zhong-Yao Shen 《World journal of microbiology & biotechnology》2009,25(8):1353-1361
Fusion proteins of d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) with Vitreoscilla Hemoglobin (VHb) and (His)6-tag were constructed and expressed in recombinant Escherichia coli. A fusing-position effect was revealed that (His)6-tag’s N-terminal fusion with TvDAAO (HDAAO) reduced the specific activity by ~29%, while the C-terminal fusion (DAAOH) remained unreduced. The N-terminal
fusion of VHb with TvDAAO and DAAOH significantly improved their activity. As in a 5 l fermentor, the activity of the triple fusion VHb-TvDAAO-(His)6 (VDAAOH) reached 2.53 U/mg dry cell at 9 h, ~58% increase than that of DAAOH together with ~40% biomass increase, confirming
the positive effect of VHb expression on cell level. After purification, the UV–visible and fluorescence spectrum of DAAOH
and VDAAOH were characterized. Enzyme kinetics studies further indicated that VDAAOH behaved a higher K
cat, but a weaker substrate affinity of K
m relative to DAAOH, revealing two distinct impacts of VHb-coupling with TvDAAO on protein level. 相似文献
5.
Jian Yu Dong-Yang Li Ying-Jiu Zhang Sheng Yang Ren-bao Li Zhong-Yi Yuan 《Journal of Molecular Catalysis .B, Enzymatic》2002,18(4-6)
To explore a new approach of high expression of
-amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis (TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 (his−mut+). By means of MM and MD plates and PCR, the recombinant P. pastoris strains (his+mut+) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction. 相似文献
6.
Trigonopsis variabilis D ‐amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7‐aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi‐synthetic cephem antibiotics. Using homology modeling and site‐directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca‐alginate) matrix using 2% alginate, 500 mM CaCl2, and 15 min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca‐alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry. 相似文献
7.
To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml−1 was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l−1. A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml−1 and GL-7-ACA acylase activity of 950 U l−1 were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly. 相似文献
8.
Pollegioni L Molla G Sacchi S Rosini E Verga R Pilone MS 《Applied microbiology and biotechnology》2008,78(1):1-16
D-Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine
adenine dinucleotide-containing flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic,
and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the α-keto acid and ammonia)
and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few
years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase
class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological
applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and
a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis
to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages
of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.
相似文献
9.
Sabine Becker Dorina Vlad Sylvia Schuster Peter Pfeiffer G. Unden 《Archives of microbiology》1997,168(4):290-296
In an oxystat, the synthesis of the fermentation products formate, acetate, ethanol, lactate, and succinate of Escherichia coli was studied as a function of the O2 tension (pO2) in the medium. The pO2 values that gave rise to half-maximal synthesis of the products (pO0.5) were 0.2–0.4 mbar for ethanol, acetate, and succinate, and 1 mbar for formate. The pO0.5 for the expression of the adhE gene encoding alcohol dehydrogenase was approximately 0.8 mbar. Thus, the pO2 for the onset of fermentation was distinctly lower than that for anaerobic respiration (pO0.5≤ 5 mbar), which was determined earlier. An essential role for quinol oxidase bd in microaerobic growth was demonstrated. A mutant deficient for quinol oxidase bd produced lactate as a fermentation product during growth at microoxic conditions (approximately 10 mbar O2), in contrast to the wild-type or a quinol-oxidase-bo-deficient strain. In the presence of nitrate, the amount of lactate was largely decreased. Therefore, under microoxic conditions,
the pO2 appears to be too high for (mixed acid) fermentation to function and too low for aerobic respiration by quinol oxidase bo.
Received: 7 February 1997 / Accepted: 2 May 1997 相似文献
10.
Elaine M. Fisher Mahmood Khan Ronald Salisbury Periannan Kuppusamy 《Cell biochemistry and biophysics》2013,67(2):451-459
We noninvasively monitored the partial pressure of oxygen (pO2) in rat’s small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30 % of blood flow for experimental rats. Noninvasive measurement of pO2 was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO2 for control rats remained stable over the 7-day period. The pO2 on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO2 post banding with a progressive decline to a final pO2 of 20.9 ± 4.5 mmHg (mean ± SE; p = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe? was used to validate the low tissue pO2. Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO2 values had a higher intensity of staining. Thus, monitoring SI pO2 using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO2 in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease. 相似文献
11.
D-Amino acid oxidase (DAAO) is a FAD-dependent enzyme that plays an important role in microbial metabolism, utilization of endogenous D-amino acids, regulation of the nervous system, and aging in mammals. DAAO from yeasts Rhodotorula gracilis and Trigonopsis variabilis are used to convert cephalosporin C into 7-aminocephalosporanic acid, the precursor of other semi-synthetic cephalosporins. This review summarizes the recent data on the enzyme localization, physiological role, gene cloning and expression, and the studies on the enzyme structure, stability, catalytic mechanism, and practical applications.Translated from Biokhimiya, Vol. 70, No. 1, 2005, pp. 51–67.Original Russian Text Copyright © 2005 by Tishkov, Khoronenkova. 相似文献
12.
Moritz Mahling Kerstin Fuchs Wolfgang M. Thaiss Florian C. Maier Martina Feger Daniel Bukala Maren Harant Martin Eichner J?rg Reutershan Florian Lang Gerald Reischl Bernd J. Pichler Manfred Kneilling 《PloS one》2015,10(4)
Tumor hypoxia can be identified by [18F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [18F]FAZA uptake and partial oxygen pressure (pO2) in carcinoma and muscle tissue of air- and oxygen-breathing mice.
Methods
CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O2). We performed 10 min static PET scans 1 h, 2 h and 3 h after [18F]FAZA injection and calculated the [18F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO2 probe in the tumor as well as in the gastrocnemius muscle to measure the pO2 and perfusion.Results
Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [18F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O2: 4.4 vs. IF-O2: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [18F]FAZA uptake in the carcinomas. Invasive pO2 probe measurements yielded enhanced intra-tumoral pO2 values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O2 9.73 mmHg, IF-O2: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).Conclusion
[18F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [18F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO2-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals. 相似文献13.
Bent Borg Jensen 《Archives of microbiology》1990,154(1):92-98
We have investigated the utilization of [14C]-fructose by whole filaments and isolated heterocysts of Anabaena variabilis ATCC 29413, a strain which is capable of fructose-dependent heterotrophic growth. The experimental conditions were chosen such that both transport and subsequent metabolism were studied. The apparent Km for fructose was 60 mM, close to the results of previous studies. Rates of fructose utilization were the same in light and darkness. When photosynthetic CO2 fixation was possible, almost all the label appeared as cell-carbon. In darkness or in the presence of DCMU appreciable amounts of label were released as CO2. Isolated heterocysts with high rates of endogenous metabolism were not capable of utilizing added fructose at significant rates. The effects of oxygen concentration on the metabolism of added fructose in darkness showed that uptake was saturated at low pO2 values. Increasing the pO2 values lead to an increase in the ratio between the lable released as CO2 and that recovred as cell-carbon. These results suggest that fructose is taken up only by the vegetative cells but carbon derived from added fructose can be released as CO2 as a result of respiration in the heterocysts. Fructose utilization was inhibited by uncouplers. The greatest inhibition was found when both (delta) (psi) and (delta) pH were abolished. High concentrations of erythrose inhibited fructose utilization. None of the other potential analogs tested had any effect. 相似文献
14.
Hohe Annette Winkelmann Traud Schwenkel Hans-Georg 《Plant Cell, Tissue and Organ Culture》1999,57(1):39-45
The effect of the relative oxygen partial pressure (pO2) in bioreactors on cell proliferation and subsequent differentiation of somatic embryos from suspension cultures of Cyclamen persicum Mill. was investigated. The growth rate of cell line 3738-VIII in growth-regulator containing medium in bioreactors at 5%
pO2 was slightly reduced in comparison to 10% and 20% pO2. Cultures growing at 40% pO2 had a lower growth rate, a markedly reduced cell viability and showed a decrease of the medium pH to 3.5. Because a pH-control
with a setpoint of 3.3 caused cell death within 4 days, it was assumed, that the reason for the poor cell proliferation and
viability in the cultures at 40% pO2 was an effect of medium acidification rather than of the high O2 partial pressure. A significantly higher number of germinating embryos was obtained from the cultures grown at 40% pO2 than from those grown in flasks or in bioreactors at 5%, 10% and 20% pO2. These results were specific for cell line 3738-VIII. Another cell line, 3736-12, did not show marked differences in cell
proliferation, viability, pH or subsequent regeneration of somatic embryos when grown at different O2 partial pressures.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
15.
Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures
of O2 (pO2; 1–80%, v/v, in N2) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO2 tested. In sub-ambient pO2 (1 and 2.5%) nodules contained specific activities of uricase (urate: O2 oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those
from air. On a cell basis, uninfected cells in nodules from 1% O2 contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in
sub-ambient O2, the activities of other enzymes of ureide synthesis were relatively unaffected by pO2. Short-term effects of pO2 on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO2 (1, 2.5 or 5%, v/v in N2) and15N2. Despite a fall in total15N2 fixation, ureide synthesis and export was maintained at a high level except in 1% O2 where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules
which allow the balance between O2 supply and demand to be maintained over a wide range of pO2, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of
fixed nitrogen.
This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian Development Assistance
Bureau postgraduate fellowship (to F.D.D.). 相似文献
16.
Yiu-Kwok Chan 《Plant and Soil》1986,90(1-3):141-150
Summary A microaerobic diazotrophic bacterium tentatively identified as aPseudomonas species was isolated from a forest soil. Its nitrogenase (C2H2 reduction) activity in liquid medium was significantly supported by phenolic compounds when compared with glucose-, mannitol- or malate-supported activity. The utilization of phenolics was dependent on substrate induction and the appropriate oxygen concentration. At a pO2 of 0.05 protocatechuate was a better carbon source for N2 fixation than glucose. In the case ofLignobacter protocatechuate was a better carbon source for N2 fixation than glucose at pO2 0.2 but not at pO2 0.05. It is suggested that certain monomeric phenols can support nitrogenase activities in many carbon-limited soil environments.Contribution No. 1484 from the Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Canada. 相似文献
17.
When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO2 they show a broad optimum between 20 and 40 kPa O2, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO2 nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO2. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO2 and decrease in resistance at below ambient pO2. Plants grown with root systems at pO2 from 5–40 kPa O2 did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO2 than at low pO2. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele. 相似文献
18.
Gas films on hydrophobic surfaces of leaves of some wetland plants can improve O2 and CO2 exchange when completely submerged during floods. Here we investigated the in situ aeration of rhizomes of cordgrass (Spartina anglica) during natural tidal submergence, with focus on the role of leaf gas films on underwater gas exchange. Underwater net photosynthesis was also studied in controlled laboratory experiments. In field experiments, O2 microelectrodes were inserted into rhizomes and pO2 measured throughout two tidal submergence events; one during daylight and one during night‐time. Plants had leaf gas films intact or removed. Rhizome pO2 dropped significantly during complete submergence and most severely during night. Leaf gas films: (1) enhanced underwater photosynthesis and pO2 in rhizomes remained above 10 kPa during submergence in light; and (2) facilitated O2 entry from the water into leaves so that rhizome pO2 was about 5 kPa during darkness. This study is the first in situ demonstration of the beneficial effects of leaf gas films on internal aeration in a submerged wetland plant. Leaf gas films likely contribute to submergence tolerance of S. anglica and this feature is expected to also benefit other wetland plant species when submerged. 相似文献
19.
d-Amino acid oxidase (DAAO) in mammal kidney regulates the renal reactive oxygen species (ROS) levels directly and plays a
leading role in the development of ROS-mediated renal pathologic damages based on its crucial role in the oxidative deamination
of d-amino acids and the consequent generation of H2O2. Quantitative measurement of DAAO activity in the process of renal ischemia, which could help to understand the molecular
mechanisms of this gripping acute renal disease, was conducted through the determination of chiral substrate by capillary
electrophoresis (CE) in our study. In this study, a chiral ligand exchange CE method was explored with Zn(II)-l-alaninamide complex as the chiral selector to investigate DAAO activity by determining the decreased concentration of the
chiral substrate of DAAO-mediated enzymatic reaction. Then, the change of DAAO activity following 60-min acute renal ischemia
in rats was observed with the proposed method. The study showed that the operation of renal ischemia resulted in a 45.49 ± 8.30%
(n = 8) decrease in the DAAO-induced consumption of substrate, indicating a sharp decrease in renal DAAO activity following
this acute renal injury. This phenomenon, with the possible reason of metabolic acidosis, could pave a new way for the study
of oxidative stress in the development of renal ischemia injury. 相似文献