Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

Studies on the alkalophilicStreptomyces with extracellular xylanolytic activity

Summary An alkalophilicStreptomyces which produced xylanase, isolated from soil, grew in a temperature range of 15–37°C. The pH optimum for growth was 10 and no growth occurred at pH 7. On a simple wheat bran medium the microorganism exhibited maximum enzyme secretion of 12 U/ml at pH 10. The enzyme had a broad pH optimum of 4.8–10 and the optimum temperature of 50°C. It was completely inactivated at 60°C in 2 h. The enzyme hydrolyzed xylan to a mixture of oligomeric products indicating that the main activity was of the endoxylanase type. The culture filtrate had no cellulase activity.     

Growth conditions of a pectinolyticAspergillus fumigatus for degumming of natural fibres

Summary Optimum growth conditions forA. fumigatus strain 4 when citric pectin was the sole carbon source were at a temperature of 45°C, pH 4.0 and an incubation time from 36 to 42h. Under these conditions no cellulase activity was found. When orange pulp was the sole carbon source, optimum polygalacturonase activities were found when the fungus was cultured for 36 h at 45°C and a pH 3.0 to 4.5.     

Regulation of product synthesis in cell cultures of Catharanthus roseus. Effect of culture temperature

A three year old, alkaloid producing cell line of Catharanthus roseus, maintained at 25°C, was grown on 2% sucrose at various temperatures from 10° to 45°C. Growth rates were maximal at 35°C but declined rapidly above 35°C and below 25°C. Maximum serpentine yields reached a peak at between 20°C and 25°C and fell sharply above and below these temperatures, while ajmalicine showed a sharp peak of accumulation at 20°C. The variable serpentine/ajmalicine ratio at different growth temperatures suggests that lower temperatures may favour ajmalicine accumulation. Both the growth rate and the rate of alkaloid accumulation at 25°C were therefore sensitive to small changes in average culture temperature.     

Release of β-galactosidase from E. coli by a plasmid containing a temperature sensitive lytic function

Summary We report the construction of a plasmid which carries the øX174 lysis gene E downstream from the lambda pL promoter. This plasmid is capable of triggering lysis inE. coli when the growth temperature is raised from 30°C to 42°C. The kinetics of release of -galactosidase and intracellular protein have been determined, as well as the decrease in cell viability upon induction of lysis. In addition, -galactosidase can be released efficiently after induction by a short sonication period. Both methods allow efficient release of -galactosidase.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

Production of active human interferon-β inE. coli I. Preferential production by lower culture temperature

Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE. coli harboring a recombinant plasmid. TheE. coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature.     

Production of human leukocyte interferon inEscherichia coli by control of growth rate in fed-batch fermentation

Summary AnE. coli harboring a vector containing double promoters, a signal sequence, and interferon gene was used. By fitting the feed rate of growth-limiting nutrients to the precalculated demand of the microorganism on the basis of a specific growth rate of 0.1 h^–1, fed-batch fermentations were performed. A cell density of 26 g/L was achieved after 46 hrs cultivation at 30°C. The culture was induced by IPTG and produced 1x10⁹ IU/L of human leukocyte interferon.     

Higher culture pH is preferable for inclusion body formation of recombinant salmon growth hormone inEsherichia coli

Summary Higher culture pH of 7.6 was shown to be preferable for the inclusion body formation of salmon growth hormone (SGH) inEscherichia coli harboring a recombinant plasmid. High-level formation of SGH inclusion bodies was achieved at 33°C (pH 7.6). Growth inhibition by soluble SGH was also observed.     

Different temperature profiles of enzyme secretion by two common strains ofTrichoderma reesei

Summary We have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichoderma reesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.     

Effect of fermentation variables on cellulase production by Trichoderma Sp.

Summary Batch cultivation ofTrichodermma reesei QM9414 was carried out in Mandels medium containing(w/v) 1% beech wood cellulose and 0.05% yeast extract at 29°C. Use of 36 hours old inoculum(10% v/v),3.2 1/min aeration rate at 400 rpm(K_La 220/h) and pH cycling strategy produced 4 g/1 cell mass and 21.5 IU/1/h FPA cellulase.     

High activity xylanase from thermotolerantStreptomyces T7: Cultural conditions and enzyme properties

Summary A thermotolerantStreptomyces T₇ produced 70–72 U/ml of extracellular xylanase activity when grown at 50°C in submerged culture, in à medium containing 5% wheat bran as a carbon source. Among the various sugars tested, maltose showed the highest activity of 8 U/ml. Pure xylan was less effective as an inducer as compared to wheat bran. Ammonium sulphate at a concentration of 0.7% was found to be optimum for maximum yield of the enzyme. The optimum period and pH for maximum production were 72th and 7.0, respectively. The culture filtrate was devoid of amylase, cellulase and B-xylosidase activity. The xylanase was exceptionally stable and did not show any loss in activity after storage at 50°C at pH 5.0 for 6 days.     

Influence of temperature on solvents production from whey

Summary The influence of temperature on solvent production from whey was investigated by using strains ofClostridium acetobutylicum andbutylicum. Higher yields of solvents were observed at 37°C or at 30°C depending on the strain used.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

Simultaneous production of cellulase complex and glucose isomerase by Streptomyces flavogriseus

Summary Fermentations of Streptomyces flavogriseus were carried out at 30° C on media containing either Avicel, hay or acid hay hydrolysate as the principal carbon source. Under these conditions the strains produced simultaneously the enzymes of the cellulase complex as well as glucose isomerase. The enzyme activities were induced by hay, hay extract and D-xylose.     

Comparison of the optimal culture conditions for cell growth and tissue plasminogen activator production by human embryo lung cells on microcarriers

Optimization of culture conditions such as the dissolved O₂ (DO) concentration, temperature and pH was attempted regarding both cell growth and the production of tissue plasminogen activator (TPA) in a microcarrier cell culture of human embryo lung cells. The growth rate was suppressed at a DO concentration below 30% saturation. From the pH range 7.2–7.6, both the specific growth rate and maximal cell concentration decreased. At a lower temperature than 37°C, although both the specific growth rate and the maximal cell concentration decreased, the cell concentration was maintained for a longer time during the production period, high TPA productivity being maintained. As the optimal conditions for culture growth, a DO concentration of 30% saturation or over, temperature of 37°C and pH of 7.4 are recommended. However, for TPA production after cell culture growth, the DO concentration should be in the range 20–30% O₂ saturation, and the temperature and pH should be lowered to 33°C and 6.8, respectively.     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

Immobilization of whole Escherichia coli containing penicillin amidase using cross-linking agents and fillers

To obtain a catalyst with good mechanical stress stability and other operational characteristics, cross-linked aggregates of whole Escherichia coli containing penicillin amidase were reinforced with surface modified precipitated silica and chitosan. The immobilized cells plus mixed fillers possess better performance characteristics i.e. higher stability at 4°C and 30°C, least protein leaching capacity and good settling characteristics. Most suitable conditions to prepare catalyst with mixed fillers were: chitosan, 0.3: silica, 0.2 g/g d.wt cell; and minimum moisture content in catalyst, 30% (w/w).     

The effect of protease on stability of cellulase and xylanase fromCellulomonas flavigena

Summary Crude preparations of extracellular cellulase and xylanase fromCellulomonas flavigena at 4°C show a rapid loss of activity. With the protease inhibitors aprotinin and -2 macroglobulin this loss of activity could be dramatically reduced. Cellulase and xylanase extracted from a protease negative mutant were also more stable. When the cellulase and xylanase was purified by DEAE sepharose from wild type strains, the protease activity could be separated, such preparations of cellulase and xylanase were extremely stable.