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1.
Zhang W Hywood Potter KJ Plantz BA Schlegel VL Smith LA Meagher MM 《Journal of industrial microbiology & biotechnology》2003,30(4):210-215
Fed-batch fermentation of a methanol utilization plus (Mut+) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually
used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant
protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve
production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective
of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut+
Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a
model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate (μ) and specific glycerol/methanol consumption rate (ν
gly, ν
MeOH) were established. A mixed feeding strategy with desired μ
gly/μ
MeOH =1, 2, 3, 4 (desired μ
MeOH set at 0.015 h−1) was employed to study growth interactions and their effect on production. The results show that the optimal desired μ
gly/μ
MeOH is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells.
Electronic Publication 相似文献
2.
An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones
could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 105 transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000
concentration and the wetness of selective plates were investigated.
Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997 相似文献
3.
Production of a sterol esterase from Ophiostoma piceae in batch and fed‐batch bioprocesses using different Pichia pastoris phenotypes as cell factory
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Víctor Barba Cedillo María Jesús Martínez Carolina Arnau Francisco Valero 《Biotechnology progress》2014,30(5):1012-1020
The potential biotechnological applications for the Ophiostoma piceae sterol esterase (OPE) are conditioned to the availability of high enzyme amounts at low prices. This enzyme is a versatile biocatalyst with different biotechnological applications. In this work a systematic study on its heterologous production in different Pichia pastoris strains and operational strategies is presented. The best results were obtained using an AOX1 defective yeast strain in a fed‐batch bioprocess using methanol as inducer substrate at a set point of 2.5 g L?1 and sorbitol as cosubstrate by means of a preprogramed exponential feeding rate at a μ = 0.02 h?1, reaching 30 U mL?1 of enzyme and a volumetric productivity of 403.5 U L?1 h?1. These values are twofold higher than those obtained with a Mut+ phenotype using methanol a sole carbon source. OPE was the main protein secreted by the yeast, 55% for Muts versus 25% for Mut+. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1012–1020, 2014 相似文献
4.
M. V. Gonchar L. B. Kostryk A. A. Sibirny 《Applied microbiology and biotechnology》1997,48(4):454-458
Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag
period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor
catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth+) in the absence of catalase activity. These Mth+
gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type
strain. In contrast to the parental C-105 strain, H2O2 does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor
strains is strongly correlated with increased levels of the alternative H2O2-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized.
Received: 9 December 1996 / Received revision: 5 May 1997 / Accepted: 25 May 1997 相似文献
5.
A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino
acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The
expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor
specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two
proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the
organism was grown under aerobic conditions.
Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997 相似文献
6.
Ottone S Nguyen X Bazin J Bérard C Jimenez S Letourneur O 《Protein expression and purification》2007,56(2):177-188
This study describes the expression in Pichia pastoris of hepatitis B surface antigens (HBsAg) corresponding to the S region of the four major subtypes: adr, adw2, ayr and ayw3 and to the preS2-S region of the two subtypes adr and adw2. The recombinant yeast strains have been selected amongst methanol utilization positive (Mut+) or sensitive strains (Muts) and cultivated to high cell density in bioreactor using a short protocol. Our results prove the efficiency of P. pastoris to produce all the major HBsAg subtypes and confirm the ability of the methanol regulated promoter of alcohol oxidase I gene (AOX) to express heterologous protein through phenotype Mut+ or Muts strains.All these recombinant HBsAg proteins, including subtype ayr, whose production has never been presented, have been highly purified using a short original sequence of steps which includes high-pressure cell disruption associated with detergent treatment, ultrafiltration and immunopurification chromatography using a mAb anti-HBs. The whole process avoids possible alterations of antigenic properties and allows to obtain with high yield, high quality reagents for in vitro diagnosis. 相似文献
7.
J Lin D Panigraphy L B Trinh J Folkman J Shiloach 《Journal of industrial microbiology & biotechnology》2000,24(1):31-35
A pilot-scale production method of recombinant human angiostatin, a 38-kD fragment of plasminogen which has been reported
to have antiangiogenic activity, has been successfully established by expressing the protein in the methylotrophic yeast Pichia pastoris. The secreted protein inhibited cultured endothelial cell proliferation in vitro and Lewis lung carcinoma growth in mice. The fermentation process was carried out using an on-line methanol controller, administering
methanol to the growing culture and keeping its concentration under 2 g L−1. The fermentation lasted 90 h, of which 70 h were growth on methanol. During growth on methanol the culture volume increased
64%, from 7 L to 11.5 L, producing 200 mg angiostatin and 5 kg of biomass. Journal of Industrial Microbiology & Biotechnology (2000) 24, 31–35.
Received 12 May 1999/ Accepted in revised form 06 September 1999 相似文献
8.
Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp 总被引:2,自引:0,他引:2
G. M. Gübitz D. Haltrich B. Latal W. Steiner 《Applied microbiology and biotechnology》1997,47(6):658-662
Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding
sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal
and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers).
Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others
acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released
(K
v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from
Penicillium simplicissimum (K
v = 0.15 l mPa−1s−1g−1) and a mannanase from Sclerotium rolfsii (K
v = 0.12 l mPa−1s−1g−1) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation
of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO
respectively. A less significant brightness increase was obtained with enzymes showing lower K
v values, such as a xylanase from Schizophyllum commune (Kv = 0.051 l mPa−1s−1g−1, 0.2% ISO) and a bacterial mannanase (K
v = 0.061 l mPa−1s−1g−1,0.5% ISO).
Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997 相似文献
9.
A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from
activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and
84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly 14C-labelled DNOC was recovered as 14CO2. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol,
2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads
in fixed-bed column reactors were 30 mmol day−1 (l reactor volume)−1, leaving an effluent concentration of less than 5 μg l−1 DNOC in the outflowing medium. The apparent K
s value of the immobilized mixed culture for DNOC was 17 μM. Degradation was inhibited at DNOC concentrations above 30 μM and
it ceased at 340 μM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing
mechanism.
Received: 27 March 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997 相似文献
10.
Dennis H. Greer 《Planta》1998,205(2):189-196
Bean (Phaseolus vulgaris L. cv. Long John) plants were grown with photoperiods of 6 and 16 h at constant photon flux density (PFD), giving a daily
photon receipt (DPR) of 17 and 48 mol · m−2 respectively. Vegetative growth was determined at regular intervals and diurnal whole-plant photosynthesis measured. Intact
trifoliate leaves were exposed to photoinhibitory treatments at PFDs of 800 and 1400 μmol · m−2 · s−1 at temperatures of 14 and 20 °C, both in the absence and presence of the inhibitors chloramphenicol and dithiothreitol. Fluorescence
and photon yields were determined at regular intervals throughout each treatment. Plants grown with photoperiods of 6 h had
significantly lower growth rates than those grown with 16-h photoperiod but no difference in net photosynthetic rates or photon
yields were found. Carbohydrate analyses confirmed short-day plants were strongly sink-limited. Long-day plants were slightly
sink-limited, with a high proportion of starch in the leaves and reduced photosynthesis between 13 and 16 h. Plants grown
in low DPR were more susceptible to photoinhibition, from sustained closure of some photosystem II reaction centres, than
plants grown in high DPR. Capacity for thermal dissipation appeared dependent on PFD while photochemical capacity was more
dependent on DPR.
Received: 6 June 1997 / Accepted: 17 September 1997 相似文献
11.
Bioconversion of (4R)-(+)-limonene to (4R)-(+)-α-terpineol by immobilized fungal mycelia of Penicillium digitatum was investigated in batch, repeated-batch and continuously fed systems. The fungi were immobilized in calcium alginate beads.
These beads remained active for at least 14 days when they were stored at 4 °C. Three different aeration rates were tested.
The highest yield was obtained at a dissolved oxygen level of 50.0 μmol/l. α-Terpineol production by this fungus was 12.83 mg
(g beads)−1 day−1, producing a 45.81% bioconversion of substrate. Repeated-batch bioconversion showed yield decreases in the second and the
third cycles. Regeneration with nutrient media after the third cycle improved the bioconversion yields. With continuous bioconversion,
the half-life was dependent on the aeration. The optimum conditions with a continuous reactor were at an aeration rate of
0.3 standard l/min and a dilution rate of 0.0144 h−1.
Received: 10 June 1997 / Received revision: 18 August 1997 / Accepted: 11 September 1997 相似文献
12.
H. E. M. Mc Mahon C. T. Kelly W. M. Fogarty 《Applied microbiology and biotechnology》1997,48(4):504-509
The α-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown
at 40 °C and 55 °C in the presence and absence of cobalt, with an inverse relationship between α-amylase production and growth
rate. Highest α-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h−1), at 40 °C in the absence of cobalt, while at the highest growth rate (0.35 h−1), at 55 °C in the presence of cobalt, α-amylase production was decreased to 150 units/ml. As growth rate increased, the rate
of specific utilisation of the carbon source maltose also increased, from 46 to 123 μg maltose (mg biomass)−1 h−1. The pattern and levels of α-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that
growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.
Received: 17 February 1997 / Received revision: 29 April 1997 / Accepted: 11 May 1997 相似文献
13.
Heterologous protein production in methylotrophic yeasts 总被引:15,自引:0,他引:15
Gellissen G 《Applied microbiology and biotechnology》2000,54(6):741-750
The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression
control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their
status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals
and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the
near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied
examples of the recent past.
Received: 9 May 2000 / Received revision: 20 June 2000 / Accepted: 23 June 2000 相似文献
14.
S. Sánchez V. Bravo E. Castro A. J. Moya F. Camacho 《Applied microbiology and biotechnology》1998,50(5):608-611
We have analysed the influence of the initial pH of the medium and the quantity of aeration provided during the batch fermentation
of solutions of d-xylose by the yeast Hansenula polymorpha (34438 ATCC). The initial pH was altered between 3.5 and 6.5 whilst aeration varied between 0.0 and 0.3 vvm. The temperature
was kept at 30 °C during all the experiments. Hansenula polymorpha is known to produce high quantities of xylitol and low quantities of ethanol. The most favourable conditions for the growth
of xylitol turned out to be: an initial pH of between 4.5 and 5.5 and the aeration provided by the stirring vortex alone.
Thus, at an initial pH of 5.5, the maximum specific production rate (μm) was 0.41 h−1, the overall biomass yield (Y
x/s
G) was 0.12 g g−1, the specific d-xylose-consumption rate (q
s
) was 0.075 g g−1 h−1 (for t = 75 h), the specific xylitol-production rate (q
Xy
) was 0.31 g g−1 h−1 (for t = 30 h) and the overall yields of ethanol (Y
E/s
G) and xylitol (Y
Xy/s
G) were 0.017 and 0.61 g g−1 respectively. Both q
s
and q
Xy
decreased during the course of the experiments once the exponential growth phase had finished.
Received: 26 March 1998 / Received revision: 30 June 1998 / Accepted: 2 July 1998 相似文献
15.
E. Kostyál E.-L. Nurmiaho-Lassila J. A. Puhakka M. Salkinoja-Salonen 《Applied microbiology and biotechnology》1997,47(6):734-741
This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft
pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached
kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor,
pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass
volatile solids)−1day−1] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination
of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached
kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft
pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification
was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible.
Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997 相似文献
16.
D. Naki C. Paech G. Ganshaw V. Schellenberger 《Applied microbiology and biotechnology》1998,49(3):290-294
Mutants that secrete increased amounts of enzyme into a selection medium can be efficiently enriched from large populations
of mutagenized microorganisms during growth in hollow fibers. Under these conditions, each colony grows in its own microenvironment
and cross-feeding between neighboring colonies is limited. We applied the technique to B. subtilis carrying a plasmid-encoded protease gene. The plasmid was subjected to random mutagenesis and clones secreting up to fivefold-increased
amounts of enzyme were selected using a medium containing bovine serum albumin as the sole nitrogen source.
Received: 22 May 1997 / Received revision: 21 October 1997 / Accepted: 7 November 1997 相似文献
17.
CO2 exchange of the endolithic lichen Verrucaria baldensis was measured in the laboratory under different conditions of water content, temperature, light, and CO2 concentration. The species had low CO2 exchange rates (maximum net photosynthesis: c. 0.45 μmol CO2 m−2 s−1; maximum dark respiration: c. 0.3 μmol CO2 m−2 s−1) and a very low light compensation point (7 μmol photons m−2 s−1 at 8°C). The net photosynthesis/respiration quotient reached a maximum at 9–15°C. Photosynthetic activity was affected only
after very severe desiccation, when high resaturation respiratory rates were measured. Microclimatic data were recorded under
different weather conditions in an abyss of the Trieste Karst (northeast Italy), where the species was particularly abundant.
Low photosynthetically active radiation (normally below 40 μmol photons m−2 s−1), very high humidities (over 80%), and low, constant temperatures were measured. Thallus water contents sufficient for CO2 assimilation were often measured in the absence of condensation phenomena.
Received: 22 September 1996 / Accepted: 26 April 1997 相似文献
18.
P. Pfeiffer B. M. Culik 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1998,168(2):143-148
We used a still-water swim channel in conjunction with open-flow oxygen and carbon dioxide respirometry to examine the energy
requirements of river-otters (Lutra lutra L.) swimming voluntarily underwater in Neumünster Zoo (Germany). While at rest on land (5 °C), river-otters had a respiratory
quotient of 0.77 and a resting metabolic rate of 4.1 W kg−1. This increased to an estimated 6.4 W kg−1 during rest in water (11–15 °C) and to 12.3 W kg−1 when the animals were feeding in the channel. River-otters swimming under water preferred a mean speed of 0.89 m s−1, and their energy requirements attained 11.6 W kg−1. Cost of transport, however, was minimal at 1.3 m s−1 and amounted to 0.95 J N−1 m−1.
Accepted: 3 November 1997 相似文献
19.
Q. Hu N. Kurano M. Kawachi I. Iwasaki S. Miyachi 《Applied microbiology and biotechnology》1998,49(6):655-662
To test the feasibility of CO2 remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO2-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through
perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of
biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively
assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential
of biomass production under extremely high CO2 concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO2 fixation rate of 16.7 g CO2 l−1 24 h−1 (or 200.4 g CO2 m−2 24 h−1) could readily be sustained at a light intensity of 2000 μmol m−2 s−1 at 25 °C, and an ultrahigh cell density of well over 80 g l−1 could be maintained by daily replacing the culture medium.
Received: 20 October 1997 / Received revision: 19 December 1997 / Accepted: 24 January 1998 相似文献
20.
Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris
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Optimization of protein production from methanol‐induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol‐feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5‐fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014 相似文献