首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.  相似文献   

2.
Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.

1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.

2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low Vmax for the latter substrate.

3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.

4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed.  相似文献   

3.
Sulfur-containing amino acids (L-cysteine or L-cystine) were reacted with D-glucose or pyruvaldehyde at various temperatures and submitted to flavor evaluation. The nuance of the aroma was changed with temperature, and the most acceptable aroma (Japanese rice cracker with sesame-like) was produced at 160°C in all the samples. Volatile compounds produced at 160°C were investigated by gas chromatography and GC–MS coupling. Many compounds such as thiazoles and thiophenes found in the volatiles of some foodstuffs were identified.  相似文献   

4.
The β-d-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β- d-glucosidase activity but also β- d-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000–48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the Km values for p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-fucoside were 1.3mm and 0.7 mm, respectively. This enzyme had also transferase activity for the β-d-fucosyl group but not for the β-d-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-d-glucosidase from other bacteria, actinomycetes, and plants.  相似文献   

5.
Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45°C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.  相似文献   

6.
Simple and speedy purification of Aspergillus oryzae metallo-proteinase was performed using Talopeptin-aminohexyl-Sepharose The properties of the metallo-proteinase were: optimum pH 6.5; pH stability, pH 5~11; optimum temperature,50°C; and molecular weight 42,000 (SDS electrophoresis). These results were similar to those of neutral protease I from Aspergillus oryzae reported by Nakadai et al. This metallo-proteinase was compared with others from microbes using the metallo-proteinase inhibitors FMPI, PLT, and Talopeptin. The metallo-proteinase is unique in the point at which FMPI and PLT gave nearly stoichiometrical inhibition.  相似文献   

7.
l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.  相似文献   

8.
An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn2+ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.  相似文献   

9.
Esterification of l-menthol by lipase is a highly selective method for the resolution of dl-menthol. The present work focuses on the reaction parameters that affect lipase-catalyzed synthesis of l-menthyl acetate in n-hexane using triacetin as acyl donor. Genetically engineered LIP2, an isoform of Candida rugosa lipase, was used as a biocatalyst in the present study. The main objectives of the work were to develop an approach that would enable a better understanding of relationships between the variables (reaction time, temperature, enzyme amount, substrate molar ratio) and the response (molar conversion) for l-menthyl acetate synthesis, and to obtain the optimum conditions for synthesis. By using central composite rotatable design (CCRD) and response surface methodology (RSM) analysis, we found that substrate molar ratio and enzyme amount were the most important variables for the reaction. Based on ridge max analysis, the optimum synthesis conditions were found to be: reaction time 2.2 days, temperature 34.3°C, enzyme amount 0.09 g and substrate molar ratio (dl-menthol:triacetin) 1:1.9, and molar conversion of dl-menthol to l-menthyl acetate was calculated to be 50%. An experiment under optimum conditions was carried out and molar conversion of 48.3% was obtained.  相似文献   

10.
D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat?K m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s?1, and 100 (mmol/L)?1 s?1 respectively.  相似文献   

11.
A protamine-degrading marine bacterium was isolated from marine soil and identified as Aevomonas salmonicida subsp. based on its taxonomical characteristics. An alanine-specific aminopeptidase, called aminopeptidase K, from an extract of the strain was purified and characterized. The aminopeptidase K was purified about 80-fold by fractionation with ammonium sulfate and column chromatography on QA-52 cellulose, Phenyl Superose and Superose 12. The purified enzyme is composed of 6 subunits of 86 kDa with a molecular mass of 520 kDa according to gel filtration and SDS–PAGE. The N-terminal sequence of the enzyme was H · Gly-Gln-GIn-Pro-Gln-Ile-Lys-Try-Tyr-His-Asp-Tyr-Asp-Ala-Pro-Asp-Tyr-Tyr-Ile-Thr-. It is inhibited by monoiodoacetate, N-ethylmaleimide, and puromycin. The Michaelis constant (Km) and the maximal rate of hydrolysis (Vmax) were, respectively, 0.28 mm and 49.4 μmol/min/mg for the l-Ala-β-naphthylamide substrate. The optimum pH and optimum temperature were 6.5 and 45°C, respectively. The purified enzyme was highly specific to l-Ala-β-naphthylamide.  相似文献   

12.
Products of strawberry jam prepared by a high hydrostatic pressure-processing method and a conventional heat-processing method were examined with regard to the changes of quality such as volatile flavor components, anthocyanins, browning index, furfural, sucrose contents, and vitamin C contents during storage at 5°C and 25°C for 1–3 months. The quality of pressure-processed jam immediately after manufacturing was superior to that of heat-processed jam in preserving the fresh flavor and natural color of raw fruits. The superior quality was maintained under low temperature storage for two to three months. However, room temperature storage resulted in rapid deterioration including off-flavor, discoloration, browning, and decomposition of sucrose and vitamin C. So, the commercial value of pressure-processed jam decreased within a brief period. On the other hand, heat-processed jam did not change in quality under room temperature storage for three months. It is supposed that dissolved oxygen and enzyme systems retained are the causative factors of rapid deterioration in pressure-processed jam.  相似文献   

13.
The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS–PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.  相似文献   

14.
l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a Km of 41 mM and a Vmax of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.  相似文献   

15.
Phosphorylation of CMP and formation of CDP-choline were tested with various haploid cells of yeasts. Most of them had more or less the ability, but a mutant (Lys–M7, alpha type) of Saccharomyces rouxii was found to lack the ability. Further study revealed the change of the temperature-sensitivity of the mutant, which could not produce CDP-choline when treated at 37°C, whereas it could at 16°C. The growth of the mutant was more sensitive to temperatures than that of the wild strain. The former did not grow at 36.3°C, while the latter grew.  相似文献   

16.
ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.  相似文献   

17.
D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 °C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.  相似文献   

18.
A putative endo-β-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, β-1,4-D-galactobiose, β-1,4-D-galactotriose, and β-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 °C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 °C.  相似文献   

19.
Sulfated polysaccharides (SPS) were extracted from three species of seaweeds of Ulvacea (Ulva pertusa, Ulva conglobata and Entromorpha prolifera) for 4 hr at various temperatures and their physicochemical properties were studied using viscometric and equilibrium sedimentation measurements in order to determine the optimum extracting condition.

Sulfated polysaccharides extracted at various temperatures from the seaweed of U. pertusa had the same physicochemical properties, while the larger molecular components of SPS was not extracted from U. conglobata and E. prolifera, at the low temperature of 30~40°C. This was confirmed by analyses of their viscosity and molecular weight and by gel filtration chromatography, in which each SPS showed two or three peaks.

The larger molecular component of SPS could be extracted at the high temperature of 80~90°C in the thermostable form.  相似文献   

20.
Acid carboxypeptidase IV from Aspergillus oryzae was purified from the rivanol precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A–50, hydroxylapatite and P-cellulose and gel filtration through Sephadex G–100. The optimum pH is at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme activity was inhibited by sulfhydryl reagents and diisopropylphosphorofluoridate, but was not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 43,000 by gel filtration method.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号