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1.
Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system. 相似文献
2.
Ki Moon Park Min Woo Song Jae Heung Lee 《Biotechnology and Bioprocess Engineering》2008,13(2):197-203
Batch culture kinetics of the red yeast, Xanthophyllomyces dendrorhous SKKU 0107, revealed reduction in biomass with glucose and lower intracellular carotenoid content with fructose. Figures were
different when compared to sucrose, which is a disaccharide of glucose and fructose. In contrast, specific growth rate constant
stayed between 0.094~0.098 h−1, irrespective of the carbon sources employed. Although the uptake rate of glucose was found to be 2.9-fold faster than that
of fructose, sucrose was found to be a more suitable carbon source for the production of carotenoids by the studied strain.
When sugar cane molasses was used, both the specific growth rate constant and the intracellular carotenoid content decreased
by 27 and 17%, respectively. Compared with the batch culture using 28 g/L sugar cane molasses, fed-batch culture with the
same strain resulted in a 1.45-fold higher cell yield together with a similar level of carotenoid content in X. dendrorhous SKKU 0107. 相似文献
3.
Abstract Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32°C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system. 相似文献
4.
Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l−1. However, high initial concentrations of oleic acid up to 50 g l−1 were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml−1 after 48 h with 5 g l−1 of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l−1 and the extracellular lipase activity was 6.3 U ml−1 at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h−1) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l−1 when the set point of specific growth rate (μset) was 0.02 h−1. High specific growth rate (0.04 and 0.08 h−1) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml−1 when μset was 0.02 h−1, while the highest lipase productivity was 0.31 U ml−1 h−1 at μset of 0.08 h−1. 相似文献
5.
Tiejun Liu Shigenobu Miura Tomohiro Arimura Min-Yi Tei Enoch Y. Park Mitsuyasu Okabe 《Biotechnology and Bioprocess Engineering》2005,10(6):522-527
Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose
concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture,
1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h−1. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam,
and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively,
compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production
ofl-lactic acid. 相似文献
6.
Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06,
0.08, and 0.1 h−1. The results show that the highest production ofl-proline was obtained at μ=0.04 h−1. The specificl-proline production rate (Qp) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at
μ=0.08 h−1. Thus, the highest productivity ofl-proline was 1.66 g L−1 h−1 at μ=0.08 h−1. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics. 相似文献
7.
Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable
specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from
a maximum value of 0.21 h−1 (at the beginning of feeding) to 0.15 h−1 prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l−1 and 320 mg l−1, respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase
2.9 times that of the conventional fed-batch method. 相似文献
8.
Liu Y Sha Q Wu S Wang J Yang L Sun W 《Journal of industrial microbiology & biotechnology》2006,33(4):274-282
A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The
epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum
temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the
enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h,
glycerol (20 g L−1) and corn steep liquor (CSL) (30 g L−1) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of
sucrose (2 g L−1 h−1) and continuous feeding of CSL (1 g L−1 h−1) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L−1; maximum EH production was 351.2±13.1 U L−1 with a specific activity of 14.3±0.5 U g−1. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study
presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of
epoxide intermediates. 相似文献
9.
Glucose alone was found to be the most effective carbon source for producing compactin. An initial glucose concentration of
40 g/L gave the highest compactin concentration of 250 mg/L. Among the various nitrogen sources, when 5 g/L of pharmamedia
and soybean meal as the sole nitrogen source were used, respectively, the compactin concentration was higher than 250 mg/L.
Especially, in the case of the mixture of 6 g/L of pharmamedia and 8 g/L of soybean meal, the compactin concentration was
400 mg/L. To select the best surfactant for effective compactin production, various surfactants were investigated. When Triton
X-100 was used, the maximum compactin concentration was 445 mg/L. With the initial concentration ranging from 1.5 to 2.0 g/L,
the compactin concentration was the highest at 465–450 g/L. The cell concentration was similar to that of the control without
the addition of Triton X-100. On the other hand, when the above 4.0 g/L of Triton X-100 were used, the cell concentration
decreased. Using the based results the continuous fed-batch cultures by adding the Triton X-100 were carried out for 10 days
in an air-lift bioreactor. When 1.5 g/L of Triton X-100 was added to the culture broth at 0, 4, and 8 days of culture, respectively,
the compactin production was increased with the increase of culture time. The maximum compactin concentration after 10 days
of culture was 1,200 mg/L, which was about 2.0-fold higher than that of the control without the addition of Triton X-100. 相似文献
10.
Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies
using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose
after batch culture, thereby giving hsBLyS at 3.7 g l−1 and a productivity of 0.11 g l−1 h−1.
Revisions requested 1 September 2005 and 11 November 2005; Revisions received 7 November 2005 and 4 January 2006 相似文献
11.
Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in
a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth
phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations,
nitrogen-growth-limiting conditions induced the accumulation of 30–28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h−1, both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h−1, carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations
provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source
concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism. 相似文献
12.
To develop the easier control method for fed-batch culture of sophorolipid production, we chose rapeseed oil as the most productive oil and compared their productivities in relation to different concentrations of glucose. The optimal concentration of glucose was 30 g/L for sophorolipid production. A fed-batch method was conducted using Candida bombicola ATCC 22214 with rapeseed oil as a secondary substrate. The feeding rate of rapeseed oil was dependent on pH and was calculated by the consumption rate of NaOH and rapeseed oil. The glucose concentration was constantly maintained between 30 and 40 g/L. As a result, we have produced a crude sophorolipid up to 365 g/L for 8 days through a feeding-rate-controlled fed-batch process. 相似文献
13.
Bum-Kyu Lee No-Hwan Park Hai Yon Piao Wook-Jin Chung 《Biotechnology and Bioprocess Engineering》2001,6(5):341-346
For the purpose of mass producingMonascus red pigments optimum medium composition and environmental conditions were investigated in submerged flask cultures. The optimum
carbon and nitrogen sources were determined to be 30 g/L of glucose and 1.5 g/L of monosodium glutamate (MSG). Of the three
metals examined, Fe2+ showed the stronges stimulatory effect on pigment production and some stimulatory effect was also found in Mn2+. Optimum pH and agitation speed were determined to be 6.5 and 700 rpm, respectively. Under the optimum culture conditions
batch fermentation showed that the maximum biomass yield and specific productivity of red pigments were 0.20 g DCW/g glucose
and, 32.5 OD500 g DCW−1 h−1, respectively. 相似文献
14.
Yuan YJ Lu ZX Huang LJ Li Y Lu FX Bie XM Teng YQ Lin Q 《Journal of industrial microbiology & biotechnology》2007,34(8):567-570
Ochrobactrum intermedium DN2 was used to degrade nicotine in tobacco waste extracts. The optimal temperature and pH of nicotine degradation by strain
DN2 was 30–37 °C and 7.0, respectively. Under these optimal conditions, the average degradation rate of nicotine in a 30L
fed-batch culture was 140.5 mg l−1 h−1. The results of this study indicate that strain DN2 may be useful for reducing the nicotine content of reconstituted tobacco. 相似文献
15.
A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed.
Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator
for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was
avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation
activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity
of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity
of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced
by IPTG. 相似文献
16.
S Sivakesava Z.N Xu Y.H Chen J Hackett R.C Huang E Lam T.L Lam K.L Siu R.S.C Wong W.K.R Wong 《Process Biochemistry》1999,34(9):597-900
Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β-
-thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting. 相似文献
17.
Ha SJ Kim SY Seo JH Moon HJ Lee KM Lee JK 《Applied microbiology and biotechnology》2007,76(1):109-116
The production yield of Coenzyme Q10 (CoQ10) from the sucrose consumed by Agrobacterium tumefaciens KCCM 10413 decreased, and high levels of exopolysaccharide (EPS) accumulated after switching from batch culture to fed-batch
culture. Therefore, we examined the effect of sucrose concentration on the fermentation profile by A. tumefaciens. In the continuous fed-batch culture with the sucrose concentration maintained constantly at 10, 20, 30, and 40 g l−1, the dry cell weight (DCW), specific CoQ10 content, CoQ10 production, and the production yield of CoQ10 from the sucrose consumed increased, whereas EPS production decreased as maintained sucrose concentration decreased. The
pH-stat fed-batch culture system was adapted for CoQ10 production to minimize the concentration of the carbon source and osmotic stress from sucrose. Using the pH-stat fed-batch
culture system, the DCW, specific CoQ10 content, CoQ10 production, and the product yield of CoQ10 from the sucrose consumed increased by 22.6, 13.7, 39.3, and 39.3%, respectively, whereas EPS production decreased by 30.7%
compared to those of fed-batch culture in the previous report (Ha SJ, Kim SY, Seo JH, Oh DK, Lee JK, Appl Microbiol Biotechnol,
74:974–980, 2007). The pH-stat fed-batch culture system was scaled up to a pilot scale (300 l), and the CoQ10 production results obtained (626.5 mg l−1 of CoQ10 and 9.25 mg g DCW−1 of specific CoQ10 content) were similar to those obtained at the laboratory scale. Thus, an efficient and highly competitive process for microbial
CoQ10 production is available. 相似文献
18.
Zhi-Yong Zheng Shan-Jing Yao Xiaobei Zhan Chi Chung Lin 《Biotechnology and Bioprocess Engineering》2009,14(1):52-59
Tryptone has multiple and complex effects on cell physiology and process performance in pulse fed-batch cultivation of recombinant
Escherichia coli. By applying feedback control of dissolved oxygen signal responding to pulse in the feed rate, the production of acetate
was avoided and the optimization of production of recombinant human epidermal growth factor (hEGF) was successfully achieved.
With the addition of an optimum amount of tryptone along with glucose in the pulse fedbatch cultivation of E. coli, the ability of the cell to divide and the stability of the plasmid within the bacteria were improved. Consequently, segregation
of the cells into a viable but non-culturable physiological state was alleviated. Addition of tryptone also enhanced cell
respiration before and after hEGF expression and thus further benefited the production of recombinant hEGF. Excessive addition
of tryptone resulted in low sensitivity of the oscillation of dissolved oxygen signal and poor operability of pulse fed-batch
cultivation as this led to an accumulation of acetate, which weakened the dissolved oxygen signal responses. Consequently,
the production of recombinant protein was considerably reduced. By combining the process performance and the positive effect
of complex media pulse addition on bacterial metabolism, the optimal production conditions of hEGF were successfully determined.
A high cell density of 91 g/L dry cell weight was obtained under these optimal production conditions. Furthermore, a high
level of 0.24 g/L hEGF was attained leading to a 32.6% increase in product yield as compared to the controls. 相似文献
19.
Stimulation of menthol production in <Emphasis Type="Italic">Mentha piperita</Emphasis> cell culture 总被引:1,自引:0,他引:1
Amrita Chakraborty Sharmila Chattopadhyay 《In vitro cellular & developmental biology. Plant》2008,44(6):518-524
Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried
out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production
in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium supplemented with 0.2 mg l−1of 2,4-dichlorophenoxy acetic acid to initiate cell suspension culture. This culture was maintained in half-strength MS medium
supplemented with 0.2 mg l−1of 2,4-dichlorophenoxy acetic acid at 15 d interval and used for further studies. Precursor feeding alone, i.e., menthone,
at 35 μM concentration showed slightly improved productivity. γ-Cyclodextrin alone at 60 μM concentration and in combination
with menthone feeding at 35 μM increased menthol yield up to 92 and 110 mg l−1 in comparison to 77 mg l−1 of control culture. Synergistic potentiation effect of menthone feeding at 35 μM and γ-cyclodextrin at 60 μM treatment followed
by in situ adsorption with RP-8 also showed potential stimulation of menthol production in M. piperita cell culture. Fungal elicitor treatment showed enhanced production level up to 140.8 mg l−1 in comparison to that of control. Further studies were carried out with the establishment of Agrobacterium tumefaciens (Ach5) gall-mediated calli, and consequently, cell suspension culture and results showed the significant enhancement of menthol
yield up to 278 mg l−1.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
20.
Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the
4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose
was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB)
homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased
to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell
concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same
feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone
(1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%,
respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions. 相似文献