甲醇抑制时重组毕赤酵母发酵的特性研究

本文对毕赤酵母进行了恒化培养研究。以甲醇为唯一碳源时,在稀释率较低时(D<0.048 h^-1),连续培养系统操作很稳定。但在稀释率高时(D>0.048h^-1),连续培养系统的定态点不止一个,实验不能维持,故采用比生长速率恒定的分批流加培养进行研究。结果表明,毕赤酵母的生长符合Andrew普遍化底物抑制模型。综合考虑水蛭素的生成、底物的消耗,在生产中维持甲醇浓度为限制性浓度(0.5 g/L),且维持比生长速率为0.02 h^-1时,水蛭素Hir65的比生成速率达到最大值0.2 mg/(g·h)且甲醇的比消耗速率为0.04 g/(g·h)。     

Oxidation of methanol,formaldehyde and formic acid by methanol-utilizing yeast

Methanol-utilizing yeast,Candida boidini 11 Bh, characterized by high tolerance to methanol during growth, displays even higher tolerance when the oxidation rate by intact cells is tested. Low respiration activity is found even at 22% v/v of methanol. The half-saturation constant was 17–18mM. The half-saturation constants for the two oxidation intermediates, formaldehyde and formic acid were 3.6–4.0 and 30–33mM, respectively. When applied together with standard concentration of methanol, very low concentrations of both intermediates stimulated the oxidation rate. These results are discussed in connection with the relationship between growth and oxidation, the tolerance to high concentrations of inhibitory products and the mechanism of inhibition.     

A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.     

Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut(+) expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(H(c))], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consumption rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (mu) determined from the model was 0.08 h(-1) at a methanol concentration of 3.65 g/L, while the actual maximum mu was 0.0709 h(-1). The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or methanol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (Y(X/MeOH)) was lower and the maintenance coefficient (m(MeOH)) was higher than compared to limited methanol conditions. The Y(X/MeOH) decreased and m(MeOH) increased with increasing methanol concentration under methanol-inhibited conditions. BoNT/A(H(c)) content in cells (alpha) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum alpha of 1.72 mg/g WCW was achieved at a mu of 0.0267 h(-1) and induction time of 12 h.     

Effect of organic solvents on the beef heart mitochondrial adenosine triphosphatase.

The effect of organic solvents on the beef heart mitochondrial ATP-base-catalyzed ATP and ITP hydrolysis was examined. It was observed that numerous organic solvents stimulated ATP hydrolysis while ITP hydrolysis was inhibited. Methanol at 20% (v/v) was found to stimulate ATP hydrolysis by over 300%, while at the same methanol concentration ITP hydrolysis was inhibited approximately 50%. In the presence of 20% methanol, ATP hydrolysis exhibited linear plots of 1/[ATP] vs. 1/v, while in the absence of methanol negative cooperativity was observed. These data can be interpreted to imply that the catalytic and regulatory sites of the mitochondrial ATPase are being dissociated 20% methanol. The effect of methanol on the hydrolysis of ATP and ITP was examined as a function of pH. It was found that, at high pH in totally aqueous solutions, the hydrolysis of ATP and ITP was inhibited, while the presence of 20% methanol either caused the hydrolytic rate to peak and remain constant above pH 8 (with ATP as substrate) or caused the rate of hydrolysis to continue to increase above pH 8 (when ITP was the substrate). These data are interpreted to indicate that an acidic group in the active site may be ionizing, limiting the ATPase-catalyzed hydrolytic rate, and, with 20% methanol, this ionization was inhibited.     

Use of a silicone tubing sensor to control methanol concentration during fed batch fermentation of Pichia pastoris

A silicone tubing sensor controlled a constant methanol concentration in a fermenter up to 72 hours without the need for on-line gas chromatography or complex feeding schemes based on dissolved oxygen spikes. Methanol concentration was controlled up to 1.0% (v/v) with control around a given set point of ± 0.24%. The length of tubing, airflow through the tubing, pump speed and medium formulation had no effect on the control of methanol concentration.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Insect cell growth evaluation during serum-free adaptation in stirred suspension cultures

Sf-9 insect cells were adapted to three different serum-free media (SF900II, EXCELL 401 and IPL/41 supplemented) in 125 ml stirred vessels by gradually reducing serum concentration from 10 to 0% (v/v). TC100 medium sup-plemented with 10% fetal bovine serum was used as control. With this procedure it was possible to obtain cells fully adapted to SF900II and EXCELL 401 in 5 weeks. The adapted cells could be frozen in serum-free medium and thawed without any decrease in specific growth rate or maximum cell concentration. Even after 4 months of culture in stirred vessels at 170 rpm the specific growth rate and maximum cell concentration (0.031 h and 4.8 × 10 cells/ml, respectively) remained constant.     

Physiological responses of a methylotrophic bacterium after sudden shifts from C-limited chemostat to C-excess batch growth conditions

The response of steady-state continuous cultures of Methylobacterium sp. RXM to the addition of methanol pulses was studied. The increase of methanol concentration in the medium did not result in cell death under any of the conditions tested. When the growth rate of the steady-state cultures was low ( D = 0.046 h⁻¹), the specific growth rate increased. When the concentration of methanol in the pulse was increased from 36 mmol l⁻¹ to 280 mmol l⁻¹, uncoupled growth occurred and the molar cell yield decreased. Conversely, steady-state cultures at high growth rate ( D = 0.2 h⁻¹) showed a decrease in both specific growth rate and molar cell yield after the addition of the methanol pulses (32 and 164 mmol 1⁻¹). For all conditions, formaldehyde and formate were excreted into the medium but the levels did not exceed 1.13 mmol 1⁻¹ Slow-growing cultures were characterized by cells with high derepressed specific activities of methanol dehydrogenase and low specific activities of formaldehyde and formate dehydrogenases, fast-growing cells had lower specific activity for methanol dehydrogenase and higher activities of formaldehyde and formate dehydrogenases, resulting in the excretion of lower concentrations of formaldehyde and formate.
It is concluded that slow-growing cultures are more stable than fast-growing cultures for low methanol concentration fluctuations, and it is expected that maximum growth yields throughout the fermentation time are better achieved under the former conditions. However, for large fluctuations in the substrate concentration, the bacterial metabolic responses were identical both for slow-growing and fast-growing cultures.     

Effect of gassing,agitation, substrate supplementation and dialysis on the growth of an extremely thermophilic archaeon Pyrococcus woesei

Summary Growth of an extremely thermophilic archaeon, Pyrococcus woesei, at 90°C in a 2–1 fermentor was significantly enhanced by gassing with N₂/CO₂ (95%/5%). Both growth and -amylase activity were also positively influenced by increasing the agitation speed up to 1200 rpm under continuous gassing at 0.2 vvm. However, increasing the agitation speed to 2400 rpm led to decreases in the maximum cell concentration and -amylase activity. Fed-batch cultivation resulted in increases in the specific growth rate, maximum cell concentration and -amylase activity. Although the latter two parameters were higher when the broth was supplemented with both starch and concentrated medium, the specific growth rate was relatively smaller. Cultivation in a dialysis reactor gave a cell concentration of 2 × 10⁹ cells/ml, which represents a 2.8-fold increase over that obtained in ordinary batch cultivation. This increase in the cell concentration was accompanied by a 5.2-fold increase in -amylase activity. Correspondence to: G. Antranikian     

重组人小分子抗体ScFv-Fc发酵条件的优化及纯化

目的:研究重组人小分子抗体ScFv-Fc在毕赤酵母中分泌表达的最佳条件,以及ScFv-Fc的纯化方法。方法:分别从甲醇浓度、pH、诱导时间等方面对毕赤酵母重组菌株产生ScFv-Fc的发酵过程进行了优化;通过硫酸铵沉淀结合protein A亲和层析柱,对ScFv-Fc的纯化方法进行了研究。结果:确定ScFv-Fc在毕赤酵母中分泌表达的最佳条件为:在pH5.2的条件下,以0.5%甲醇诱导72 h。经过protein A亲和层析柱纯化后,ScFv-Fc纯度可达94%以上。结论:确定了ScFv-Fc在毕赤酵母中分泌表达的最佳条件以及纯化方法,为重组抗体分子诊断、治疗试剂的开发以及抗体的人源化奠定了物质基础。     

Oxygen uptake and citric acid production by Candida lipolytica Y 1095

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L . h, and volumetric oxygen uptake rate, 343 mg O(2)/L . h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell . h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell . h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell . h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L . h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions. (c) 1994 John Wiley & Sons, Inc.     

Cryoenzymology of Bacillus cereus beta-lactamase II

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cultural properties and mass-energy balances in methanol fermentation by Methylomonas methanolovorans

The cultural properties of an obligate methanol utilizer, Methylomonas methanolovorans, were investigated in batch and continuous cultures, and the problems of mass-energy balances were examined. Among the culture data, an exponential increase of growth lag with increased methanol concentration, as well as the inhibition kinetics in the relation between attainable maximum specific growth rate (mu(m) <== 0.52) and methanol concentration are of interest. In the latter case, the inhibition constant (K(i)) and the index number were 40 g/L, and 3 (dimensionless), respectively. The maximum yield coefficient (Y) in both batch and chemostat cultures was around 0.52. An analysis of the behavior of respiratory activity (Q(o2)) in response to the dissolved oxygen concentration (DO) indicated that the oxygen-terminal entity should be regarded as a single one with a saturation constant for DO of 32 mug/L (1.1 x 10(-6)M). Chemostat data showed that the saturation constant for methanol is as low as 2.2 mg/L or 7 x 10(minus;5)M. A linear relationship was observed between the respiratory activity (mol O(2)g(-1)h(-1)) and the specific growth rate (mu i h(-1)), with the relationship Q(o2) = 0.0504mu + 0.00112. The theory of mass and energy balances used by Roels has been reformed to give useful relationships between RQ or the cell yield and mu. In the case of M. methanolovorans, the relations can be greatly simplified since the influence of metabolic by-product formation was negligible. Experimental RQ values (theoretical values for Y = 0.52 and 0.445) at varying mu-values were compared with theoretical ones; despite considerable fluctuations, the results were regarded to conform with theory. By use of mass balance equations and enthalpy data of known compounds, the heat evolution in methanol fermentation was estimated indirectly to be 612 kcal/100 g biomass formed. The Y(ATP) problems are also discussed.     

Permeabilization of metabolites from biologically viable soybeans (Glycine max)

Chemical permeabilization has been widely studied for the release useful metabolites from many types of plant cells and tissues. In this study, the effect of 0-30% (v/v) of aqueous methanol solutions were used to permeabilize soybeans for the release of two isoflavonoids: daidzein and genistein. The release of these metabolites increases with increasing methanol concentrations. The amounts of daidzein and genistein released can increase up to 40- and 86-fold, respectively, when incubated in a 30% (v/v) methanol solution for 24 h compared with those incubated with water only. The effect of methanol on the release rates is primarily due to an increase in solubility of the stored daidzein and genistein (14- to 18-fold) inside the seeds, thus maximizing the concentration gradients for metabolite release. However, the viability of the seeds dropped with increase in methanol concentrations and the incubation time. The viability of soybeans (indicated by their ability to germinate) after permeabilization treatment with 0-20% (v/v) methanol solutions was maintained above 80% throughout the 24 h, whereas no seeds were found to be viable when 30% (v/v) methanol solution was used. The permeability coefficients (P) of daidzein and genistein were found to increase as the methanol concentration used was increased. These P values were estimated to range from 1.1 x 10(-)(9) to 1.9 x 10(-)(8) m/s and 1.0 x 10(-)(9) to 1.7 x 10(-)(8) m/s, respectively. The increase in P can be attributed primarily to an increase in the partition coefficient of the metabolites in the soybean seedcoats. An empirical correlation is proposed in which the log P values are described as a function of the metabolite molecular weights and the partition coefficients of the metabolites between octanol and water, K(oct/water), which was modified to include the effect of methanol present. Knowledge obtained from this study will help provide useful selection criteria for chemical permeabilization of plant tissues, such as seeds, with minimal loss in their viability.     

GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns

Strain tolerance to toxic metabolites remains a limiting issue in the production of chemicals and biofuels using biological processes. Here we examined the impact of overexpressing the autologous GroESL chaperone system with its natural promoter on the tolerance of Escherichia coli to several toxic alcohols. Strain tolerance was examined using both a growth assay as well as viable cell counts employing a CFU (colony-forming unit) assay. GroESL over expression enhanced cell growth to all alcohols tested, including a 12-fold increase in total growth in 48-h cultures under 4% (v/v) ethanol, a 2.8-fold increase under 0.75% (v/v) n-butanol, a 3-fold increase under 1.25% (v/v) 2-butanol, and a 4-fold increase under 20% (v/v) 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in CFU numbers compared to a plasmid control strain after 24 h of culture under 6% (v/v) ethanol, and a 3.5-fold and 9-fold increase for culture under 1% (v/v) n-butanol and i-butanol, respectively. The toxicity of the alcohols was examined against their octanol–water partition coefficient, a measure commonly used to predict solvent toxicity. For both the control and the GroESL overexpressing strains, the calculated membrane concentration of each alcohol based on the octanol–water partition coefficient could be correlated, but with different patterns, to the impact of the various alcohols on cell growth, but not on cell viability (CFUs). Our data suggest a complex pattern of growth inhibition and differential protection by GroESL overexpression depending on the specific alcohol molecule. Overall, however, GroESL overexpression appears to provide molecule-agnostic tolerance to toxic chemicals.     

Enhancement of CO2 tolerance of Chlorella vulgaris by gradual increase of CO2 concentration

Summary Chlorella vulgaris UTEX259 was cultivated using two different methods of gas supply. In one method the CO₂ concentration in bubbled gas was held constant and in the other method it was increased gradually. Algal growth was almost linear after a short period of lag phase in both methods. With the constant CO₂ concentration, the CO₂ fixation rate in the linear growth phase decreased over 10%(v/v) CO₂, while the rate increased up to 6% CO₂. However, the rate was enhanced by using the latter incremental increase method, especially under a higher concentration of CO₂. The maximum rate of CO₂ fixation was 52 mg CO₂/l·h at 20% CO₂ during the gradual increase of CO₂ concentration.     

Methanol-induced tertiary and secondary structure changes of granulocyte-colony stimulating factor

We have studied methanol-induced conformational changes in rmethuG-CSF at pH 2.5 by means of circular dichroism (CD), fluorescence and infrared (IR) spectroscopy, and 8-anilino-1-naphthalene sulfonic acid (ANS) binding. Methanol has little effect on the secondary and tertiary structures of rmethuG-CSF when its concentration is in the range of 0 to 20% (v/v). At 30% (v/v) methanol, rmethuG-CSF has ANS binding ability. In the methanol concentration range of 30 to 70% (v/v) the amount of alpha-helix decreases a little, and the tertiary structure decreases significantly. At methanol concentrations above 70% (v/v), a transition to a more helical state occurs, while there is little change in the tertiary structure, and no ANS binding ability. Thermal denaturation studies involving CD have demonstrated that as the methanol concentration increases the melting temperature and the cooperativity of transition decrease, and the transition covers a much wider range of temperature. It seems that the decreased cooperativity means an increase in the concentration of partially folded intermediate states during the unfolding of rmethuG-CSF.     

Methanol as alternative carbon source for quicker efficient production of the microalgae Chlorella minutissima: Role of the concentration and frequence of administration

Autotrophic cultures of the marine microalgae Chlorella minutissima were performed at 13 000 lux continuous illumination in 1 l chambers fertilised with 0.25 g l⁻¹ F2 medium and different doses of methanol. This was administered in two ways during two parallel experimental series of 10 days: 0.05, 0.1, 0.5, 1.0 and 5.0% methanol (v/v) in one unique dose at the beginning of the culture and 1/10 of these (i.e. 0.005, 0.01, 0.05, 0.1 and 0.5% methanol (v/v)) in daily doses for the 10-day culture period. Low concentrations of methanol induced a faster increase of cell density and dry weight than control, while high concentrations induced symptoms of toxicity. The higher cell densities and quicker growth were observed in the experiments with daily administration of 0.005 and 0.1% (v/v) methanol, while those with one dose presented an initial boosted growth but a final cell density lower than control. The role of methanol as alternative carbon source for microalgae, as well as its possible impact on the quality of biomass production and on the environment, are discussed.