共查询到20条相似文献,搜索用时 15 毫秒
1.
Pseudomonas putida JS444, isolated from p-nitrophenol (PNP) contaminated waste sites, was genetically engineered to simultaneously degrade organophosphorus pesticides (OP) and PNP. A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringae was used to target the organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida JS444, reducing the potential substrate uptake limitation. Engineered cells were capable of targeting OPH onto the cell surface as demonstrated by western blotting, cell fractionation, and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without instability problems associated with the engineered Moraxella sp. The initial hydrolysis rate was 7.90, 3.54, and 1.53 micromol/h/mg dry weight for paraoxon, parathion, and methyl parathion, respectively. The excellent stability in combination with the rapid degradation rate for organophosphates and PNP make this engineered strain an ideal biocatalyst for complete mineralization of organophosphates. 相似文献
2.
Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Chao Yang Yaran Zhu Jijian Yang Zheng Liu Chuanling Qiao Ashok Mulchandani Wilfred Chen 《Applied microbiology》2008,74(24):7733-7739
Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites. 相似文献
3.
Yang C Cai N Dong M Jiang H Li J Qiao C Mulchandani A Chen W 《Biotechnology and bioengineering》2008,99(1):30-37
Methyl parathion hydrolase (MPH) has been displayed on the surface of microorganisms for the first time using only N- and C-terminal domains of the ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. A shuttle vector pINCM coding for INPNC-MPH was constructed and used to target MPH onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, overcoming the potential substrate uptake limitation. Over 90% of the MPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of the INPNC-MPH fusion was further verified by Western blot analysis and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without growth inhibition. Compared to organophosphorus hydrolase-displaying systems reported, changes in substrate specificity highlight an important potential use of the engineered strain for the clean-up of specific organophosphate nerve agents. 相似文献
4.
An operon encoding enzymes responsible for degradation of the EPA priority contaminant para‐nitrophenol (PNP) from Pseudomonas sp. ENV2030 contains more genes than would appear to be necessary to mineralize PNP. To determine some necessary genes for PNP degradation, the genes encoding the proposed enzymes in the degradation pathway (pnpADEC) were assembled into a broad‐host‐range, BioBricks‐compatible vector under the control of a constitutive promoter. These were introduced into Escherichia coli DH10b and two Pseudomonas putida strains, one with a knockout of the aromatic transport TtgB and the parent with the native transporter. The engineered strains were assayed for PNP removal. E. coli DH10b harboring several versions of the refactored pathway was able to remove PNP from the medium up to a concentration of 0.2 mM; above which PNP was toxic to E. coli. A strain of P. putida harboring the PNP pathway genes was capable of removing PNP from the medium up to 0.5 mM. When P. putida harboring the native PNP degradation cluster was exposed to PNP, pnpADEC were induced, and the resulting production of β‐ketoadipate from PNP induced expression of its chromosomal degradation pathway (pcaIJF). In contrast, pnpADEC were expressed constitutively from the refactored constructs because none of the regulatory genes found in the native PNP degradation cluster were included. Although P. putida harboring the refactored construct was incapable of growing exclusively on PNP as a carbon source, evidence that the engineered pathway was functional was demonstrated by the induced expression of chromosomal pcaIJF. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 相似文献
5.
Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile
monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of
the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed
that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional
to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP.
Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized
metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous
potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations
with inexpensive instrumentation based on detecting green fluorescence.
Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999 相似文献
6.
The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding
family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal
GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the
process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain
areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire
cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and
60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds
was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by
using a fluorescence-labeled CBM. 相似文献
7.
A phenanthrene-utilizing bacterium was anaerobically isolated and identified as Rhodopseudomonas palustris. It tolerated up to 100 mg phenanthrene l−1 and degraded 50% of 50 mg phenanthrene l−1 over 10 days. The presence of phenanthrene caused a prolonged lag phase (2–3 days) in cell growth and affected the photopigments
biosynthesis, while DMSO (the solvent for phenanthrene) had no impact on cell growth. The cell surface hydrophobicity of the
isolate was also increased. 相似文献
8.
Xiangyu Cao Chao Yang Ruihua Liu Qiang Li Wei Zhang Jianli Liu Cunjiang Song Chuanling Qiao Ashok Mulchandani 《Biodegradation》2013,24(2):295-303
A genetically engineered microorganism (GEM) capable of simultaneously degrading organophosphate and organochlorine pesticides was constructed for the first time by display of organophosphorus hydrolase (OPH) on the cell surface of a hexachlorocyclohexane (HCH)-degrading Sphingobium japonicum UT26. The GEM could potentially be used for removing the two classes of pesticides that may be present in mixtures at contaminated sites. A surface anchor system derived from the truncated ice nucleation protein (INPNC) from Pseudomonas syringae was used to target OPH onto the cell surface of UT26, reducing the potential substrate uptake limitation. The surface localization of INPNC–OPH fusion was verified by cell fractionation, western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH was demonstrated by OPH activity assays. Surface display of INPNC–OPH fusion (82 kDa) neither inhibited cell growth nor affected cell viability. The engineered UT26 could degrade parathion as well as γ-HCH rapidly in minimal salt medium. The removal of parathion and γ-HCH by engineered UT26 in sterile and non-sterile soil was also studied. In both soil samples, a mixture of parathion (100 mg kg?1) and γ-HCH (10 mg kg?1) could be degraded completely within 15 days. Soil treatment results indicated that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of multiple pesticide-contaminated environments. 相似文献
9.
Takeshi Fukuda Kouta Tsuchiyama Hirokazu Makishima Katsumi Takayama Ashok Mulchandani Kouichi Kuroda Mitsuyoshi Ueda Shin-ichiro Suye 《Biotechnology letters》2010,32(5):655-659
Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor
system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions
with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater
OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity
than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of
OPH in the cell surface-expression systems. 相似文献
10.
Li Hua Jin Kyuhyuk Kwun Jung-heon Lee 《World journal of microbiology & biotechnology》2008,24(9):1657-1662
A fed-batch fermentation process for the production of organophosphorus hydrolase (OPH) (EC 3.1.8.1) by E. coli pET812 was developed in this research. With batch fermentation, the maximum OPH concentrations attained by batch fermentation
were as low as 4 × 105 U/l because cell growth and OPH production were inhibited by a high initial concentration of glucose. To develop a fed-batch
fermentation process for obtaining higher concentrations of OPH, highly concentrated glucose solution (500 g/l) was added
intermittently or continuously to increase the carbon source concentration. Eventually, 3.2 × 106 U/l of OPH was produced with fed-batch fermentation in 24 h. This was eight times higher than the yield with conventional
batch fermentation. A total concentration of 399–441 mg of OPH was produced/l, which was four times higher than that reported
when using E. coli. Nearly half (44%) of the produced OPH was secreted into the culture solution. 相似文献
11.
12.
Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethyl-benzene as the sole carbon and energy
source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation
of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene
concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μmax) under the optimum conditions were 0.19+0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87+0.13 h−1, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene
isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium
(each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70–80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of
P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability
to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene. 相似文献
13.
Isam A. Mohamed Ahmed Jiro Arima Tsuyoshi Ichiyanagi Emi Sakuno Nobuhiro Mori 《World journal of microbiology & biotechnology》2010,26(8):1455-1464
Soil isolates, identified as Pseudomonas sp. strain A9 and Pseudomonas sp. strain B9b (based on the phenotypic features and phylogenetic analysis) were found to degrade homocholine aerobically.
Morphological characterization using the optical microscope under light and phase contrast conditions showed that cells of
strain A9 formed short rods measuring approximately 0.5–1 × 1.5–2.0 μm in size while those of B9b formed long rods of 0.5–1 × 2.5–3.0 μm
during the early growth phase on both nutrient broth and basal-homocholine (basal-HC) media. Strain A9 was able to grow on
basal-HC medium at a wide range of temperatures (4–41°C) whereas strain B9b was not able to grow at either 4 or 41°C. Comparative
16S rRNA sequencing studies indicated that strain A9 fell into the Pseudomonas putida subclade whereas strain B9b located in Pseudomonas fulva subclade. Washed cells of strains A9 and B9b degraded homocholine completely within 6 h with concomitant formation of several
metabolites. Analysis of the metabolites by capillary electrophoresis, fast atom bombardment–mass spectrometry, and gas chromatography–mass
spectrometry, showed trimethylamine (TMA) as the major metabolite beside β-alanine betaine and trimethylaminopropionaldehyde.
Therefore, the possible degradation pathway of homocholine in the isolated strains is through successive oxidation of the
alcohol group (–OH) to aldehyde (–CHO) and acid (–COOH), and thereafter the cleavage of β-alanine betaine C–N bonds yielding
trimethylamine and an alkyl chain. 相似文献
14.
Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered Escherichia coli Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents. 相似文献
15.
Zahir Ahmad Zahir Usman Ghani Muhammad Naveed Sajid Mahmood Nadeem Hafiz Naeem Asghar 《Archives of microbiology》2009,191(5):415-424
Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains
isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under
axenic conditions at 1, 5, 10 and 15 dS m−1. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m−1. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain
yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m−1. Similarly, chlorophyll content and K+/Na+ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of
these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene.
The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction. 相似文献
16.
Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of
the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation
of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection
between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively,
under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional
symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a
peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A.
thaliana covering the entire range of its genetic diversity, as well as Arabidopsis
halleri, were found to be susceptible towards Cuscuta
reflexa. 相似文献
17.
18.
Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study,
Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally
diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse
azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater
bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by
enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads
to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows
a ping-pong bi–bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance
liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains. 相似文献
19.
Anber Hassanein Latifa Hamama Karine Loridon Noëlle Dorion 《Plant cell reports》2009,28(10):1521-1530
Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses
from a 33-μF capacitor in a 250-V cm−1 electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient
expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast
suspension conductivity of >1,500 μS cm−1 allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing
uidA and nptII genes. When selection (50 mg l−1 kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l−1 kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant
rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated
plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones. 相似文献