Beatriz Vahan Kilikian

Recombinant gene expression in Escherichia coli cultivation using lactose as inducer.

The use of lactose as inducer of foreign gene expression under control of the lac UV5 promoter was investigated in recombinant Escherichia coli. Chicken muscle troponin C (TnC) gene was transcripted by T7 RNA polymerase and expressed in bioreactor cultivations after a feed-forward controlled fed-batch growth phase. Cell concentrations of 22 g l-1 dry cell weight (DCW)--before induction started--were used to achieve a TnC content of 19.5% of total cell protein through an induction strategy that combined the addition of a specific lactose amount of 4.7 g g-1 DCW divided into three pulses and the addition of yeast extract (1 g l-1) together with the second and the third lactose pulses. The results presented suggest that the residual lactose concentration plays an important role on the production of the heterologous protein.

Process strategies to improve heterologous protein production in Escherichia coli under lactose or IPTG induction.

Abstract Cells of Escherichia coli BL21 bearing the chicken muscle Troponin C (TnC) gene under the control of the lac UV5 promoter were induced under different cultivation conditions and the consequences on growth and cell protein content were investigated. The type of inducer molecule (lactose or IPTG), the average carbon source concentration (0.0–10.6 g l −1 ), the specific growth rate at the beginning of induction (0.20–0.54 h −1 ), and the cell concentration at the beginning of induction (3.2–36.5 g l −1 ) were varied. The highest value for the intracellular level of the target protein (TnC X ), 110 mg per gram dry cell weight (DCW), was achieved when isopropyl-β- d -thiogalactoside (IPTG) was the inducer. Under lactose induction, a value of 96 mg per gram DCW was attained. However, the high metabolic load imposed by IPTG, when compared with lactose induction, as assessed by the cell protein content and stability, indicates that lactose is probably the most appropriate inducer for the synthesis of this heterologous protein.

Separation and purification of glucoamylase in aqueous two-phase systems by a two-step extraction

Extraction in two steps of glucoamylase was studied in poly(ethylene glycol) (PEG) and potassium phosphate systems at pH values of 6, 7 and 9. Ten different conditions using PEG 300, 600, 1500, 4000 and 6000 were studied. The bottom phase of the first extraction step, with the enzyme, was reused in an appropriate concentration of PEG to form the second extraction step. The optimal partitioning conditions for glucoamylase separation were obtained in PEG 4000 (first step), PEG 1500 (second step) at pH 7 and resulted in a three-fold increase in glucoamylase purification.

Acetic acid accumulation in aerobic growth of recombinant Escherichia coli

Abstract A correlation between μ H Ac (specific acetic acid accumulation rate) and μ (specific growth rate) for a recombinant Escherichia coli BL21 strain was defined under typical conditions to achieve high cell densities (fed-batch process, dissolved oxygen concentration higher than 30% saturation, semi-synthetic medium). The feeding rate of glucose was continuously adjusted in order to support constant values of μ (0.4, 0.3, 0.2, 0.1 and 0.05 h −1 ) during each of the five runs. An exponential correlation between μ and μ H Ac showed significant increments in μ H Ac for μ values higher than 0.30 h −1 . The metabolic activity of acetic acid synthesis was not suppressed, even at μ as low as 0.05 h −1 , a result which is not in agreement with previous published data.

Bioaffinity extraction of glucoamylase in aqueous two-phase systems using starch as free bioligand.

Aqueous two-phase systems with bioligands bound to the polymer, usually polyethylene glycol (PEG), have high selectivity. However, such kind of systems can be costly since some specific synthetic ligands are expensive, making them non-viable for bioaffinity extraction of low-cost proteins. The use of free bioligands is an alternative for decreasing the high cost of these systems. This work describes the use of starch as a free bioligand on the partition of glucoamylase in PEG 300-phosphate system. The results were separated into two parts. In the first part, analysis of the starch distribution between the phases showed one-sided distribution to the bottom phase. In the second part, filtered broth from submerged cultivation of Aspergillus awamori, containing glucoamylase and contaminants, was submitted to the extraction in the system with starch. In the system without starch, glucoamylase and contaminants are partitioned in the upper phase. Upon starch addition, the partition coefficient of glucoamylase was decreased nine-fold without altering the partition of contaminants. These results indicate the possibility of separation of enzymes with high-molecular-mass and hydrophilic substrates, like glucoamylase, cellulase and pullulanase, from their contaminants in a one-step extraction. Since systems made of low-molecular-mass PEG partitioned almost all of the proteins to the upper phase, the separation can be achieved by extraction of the target enzyme in the bottom phase, as in the case of the presented study.

Influence of carbon and nitrogen sources on glucoamylase production byAspergillus in batch process

SummaryGlucoamylase production kinetics was greatly affected by medium composition. While maltose and cassava flour induced glucoamylase synthesis, fructose clearly repressed it, reaching a maximum enzyne activity value (Am) of only 6% of those obtained with the former carbon sources. Among the nitrogen sources the best result was obtained with urea, reaching Am values of about 40% higher than those obtained with ammonium sulphate.

Effect of yeast extract on Escherichia coli growth and acetic acid production

Fed batch cultures were performed to investigate the effect of yeast extract concentration on the kinetics of growth and acetic acid production of recombinant Escherichia coli BL21 in a synthetic medium. Three runs were performed with 40g/l total glucose concentration. The yeast extract/glucose ratio (YE/G; w/w), was 0.1, 0.05 and 0.025 in the feed. These decreasing YE/G values did not affect growth kinetics, but reduced the final cell concentration by about 10%, and also reduced the cell yield. Experiments with 60g/l total glucose concentration, one with a YE/G of 0.025 in the feed and the other without yeast extract, showed final acetic acid concentrations of 5.1 and 0.5g/l respectively, without any difference in cellular concentration. Although there was no significant influence on growth kinetics and final cellular concentration, the cell fermentative capacity was enhanced by yeast extract. The feed medium without yeast extract was the best condition for control purposes in high cell density cultures and for recombinant gene expression.