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1.
研究了从玉米(K02)、披碱草(K09)、多年生黑麦草(K11)、匍匐翦股颖(K12)根际土壤中筛选得到的4株胶质芽胞杆菌(Bacillus mucilaginosus),并对其培养条件以及典型生长曲线进行研究。结果表明,菌株在10~45℃的范围内均可生长,最适生长温度25~30℃;最适生长初始pH值7.5~8.0;菌株K02、K12的最佳通气量为220 mL,菌株K09、K11的最佳通气量为200 mL;并在上述基础上经过进一步试验得到了菌株的典型生长曲线。试验数据对于了解掌握菌株的生长规律以及作为生物菌肥加以利用具有重要的意义。 相似文献
2.
利用PCR技术,从扣囊复膜孢酵母的总DNA中扩增得到β-葡萄糖苷酶(β-Glucosidase)基因(BGL1),长度为2596 bp,连接到pGEM-T载体上,用限制性内切酶切下目的基因,插入到巴斯德毕赤酵母表达载体pPIC9K中,使之位于α-因子信号肽下游,且与之同框,构建成重组质粒pSHL9K.通过电转化将重组质粒pSHL9K插入到Pichia pastoris GS115菌株染色体中,获得高效表达BGL1基因的毕赤酵母重组工程菌株.重组酶的最适温度为50℃,最适pH为5.4.培养基中β-葡萄糖苷酶活性最高可达47U/mL. 相似文献
3.
Purification and characterization of an exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici 总被引:1,自引:0,他引:1
Abstract An exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici , a fungus that produces root rot, was purified by gel filtration and ion exchange chromatography. It had a M r 68 K, a pH optimum of 5.6 and an optimum temperature of 60°C. This polygalacturonase was inhibited by calcium ions and had a K m of 0.64 mM using sodium polypectate as substrate. The exo mode of action of this enzyme was revealed by thin-layer chromatography of hydrolysed substrate. 相似文献
4.
本研究旨在探讨福建荔枝最重要品种兰竹的叶片营养元素适宜含量。统计分析表明,不同地点、年份对同一品种叶片元素含量存在明显的差异;荔枝叶片含氮量变异系数最小,并按磷、镁、钙、钾依次增大。本研究初步提出的丰产兰竹荔枝秋梢叶片营养元素适宜含量为:氮1.5—2.2%,磷0.12—0.18%,钾0.7—1.4%,钙0.3—0.8%,及镁0.18一0.88%;其叶片氮、磷、钾、钙、镁的适宜比例是1:0.08:0.57:0.30:0.12。上列指标可供荔枝营养诊断指导施肥之参考。 相似文献
5.
D P Briskin 《Archives of biochemistry and biophysics》1986,248(1):106-115
The phosphorylation reaction for the plasma membrane ATPase of red beet (Beta vulgaris L.) was examined in order to further understand the mechanism of this enzyme. The level of steady-state phosphorylation had a pH optimum of about 6.0 while ATPase activity (32Pi production) measured under identical conditions had a pH optimum of 7.0. Phosphoenzyme decomposition was accelerated as both the pH and temperature were increased. The former effect may account for the observed difference between the pH optimum for phosphorylation and ATPase. Although the kinetics of K+ stimulation of ATP hydrolysis have been observed to be complex, the kinetics of K+ stimulation of phosphoenzyme turnover were observed to be simple Michaelis-Menten. An antagonism was observed between MgATP and K+ for the stimulation of phosphoenzyme turnover. Increased MgATP concentration reduced the degree of K+ stimulation of phosphoenzyme turnover and ATPase activity. These effects could be explained by the observation that two forms of phosphoenzyme occur during ATP hydrolysis. One form is discharged by ADP while the other form is ADP insensitive. Potassium stimulation of phosphoenzyme breakdown occurs primarily because of effects on the ADP-insensitive phosphoenzyme form. These results are consistent with a mechanism of ATP hydrolysis involving interconversions of conformational states. 相似文献
6.
Chlorferon and diethylthiophosphate (DETP) are the hydrolysis products of coumaphos, an organophosphate pesticide. In this research, two consortia of bacterial cultures, one responsible for degrading chlorferon and the other for degrading DETP, were selectively enriched from waste cattle dip solution. The enriched cultures were used as inocula to grow biomass for biodegradation studies. For chlorferon degradation, the optimum biomass concentration was found to be 80g/L, and pH 7.5 was selected as the optimal operating pH. Chlorferon degradation was characterized by substrate inhibition kinetics with parameter values estimated to be V(m)=0.062+/-0.011mg/(g-biomass)h, K(m)=21+/-7mg/L, and K(Si)=118+/-45mg/L. For DETP degradation, the optimum biomass concentration was found to be 60g/L, and the optimum pH was in the range of 7.5-8. DETP degradation was characterized by Michaelis-Menten kinetics with parameter values estimated to be V(m)=1.52+/-0.10mg/(g-biomass)h and K(m)=610+/-106mg/L. 相似文献
7.
Two extracellular keratinases of Scopulariopsis brevicaulis were purified and partially characterized. The enzymes were isolated by the techniques of gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These keratinases (K I & K II) were purified approximately 33 and 29 fold, respectively. SDS-PAGE of the products of gel filtration chromatography (K I & II) produced only one band each, suggesting homogeneity. The optimum pH for both keratinases was 7.8, while the optimum temperatures were 40°C (K I) and 35°C (K II). Estimated molecular weights were 40–45 KDa and 24–29 KDa for K I & K II respectively. Both keratinases were inhibited by phenylmethylsulfonyl fluoride which suggests a serine residue at or near an active site. 相似文献
8.
The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease responsible for processing the viral polyprotein into functional proteins. The NIa protease exhibits an unusual optimum proteolytic activity at about 16 degrees C. In order to understand the origin of the low-temperature optimum activity, the effects of temperature and salt ions on the catalytic activity and the structure of the NIa protease have been investigated. The analysis of the temperature dependence of k(cat) and K(m) revealed that K(m) decreases more drastically than k(cat) as temperature decreases. The thermodynamic analysis showed that the decrease of K(m) is driven entropically, suggesting a possibility that the substrate binding might need a large entropy cost. The secondary structure of the NIa protease was significantly perturbed at temperatures between 20 and 40 degrees C and the protease was unfolded at very low concentrations of guanidine hydrochloride with a transition midpoint of 0.8 M. These results suggest that the NIa protease is highly flexible in structure. Interestingly, salt ions including NaCl, KCl, CaCl(2) and MgCl(2) stimulated the proteolytic activity by 2-6-fold and increased the optimum temperature to 20-25 degrees C. This stimulatory effect of the salt ions was due to the lowering of K(m). The salt ions promoted the structural rigidity as evidenced in the higher resistance to the heat-induced unfolding in the presence of the salt ions. The increase in rigidity may lead to the lowering of K(m) possibly by reducing the entropic cost for substrate binding. Taken together, these results suggest that the NIa protease is highly flexible in structure and the low-temperature optimum activity might possibly be attributed to lowered entropy cost for substrate binding at lower temperatures. 相似文献
9.
In the presence of vanadate, the optimum pH of renal (Na+, K+)-ATPase in rats is reduced and lies in the range of intracellular pH. This explains the difference in optimum pH observed with ATP extracted from equine muscle. Removal of vanadate from such ATP (with noradrenaline) raises the optimum to the accepted range obtained with synthetic ATP. Changes in the sensitivity of the enzyme to potassium concentration contribute to the alterations in optimum pH. The optimum pH of Mg-ATPase is unaffected by vanadate. Since vanadate may be an intracellular regulator of (Na+, K+)-ATPase changes of optimum pH in relation to intracellular pH could well contribute to the regulation of sodium pump activity. 相似文献
10.
The role of site 342 of endoglucanase II from Trichoderma reesei in catalytic efficiency and pH optima was investigated by site saturation mutagenesis. The mutations identified in this study can be divided into three separate classes according to their amino acid features. When Asn342 was substituted by hydrophobic and non-polar amino acids, most variants exhibited an up-shift in pH optimum and their catalytic efficiency was similar to that of the wild-type at their optimal pH. N342R variant had a pH optimum at 6.2. N342K variant did not give an up-shift in pH optimum, although K and R are both amino acids carrying positive charges. Molecular modelling indicated that residue 342 was located at the C-terminus of one of the α-helices near two catalytic residues. Hydrophobic side chains and more H-bonds would make the helix more rigid, which might affect the stability and activity of the enzyme at higher pH. 相似文献
11.
Kim T Mullaney EJ Porres JM Roneker KR Crowe S Rice S Ko T Ullah AH Daly CB Welch R Lei XG 《Applied and environmental microbiology》2006,72(6):4397-4403
Environmental pollution by phosphorus from animal waste is a major problem in agriculture because simple-stomached animals, such as swine, poultry, and fish, cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from Aspergillus niger PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of the stomach (3.5), where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in Pichia pastoris yeast. The wild-type (WT) PhyA showed the unique bihump, two-pH-optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to the more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (P < 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feed trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase under stomach pH conditions by rational protein engineering. 相似文献
12.
Shifting the pH Profile of Aspergillus niger PhyA Phytase To Match the Stomach pH Enhances Its Effectiveness as an Animal Feed Additive 总被引:1,自引:0,他引:1
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Taewan Kim Edward J. Mullaney Jesus M. Porres Karl R. Roneker Sarah Crowe Sarah Rice Taegu Ko Abul H. J. Ullah Catherine B. Daly Ross Welch Xin Gen Lei 《Applied microbiology》2006,72(6):4397-4403
Environmental pollution by phosphorus from animal waste is a major problem in agriculture because simple-stomached animals, such as swine, poultry, and fish, cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from Aspergillus niger PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of the stomach (3.5), where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in Pichia pastoris yeast. The wild-type (WT) PhyA showed the unique bihump, two-pH-optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to the more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (P < 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feed trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase under stomach pH conditions by rational protein engineering. 相似文献
13.
Some properties of phytase from cotton plant seeds were studied. The phytase activity was shown to increase during seed germination. The enzyme from cotton sprouts is not activated by Ca2+, Mg2+ and K+ and has the pH optimum of 5,0 and temperature optimum of 50 degrees. The molecular weight of the enzyme as determined by gel-filtration is 36000. 相似文献
14.
Abstract A glycerol:NADP+ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K m = 0.01 mM) or NADP+ ( K m = 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination. 相似文献
15.
K Ishikawa I Matsui K Honda S Kobayashi H Nakatani 《Archives of biochemistry and biophysics》1991,289(1):124-129
Porcine pancreatic alpha-amylase (EC 3.2.1.1; abbreviated PPA), which hydrolyzes alpha-D-(1,4) glucosidic bonds in starch and amylose, displays an optimum at pH 6.9 for the majority of substrates. The optimum pH, however, shifted to 5.2 for the hydrolysis of some low molecular substrates (Ishikawa, K., et al., 1990, Biochemistry 29, 7119-7123). Details of the substrate-dependent shift of the optimum pH in PPA were studied by use of a series of maltooligosaccharides with 14C-labeled reducing end glucose as substrates. The optimum pH for maltotriose was 5.2, whereas that for maltopentaose and maltohexaose was unchanged at pH 6.9. The pH profile for the intermediate size substrate maltotetraose showed abnormality; the apparent optimum pH was broadened between 5.5 and 6.5 and the bond cleavage pattern depended on pH, unlike that for the other substrates examined. These results were independent of either buffer systems or substrate concentration. Analyses of the hydrolysates of the maltooligosaccharides revealed that the shift of the optimum pH to the neutral region occurred only when the fifth subsite of PPA in the productive binding modes was occupied by a glucosyl residue of a substrate. The three-catalytic residue model of PPA deduced from the analysis of the hydrolysis of some modified maltooligosaccharides (p-nitrophenyl-alpha-D-maltoside, gamma-cyclodextrin, maltopentaitol, and maltohexaitol) (Ishikawa, K., et al., 1990, Biochemistry 29, 7119-7123) was successfully adapted to the linear maltooligosaccharides used in this work. These results indicate that the different productive binding modes of the linear oligosaccharide substrates affect directly the catalytic power and the optimum pH of PPA. 相似文献
16.
Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations. 相似文献
17.
经PEG-1000处理的高粱根,用不连续蔗糖密度梯度制备获得反转密闭的纯化质膜囊泡,显示一种特殊的ATP酶活力,它不同于H+-ATP酶,其最适PH为7.5,具有高ATP亲和力和较低的K+转运能力。环己酰亚胺和钒酸钠能抑制其活力,表明它是新合成的P-型ATP酶。 相似文献
18.
目的:优选出羊胎免疫调节因子提取的最佳工艺。方法:采用正交实验设计L8(27),以淋巴细胞转化实验为考察指标进行实验,对提取过程中的6个指标进行了优化,确定了最佳提取工艺。结果:羊胎免疫调节因子最佳提取工艺为A1B2C2D1E2F2,即2月龄内羊胎粉碎10 m in,加1:4双蒸水常温下8000 r.m in-1离心后10K超滤膜超滤即得。 相似文献
19.
Phosphorylation of fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) by the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle decreased the K0.5 for fructose-bisphosphate from 21 to 11 microM. When the phosphorylated fructose-bisphosphatase was treated with trypsin the K0.5 increased to 22 microM. The K0.5 also increased when the phosphoenzyme was treated with a partially purified phosphatase from rat liver. There was no difference between the unphosphorylated and phosphorylated enzyme with respect to pH dependence, the pH optimum being about 7.0 for both. Limited treatment of fructose-bis-phosphatase with subtilisin, which cleaves the enzyme at its unphosphorylatable N-terminal part, increased the pH optimum more than limited treatment with trypsin, which releases the phosphorylated peptide at the C-terminal part of fructose-bisphosphatase. The phosphorylated site on the phosphorylated fructose-bisphosphatase was more easily split off by trypsin treatment than the corresponding unphosphorylated site. The results suggest in addition to the glucagon-induced phosphorylation of fructose-bisphosphatase described by Claus et al. [1] that the phosphorylation-dephosphorylation of fructose-bisphosphatase could be of importance for the hormonal regulation of the enzyme in vivo. 相似文献
20.
M De Bortoli C Giunta 《Bollettino della Società italiana di biologia sperimentale》1980,56(22):2381-2385
In Xenopus laevis, the renal Na+/K+-dependent ATPase is a very important enzyme involved in osmoregulatory processes and active transport. The enzyme was obtained from a microsome fraction purified by sucrose discontinuous gradient (10%, 15%, 29.4%) ultracentrifugation after SDS treatment, and concentrated in the denser layer. The assayed biochemical parameters and their values are: 1) Km (ATP): 0.24 mM; 2) K1/2 (Na+): 20.6 mM; 3) K1/2 (K+) 1.6 mM; 4) Ki (ouabain): 0.025 micrometer; 5) optimum pH: 7.2; 6) optimum temperature:" two peaks at 37 degrees C and 45 degrees C. 相似文献