共查询到20条相似文献,搜索用时 15 毫秒
1.
Di Gennaro P Ferrara S Bestetti G Sello G Solera D Galli E Renzi F Bertoni G 《Applied microbiology and biotechnology》2008,79(4):617-625
Novel expression systems for the development of whole-cell biocatalysts were generated. Their novelty consists both in the host, Pseudomonas putida, and in the ability to auto-induce the expression of genes of interest at the exhaustion of the carbon source used for the biomass growth. The auto-induction relies on new expression vectors developed in this study and based on the activator TouR from Pseudomonas sp. OX1, which was shown to mediate the activation of target promoters in an effector-independent growth-phase-dependent manner when the carbon source is exhausted at the onset of the stationary phase. We validated the suitability of these expression systems through the production of (S)-styrene oxide by the styrene monooxygenase from Pseudomonas fluorescens ST. The yields of epoxides produced by these biocatalysts in flask experiments showed to be as efficient as those currently available based on inducible Escherichia coli systems. In addition, a larger scale of biomass production showed no reduction of biocatalysis efficiency. Therefore, the systems developed in this study constitute a valid alternative to current expression systems to use in bioconversion processes. 相似文献
2.
Jan Schüürmann Paul Quehl Gunter Festel Joachim Jose 《Applied microbiology and biotechnology》2014,98(19):8031-8046
Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes. 相似文献
3.
Gene therapy is a promising and rapidly developing field of modern medicine and is expected to improve or even cure the diseases that are incurable with classical therapies. The logics of the development of gene therapy in the nearest future will require the systems wherein a regulation is possible for expression of therapeutic genes. The review considers the currently available regulated gene therapeutic systems, which can be divided into two main classes. One includes the systems wherein external inducers are used to trigger therapeutic gene expression. Systems of the other class are autoregulated and function without an external inducer. The most important first-class expression systems are based on the regulation by tetracycline, rapamycin derivative-induced dimerization, steroid hormones, regulatory RNAs, and physical factors. The most important systems of the second class are regulated by oxygen or glucose levels. 相似文献
4.
Braun M Sun B Anselment B Weuster-Botz D 《Applied microbiology and biotechnology》2012,95(6):1457-1468
Ursodeoxycholic acid is an important pharmaceutical so far chemically synthesized from cholic acid. Various biocatalytic alternatives have already been discussed with hydroxysteroid dehydrogenases (HSDH) playing a crucial role. Several whole-cell biocatalysts based on a 7α-HSDH-knockout strain of Escherichia coli overexpressing a recently identified 7β-HSDH from Collinsella aerofaciens and a NAD(P)-bispecific formate dehydrogenase mutant from Mycobacterium vaccae for internal cofactor regeneration were designed and characterized. A strong pH dependence of the whole-cell bioreduction of dehydrocholic acid to 3,12-diketo-ursodeoxycholic acid was observed with the selected recombinant E. coli strain. In the optimal, slightly acidic pH range dehydrocholic acid is partly undissolved and forms a suspension in the aqueous solution. The batch process was optimized making use of a second-order polynomial to estimate conversion as function of initial pH, initial dehydrocholic acid concentration, and initial formate concentration. Complete conversion of 72?mM dehydrocholic acid was thus made possible at pH?6.4 in a whole-cell batch process within a process time of 1?h without cofactor addition. Finally, a NADH-dependent 3α-HSDH from Comamonas testosteroni was expressed additionally in the E. coli production strain overexpressing the 7β-HSDH and the NAD(P)-bispecific formate dehydrogenase mutant. It was shown that this novel whole-cell biocatalyst was able to convert 50?mM dehydrocholic acid directly to 12-keto-ursodeoxycholic acid with the formation of only small amounts of intermediate products. This approach may be an efficient process alternative which avoids the costly chemical epimerization at C-7 in the production of ursodeoxycholic acid. 相似文献
5.
Ying Zhou Takeshi Minami Kohsuke Honda Takeshi Omasa Hisao Ohtake 《Applied microbiology and biotechnology》2010,87(2):647-655
Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed
by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (ΔcpxA, ΔgcvR, ΔglnL, and an unknown-gene-deleted one (Δuk)) were able to increase the CYP154A1 activity by approximately 1.4–1.7 times compared with that of the control strain. When
new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (ΔcpxA, ΔgcvR, and ΔglnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Δuk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of ΔcpxA, ΔgcvR, and ΔglnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results
indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge,
this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell
biocatalyst. 相似文献
6.
During the last decades a large number of flavin-dependent monooxygenases have been isolated and studied. This has revealed that flavoprotein monooxygenases are able to catalyze a remarkable wide variety of oxidative reactions such as regioselective hydroxylations and enantioselective sulfoxidations. These oxidation reactions are often difficult, if not impossible, to be achieved using chemical approaches. Analysis of the available genome sequences has indicated that many more flavoprotein monooxygenases exist and await biocatalytic exploration. Based on the known biochemical properties of a number of flavoprotein monooxygenases and sequence and structural analyses, flavoprotein monooxygenases can be classified into six distinct flavoprotein monooxygenase subclasses. This review provides an inventory of known flavoprotein monooxygenases belonging to these different enzyme subclasses. Furthermore, the biocatalytic potential of a selected number of flavoprotein monooxygenases is highlighted. 相似文献
7.
8.
9.
10.
Regulated expression of a cell division control-related protein kinase during development 总被引:1,自引:0,他引:1
V J Kidd W Luo J L Xiang F Tu J Easton S McCune M L Snead 《Cell growth & differentiation》1991,2(2):85-93
Protein kinases are important signaling molecules that are known constituents of cellular pathways critical for normal cellular growth and development. We have recently identified a new protein kinase, p58, which contains a large domain that is highly homologous to the cell division control p34cdc2 protein kinase. This new cell division control-related protein kinase was originally identified as a component of semipurified galactosyltransferase; thus, it has been denoted galactosyltransferase-associated protein kinase. In vitro, this protein kinase has been shown to phosphorylate a number of substrates, including histone H1, casein, and galactosyltransferase. In vivo, we have found that this protein kinase affects galactosyltransferase enzyme activity and that it is apparently involved in some aspect of normal cell cycle regulation. In this report, we find that the p58 gene is evolutionarily well conserved and expressed ubiquitously, but to varying extents, in adult tissues. In developmentally staged embryos, p58 expression was elevated early in embryogenesis and then decreased dramatically. In the murine submandibular gland, p58 expression was elevated between day 14 and day 16 post coitus. Expression in the submandibular gland appeared to parallel the proliferation and differentiation of specific cell types as judged by in situ hybridization. These studies indicate that the p58 protein kinase may have a critical function during normal embryonic development and that this protein kinase continues to be expressed in differentiated adult tissues. 相似文献
11.
Nicoli S Gilardelli CN Pozzoli O Presta M Cotelli F 《Gene expression patterns : GEP》2005,5(4):539-544
12.
Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole-cell biocatalysts. 总被引:2,自引:0,他引:2
H L Holland 《Steroids》1999,64(3):178-186
Recent advances in microbial steroid hydroxylation are covered, including new biocatalysts and substrate groups and new methodologies such as the use of low-water systems, immobilised biocatalysts, genetically constructed biocatalysts and enzyme mimics. Mechanistic factors that control the regiochemistry and stereochemistry of steroid hydroxylation are also discussed. 相似文献
13.
During early postnatal development there was an increase in the specific activity of a number of oxidative enzymes localized on the outer and inner mitochondrial membrane. The succinic oxidase complex of the inner mitochondrial membrane, whose activity in 1-day-old rats was 50% of the value in adult animals, attained the maximum on about the 10th day after birth. Activity of the choline and the proline oxidase complex, both of which are also localized in the inner mitochondrial membrane, was minimal in 1-day-old rats and went on rising after the 10th day. Rotenone-insensitive NADH-cytochrome c reductase activity, which is localized on the outer mitochondrial membrane, remained stable up to the 10th day, and rose between the 10th and the 90th day. Developmental changes in monoaminooxidase activity, which is likewise localized on the outer mitochondrial membrane, followed a similar course to the choline and proline oxidase complexes. The amount of cytochromes a+alpha3 and cytochrome b in isolated mitochondria did not alter during development. The protein spectrum of the mitochondrial particles, determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, likewise displayed no marked changes during postnatal development. The above findings show that the metabolic functions of the mitochondria mature during development and that changes in the different enzymes have their own characteristic time course. 相似文献
14.
The world economy is moving toward the use of renewable and nonedible lignocellulosic biomasses as substitutes for fossil sources in order to decrease the environmental impact of manufacturing processes and overcome the conflict with food production. Enzymatic hydrolysis of the feedstock is a key technology for bio-based chemical production, and the identification of novel, less expensive and more efficient biocatalysts is one of the main challenges. As the genomic era has shown that only a few microorganisms can be cultured under standard laboratory conditions, the extraction and analysis of genetic material directly from environmental samples, termed metagenomics, is a promising way to overcome this bottleneck. Two screening methodologies can be used on metagenomic material: the function-driven approach of expression libraries and sequence-driven analysis based on gene homology. Both techniques have been shown to be useful for the discovery of novel biocatalysts for lignocellulose conversion, and they enabled identification of several (hemi)cellulases and accessory enzymes involved in (hemi)cellulose hydrolysis. This review summarizes the latest progress in metagenomics aimed at discovering new enzymes for lignocellulose saccharification. 相似文献
15.
Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids 总被引:1,自引:0,他引:1
Shogo Arai Kazunori Nakashima Takanori Tanino Chiaki Ogino Akihiko Kondo Hideki Fukuda 《Enzyme and microbial technology》2010,46(1):S50
The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF4] or [Bmim][BF4] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF4] for 72 h. The stability of w-ROL in [Bmim][BF4] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts. 相似文献
16.
Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts 总被引:1,自引:0,他引:1
The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD,
EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts
of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing
both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such
as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we
introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant
genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA,
and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced
stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had
enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple
antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for
developing transgenic plants with enhanced tolerance to multiple environmental stresses. 相似文献
17.
Spectrin is the major component of the erythrocyte membrane skeleton and exists as a 526 kDa alphabeta heterodimer. The 246 kDa beta-chain of human spectrin is phosphorylated near the C-terminus, but the exact phosphorylation sites are unknown and the role of this phosphorylation is not fully characterized. In this study, we produced a monoclonal antibody, Sp316, capable of recognizing the C-terminal region of beta-spectrin regardless of its phosphorylation state and used it to purify the phosphorylated region after 2-nitro-5-thiocyanobenzoic acid cleavage of spectrin. Two-dimensional gels, mass spectrometry, and reversed-phase high-performance liquid chromatography were used to characterize these phosphorylation states. Only about 1.5% of spectrin isolated from fresh blood is unphosphorylated, about 9% has more than four phosphates per molecule, and the majority of the protein has one to four phosphates per molecule. A total of six phosphorylation sites were identified by tandem mass spectrometry. Quantitative analysis of the phosphorylation states by reversed-phase high-performance liquid chromatography revealed that phosphorylation of beta-spectrin occurs in a sequential manner where each specific site is completely phosphorylated before the next site is modified. The first phosphorylation event occurs on Ser-2114, followed by Ser-2125, Ser-2123, Ser-2128, Ser-2117, and Thr-2110. The identification of the specific phosphorylated beta-spectrin residues and the ordered sequence of phosphorylation events in vivo should provide an invaluable basis for further studies of the role of these posttranslational modifications in spectrin function in situ. 相似文献
18.
Three different developmental patterns have been found in the heart muscle mitochondria: (a) Activity of inner membrane enzymes, succinate-cytochrome c reductase and rotenone-sensitive NADH-cytochrome c reductase, was found to increase rapidly after birth till the 25th day; no further increase was found till the 60th day. Both brances of the respiratory chain, i.e. NADH-dependent and flavoprotein-linked were found to develop in parallel. (b) Activity of retoenone insensitive-NADH cytochrome c reductase, an outer membrane enzyme, did not show any change during developement. (c) Activity of monoamine oxidase, another outer membrane enzyme, was found to increase after the 10th day of postnatal life and the increase in activity continued till the 60th day. 相似文献
19.
Macpherson PC Thayer RE Rodgers C Taylor AW Noble EG 《Acta physiologica Hungarica》1999,86(2):111-125
The present study was initiated to determine the time course of changes in the profile of selected skeletal muscle myofibril proteins during compensatory overload. Whole muscle isometric contractile properties were measured to assess the physiological consequences of the overload stimulus. Compensatory overload of plantaris muscle of rats was induced by surgical ablation of the synergistic soleus and gastrocnemius muscles. Myosin light chain (LC) and tropomyosin (TM) compositions of control (CP) and overloaded plantaris (OP) muscles were determined by electrophoresis and myofibrillar ATPase assays were performed to assess changes in contractile protein interactions. Within one week of overload decreases in the alpha:beta TM ratio and myofibrillar ATPase activity were observed. Following 30 days of overload, a transition in type II to type I fibres was associated with an increase in slow myosin LC1. Interestingly, after 77 days of overload, the TM subunit ratio returned to one resembling a fast twitch muscle. It is proposed that the early and transitory changes in the TM subunits of OP, as well as the rapid initial depression in maximum tetanic isometric force and myofibrillar ATPase activity may be explained as a result of muscle fibre degeneration-regeneration. We propose that alterations in protein expression induced by compensatory overload reflect both degenerative-regenerative change and increased neuromuscular activity. 相似文献
20.
Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems 总被引:1,自引:0,他引:1
Differential screening of a stage-specific cDNA library of Indica rice has been used to identify two genes expressed in pre-pollination stage panicles, namely OSIPA and OSIPK coding for proteins similar to expansins/pollen allergens and calcium-dependent protein kinases (CDPK), respectively. Northern
analysis and in situ hybridizations indicate that OSIPA expresses exclusively in pollen while OSIPK expresses in pollen as well as anther wall. Promoters of these two anther-specific genes show the presence of various cis-acting elements (GTGA and AGAAA) known to confer anther/pollen-specific gene expression. Organ/tissue-specific activity and
strength of their regulatory regions have been determined in transgenic systems, i.e., tobacco and Arabidopsis. A unique temporal activity of these two promoters was observed during various developmental stages of anther/pollen. Promoter
of OSIPA is active during the late stages of pollen development and remains active till the anthesis, whereas, OSIPK promoter is active to a low level in developing anther till the pollen matures. OSIPK promoter activity diminishes before anthesis. Both promoters show a potential to target expression of the gene of interest
in developmental stage-specific manner and can help engineer pollen-specific traits like male-sterility in plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Accessions: OSIPA cDNA, AF220610; OSIPK cDNA, AF312920; OSIPA partial gene and upstream promoter region, AY166659; OSIPK gene-specific and upstream sequence, AY168440. 相似文献