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1.
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to assess the cell envelope fluidity of Corynebacterium glutamicum 2262 during a temperature-triggered glutamate producing process. Because the fluorescence lifetime of TMA-DPH was shown to be constant all over the process, fluorescence anisotropy can be considered as a good index of cell envelope fluidity. When the temperature of the fed-batch culture was increased from 33 to 39°C to induce glutamate excretion, the fluorescence anisotropy values decreased from 0.212 ± 0.002 to 0.186 ± 0.002 (corresponding to an increase in the cell fluidity), while the specific glutamate production rate reached its maximal value. The increase in fluidity of the C. glutamicum cell envelope was not due to a physical effect related to the temperature elevation, but rather to an alteration of the composition of the cell envelope. Using a mutant devoid of corynomycolates, significant differences in fluorescence anisotropy values were obtained compared to the wild-type strain, suggesting that TMA-DPH is mainly anchored into the corynomycomembrane. Differences in fluorescence anisotropy were also observed when the bacteria were cultivated at 33, 36, 38, and 39°C in batch cultures, and a linear relationship was obtained between the maximum specific glutamate production rate and the measured fluidity. When using the glutamate non-producing variant of C. glutamicum 2262, the fluorescence anisotropy remained constant at 0.207 ± 0.003 whatever the applied temperature shift. This suggests that the fluidity of the Corynebacteria mycomembrane plays an important role in glutamate excretion during the temperature-triggered process.  相似文献   

2.
Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.  相似文献   

3.
The cell wall structure of the Gram-positive Corynebacterium glutamicum was evaluated by electron microscopy of thin sections after freeze-substitution and conventional fixation with glutaraldehyde. For the cell wall an overall thickness of approximately 32 nm was determined, with 8.5 nm corresponding to an outer layer, 6.5 nm to an electron translucent region (ETR) as found in mycobacteria and 17 nm to the peptidoglycan. Knob-like surface structures previously observed in freeze-fracture experiments were detected when cells were conventionally processed with a fixation using glutaraldehyde. By mild treatment with detergents approximately 20 proteins were extracted from the cell wall. From seven of these N-terminal amino acid sequences were determined.  相似文献   

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过氧化氢酶能催化过氧化氢分解为水和氧气,在工业上有着较为广泛的应用。然而,纺织和造纸工业的特殊的高碱性环境,使得开发碱性过氧化氢酶有着重要的应用价值。利用大肠杆菌表达来自于谷氨酸棒杆菌的过氧化氢酶,对其表达条件进行了优化,并通过镍柱亲和层析的方法分离纯化重组蛋白,然后表征纯酶的酶学性质。最适表达条件为:诱导剂IPTG浓度0.2 mmol/L,诱导温度25℃,诱导时间11 h。过氧化氢酶比酶活达到55 266 U/mg,具有较高的催化活性。该酶具有相当宽泛的p H值适应范围,在p H 4.0–11.5范围内均具有较高的酶活性,并在p H 11.0条件下表现出最高的酶活性。将纯酶在p H 11.0的溶液中处理3 h时剩余酶活为93%,说明该酶在高碱条件下有良好的稳定性。该酶最适温度为30℃,在25–50℃热稳定性较好。其动力学参数Km为25.89mmol/L,Vmax为185.18mmol/(min?mg)。抑制剂十二烷基硫酸钠(SDS)、尿素、Na N3、β-巯基乙醇、EDTA对酶活有不同程度的抑制作用。来源于谷氨酸棒杆菌的过氧化氢酶具有较高的催化效率、良好的碱耐受性,在工业生产中有较好...  相似文献   

6.
利用美国Thomson公司旗下的Thomson Data Analyzer和微软公司的Excel等分析工具,从时间、地域、专利权人、技术领域等方面,对德温特专利数据库中全球谷氨酸棒状杆菌领域专利的生命周期、区域布局、竞争机构和技术趋势进行了分析。以期为谷氨酸棒状杆菌技术发展态势提供情报支持,辅助企业、高校制定和调整技术发展战略、完善技术发展布局,在当今激烈的市场竞争环境下保持较高的技术优势。  相似文献   

7.
Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K m of 0.6 mM and a V max of 15 nmol min−1 (mg dw)−1. For the co-transported sodium ions, a relatively low K m of 3.3 mM was determined.  相似文献   

8.
利用生物信息学手段,在GenBank数据库进行氨基酸的同源性检索分析,发现来自谷氨酸棒杆茵(Corynebacterium glutamicum)一功能未确定的ORF序列被注释为假设的海藻糖酶(putative trehalose sesynthase),它与已报道的海藻糖合成酶的氨基酸序列有60%以上的同源性。本研究把这段ORF克隆到大肠杆茵进行表达及进行功能鉴定。实验表明这段ORF序列为一新的海藻糖合成酶基因,其表达产物能将麦芽糖分子转化成海藻糖分子。重组酶性质的初步研究表明重组酶在pH7.0~7.5,30℃转化麦芽糖效率最高。  相似文献   

9.
琥珀酸是一种具有重要应用价值的四碳平台化合物。微生物法发酵生产琥珀酸以其社会、环境和经济优势展现出良好的发展前景。谷氨酸棒杆菌被广泛应用于氨基酸、核苷酸等高附加值化学品的工业化生产,在厌氧条件下细胞处于生长停滞状态,但仍能高效利用碳源合成有机酸,通过代谢工程改造的谷氨酸棒杆菌有望成为理想的琥珀酸生产菌株。结合近年来谷氨酸棒杆菌生产琥珀酸取得的最新成果,本文综述了构建高产琥珀酸工程菌株的代谢工程策略、底物的扩展利用,并展望了将来的研究方向。  相似文献   

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Construction and application of new Corynebacterium glutamicum vectors   总被引:1,自引:0,他引:1  
The construction of new Corynebacterium glutamicum/Escherichia coli shuttle vectors with improved cloning properties and an increased chloramphenicol resistance (50 g ml–1 MIC) is described. A modified glnB gene encoding a Strep-tag II domain modified PII protein was expressed in C. glutamicum and streptavidin affinity chromatography was used to purify this protein.  相似文献   

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周宁一 《微生物学通报》2016,43(11):2539-2539
正自从1957年Kinoshita等首次描述谷氨酸棒杆菌(Corynebacterium glutamicum)为谷氨酸产生菌[1]以来,其已成为用于氨基酸生产的主要菌株。目前,全世界每年利用谷氨酸棒杆菌生产约100万t L-谷氨酸用于食品调味剂和约45万t L-赖氨酸用作食品添加剂[2]。通过谷氨酸棒状杆菌发酵获得谷氨酸的发酵水平已较高,通过进一步优化工艺来提高产量具有较大困难[3]。  相似文献   

14.
AIM: The ultimate aim is to elucidate the molecular mechanisms for glutamate overproduction by Corynebacterium glutamicum. METHODS AND RESULTS: Gene expression in response to the conditions inducing glutamate overproduction was investigated by using a DNA microarray technique. Most genes involved in the EMP pathway, the PPP, and the TCA cycle were downregulated, while five genes that were highly upregulated (NCgl0917, NCgl2944, NCgl2945, NCgl2946, and NCgl2975) were identified under all the three conditions for overproduction that are studied here. Gene products of NCgl2944, NCgl2945, and NCgl2946 were highly homologous to each other, did not resemble any other protein, and have remained uncharacterized thus far. The product of NCgl0917 showed a similarity to a few hypothetical and uncharacterized proteins. NCgl2975 was homologous to metal-binding proteins. CONCLUSIONS: The decrease in the activity of 2-oxoglutarate dehydrogenase complex, a key enzyme that is downregulated during glutamate overproduction, can be mainly attributed to the downregulation of odhA and sucB. Five highly upregulated genes were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Although fermentative production of glutamate has been carried out for more than 45 years, information on the molecular mechanisms of glutamate overproduction is still limited. This study further elucidates these mechanisms.  相似文献   

15.
ABSTRACT

We identified L-cysteine exporter candidates of Corynebacterium glutamicum and investigated the effect of overexpression of the potential L-cysteine exporter genes on L-cysteine production in a recombinant strain of C. glutamicum. Overexpression of NCgl2566 and NCgl0580 resulted in enhanced L-cysteine production in an L-cysteine-producing recombinant strain of C. glutamicum.  相似文献   

16.
The tetracycline resistance region of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium striatum M82B was analyzed in Corynebacterium glutamicum ATCC 13032 and confined to a 4.4-kb SphI-Sa/I DNA fragment. Nucleotide sequence analysis revealed two open reading frames, termed tetA and tetB, specifying proteins of 513 and 528 amino acids, respectively. The deduced amino acid sequences of tetAB displayed similarity to ATP-binding cassette transporters including StrV and StrW of Streptomyces glaucescens which are proposed to play a role in the export of streptomycin-like aminoglycosides. An antibiotic susceptibility screening in C. glutamicum showed that the tetAB genes confer resistance to tetracycline, oxytetracycline and to the structurally and functionally unrelated beta-lactam antibiotic oxacillin.  相似文献   

17.
Transmembrane threonine fluxes (i.e., uptake, diffusion, and carrier-mediated excretion) all contribut-ing to threonine production by a recombinant strain of Corynebacterium glutamicum, were analyzed and quantitated. A threonine-uptake carrier that transports threonine in symport with sodium ions was identified. Under production conditions (i.e., when internal threonine is high), this uptake system catalyzed predominantly threonine/threonine exchange. Threonine export via the uptake system was excluded. Threonine efflux from the cells was shown to comprise both carrier-mediated excretion and passive diffusion. The latter process was analyzed after inhibition of all carrier-mediated fluxes. Threonine diffusion was found to proceed with a first-order rate constant of 0.003 min–1 or 0.004 μl min–1 (mg dry wt.)–1, which corresponds to a permeability of 8 × 10–10 cm s–1. According to this permeability, less than 10% of the efflux observed under optimal conditions takes place via diffusion, and more than 90% must result from the activity of the excretion carrier. In addition, the excretion carrier was identified by (1) inhibition of its activity by amino acid modifying reagents and (2) its dependence on metabolic energy in the form of the membrane potential. Activity of the excretion system depended on the membrane potential, but not on the presence of sodium ions. Threonine export in antiport against protons is proposed. Received: 25 August 1995 / Accepted: 18 October 1995  相似文献   

18.
A bla(VIM-2) metallo-beta-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the bla(VIM-2) determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a bla(VIM-2) determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the bla(VIM-2)-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among bla(VIM-2)-containing integrons in clinical isolates of P. aeruginosa from Europe.  相似文献   

19.
Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.  相似文献   

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