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运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a( )上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。 相似文献
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血色红假单胞菌Form Ⅱ Rubisco基因的克隆及序列分析 总被引:1,自引:0,他引:1
根据GenBank中已登录的FormⅡRubisco(cbbM)基因序列,设计一对引物R1和R2,并在引物5′端分别加上SmaI和NotI位点。以血色红假单胞菌1.2352的染色体为模板,通过PCR扩增目的片段,克隆后测序。核苷酸序列测定结果表明,该菌株的cbbM基因全长为1.386bp,共编码461个氨基酸,推导的氨基酸序列与其它生物相应序列的同源性在76%~90%之间。表明了cbbM基因在进化上的保守性。 相似文献
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利用D_泛解酸内酯水解酶N末端序列,并根据NCBI中公布的D_泛解酸内酯水解酶cDNA序列设计了一个特异引物,该引物结合Oligo(dT) 1 5,以串珠镰孢霉(Fusariummoniliforme)CGMCC 0 5 36mRNA反转录得到的总cDNA为模板进行扩增,获得约1 5kb左右的片段,将其克隆到T载体上进行测序,对测得的序列进行分析,重新设计了一对引物,并在引物两端分别加上限制酶EcoRⅠ和SalⅠ的识别位点序列,利用热启动PCR成功地扩增出了D_泛解酸内酯水解酶基因,基因片段长度为114 6bp ,该序列同来源于尖镰孢霉菌(F .oxysporum)AKU 370 2菌株的编码D_泛解酸内酯水解酶cDNA结构基因的同源性为90 0 6 %。将所得片段定向克隆到pTrc99a载体中,转化至JM10 9感受态细胞,筛选出了阳性克隆。经IPTG诱导阳性菌,进行SDS_PAGE电泳,检测出在约4 0kD处有一蛋白表达带。对两株重组基因工程菌的比活力进行测定,结果分别为37U和4 1U。 相似文献
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传染性支气管炎病毒核衣壳蛋白基因克隆及在E.coli中的表达 总被引:6,自引:0,他引:6
核衣壳蛋白基因 (N基因 )是传染性支气管炎病毒的重要结构基因 .根据已报道的序列设计引物 ,利用RT PCR技术从病毒RNA中扩增和克隆到了N基因的cDNA ,并测定了核苷酸序列 .克隆的N基因片段ORF全长 12 30bp ,编码 4 0 9个氨基酸 .将该片段序列与其他IBV病毒株比较 ,核苷酸的同一性为 87 0 %~ 98 6 %,氨基酸的同一性为 91 0 %~ 98 1%.将该cDNA亚克隆到pBV2 2 0表达载体 ,转化大肠杆菌DH5α菌株 ,Western印迹检测 ,获得了分子量约 4 5kD表达蛋白 相似文献
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摘要:【目的】repC为质粒复制必需的起始蛋白基因。本研究旨在对华癸中生根瘤菌菌株HN3015及其质粒消除突变株进行repC基因的克隆和鉴定。【方法】采用通用引物RC1和RC3进行repC基因的PCR扩增,扩增产物克隆到载体pMD-18T,然后测序。利用Southern 杂交对repC基因定位。利用在线软件分析基因的序列特征,BLAST 工具进行同源性搜索;ExPASy推断其氨基酸的序列;ClustalW进行同源核苷酸和氨基酸序列的多重比较分析;PredictProtein 进行蛋白二级结构分析。【结果】 相似文献
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Fillinger S Ruijter G Tamás MJ Visser J Thevelein JM d'Enfert C 《Molecular microbiology》2001,42(1):145-157
In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3-phosphate dehydrogenase (G3PDH)-encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3-phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin-interacting agent calcofluor and the membrane-destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi. 相似文献
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L A Madden A J Anderson D T Shah J Asrar 《International journal of biological macromolecules》1999,25(1-3):43-53
We have identified a range of compounds which, when present during poly(3-hydroxybutyrate) [P(3HB)] accumulation by Ralstonia eutropha (reclassified from Alcaligenes eutrophus), can act as chain transfer agents in the chain termination step of polymerization. End-group analysis by 31P NMR of polymer derivatized with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane revealed that all these compounds were covalently linked to P(3HB) at the carboxyl terminus. All chain transfer agents possessed one or more hydroxyl groups, and glycerol was selected for further investigation. The number-average molecular mass (Mn) of P(3HB) produced by R. eutropha from glycerol was substantially lower than for polymer produced from glucose, and we identified two new end-group structures. These were attributed to a glycerol molecule bound to the P(3HB) chain via the primary or secondary hydroxyl groups. When a primary hydroxyl group of glycerol is involved in chain transfer, the end-group structure is in both [R] and [S] configurations, implying that chain transfer to glycerol is a random transesterification and that PHA synthase does not catalyse chain transfer. 3-Hydroxybutyric acid is the most probable chain transfer agent in vivo, with propagation and termination reactions involving transfer of the P(3HB) chain to enzyme-bound and free 3-hydroxybutyrate, respectively. Only carboxyl end-groups were detected in P(3HB) extracted from exponentially growing bacteria. It is proposed that a compound other than 3-hydroxybutyryl-CoA acts as a primer in the initiation of polymer synthesis. 相似文献
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产甘油假丝酵母甘油代谢关键酶的研究 总被引:15,自引:2,他引:15
本文对产甘油假丝酵母的甘油代谢关键酶进行了研究,发现产甘油假丝酵母同化甘油能力极弱,少量葡萄糖明显改善其同化甘油的能力;线粒体3磷酸甘油脱氢酶受3磷酸甘油的强烈诱导,受葡萄糖代谢的阻遏。在甘油发酵过程中,产甘油假丝酵母胞浆3磷酸甘油脱氢酶酶活处于较高水平并在36h和60h时出现两次酶活高峰,其中第一次酶活峰值水平决定产甘油假丝酵母的甘油合成和积累水平,成为甘油高速积累期(18~48h)甘油合成的关键性的限速酶。在甘油发酵18~48h内,3磷酸甘油酯酶的酶活处于高水平,并在36h时出现酶活峰值;处于缓慢甘油积累阶段的48~72h间,3磷酸甘油酯酶已处于低水平表达,此时,3磷酸甘油酯酶则成为甘油合成的限速酶。产甘油假丝酵母稳定并高表达其胞浆3磷酸甘油脱氢酶基因并且其所表达的3磷酸甘油酯酶酶活远高于胞浆3磷酸甘油脱氢酶这一特征是其高产甘油根本所在。 相似文献
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Nikolay Vassilev Bettina Eichler-Löbermann Antonia Reyes Requena Vanessa Martos Ana Lopez Maria Vassileva 《Reviews in Environmental Science and Biotechnology》2016,15(4):627-638
Biodiesel derived from renewable biological sources has in recent years emerged as an alternative fuel for transportation sector. Similarly, glycerol, the main co-product of biodiesel production, has received considerable attention as feedstock for producing various value added products including biofertilizers. The aim of this review is to highlight the value-added utilization of glycerol as a substrate in fermentation processes for P-solubilization by free and immobilized cells and as a formulation agent in preparation of commercial products. In the majority of cases, glycerol demonstrates multiple traits and, therefore some routine techniques for P-solubilization when based on glycerol can be attractive from technical and economic point of view. 相似文献
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AIMS: Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase. METHODS AND RESULTS: These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels. CONCLUSION: The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type. SIGNIFICANCE AND IMPACT OF THE STUDY: These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol. 相似文献
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1-O-methyl-rac-glycerol (1-O-MG), also known as 3-methoxy-1,2-propanediol is a lipophilic derivative of glycerol, and has been studied as a new cryoprotective agent (CPA) for mononuclear blood cells (MNC), a well-established experimental model in cryopreservation. The chemical modification of the glycerol molecule results in improved cryobiological properties, such as membrane permeability, thus allowing easier handling in the freeze/thaw process. The optimum preincubation period for MNC and 1-O-MG before freezing is 5 min at 4 degree C, resulting in 86% recovery of viable cells, whereas optimal recovery of glycerol-frozen MNC is only guaranteed after 30 min of preincubation at room temperature (74% viable recovery). The optimal concentration of 1-O-MG is 10% (v/v). Although this new agent offers no improvement in cryoprotective properties over dimethyl sulfoxide (Me2SO) there may be possible pharmacological advantages when used in humans. It is, however, obviously superior to glycerol with regard to its permeation kinetics. 1-O-MG might therefore also be of interest in the cryoprotection of other hematopoietic cells and biological tissues. 相似文献
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D P Lee A S Deonarine M Kienetz Q Zhu M Skrzypczak M Chan P C Choy 《Journal of lipid research》2001,42(12):1979-1986
The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions. 相似文献
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Glycerol production by microbial fermentation: a review 总被引:18,自引:0,他引:18
Microbial production of glycerol has been known for 150 years, and glycerol was produced commercially during World War I. Glycerol production by microbial synthesis subsequently declined since it was unable to compete with chemical synthesis from petrochemical feedstocks due to the low glycerol yields and the difficulty with extraction and purification of glycerol from broth. As the cost of propylene has increased and its availability has decreased especially in developing countries and as glycerol has become an attractive feedstock for production of various chemicals, glycerol production by fermentation has become more attractive as an alternative route. Substantial overproduction of glycerol by yeast from monosaccharides can be obtained by: (1) forming a complex between acetaldehyde and bisulfite ions thereby retarding ethanol production and restoring the redox balance through glycerol synthesis; (2) growing yeast cultures at pH values near 7 or above; or (3) using osmotolerant yeasts. In recent years, significant improvements have been made in the glycerol production using osmotolerant yeasts on a commercial scale in China. The most outstanding achievements include: (1) isolation of novel osmotolerant yeast strains producing up to 130 g/L glycerol with yields up to 63% and the productivities up to 32 g/(L day); (2) glycerol yields, productivities and concentrations in broth up to 58%, 30 g/(L day) and 110-120 g/L, respectively, in an optimized aerobic fermentation process have been attained on a commercial scale; and (3) a carrier distillation technique with a glycerol distillation efficiency greater than 90% has been developed. As glycerol metabolism has become better understood in yeasts, opportunities will arise to construct novel glycerol overproducing microorganisms by metabolic engineering. 相似文献
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目的为皮上划痕人用布氏菌活疫苗筛选存活率高、无明胶冻干稳定剂。方法以冻干活菌存活率为指标,对甘油、甘露醇、蔗糖、葡萄糖、乳糖、谷氨酸钠、甘氨酸、谷氨酸、脯氨酸和硫脲等10种稳定剂通过单因素筛选法,筛选出冻干存活率高的4种单因素稳定剂成分;将4种单因素稳定剂成分进行正交试验优化,筛选出最优稳定剂组合。结果单因素试验结果显示,甘油、葡萄糖、谷氨酸钠和硫脲4种稳定剂成分冻干后活菌存活率较高,对布氏菌活疫苗具有良好保护效果。通过正交试验筛选出最优稳定剂配方中四组分的质量分数分别为甘油1.5%、葡萄糖5%、硫脲1.5%、谷氨酸钠1.0%,该配方的冻干存活率可达81.5%。结论无明胶冻干稳定剂对布氏菌活疫苗具有较好的保护作用。 相似文献
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Glycerol metabolism and osmoregulation in the salt-tolerant yeast Debaryomyces hansenii. 总被引:8,自引:2,他引:6 下载免费PDF全文
A glycerol-nonutilizing mutant of the salt-tolerant yeast Debaryomyces hansenii was isolated. When subjected to salt stress the mutant produced glycerol, and the internal level of glycerol increased linearly in proportion to increases of external salinity as in the wild-type strain. However, at increased salinity the mutant showed a more pronounced decrease of growth rate and growth yield and lost more glycerol to the surrounding medium than did the wild type. Uptake experiments showed glycerol to be accumulated against a strong concentration gradient, and both strains displayed similar kinetic parameters for the uptake of glycerol. An examination of enzyme activities of the glycerol metabolism revealed that the apparent Km of the sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5) was increased 330-fold for sn-glycerol 3-phosphate in the mutant. Based on the findings, a scheme for the pathways of glycerol metabolism is suggested. 相似文献