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1.
枯草芽孢杆菌葡萄糖脱氢酶基因的克隆及高效表达   总被引:1,自引:0,他引:1       下载免费PDF全文
扩增了枯草芽孢杆菌中的葡萄糖脱氢酶基因(gdh),构建了诱导型表达载体pET-gdh,导入E.coliBL21(DE3)后获得了高效表达葡萄糖脱氢酶基因的重组菌BL21/pET-gdh。经过诱导时间、诱导温度参数的优化,IPTG诱导后重组菌BL21/pET-gdh的葡萄糖脱氢酶比酶活高达9.65 U/mg蛋白,SDS-PAGE电泳分析表明,重组蛋白质表达量占全菌胞内可溶性蛋白的53%,实现了葡萄糖脱氢酶基因的高效表达,为氧化还原酶催化系统提供高效率的NADP+与NADPH循环奠定了基础。  相似文献   

2.
源自酿酒酵母的老黄酶基因oye2和源自假丝酵母的甲酸脱氢酶基因fdhcb分别成功地在大肠杆菌BL21 (DE3)中过量表达.重组表达的老黄酶OYE2和甲酸脱氢酶FDHCB经Ni-NTA亲和层析获得相应的纯酶.以这两种纯酶构建不对称还原柠檬醛生成香茅醛的催化体系,并优化了其反应条件.优化后的反应体系含有25 mmol/L柠檬醛,100 mmol/L甲酸钠,0.42mg/mL老黄酶OYE2,0.2 U/mL甲酸脱氢酶FDHCB,0.5 mmol/L NAD+以及50 mmol/LPIPES缓冲液(pH 7.0).在pH 7.0和30 ℃下反应10 h,香茅醛得率达92.0%,与没有甲酸脱氢酶辅助辅酶循环时的得率(9.8%)相比,香茅醛得率提高了约9.4倍.  相似文献   

3.
甲酸脱氢酶(FDH)是工业上常用的辅酶NADH再生酶,而天然的FDH主要缺陷是酶活和催化效率低。作者通过序列比对7种不同来源的FDH,发现其中来源于Candida boidinii的FDH的第93位氨基酸为缬氨酸(V),而其余6种来源均为异亮氨酸(I)。进一步根据疏水性大小,构建4个突变体酶V93L、V93I、V93A和V93G。酶学性质研究表明,除了V93L外,突变体酶的酶活随着第93位氨基酸残基的疏水性增强而提高。其中,V93I的比酶活提高22.6%、催化效率(NAD+)提高了133%。利用V93I偶联L-亮氨酸脱氢酶用于不对称还原2-氧代戊酸合成L-正缬氨酸,结果表明V93I能够提高2-氧代戊酸的转化效率。  相似文献   

4.
亮氨酸脱氢酶催化2-酮丁酸生成L-2-氨基丁酸需要辅酶NADH参与,构建Escherichia coli (Leu DH/FDH),通过共表达亮氨酸脱氢酶和甲酸脱氢酶实现了辅酶NADH胞内循环再生。通过产酶条件优化,提高该菌株催化制备L-2-氨基丁酸的效率。结果表明,在5 L发酵罐上,在诱导温度为22℃、诱导剂乳糖质量浓度为8. 0 g/L和诱导时间为17 h的条件下,亮氨酸脱氢酶和甲酸脱氢酶的酶活分别达到79. 2 U/g和216. 1 U/g,催化效果最佳。利用该菌株全细胞为催化剂,耦合苏氨酸脱氨酶,在1 L反应体系中进行了催化反应,L-苏氨酸质量浓度为180 g/L时,无需额外添加辅酶,8 h反应后底物转化率达到99%,L-2-氨基丁酸e. e.值99. 5%以上,时空产率19. 3 g/(L·h)。该研究为建立高效、低成本的L-2-氨基丁酸工业化生产方法提供了基础。  相似文献   

5.
重组L-乳酸脱氢酶在大肠杆菌中的表达、纯化及活性研究   总被引:1,自引:0,他引:1  
乳酸脱氢酶是生物法转化苯丙酮酸为苯乳酸的一种有效的酶。实验克隆到一种新型L-乳酸脱氢酶基因ldhL,来源于Lactobacillus plantarumSK-2(植物乳杆菌SK-2),GenBank接受号为FJ392647。以pET-22b(+)为载体质粒,E.coliBL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白的过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE电泳分析,约在37ku处出现显著的特征蛋白条带;重组LDH的比酶活为0.06U/mg。  相似文献   

6.
重组葡萄糖脱氢酶的酶学性质及其偶联辅酶再生   总被引:1,自引:0,他引:1  
为解决生物催化氧化还原反应中辅酶循环问题,人工合成密码子优化后的嗜酸热源体Thermoplasma acidophilum葡萄糖脱氢酶基因Sygdh,于E.coli BL21(DE3)中表达,粗酶液经镍柱亲和层析获得纯化的重组葡萄糖脱氢酶SyGDH。SDS-PAGE显示相对分子质量为41.0 kDa。酶学性质分析表明:该酶的最适pH值为7.5,在pH 6.0~8.0稳定;最适反应温度为40℃,在55℃以下稳定;最适条件下其比活性达4.5 U/mg;Zn2+对其有明显激活作用;该酶对NADP+的亲和力大于NAD+且对大多数有机溶剂有良好的耐受性;对D-葡萄糖的Km和Vmax值分别为28.2 mmol/L和6.5 U/mg。在葡萄糖脱氢酶与羰基还原酶偶联构建的NADPH辅酶循环体系中,以4-氯乙酰乙酸乙酯为底物,羰基还原酶催化产物4-氯-3-羟基丁酸乙酯的产率为99.0%,是未添加葡萄糖脱氢酶时产物产率的3.11倍,表明重组SyGDH具有为生物催化氧化还原反应提供辅酶NADPH再生的能力。  相似文献   

7.
将来自于Lactobacillus plantarum的突变D-乳酸脱氢酶(D-LDH~(Y52V))和Candida boidinii的甲酸脱氢酶(FDH)进行融合,重组成双功能融合蛋白,为生物法合成(R)-2-羟基-3-苯基丙酸(D-PLA)提供新的方法。利用重叠延伸PCR技术,将对底物苯丙酮酸(PPA)亲合力更高的突变蛋白D-LDH~(Y52V)与FDH通过连接肽(Gly4Ser)3融合,将融合基因克隆至表达载体p ET-28a(+),并转化Escherichia coli BL21(DE3),经IPTG诱导得到高效表达。SDS-PAGE电泳分析结果表明,融合蛋白分子质量为79.7 k Da,在重组菌中获得了正确表达;酶活性测定结果表明,融合蛋白具有D-LDH和FDH的双重生物学活性。在不对称转化PPA合成(R)-2-羟基-3-苯基丙酸(DPLA)的应用中,融合蛋白体系比D-LDH~(Y52V)单独表达体系的D-PLA产量提高了40.71%。通过5 h底物分批补加发酵,D-PLA产量为17.34 g/L,底物转化率为77.64%,生产强度3.47 g/(L·h)。双功能融合蛋白增强了辅酶再生的效率,有效促进了(R)-2-羟基-3-苯基丙酸的不对称合成。  相似文献   

8.
甲酸脱氢酶(formate dehydrogenase,FDH)是NADH循环再生的最佳酶之一,广泛应用于食品、医药和化工等行业。但是野生型甲酸脱氢酶普遍存在酶活低、催化效率差等缺点,导致产品转化率较低,影响产品的工业化生产。为了获得具有更佳催化性能的甲酸脱氢酶,作者以博伊丁假丝酵母(Candida boidinii)来源的甲酸脱氢酶为模板,利用HOTSPOT WIZARD v3.1进行三维结构模拟预测,构建了P68G、Q197K两个突变体,比酶活较野生型分别提高了11%和33%。这是由于P68G氨基酸残基侧链的苯环被氢取代,减少了甲酸盐底物进入口袋的空间位阻;而Q197K侧链酰胺基突变为胺丁基增强了酶的柔性。然而这两个突变点对甲酸脱氢酶的热稳定性产生了负面影响,因此在I239位引入半胱氨酸突变与C262构成二硫键以提高其热稳定性,最终获得一株热稳定性显著提高,比酶活较野生型提高31%、较I239C提高45%的突变株CbFDH Q197K/I239C。通过半理性预测蛋白质结构提高了甲酸脱氢酶的活力和热稳定性,为高效构建性能稳定、还原力强的甲酸脱氢酶提供了的理论基础。  相似文献   

9.
以北京棒杆菌E31染色体DNA为模板,用PCR法扩增了高丝氨酸脱氢酶(HD)基因。将结构基因装载于pET-28a质粒的T7启动子下游,得到了重组表达质粒pET-HD。将其转入大肠杆菌(E.coli)BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导,获得了高效的表达。含表达质粒的菌体胞内粗提液经酶活分析表明,高丝氨酸脱氢酶的活性为不含重组质粒的对照菌的10倍。  相似文献   

10.
乙醛脱氢酶和乙醇脱氢酶是嗜热厌氧乙醇菌Thermoanaerobacter ethanolicus乙醇代谢途径中的关键酶。根据高同源性的NADP(H)依赖型Ⅱ型乙醇脱氢酶(S-ADH)的序列设计引物,从T.ethanolicusJW200基因组中PCR扩增出编码S-ADH的基因,插入带组氨酸标签的pET-20(b),测序获得基因大小为1 059 bp,并在大肠杆菌JM109(DE3)中表达。表达产物经Ni亲和柱提纯达电泳纯。带组氨酸标签的重组NADP(H)依赖型乙醇脱氢酶具乙醇脱氢酶和乙醛脱氢酶双活性,能将乙酰辅酶A转化成乙醇.带组氨酸标签的重组酶酶学性质为乙醛脱氢酶活性最适值为pH 8.4,最适温度70℃;乙醇脱氢酶活性正、逆反应分别最适pH 8.0,最适T 55℃;最适pH8.9,最适为T 60℃。以乙酰辅酶A(Aceyl-CoA)为底物,该酶的Km为3.32 mmol/L,Vmax为12.5μmol/(min.mg)。T.ethanolicusJW200中双活性S-ADH的首次发现,建立了该菌乙醇代谢途径中从乙酰辅酶A到的乙醇的另一条通路。  相似文献   

11.
Fe2+对太湖蓝藻厌氧发酵产甲烷过程中关键酶的影响   总被引:1,自引:0,他引:1       下载免费PDF全文
通过研究不同质量浓度Fe2+对太湖蓝藻厌氧消化过程中相关酶活性的影响,以进一步提高其厌氧产甲烷的能力。结果表明,当添加Fe2+3 mg/L时,反应瓶中甲烷积累产量最高,为986.7mL,比空白对照组提高了43倍。同时,当Fe2+质量浓度为0.5 mg/L时,脱氢酶活性达到118 U,比空白对照组提高了76%;当Fe2+质量浓度为0.5 mg/L时,BAA-蛋白水解酶活性为3126.9 U,比空白对照组提高了83.4%;当Fe2+质量浓度为1 mg/L时,β-葡萄糖苷酶活性达到47 493 U,比空白对照组提高了6.3倍;当Fe2+质量浓度为3 mg/L时,F420摩尔质量为0.2 mmol/g,比空白对照组提高了4.3倍。  相似文献   

12.
This paper describes the development and performance of a new rapid amperometric biosensor for fructose monitoring in food analysis. The biosensor is based on the activity of fructose dehydrogenase (FDH) immobilised into a carbon nanotube paste electrode according to two different procedures. The direct wiring of the FDH in a highly original osmium-polymer hydrogel was found to offer a better enzyme entrapment compared to the immobilisation of the enzyme in an albumin hydrogel. The optimised biosensor required only 5 U of FDH and kept the 80% of its initial sensitivity after 4 months. During this time, the biosensor showed a detection limit for fructose of 1 μM, a large linear range between 0.1 and 5 mM, a high sensitivity (1.95 μA cm−2 mM), good reproducibility (RSD = 2.1%) and a fast response time (4 s).  相似文献   

13.
该研究探讨了姜黄植物饮料(以下简称“姜黄饮”)对KM小鼠的解酒作用及可能的作用机制。构建高浓度酒精致小鼠醉酒模型,通过小鼠防醉试验行为学变化、醉酒小鼠血液乙醇浓度、体内乙醇代谢关键酶的含量或活性及胃肠组织的变化,评价姜黄饮对小鼠的解酒作用。结果显示,姜黄饮高剂量组醉酒潜伏期为235.00 min,与模型组相比显著延长(p<0.05);醒酒时间为232.00 min,与模型组相比显著缩短(p<0.05)。血液乙醇含量为4.21 mg/mL,与模型组相比极显著降低(p<0.01);乙醇脱氢酶、乙醛脱氢酶活力分别为3.74 U/mg prot和8.36 U/mg prot,与模型组相比显著提高(p<0.05);辅酶Ⅰ和还原型辅酶Ⅰ含量分别为0.08 nmol/mg prot和0.39 U/mg prot,NADH/NAD+比值与模型组相比极显著提高(p<0.01);细胞色素P450、谷胱甘肽过氧化物酶含量分别为78.51 pg/mg和1341.00 pg/mg,与模型组相比极显著提高(p<0.01)。胃部、肠组织观察和病理切片结果显示,姜黄饮低、高剂量组可减轻乙醇对小鼠肠道引起的损伤,减少小鼠肠道出血和水肿。上述结果表明,姜黄饮对醉酒小鼠有明显的解酒作用,其作用机制可能与其增强机体乙醇代谢路径关键酶及抗氧化酶的活性,加快体内乙醇代谢速度,保护肝脏及胃肠道有关。  相似文献   

14.
Variability of expression of formate dehydrogenase (FDH) caused by uptake of C-1 compounds was examined by using Arabidopsis thaliana as a model plant. Effects of uptake of several C-1 compounds were evaluated by Northern blot analysis using cDNA of A. thaliana FDH prepared by cloning on the basis of known sequence. As a result, expression of the FDH gene in A. thaliana was not intensely influenced by formic acid, an inherent substrate for FDH, but strongly induced by its reduced form, formaldehyde.  相似文献   

15.
Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.  相似文献   

16.
L-carnitine dehydrogenase (CDH) was partially purified from Pseudomonas putida IAM12014 for the stereospecific reduction of 3-dehydrocarnitine to L-carnitine. CDH and glucose dehydrogenase (GDH) were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of L-carnitine from 3-dehydrocarnitine with NADH regeneration. In the NFMBR, NAD was partially immobilized through rejection by the nanofiltration membrane and effectively regenerated by the conjugation reaction of CDH and GDH. Since 3-dehydrocarnitine was unstable at neutral pH, it was maintained under acidic conditions (pH 0.7) and supplied to the NFMBR separately from the other substrates, glucose and coenzyme NAD. As 50 mM 3-dehydrocarnitine in HCl solution, 0.05 mM NAD, and 100 mM glucose in 0.5 M Tris buffer (pH 8) were continuously supplied to the NFMBR with immobilized CDH (200 U/ml) and GDH (200 U/ml) at the retention time of 80 min and temperature of 25 degrees C, the maximum conversion, reactor productivity, and NAD regeneration number were 78%, 113 g/l/d, and 780, respectively. The half-life of the NFMBR was longer than 500 h.  相似文献   

17.
该研究从库巴德巴利酵母FBKL2.0130中克隆D-阿拉伯糖醇脱氢酶基因ARD,利用闪电克隆技术构建重组质粒Yep-PAK,并将重组质粒导入酿酒酵母(Saccharomyces cerevisiae)W303-1A中,得到重组菌株W303-ARD并成功表达。并对重组菌株和亲本菌株的生长性能进行测定,并对重组D-阿拉伯糖醇脱氢酶酶学性质进行研究。结果表明,与亲本菌株相比,重组菌株的D-阿拉伯糖醇脱氢酶的还原活力从(32.7±0.803) U/mL提高至(122.4±1.012) U/mL,氧化活力从(18.46±0.105) U/mL提高至(97.30±0.826)U/mL。重组D-阿拉伯糖醇脱氢酶的最适反应温度为30 ℃,在25~40 ℃范围内氧化、还原活力均保持稳定。还原反应的最适pH值为9.0,在pH 9.0~11.0范围内比较稳定;氧化反应的最适pH值为7.0,在pH 7.0~8.0的范围内能保持较高的活性。Cu2+、Ba2+、Zn2+均能抑制重组酶活力;Mg2+对重组酶活力无明显影响;Ca2+、Mn2+、Fe3+均能提高重组酶活力。  相似文献   

18.
本研究从马槟榔种子中扩增得到天然植物甜蛋白MabinlinⅡ的全长cDNA序列(M1),并通过特异引物PCR去除部分5'端序列得到修饰型的基因序列(M2).将M1与M2分别克隆至表达载体pET43.1a(+),并分别转化表达宿主菌Escherichia coli BL21(DE3)和Rosetta(DE3),成功构建出...  相似文献   

19.
Allitol was produced from D-fructose via a new NADH-regenerating enzymatic reaction system using D-tagatose 3-epimerase (D-TE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH). D-fructose was epimerized to D-psicose by the D-TE of Pseudomonas cichorii ST-24 and the D-psicose was subsequently reduced to allitol by the RDH of an RDH-constitutive mutant, X-22, derived from Klebsiella pneumoniae IFO 3321. NADH regeneration for the reduction of D-psicose by the RDH was achieved by the irreversible formate dehydrogenase reaction, which allowed the D-psicose produced from d-fructose to be successively transformed to allitol with a production yield from D-fructose of almost 100%. The reactions progressed without any by-product formation. After separation of the product from the reaction mixture by a simple procedure, a crystal of allitol was obtained in a yield exceeding 90%. This crystal was characterized and determined to be allitol by HPLC analysis, its IR and NMR spectra, its melting point, and optical rotation measurement.  相似文献   

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