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1.
Constitutive expression of human angiostatin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture 总被引:1,自引:0,他引:1
Zhang AL Zhang TY Luo JX Chen SC Guan WJ Fu CY Peng SQ Li HL 《Journal of industrial microbiology & biotechnology》2007,34(2):117-122
A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression
of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method
by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained
at 25–30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption
of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16
melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression
of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale
expression of angiostatin and other heterologous proteins.
Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal
Government of China (99-Z-004-001). 相似文献
2.
N Kiran Sree M Sridhar K Suresh I M Banat L Venkateswar Rao 《Journal of industrial microbiology & biotechnology》2000,24(3):222-226
A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS3) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation.
Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L−1 at 30°C. The maximum amount of ethanol produced by immobilized VS3 using 150 g L−1 glucose was only 44 g L−1 after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L−1. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount
of ethanol produced by free cells decreased from 72 g L−1 to 25 g L−1 after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L−1. The maximum amount of ethanol produced by immobilized VS3 cells using 150, 200 and 250 g glucose L−1 was 72.5, 93 and 87 g ethanol L−1 at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226.
Received 16 September 1999/ Accepted in revised form 22 December 1999 相似文献
3.
B H Chung Y J Choi S H Yoon S Y Lee Y I Lee 《Journal of industrial microbiology & biotechnology》2000,24(2):94-99
Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated
in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the
mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production.
Under this condition, 2.8 g L−1 of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification
of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein
expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography,
refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities
of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99.
Received 13 January 1999/ Accepted in revised form 02 October 1999 相似文献
4.
Exopolysaccharide (EPS) production was compared among three strains of lactobacilli. Lactobacillus rhamnosus strain 9595M can be classified among the highest EPS-producing strains of lactic acid bacteria reported to date with a maximum
EPS production of 1275 mg L−1. Under controlled pH, no significant differences in the quantity of EPS produced could be detected between carbon source
(glucose or lactose) or fermentation temperature (32 or 37°C). In milk, strains ATCC 9595M and R produced more than 280 mg L−1 EPS whereas strain Type V produced less than 80 mg L−1 EPS. Journal of Industrial Microbiology & Biotechnology (2000) 24, 251–255.
Received 10 September 1999/ Accepted in revised form 22 December 1999 相似文献
5.
The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal,
groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity
(108 U g−1 dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a
corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH2PO4 (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100
(0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C
respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on
incubation at 55°C for 1 h. In the presence of 1 mM K+ and Zn2+, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on
wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243.
Received 07 June 1999/ Accepted in revised form 18 December 1999 相似文献
6.
Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m2). Cell mass at the MBR reached 22.18 g L−1 as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the
vial culture was μ = 0.385 h− and at the batch culture was μ = 1.13 h− in the exponential period and μ = 0.31 h−1 in the stationary period. In the fed-batch mode was μ = 1.102 h−1 for 6 h with inoculation and declined to μ = 0.456 h−1 with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h−1. The number of viable cells was 6.01 × 1012 cfu L−1 at the batch culture, but increased to 1.15 × 1014 cfu L−1 at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by
6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated
into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR
culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR. 相似文献
7.
Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation 总被引:3,自引:0,他引:3
1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than
the glycerol fermentation. μ
max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l−1). For glycerol at 20 g l−1, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l−1. However, for an initial 1,3-propanediol concentration of 50 g l−1 and glycerol at 70 g l−1, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l−1(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol
concentration higher than the initial inhibitory concentration (65 g l−1). The addition of 1,2-propanediol or 2,3-butanediol (50 g l−1) in the presence of glycerol (50–100 g l−1), showed that 2-diols reduced the μ
max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol
mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking
into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for
optimising production were considered.
Received: 15 November 1999 / Revision received: 1 February 2000 / Accepted: 4 February 2000 相似文献
8.
Biotechnology of succinic acid production and markets for derived industrial products 总被引:29,自引:4,他引:25
Succinic acid, derived from fermentation of agricultural carbohydrates, has a specialty chemical market in industries producing
food and pharmaceutical products, surfactants and detergents, green solvents and biodegradable plastics, and ingredients to
stimulate animal and plant growth. As a carbon-intermediate chemical, fermentation-derived succinate has the potential to
supply over 2.7 × 108 kg industrial products/year including: 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, adipic acid, n-methylpyrrolidone and linear aliphatic esters. Succinate yields as high as 110 g/l have been achieved from glucose by the
newly discovered rumen organism Actinobacillus succinogenes. Succinate fermentation is a novel process because the greenhouse gas CO2 is fixed into succinate during glucose fermentation. New developments in end-product recovery technology, including water-splitting
electrodialysis and liquid/liquid extraction have lowered the cost of succinic acid production to U.S. $ 0.55/kg at the 75 000
tonne/year level and to $ 2.20/kg at the 5000 tonne/year level. Research directions aimed at further improving the succinate
fermentation economics are discussed.
Received: 27 October 1998 / Received revision: 22 January 1999 / Accepted: 22 January 1999 相似文献
9.
P Buzzini 《Journal of industrial microbiology & biotechnology》2000,24(1):41-45
A multivariate statistical approach was employed for the optimization of conditions for carotenoid production by Rhodotorula glutinis DBVPG 3853 from a substrate containing concentrated rectified grape must as the sole carbohydrate source. Several experimental
parameters (carbohydrate, yeast autolysate and salt concentrations, and pH) were tested at two levels by following a fractional
factorial design. Carotenogenesis was most sensitive to both initial pH and yeast autolysate concentration. A Central Composite
Design experiment was then performed by obtaining both second-order polynomial models and isoresponse diagrams where initial
pH and yeast autolysate concentration were considered as variables. In this way it was possible to determine the conditions
(pH = 5.78, yeast autolysate = 4.67 g L−1) which maximize both the concentration of total carotenoids and that of β-carotene (6.9 mg L−1 and 1100 μg L−1 of culture fluid, respectively, after 120 h of fermentation). Journal of Industrial Microbiology & Biotechnology (2000) 24, 41–45.
Received 23 February 1999/ Accepted in revised form 14 September 1999 相似文献
10.
Extracts from brown seaweeds could possibly be fermented to ethanol, particularly seaweeds harvested in the autumn, which
contain high levels of easily extractable laminaran and mannitol. Few microorganisms are able to utilise mannitol as a substrate
for ethanol production and Zymobacter palmae was tested for this purpose. Bacterial growth as well as ethanol yield depended on the amount of oxygen present. Strictly
anaerobic growth on mannitol was not observed. At excessive aeration, a change in the fermentation pattern was observed with
high production of acetate and propionate. Under oxygen-limiting conditions, the bacteria grew and produced ethanol in a synthetic
mannitol medium with a yield of 0.38 g ethanol (g mannitol)−1. Z. palmae was also successfully applied for fermentation of mannitol from Laminaria hyperborea extracts. Journal of Industrial Microbiology & Biotechnology (2000) 24, 51–57.
Received 27 June 1999/ Accepted in revised form 23 September 1999 相似文献
11.
Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey 总被引:3,自引:0,他引:3
Batch and continuous cultivation of Anaerobiospirillum succiniciproducens were systematically studied for the production of succinic acid from whey. Addition of 2.5 g l−1 yeast extract and 2.5 g l−1 polypeptone per 10 g l−1 whey was most effective for succinic acid production from both treated and nontreated whey. When 20 g l−1 nontreated whey and 7 g l−1 glucose were used as cosubstrates, the yield and productivity of succinic acid reached at the end of fermentation were 95%
and 0.46 g (l h)−1, respectively. These values were higher than those obtained using nontreated whey alone [93% and 0.24 g (l h)−1 for 20 g l−1 whey]. Continuous fermentation of A. succiniciproducens at an optimal dilution rate resulted in the production of succinic acid with high productivity [1.35 g (l h)−1], high conversion yield (93%), and higher ratio of succinic acid to acetic acid (5.1:1) from nontreated whey.
Received: 23 July 1999 / Received revision: 17 November 1999 / Accepted: 24 December 1999 相似文献
12.
The influence of culture chamber capacity, medium volume and culture density on the growth yields of lettuce (Lactuca sativa L.) and spearmint (Mentha spicata L.) shoots were determined in an environment containing either 350 or 10,000 μmol mol–1 CO2 after 8 weeks of incubation. High positive correlations occurred between the culture vessel capacity and spearmint fresh
weight, leaf number, root number, and shoot number. Similarly, high positive correlations occurred between culture vessel
capacity and lettuce fresh weight, leaf number, and root number. Higher fresh weights, leaf numbers, and root numbers were
obtained from lettuce and spearmint shoots when cultured in 1-quart Mason jars containing 100- or 150-ml aliquots of medium
compared to jars containing 25- or 50-ml aliquots of medium within an environment containing either 350 or 10,000 μmol mol–1 CO2. High culture density decreased growth yields, and this phenomenon could only be slightly off-set by the employment of an
elevated CO2 environment or larger culture vessels.
Received: 22 December 1998 / Revision received: 2 July 1999 / Accepted: 12 July 1999 相似文献
13.
The growth behavior of Clostridium thermobutyricum JW171K and its production of butyric acid were investigated under continuous cultivation in a recently developed rotary fermentor.
Using low dilution rates (up to 40 times the shortest doubling time), the continuous culture conditions caused metabolic shifts
from butyrate formation to the production of acetate. Using an 18-h volumetric retention time, no true steady state in butyrate
formation was achieved after 22 days, although the optical density was stable. Acetate and butyrate were formed in an oscillatory
mode with an alternating predominance between these two products, indicating an oscillation between the less exergonic acetate-forming
but higher ATP (4ATP mol−1 glucose) forming mode, and the more exergonic butyrate and 3ATP mol−1 glucose forming mode. During the continuous culture drastic changes in cell morphology occurred and, at the lower dilution
rates, long, granulose-containing, filamentous cells with rounded protuberances and swellings were observed. A maximal butyrate
concentration of 18.4 g L−1 and a productivity of about 2.4 g L−1 per h (at 25–27 mM concentration in the broth) were obtained. Journal of Industrial Microbiology & Biotechnology (2000) 24, 7–13.
Received 26 April 1999/ Accepted in revised form 16 August 1999 相似文献
14.
M. C. Loewen X. Liu P. L. Davies A. J. Daugulis 《Applied microbiology and biotechnology》1997,48(4):480-486
Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins
are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its
ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production
was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase
yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and
dissolved oxygen, (2) mixed feeding of methanol and glycerol with Muts clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones. The mixed-feeding/Muts strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l.
Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997 相似文献
15.
Analysis and optimization of a two-substrate fermentation for xylitol production using Candida tropicalis 总被引:1,自引:0,他引:1
Xylitol, a functional sweetener, was produced from xylose using Candida tropicalisATCC 13803. A two-substrate fermentation was designed in order to increase xylitol yield and volumetric productivity. Glucose
was used initially for cell growth followed by conversion of xylose to xylitol without cell growth and by-product formation
after complete depletion of glucose. High glucose concentrations increased volumetric productivity by reducing conversion
time due to high cell mass, but also led to production of ethanol, which, in turn, inhibited cell growth and xylitol production.
Computer simulation was undertaken to optimize an initial glucose concentration using kinetic equations describing rates of
cell growth and xylose bioconversion as a function of ethanol concentration. Kinetic constants involved in the equations were
estimated from the experimental results. Glucose at 32 g L−1 was estimated to be an optimum initial glucose concentration with a final xylose concentration of 86 g L−1 and a volumetric productivity of 5.15 g-xylitol L−1 h−1. The two-substrate fermentation was performed under optimum conditions to verify the computer simulation results. The experimental
results were in good agreement with the predicted values of simulation with a xylitol yield of 0.81 g-xylitol g-xylose−1 and a volumetric productivity of 5.06 g-xylitol L−1 h−1.
Received 16 June 1998/ Accepted in revised form 28 February 1999 相似文献
16.
Acetobacter aceti have been grown on ethanol under inhibitory conditions created by high concentrations of phenol. A defined medium with no
vitamin or amino acid supplements has been used such that ethanol was the sole carbon substrate. The culture temperature was
maintained at 30 °C while the pH was manually controlled to fall within the range 4.5–6.0 during ethanol consumption. Growth
on ethanol at a few thousand milligrams per litre (below the known inhibitory level) resulted in a maximum specific growth
rate of 0.16 h−1 with a 95% yield of acetic acid, followed immediately by acetic acid consumption at a growth rate of 0.037 h−1. Phenol was found to inhibit growth by decreasing both the specific growth rate and the biomass yield during ethanol consumption.
On the other hand, the yield of acetic acid during ethanol consumption and the yield of biomass during acetic acid consumption
remained constant, independent of phenol inhibition. A model is presented and is shown to represent the phenol-inhibited growth
behaviour of A. aceti during both ethanol and acetic acid consumption.
Received: 6 November 1998 / Received revision: 8 February 1999 / Accepted: 12 February 1999 相似文献
17.
L. Escalante I. Ramos I. Imriskova E. Langley S. Sanchez 《Applied microbiology and biotechnology》1999,52(4):572-578
The effect of glucose on growth and anthracycline production by Streptomyces peucetius var. caesius was examined in a chemically defined medium. Glucose concentrations above 100 mM inhibited anthracycline synthesis in the
original strain without causing significant change in growth and final pH values. This effect was observed when the carbohydrate
was added initially or after 24 h fermentation, but not when added during the stationary growth phase. When the microorganism
was pregrown in 100 mM glucose and then transferred to a resting cell system with 444 mM glucose, no significant differences
in antibiotic production were observed compared to the control without glucose. The negative effect of glucose on antibiotic
synthesis was not observed in a mutant (2-dogR–21) resistant to growth inhibition by 2-deoxyglucose. Glucose consumption by this mutant was approximately 30% of that utilized
by the original strain. Compared to the original strain, the mutant 2-dogR–21 exhibited a reduction of 50% in glucose transport and an 85% decrease in glucose kinase activity. The experimental evidence
obtained suggests that glucose represses anthracycline formation in a transitory manner and that this effect is related to
glucose transport and phosphorylation.
Received: 15 January 1999 / Received revision: 7 April 1999 / Accepted: 1 May 1999 相似文献
18.
H-J Son K-K Kim H-S Kim Y-K Kim S-J Lee 《Journal of industrial microbiology & biotechnology》2000,24(1):36-40
Pseudomonas sp EL-2 was cultivated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from a structurally unrelated
carbon source, glucose, by a fed-batch culture technique. Variation of the carbon to nitrogen (C/N) ratio of the medium produced
optimal P(3HB-co-3HV) production at a C/N ratio of 95. Production of P(3HB-co-3HV) was favored by a dissolved oxygen tension
of 40%. A maximum biomass concentration of 38 g L−1 containing 53% P(3HB-co-3HV) was achieved after 45 h of cultivation. This corresponds to a volumetric productivity of 0.84 g
L−1 h−1. The copolymer contained 7.5 mol% 3-hydroxyvalerate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 36–40.
Received 28 January 1999/ Accepted in revised form 11 September 1999 相似文献
19.
S H de Kock J C du Preez S G Kilian 《Journal of industrial microbiology & biotechnology》2000,24(4):231-236
Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose
oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations
in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous
respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the
three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L−1 from 20 g glucose L−1 with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast,
in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L−1) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics
and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation
of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236.
Received 09 August 1999/ Accepted in revised form 18 December 1999 相似文献
20.
Anaerobic bioconversion of cellulose by Ruminococcus albus, Methanobrevibacter smithii, and Methanosarcina barkeri 总被引:1,自引:0,他引:1
A system is described that combines the fermentation of cellulose to acetate, CH4, and CO2 by Ruminococcus albus and Methanobrevibacter smithii with the fermentation of acetate to CH4 and CO2 by Methanosarcina barkeri to convert cellulose to CH4 and CO2. A cellulose-containing medium was pumped into a co-culture of the cellulolytic R. albus and the H2-using methanogen, Mb. smithii. The effluent was fed into a holding reservoir, adjusted to pH 4.5, and then pumped into a culture of Ms. barkeri maintained at constant volume by pumping out culture contents. Fermentation of 1% cellulose to CH4 and CO2 was accomplished during 132 days of operation with retention times (RTs) of the Ms. barkeri culture of 7.5–3.8 days. Rates of acetate utilization were 9.5–17.3 mmol l−1 day−1 and increased with decreasing RT. The K
s for acetate utilization was 6–8 mM. The two-stage system can be used as a model system for studying biological and physical
parameters that influence the bioconversion process. Our results suggest that manipulating the different phases of cellulose
fermentation separately can effectively balance the pH and ionic requirements of the acid-producing phase with the acid-using
phase of the overall fermentation.
Received: 7 December 1999 / Received revision: 28 April 2000 / Accepted: 19 May 2000 相似文献