共查询到20条相似文献,搜索用时 15 毫秒
1.
A new and simple method for the purification of extracellular levansucrase from Zymomonas mobilis from highly viscous fermentation broth was developed. After incubation of the fermentation broth with a fructose-polymer cleaving enzyme preparation (Fructozyme, Novozymes, DK) for 48 h, levansucrase precipitated as aggregates and was redissolved in a 3 M urea solution. By ongoing size-exclusion chromatography on Sephacryl S-300 the final levansucrase preparation was purified 100-fold and exhibited a specific activity of 25-35 U/mg(protein). The levansucrase was stable in 3 M urea solution for at least four months without inactivation. To maximize the enzyme yield the dynamic changes of extracellular levansucrase activity during fermentation were investigated. The highest levansucrase activity was observed during the logarithmic phase of growth (15-19 h of fermentation). 相似文献
2.
Dina Rairakhwada Jeong-Woo Seo Mi-young Seo Ohsuk Kwon Sang-Ki Rhee Chul Ho Kim 《Journal of industrial microbiology & biotechnology》2010,37(2):195-204
Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of d-glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase
expressed in Escherichia coli. Optimum pH for d-glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30°C and 4°C, respectively.
The K
m and V
max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression
of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His6-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology
(RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum
levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28°C, starting induction with 0.735%
xylose when A
600 was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain
and the native strain, respectively. 相似文献
3.
M. A. Perez Oseguera L. Guereca A. Lopez-Munguia 《Applied microbiology and biotechnology》1996,45(4):465-471
A polysaccharide-producing bacterium was isolated from cane sugar. It was identified asBacillus circulans and produced levansucrase at pH and temperature optima of 5–7 and 40°C respectively. The enzyme is extracellular and inducible with sucrose. It possesses initial hydrolytic and transferase activities that can be altered by modifying reaction conditions. Levansucrase was recovered from the fermentation broth by extraction with polyethylene glycol (1500 Da). Further purification resulted in an enzyme with a molecular mass of 52 kDa and a pI of 4.7. At high sucrose concentration (300 mM), the transferase activity but not the hydrolase activity were inhibited. Levan increased the transferase activity but had no effect on the hydrolytic activity. The levansucrase had high transferase activity with maltose, galactose and lactose and moderate activity towards sorbitol and glycerol. 相似文献
4.
Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus 总被引:1,自引:0,他引:1
M. Hensing H. Vrouwenvelder C. Hellinga R. Baartmans H. van Dijken 《Applied microbiology and biotechnology》1994,42(4):516-521
The production of extracellular inulinase (\-1,2-d-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40° C. At both temperatures, the final biomass concentration exceeded 100 g·l–1 and more than 2 g enzyme. L–1 of culture supernatant was produced. The biomass yield on O2 at 40° C was substantially lower than at 30°C. Nevertheless, at 40° C a growth rate of 0.20 h–1 could be maintained for a longer period than at 30° C. The unexpected higher O2-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40°C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter. 相似文献
5.
Biedendieck R Beine R Gamer M Jordan E Buchholz K Seibel J Dijkhuizen L Malten M Jahn D 《Applied microbiology and biotechnology》2007,74(5):1062-1073
Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His6- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive
bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin
amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene
sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant
proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins.
After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His6-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified
levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru.
R. Biedendieck and R. Beine contributed equally to this work. 相似文献
6.
Gregory D. Gibb Donald E. Ordaz William R. Strohl 《Applied microbiology and biotechnology》1989,31(2):119-124
Summary
Streptomyces C5-A13, a non-sporulating, pleiotropic mutant of the anthracycline-producing strain, Streptomyces C5, overproduces extracellular proteolytic activity against the substrate azocasein. This extracellular protease activity was produced primarily during the stationary phase. This appears to be an effect related to growth rate rather than to glucose repression, because only very high concentrations of glucose appear to inhibit protease synthesis. Production of extracellular protease activity was stimulated by the presence of carbonate anions in the medium. The optimal concentration of soluble carbonate was 60–80 mM and the stimulation by carbonate was shown not to be due to a pH effect. Approximately 3200–3500 units of extracellular azocaseinase activity were produced per millititre of culture broth using partially optimized fed-batch fermentation processes. This value represents about ninefold greater activity than produced under shake flask conditions. 相似文献
7.
A. Bódalo J. Bastida J. L. Gómez E. Gómez M. L. Asanza I. Alcaraz 《Biotechnology letters》1995,17(8):859-862
Summary The bacterial strain Pseudomonas sp. BA2 did not develop l-aminoacylase activity in the absence of N-acetyl-dl-alanine. The maximum growth rate and enzyme production were obtained when the acetylated amino acid was used as the sole carbon and nitrogen source. A maximum biomass of A660=1.543, after 24 h of cultivation, was obtained. The l-aminoacylase activity reached the maximum value (5.6 U ml–1 broth) in the stationary growth phase. 相似文献
8.
Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg2+, SDS, p-chloromercuribenzoate, Cu2+ and Mn2+ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca2+, Co2+ and Mg2+ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg2+ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin. 相似文献
9.
We have compared the characteristics of nitrate uptake by Aphanothece halophytica grown under non-stress and salt-stress conditions. Both cell types showed essentially similar patterns of nitrate uptake toward ammonium, nitrite, and DL-glyceraldehyde. Although the affinities of nitrate to non-stress cells and salt-stress cells were not significantly different, i.e., Ks = 416 and 450 µM, respectively, the Vmax value for non-stress cells was about twofold of that for salt-stress cells (9.1 vs 5.3 µmol min–1 mg–1 Chl). Nitrate uptake by A. halophytica was found to be dependent on Na+. Ammonium inhibited nitrate uptake, and the presence of methionine sulfoximine could not release the inhibition by ammonium. Nitrite appeared to competitively inhibit nitrate uptake with a Ki value of 84 µM. Both chloride and phosphate anions did not affect nitrate uptake. DL-Glyceraldehyde, an inhibitor of CO2 fixation, caused a reduction in the uptake of nitrate.Received: 22 October 2002 / Accepted: 6 December 2002 相似文献
10.
Rebecca C. Garland Carl F. Cori Hsia-Fei W. Chang 《Molecular and cellular biochemistry》1976,12(1):23-31
Summary Iodoacetamide, N-ethylmaleimide, p-hydroxy-mercuribenzoate (p-MB) and HgCl2 were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2mm. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M2) or crude microsomes exposed to deoxycholate.14C-labelled N-ethylmaleimide was not bound by the M2 protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M2 fraction (specific activity: 2–4µmoles Pi/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 × 10–5
m p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3µmoles14C-p-MB were incorporated into 100 mg M2 protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M2 protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA > barbital > tryptophan > histidine > lysine > other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the Km for glucose-6-phosphate was increased and the Vmax reduced in the presence of p-MB. HgCl2 was a more effective inhibitor than p-MB with a Ki of 6 × 10–6
m. We conclude that a reaction of p-MB with M2 sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.Supported by U.S. Public Health Service Grant No. AM11448-08 and General Research Support Grant No. RR05486-12. 相似文献
11.
Her SL Duan KJ Sheu DC Lin CT 《Journal of industrial microbiology & biotechnology》2004,31(9):427-432
A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9×109 and 3.4×109 cfu ml–1 for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l–1 h–1, respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l–1 h–1, respectively. The results indicate a possible industrial process to culture Bifidobacteria sp. 相似文献
12.
During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l–1 to 0.6 g l–1 KH2PO4 reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l–1 to 71 g l–1 and from 1.36 g l–1 to 0.18 g l–1, respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l–1 KH2PO4 was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production. 相似文献
13.
Shakuntala Ghorai Sumana Mukherjee Suman Khowala 《Biotechnology and Bioprocess Engineering》2011,16(2):297-304
Increased production, secretion, and activity of β-glucosidase in the filamentous fungus Termitomyces clypeatus was achieved in presence of the glycosylation inhibitor 2-deoxy-d-glucose (0.05%, w/v) during submerged fermentation. Enzyme activity increased to 163 U/mL by adding mannose (2 mg/mL) to
the medium. Such a high enzyme activity has not been achieved without mutation or genetic manipulation. The Km and Vmax of the enzyme in culture medium were determined to be 0.092 mM and 35.54 U/mg, respectively, with p-nitrophenyl β-d-glucopyranoside as substrate, confirming its high catalytic activity. The enzyme displayed optimum activity at pH 5.4 and
45°C. The enzyme was fairly stable between acidic to alkaline pH and retained about 75 ∼ 65% residual activities between pH
4 and 10.6 and demonstrated full activity at 45°C for 3 days. The enzyme was also stable in the presence of Zn2+ and Mg2+ and 80% of the residual activity was observed in the presence of Mn2+, Ca2+, K+, Cu2+, EDTA, and sodium azide. Around 70% of the activity was retained in the presence of 2 M guanidium HCl and 3 M urea, whereas
the activity was 5 and 2 times higher in the presence of 4 mM beta-mercaptoethanol and 50 mM DTT, respectively. The enzyme
obtained from the culture filtrate showed potential cellulose saccharifying ability which increased further when supplemented
with commercial cellulase. Thus, this enzyme could be used without any additional downstream processing for commercial cellulase
preparation and production of bioethanol or for other biotechnological applications. 相似文献
14.
S M Kotwal M I Khan J M Khire 《Journal of industrial microbiology & biotechnology》1995,15(2):116-120
The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu2+, Ni2+ and Hg2+ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg2+ strongly inhibited enzyme activity. TheK
m andV
max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min–1 mg–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min–1 mg–1, respectively. 相似文献
15.
Ma CQ Xu P Qiu JH Zhang ZJ Wang KW Wang M Zhang YN 《Applied microbiology and biotechnology》2004,66(1):34-39
A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l–1 cell concentration in the broth was obtained with 20 g l–1 (178 mM) sodium lactate and 4 g l–1 NH4Cl in the medium; and the biotransformation ability was 2.51 units ml–1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g–1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l–1 h–1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform. 相似文献
16.
Liu F Tachibana S Taira T Ishihara M Yasuda M 《Journal of industrial microbiology & biotechnology》2004,31(1):23-28
Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The Km, Vmax, Kcat, and Kcat/Km values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min–1, 291 s–1, and 339 mM–1 s–1, respectively. 相似文献
17.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
18.
Many mammalian cells regulate their volume by the osmotic movement of water directed by anion and cation flux. Ubiquitous volume-dependent anion currents permit cells to recover volume after swelling in response to a hypotonic environment. This study addressed competition between glutamate (Glu) and Cl– permeation in volume-activated anion currents in order to provide insight into the ionic requirements for volume regulation, volume-dependent anion channel activity and to the architecture of the channel pore. The effect of changing the intracellular molar fraction (MF) of Glu and Cl– on conductance and relative anion permeability was evaluated as a function of the extracellular permeant anion and/or the ionic strength. Relative permeability of Glu to Cl– was determined by measuring reversal potentials under defined ionic conditions. Under conditions with high (150 mM) or low (50 mM) ionic strength solutions on both sides of the membrane, Cl– was always more permeable than Glu. When a transmembrane ionic strength gradient (150 mM extracellular: 50 mM intracellular) was set to drive water into the cell, and in the presence of extracellular Cl–, Glu became up to 16-fold more permeable than Cl–. Replacement of extracellular Cl– with Glu abolished this effect. These results indicate that it is possible for Glu to move into the extracellular environment during volume-regulatory events and they support the emerging role of glutamate as a modulator of anion channel activity. 相似文献
19.
Bacillus subtilis strain 316 M was found to produce extracellular alkaline serine proteinase and lectin. The characteristics of proteinase and lectin accumulation during the growth of the producer organism were found to be similar. The maxima of proteolytic and lectin activities were close and observed at 16 h and 14 h of B. subtilis 316 M batch cultivation, respectively. Alkaline serine proteinase was purified by ion exchange chromatography directly from the culture fluid. Proteinase (eluate) purified 40-fold possessed 60–90 units/ml of caseinolytic activity and 240–320 units/ml of elastolytic activity. Eluate obtained after enzyme sorption on the ion exchanger was used for lectin isolation followed by ammonium sulphate precipiration. Lectin purified 12.3-fold was shown to have a high carbohydrate specificity to N-glycolylneuraminic, N-acetylneuraminic, N-acetylmuramic and d-galacturonic acids with minimal inhibiting concentrations of 2.5–7.5 mm. *** DIRECT SUPPORT *** AG903053 00002 相似文献
20.
Christiane Guimont Philippe Henry Guy Linden 《Applied microbiology and biotechnology》1993,39(2):216-220
Streptococcus thermophilus strain CNRZ 455 produces a type II restriction endonuclease designated Sth455I. This enzyme was isolated from cell extracts by anionic and cationic exchange chromatography. This yielded an enzyme preparation free of non-specific nucleases. The optimal reaction conditions for Sth455I are: MgCL2, 30 mm; pH range, 8–9; incubation temperature, 37–42°C; and a high NaCl concentration, 100–200 mm. The results of single- and double-digestion experiments indicates that Sth455I is an isoschizomer of BstNI and EcoRII showing different sensitivity to methylation. The enzyme exhibits restriction activity on the DNA of three bacteriophages of S. thermophilus and no activity on the phage lytic for strain CNRZ 455. The restriction/modification system associated with this strain is discussed. 相似文献