Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures. (c) 1993 John Wiley & Sons, Inc.     

Loss of antibody productivity is highly reproducible in multiple hybridoma subclones

An immunoglobulin G (IgG(2b)) producing hybridoma cell line (S3H5/gamma2bA2) was cloned and subcloned. Twenty subclones were grown in parallel while being adapted in a stepwise fashion to serum-free medium. Following adaptation to serum-free medium, it was found that 16 of the 20 subclones remained at a relatively constant proportion of nonproducing cells. Three of the remaining subclones transiently deviated from this balance but eventually returned toward this population composition. One subclone continued to lose productivity. A population balance was reached at approximately 8% of the population being nonproducers. The loss of antibody productivity was thus highly reproducible. (c) 1993 John Wiley & Sons, Inc.     

Catalytic and interfacial aspects of enzymatic polymer synthesis in reversed micellar systems

The enzyme horseradish peroxidase, when encapsulated in reversed micelles, is capable of catalyzing the synthesis of phenolic and aromatic amine polymers. The synthesis of polyethylphenol is specifically considered in this article and is found to be extremely feasible in the micellar system. Polymer chain growth can be controlled to some degree by manipulating the ability of the solvent to sustain chain solubility; this is effectively done by adjusting the surfactant concentration. This results in a degree of control of polymer molecular weight. The synthesized polymer drops out of solution and can be easily recovered. (c) 1993 John Wiley & Sons, Inc.     

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.     

Stability of bilirubin oxidase in organic solvent media: a comparative study on two low-water systems

The storage stability of bilirubin oxidase was studied in water-in-oil CTAB microemulsions with a chloroformrich continuous organic phase. The kinetics of the inactivation process were best described by a double exponential equation. Approximately half of enzymatic activity was lost during a "fast" phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady-state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the K(m) of the enzyme for bilirubin. Stability was also studied in a liquid/solid two-phase system; it was found that the inactivation process in this system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the "fast" phase rate observed in the water-in-oil microemulsion system. Inactivation of the enzyme slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the "slow" phase observed in water-in-oil microemulsions. (c) 1993 Wiley & Sons, Inc.     

A three-phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture

The use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using (15)NH(4)Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis-appearance of mRNA consistent with the three phases indicated by the fermentation parameters. (c) 1993 John Wiley & Sons, Inc.     

Relationship between cardiac output and oxygen uptake at the onset of exercise

The purpose of the present study was to assess the relationship between the rapidity of increased gas exchange (i.e. oxygen uptake ) and increased cardiac output ( ) during the transient phase following the onset of exercise. Five healthy male subjects performed multiple rest-exercise or light exercise (25 W)-exercise transitions on an electrically braked ergometer at exercise intensities of 50, 75, or 100 W for 6 min, respectively. Each transition was performed at least eight times for each load in random order. The was obtained by a breath-by-breath method, and was measured by an impedance method during normal breathing, using an ensemble average. On transitions from rest to exercise, rapidly increased during phase I with time constants of 6.8–7.3 s. The also showed a similar rapid increment with time constants of 6.0–6.8 s with an apparent increase in stroke volume (SV). In this phase I, increased to about 29.7%–34.1% of the steady-state value and increased to about 58.3%–87.0%. Thereafter, some 20 s after the onset of exercise a mono-exponential increase to steady-state occurred both in and with time constants of 26.7–32.3 and 23.7–34.4 s, respectively. The insignificant difference between and time constants in phase I and the abrupt increase in both and SV at the onset of exercise from rest provided further evidence for a cardiodynamic contribution to following the onset of exercise from rest.     

Effects of prolonged cycle ergometer exercise on maximal muscle power and oxygen uptake in humans

The mechanical power (W_tot, W·kg^–1) developed during ten revolutions of all-out periods of cycle ergometer exercise (4–9 s) was measured every 5–6 min in six subjects from rest or from a baseline of constant aerobic exercise [50%–80% of maximal oxygen uptake (VO_2max)] of 20–40 min duration. The oxygen uptake [VO₂ (W·kg^–1, 1 ml O₂ = 20.9 J)] and venous blood lactate concentration ([la]_b, mM) were also measured every 15 s and 2 min, respectively. During the first all-out period, W_tot decreased linearly with the intensity of the priming exercise (W_tot = 11.9–0.25·VO₂). After the first all-out period (i greater than 5–6 min), and if the exercise intensity was less than 60% VO_2max, W_tot, VO₂ and [la]_b remained constant until the end of the exercise. For exercise intensities greater than 60% VO_2max, VO₂ and [la]_b showed continuous upward drifts and W_tot continued decreasing. Under these conditions, the rate of decrease of W_tot was linearly related to the rate of increase of V [(d W_tot/dt) (W·kg^–1·s^–1) = 5.0·10^–5 –0.20·(d VO₂/dt) (W·kg^–1·s^–1)] and this was linearly related to the rate of increase of [la]_b [(d VO₂/dt) (W·kg^–1·s^–1) = 2.310^–4 + 5.910^–5·(d [la]_b/dt) (mM·s^–1)]. These findings would suggest that the decrease of W_tot during the first all-out period was due to the decay of phosphocreatine concentration in the exercising muscles occurring at the onset of exercise and the slow drifts of VO₂ (upwards) and of W_tot (downwards) during intense exercise at constant W_tot could be attributed to the continuous accumulation of lactate in the blood (and in the working muscles).     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。