Paper strips inoculated with spores of Bacillus stearothermophilus ATCC 7953 were conventionally dried (lot 1) and lyophilized (lot 2); stored in defined environments of 32 and 86% relative
humidity at 10, 25 and 33°C for 210 d; and submitted to moist heat treatments at 121°C. A significant decrease in thermal
resistance from initial starting levels was found for lyophilized bioindicators stored at 86% relative humidity. The respective
average D121°C values were 1.55 ± 0.05 and 1.37 ± 0.10 min for lyophilized bioindicators stored at 32 and 86% relative humidity; and 1.65±0.15min
and 1.57 ± 0.11 min for dried bioindicators stored in the same environments. 相似文献
l-Glutamine amidohydrolase (l-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent
and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on l-glutaminase. In this article, we report the continuous production of extracellular l-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized
in the order-support (Na-alginate) concentration, concentration of CaCl2 for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production
medium, temperature of incubation, and retention time. Parameters optimized under batch mode for l-glutaminase production were incorporated into the continuous production studies. Beads with 12×108 spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed
into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from
the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous
production of l-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under
the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates
that continuous production of the enzyme by Ca-alginate-immobilized spores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of l-glutaminase. 相似文献
Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase
by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (25–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose,
peptone, yeast extract, pH, and K2HPO4), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas
peptone, yeast extract, and K2HPO4 had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According
to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized
in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose,
10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K2HPO4. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization. 相似文献
For optimum fermentation, fermenting xylose into acetic acid by Clostridium thermoaceticum (ATCC 49707) requires adaptation of the strain to xylose medium. Exposed to a mixture of glucose and xylose, it preferentially
consumesxylose over glucose. The initial concentration of xylose in the medium affects the final concentration and the yield
of acetic acid. Batch fermentation of 20 g/L of xylose with 5g/L of yeast extract as the nitrogen source results in a maximum
acetate concentration of 15.2 g/L and yield of 0.76 g of acid/g of xylose. Corn steep liquor (CLS) is a good substitute for
yeast extract and results in similar fermentation profiles. The organism consumes fructose, xylose, and glucose from a mixture
of sugars in batch fermentation. Arabinose, mannose, and galactose are consumed only slightly. This organism loses viability
on fed-batch operation, even with supplementation of all the required nutrients. In fed-batch fermentation with CSL supplementation,
d-xylulose (an intermediate in the xylose metabolic pathway) accumulates in large quantities. 相似文献
The amino acid sequences of xylanase B (XlnB) and xylanase C (XlnC) from Streptomyces lividans show significant homology. However, the temperature optima and stabilities of the two enzymes are quite different. XlnB exhibits
an optimum temperature of 40 °C and retains 50% of its maximum activity at 43 °C, whereas the corresponding values for XlnC
are 60 and 70 °C. To analyze these properties further, as well as to study the effect of the exchange of homologous segments in the C-terminal region, four chimeras designated as BSC, BFC, CSB, and CFB were constructed by substituting
segments from the C-terminal homologous region of XlnB gene with that of XlnC and in turn substituting XlnC gene with that
of XlnB. The purified chimeric enzymes were characterized with respect to pH/temperature activity, stability, and kinetic
parameters. Most of enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were
optimally active at 45–55 °C and pH 7.0. Both Km and kcat values of chimeric enzymes for p-nitrophenyl-β-d-cellobioside were admixtures of both parental enzymes, except that the kcat value of chimeric BFC (2.79 s−1) was higher than that of parental XlnC (1.99 s−1). Notably, thermal stability of chimeric BSC and BFC was increased by 25 and 13 °C separately, as compared to one of parental XlnB, whereas
the thermal stability of chimeric CSB and CFB was decreased by 23 and 21 °C, respectively, as compared to another parental
XlnC. These results suggest that homologous C-terminal region in S. lividans GH11 xylanase appears to play an important role in determining enzyme characteristics, and exchanging of different segments of gene in this region might significantly alter or improve the enzymatic properties such as thermal
stability. 相似文献
γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter synthesized in the central nervous system from glutamate
by glutamate decarboxylase (GAD). It has applications in the production of many drugs. The technology of GABA synthesis by
treating L-glutamic acid with the cells of the gene-engineered GAD superproducer strain of Escherichia coli GAD K10 was developed. Cell growing in the presence of 0.02 mM pyridoxal phosphate (PLP) causes the 2- to 2.5-fold increase of total productivity of the cells. The best way to prepare
the cells for the reaction was their thermal activation by pretreatment for 1 h at 53°C. The optimal conditions for this reaction
were 37°C and pH 4.6. The rate of the enzymatic reaction is the function of acetate concentration with the maximum at 0.5
M acetate. The total amount of GABA synthesized using 1 g of wet cells reached 23–25 g. The final concentration of GABA in
the reaction medium was 280–300 g/L. The yield of the product was about 99%. 相似文献
Inhibition of radial growth and spore germination of Aspergillus niger in media with added chitosan were detected. The highest radial growth inhibition (73%) was determined at 24 h with 3 g · L?1 of chitosan, and the percent inhibition of spore germination was 40% after 13 h of inoculation. Further, the CC50, that is, the concentration at which spore germination is inhibited by 50%, was estimated by probit analysis (3.5 g · L?1). The activation energies, EA were estimated by an Arrhenius model in control and amended chitosan media, obtaining 35.6 and 36.6 kcal · mol?1, respectively. These values were in the same order of magnitude because chitosan as inhibitor was more effective at low temperature (≤ 18 °C). Hence synergism of temperature and chitosan were only observed at 12 and 18 °C. Therefore, the maximal percentage of germinated spores, Smax was also affected by low temperatures in chitosan‐amended media with estimated values lower than 70% at temperatures < 37 °C whereas in control media Smax reached values close to 100%. Scanning electron micrographs showed that chitosan produced spore aggregation and morphological anomalies affecting swelling, germ tube emergence, and polarization.
Chemical modification was evaluated to stabilize pig kidney d-amino acid oxidase (pkDAAO), which is required for analytical determination of d-amino acids. Optimization of modification conditions was performed to obtain high recovery yield and stability, and chemical
modification at 30°C for 12 h with a highly concentrated enzyme solution gave dextran-conjugated pkDAAO with a 70% yield of
activity. pkDAAO was stable at less than 55°C at pH 6.0, while the conjugated enzyme was stable even at 70°C. In addition,
the conjugated enzyme showed decreased Km values for d-amino acids. Because of these outstanding charcteristics, this new material is expected to be available for use as a liquid
assay reagent. 相似文献
Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-dl-alanine. The effects of pH, temperature, and Co2+ concentration on the activity of free and immobilized enzymes were in vestigated along with the operational and the thermal
stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature
for the hydrolysis of N-acyl-l-alanine in the dl-isomer mixture were 8.0 and 65°C, respectively. Co2+ was an activator for the immobilized enzyme in a similarroleas for the free enzyme. Nosignificant loss of activity was observed
for at least 300 h of continuous operation. The yield of l-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation,
but could be completely reactivated by regeneration. 相似文献
The resistance to pulsed light (PL) of spores of Bacillus subtilis strain 168 and of strains with mutations increasing sensitivity to UV‐C or affecting spore structure was evaluated and compared to resistance to continuous UV‐C and moist heat, in order to reveal original mechanisms of inactivation by PL. Spores of B. subtilis strain 168 (1A1) and eight mutant strains (sspA, sspB, sspAB, cotA, gerE, cotE, uvrA and recA) were exposed to PL (up to 1.77 J cm?2), continuous UV‐C (up to 147 mJ cm?2) and moist heat at 90°C. Spores of the strains lacking proteins linked to coat formation or structure (cotA, gerE and cotE) were markedly more sensitive to PL than 1A1, while their sensitivity to continuous UV‐C or to moist heat was similar to the one of strain 1A1. Coat proteins had a major contribution to the resistance of B. subtilis spores to PL irradiation characterized by short‐time and high‐energy pulses of white light in the wavelengths 200–1100 nm. In contrast the role of coat proteins to UV‐C or to moist heat resistance was marginal or null. 相似文献
Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed
by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted
in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa.
The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up. 相似文献
-1,3-d-Glucan is a biologically active component mainly from fungi that has been shown in several studies to be related to respiratory health outcomes from damp building exposures. Here, we report the development and application of a method for the analysis of the glucan extracted in 0.5 N NaOH solution making use of an available preparation of Limulus amebocyte lysate (LAL). The method yields reproducible-1,3-d-glucan measurements from samples of outdoor air, yeast cells, fungal spore preparations and ragweed pollen, and is more sensitive than competing measurements. The LAL-based measurement compared favourably to that based on size-exclusion chromatography using UV and refractive index detection. Growth conditions of the fungi did not materially change the concentrations of glucan in spores indicating that this is a stable property. Glucan content was proportional to spore surface area; however, some species contain higher relative spore glucan contents. 相似文献
Wheat straw hydrolysate produced by enzymatic hydrolysis of hydrothermal pretreated wheat straw at a very high solids concentration of 30% dry matter (w/w) was used for testing the effect of nutrients on their ability to improve fermentation performance of Saccharomyces cerevisiae. The nutrients tested were MgSO4 and nitrogen sources; (NH4)2SO4, urea, yeast extract, peptone and corn steep liquor. The fermentation was tested in a separate hydrolysis and fermentation process using a low amount of inoculum (0.33 g kg?1) and a non-adapted baker’s yeast strain. A factorial screening design revealed that yeast extract, peptone, corn steep liquor and MgSO4 were the most significant factors in obtaining a high fermentation rate, high ethanol yield and low glycerol formation. The highest volumetric ethanol productivity was 1.16 g kg?1 h?1 and with an ethanol yield close to maximum theoretical. The use of urea or (NH4)2SO4 separately, together or in combination with MgSO4 or vitamins did not improve fermentation rate and resulted in increased glycerol formation compared to the use of yeast extract. Yeast extract was the single best component in improving fermentation performance and a concentration of 3.5 g kg?1 resulted in high ethanol yield and a volumetric productivity of 0.6 g kg?1 h?1. 相似文献
A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium
containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH4)H2PO4 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract
of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum
temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively.
Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate
the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application. 相似文献
Complexes of boron trichloride, boron tribromide, and ethylaluminumdichloride with various acetates were directly observed by 1H-NMR. Complexes of secondary and tertiary acetates which model macromolecular active species in polymerization of styrene and isobutene are stable at ?75°C, but decompose at temperatures above ?30°C to yield corresponding chlorides or bromides. The stability of complexes depends on the Lewis acid, the alkyl group in the ester, and the structure of acetate. Rates of the bimolecular exchange of complexes with excess acetate were calculated from dynamic NMR to be kex = 2 × 101 L mol?1 s?1 (?65°C) and kex = 5 × 104 L mol?1 s?1 (?75°C) for 1-phenylethyl acetate with BCl3 and EtAlCl2, respectively. 相似文献
This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis
by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of d- and l-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced
growth rate. At a specific growth rate of 0.048/h, the average yield of dl-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield
of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined
salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, d-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. d-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence
of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption
was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing
the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase
in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic
flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant
Z. mobilis strains, relative to increased byproduct synthesis, is discussed. 相似文献
The ratios of stable carbon isotopes (13C/12C) of ganoderma fruiting body, ganoderma spore, ganoderma spore lipid (GSL) and individual fatty acids in GSL were determined
by gas chromatography–stable isotope ratio mass spectrometry and elemental analysis–stable isotope ratio mass spectrometry.
These values fall into a range from −26.9 to −23.3‰, suggesting that the cut log as the Ganoderma-cultivated substrate in Fujian, China, may belong to C3 plants. Eighteen fatty acids were identified and their abundances
measured by gas chromatography–mass spectrometry in the six GSL samples with C16:0, C18:0, C18:1 and C18:2 as major constituents, and C16:1 is evidently enriched compared with the other edible vegetable oils. On the basis of the compositions of fatty acids and
stable carbon isotopes in GSL, we have developed a novel method to detect the adulteration of GSL products with cheaper edible
vegetable oils. An example of ideal blending between GSL and C4 or C3 vegetable oil is further provided to expound the discrimination
procedures and corresponding sensitive indicators. Simultaneously, the carbon isotope fractionation in the biosynthesis of
individual fatty acids was observed, revealing that the formation of C18:0 from C16:0 in ganodema spores had no conspicuous 13C enrichment of +0.4‰ for Ganoderma sinensis spore and +0.1‰ for G. lucidum spore; the desaturation of C18:0 to C18:1 resulted in a distinct 13C depletion of −1.4‰ for G. sinensis spore and −0.9‰ for G. lucidum spore; and the next desaturation from C18:1 to C18:2 displayed no evident 13C fractionation of −0.1‰ for G. sinensis spore and −0.2‰ for G. lucidum spore.
Figure
Ganoderma lucidum has been widely used in traditional Chinese medicines. Ganoderma spore lipid (GSL) extracted from the spores of G. lucidum has been approved as a health food supplement. However, because of rarity, GSL has become a target for adulteration with
cheaper vegetable oils. 相似文献
Pleurotus ostreatus mushroom mycelium was cultivated in submerged culture in shake-flask experiments with acid extract from peat and yeast extract
as nutrient sources. Different concentrations of water-diluted peat extract were tested in an attempt to overcome the effect
of growth inhibitors apparently present in nondiluted peat extracts. The best results were obtained with a ratio of one part
of peat extract diluted with one part of water. Several operating variables were studied to optimize the growth of mycelial
biomass ofP. ostreatus. The best results produced approximately 5 g/L dry biomass with a yield of 60% and an efficiency of 33%. These results were
obtained in 8 d at 5% (v/v) inoculum ratio, 28°C, pH of 5.0, and 150 rpm. 相似文献
Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12 h, with maximum activity observed at pH 4.5. The highest enzyme activity seen at 55 °C and thermal stability measurements demonstrated that more than 30% activity remained after 30 min incubation at 60 °C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis.
Summary A new, simple, and accurate micellar electrokinetic chromatographic (MEKC) method is described for quantification of hydrocortisone,
hydrocortisone hemisuccinate, hydrocortisone acetate, mystatin, oxytetracycline, Zn-bacitracin, polymyxin B, and lidocaine
in ocular and cutaneous pharmaceutical products. The separation was performed at 25°C and 25 kV, with 15mm phosphate +15mm borate buffer, pH 8.2, and 60mm sodium dodecylsulfate (SDS) in 10∶1 (%,v/v) methanol-water as background electrolyte. Under these conditions the analysis time is approximately 23 min. The method has
been used for quantification of these compounds in different commercial pharmaceutical products and gave good results when
compared with reference spectrophotometric and HPLC methods. 相似文献