From laboratory to pilot plant E. coli fed-batch cultures: optimizing the cellular environment for protein maximization

For recombinant protein production in E. coli fed-batch cultures, post-induction conditions have great influence in the quantity and quality of the product. The present paper covers the effect of different factors affecting the cellular environment in recombinant aldolase (rhamnulose-1-phosphate aldolase, RhuA) production. An operational mode employing an exponential addition profile for constant specific growth rate has been analyzed, in order to understand and define possible modifications with influence on post-induction cellular behavior. A constant addition profile has been demonstrated to render higher specific aldolase production than the exponential addition profile, probably due to a more constant environment for the cells. On the other hand, amino acid (leucine) supplementation has proven to increase protein quality in terms of activity units (U) per unit mass of RhuA (U mg^?1 RhuA), alleviating metabolic overload. Based on the above, a production process was set up and scaled up to pilot plant. Resulting production was double that of a standard laboratory operation, 45,000 U L^?1, and almost all the protein retained the 6xHis-tag with the highest quality, 11.3 U mg^?1 RhuA.     

Induction strategies in fed-batch cultures for recombinant protein production in Escherichia coli: Application to rhamnulose 1-phosphate aldolase

Different induction strategies for fed-batch recombinant protein production under the control of the strong T5 promoter in Escherichia coli have been investigated. Since the production of recombinant rhamnulose 1-phosphate aldolase is growth-related, the productivity of the process can be strongly reduced due to the negative effect of protein expression on cell growth. IPTG pulse induction as well as inducer dosage have been applied and their advantages and drawbacks highlighted. Both strategies led to high levels of the recombinant protein, 1000 AU g DCW⁻¹. Inducer concentration and inducer to biomass ratio were identified as the parameters influencing the rate of protein production and final enzymatic activity per gram of biomass. In pulse induction, the maximum enzymatic activity was found at inducer concentration of 70 μM. In continuous induction experiments, inducer concentrations between 4 and 12 μM were identified as the working range in which cell growth and recombinant protein accumulation occurred simultaneously. On the other hand, the amount of IPTG per gram of biomass required was 1.6 μmol IPTG gDCW⁻¹ in pulse induction and between 0.3 and 0.5 μmol IPTG g DCW⁻¹ in continuous induction.     

Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137

The influence of organic acids on growth and dithiolopyrrolone antibiotic production by Saccharothrix algeriensis NRRL B-24137 was studied. The production of dithiolopyrrolones depends upon the nature and concentration of the organic acids in the culture medium. Study of the nature of organic acids showed that the most effective organic acids for thiolutin specific production were maleic, 4-hydroxybenzoic, benzentetracarboxylic, pantothenic, pivalic and pyruvic acids (which yielded almost five-fold over the starting medium) and pimelic acid (more than three-fold). 4-Bromobenzoic acid showed the best production of senecioyl-pyrrothine (59 mg g⁻¹ DCW). Tiglic acid showed the best production of tigloyl-pyrrothine (22 mg g⁻¹ DCW). The highest yield of isobutyryl-pyrrothine (7.6 mg g⁻¹ DCW) was observed in the presence of crotonic acid. Sorbic acid yielded the best production of butanoyl-pyrrothine (26 mg g⁻¹ DCW). Methacrylic, butyric, pyruvic and 4-bromobenzoic acids also exhibited the best production of butanoyl-pyrrothine (27–11-fold).Study of organic acid concentration showed that among the selected organic acids, pimelic acid yielded the highest specific production of thiolutin (91 mg g⁻¹ DCW) at 7.5 mM; and senecioyl-pyrrothine (11 mg g⁻¹ DCW), tigloyl-pyrrothine (9 mg g⁻¹ DCW) and butanoyl-pyrrothine (3.5 mg g⁻¹ DCW) at 5 mM. Pyruvic acid at 1.25 mM enhanced the production of senecioyl-pyrrothine (4.3 mg g⁻¹ DCW). The maximum production of tigloyl-pyrrothine (18.6 mg g⁻¹ DCW) was observed in the presence of tiglic acid at 2.5 mM. Maximum production of isobutyryl-pyrrothine was observed in the presence of 7.5 mM tiglic acid. In addition, methacrylic acid (at 5 mM) and butyric acid (at 2.5 mM) enhanced the production of butanoyl-pyrrothine (26 and 20 times, respectively).The above results can be employed in the optimisation of the culture medium for the production of dithiolopyrrolone in higher quantities.     

Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH₄⁺ and low concentrations of Co²⁺. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent K_m was calculated to be 4.4 ± 0.07 mM, while V_max was found to be 100 ± 0.02 μM min⁻¹ mg protein⁻¹. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.     

Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs) were produced in carbon-limited, single-stage, fed-batch fermentations of Pseudomonas putida KT2440 by co-feeding nonanoic acid (NA) and glucose (G) to enhance the yield of PHA from NA. An exponential (μ = 0.25 h⁻¹) followed by a linear feeding strategy at a NA:G ratio of 1:1 (w/w) achieved 71 g l⁻¹ biomass containing 56% PHA. Although the same overall PHA productivity (1.44 g l⁻¹ h⁻¹) was obtained when NA alone was fed at the same specific growth rate, the overall yield of PHA from NA increased by 25% (0.66 g PHA g NA⁻¹ versus 0.53 g g⁻¹) with glucose co-feeding. Further increasing glucose in the feed (NA:G = 1:1.5) resulted in a slightly higher yield (0.69 g PHA g NA⁻¹) but lower PHA content (48%) and productivity (1.16 g l⁻¹ h⁻¹). There was very little change in the PHA composition.     

Metabolic rate, maximum metabolism, and advantages of torpor in the fat mouse Steatomys pratensis natalensis Roberts, 1921 (Dendeomurinae)

1. The fat mouse Steatomys pratensis natalensis (mean body mass 37.4±0.43 (se)) has a low euthermic body temperature T_b=30.1–33.8 °C and a low basal metabolic rate (BMR)=0.50 ml O₂ g⁻¹ h⁻¹.

2. Below an ambient temperature (T_a)=15 °C, the mice were hypothermic.

3. The lowest survivable T_a=10 °C.

4. Torpor is efficient in conserving energy between T_a=15–30 °C, below T_a=15 °C, the mice arouse.

5. Euthermic and torpid mice were hyperthermic at T_a=35 °C.

6. Thermal conductance was 0.159 ml O₂ g⁻¹ h⁻¹ °C⁻¹, 98.8% of the expected value.

7. Non-shivering thermogenesis (NST) was 2.196 ml O² g⁻¹ h⁻¹ (3.69×BMR).

8. Maximal oxygen consumption, however, was 3.83 ml O₂ g⁻¹ h⁻¹ (6.44×BMR), indicating that other methods of heat production are additive.

9. Because fat mice conserve energy by torpor only between T_a=15–30 °C, we suggest that torpor may be a more important mechanism for surviving food shortages than for surviving cold weather.

Cyanobacteria and cyanobacterial toxins in the alkaline crater lakes Sonachi and Simbi, Kenya

The phytoplankton communities and the production of cyanobacterial toxins were investigated in two alkaline Kenyan crater lakes, Lake Sonachi and Lake Simbi. Lake Sonachi was mainly dominated by the cyanobacterium Arthrospira fusiformis, Lake Simbi by A. fusiformis and Anabaenopsis abijatae. The phytoplankton biomasses measured were high, reaching up to 3159 mg l⁻¹ in L. Sonachi and up to 348 mg l⁻¹ in L. Simbi. Using HPLC techniques, one structural variant of the hepatotoxin microcystin (microcystin-RR) was found in L. Sonachi and four variants (microcystin-LR, -RR, -LA and -YR) were identified in L. Simbi. The neurotoxin anatoxin-a was found in both lakes. To our knowledge this is the first evidence of cyanobacterial toxins in L. Sonachi and L. Simbi. Total microcystin concentrations varied from 1.6 to 12.0 μg microcystin-LR equivalents g⁻¹ DW in L. Sonachi and from 19.7 to 39.0 μg microcystin-LR equivalents g⁻¹ DW in L. Simbi. Anatoxin-a concentrations ranged from 0.5 to 2.0 μg g⁻¹ DW in L. Sonachi and from 0 to 1.4 μg g⁻¹ DW in L. Simbi. In a monocyanobacterial strain of A. fusiformis, isolated from L. Sonachi, microcystin-YR and anatoxin-a were produced. The concentrations found were 2.2 μg microcystin g⁻¹ DW and 0.3 μg anatoxin-a g⁻¹ DW. This is the first study showing A. fusiformis as producer of microcystins and anatoxin-a. Since A. fusiformis occurs in mass developments in both lakes, a health risk for wildlife can be expected.     

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09 ± 0.02 μmol O₂ h^− 1 g^− 1; summer: 0.31 ± 0.06 μmol O₂ h^− 1 g^− 1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content / ascorbate content ratio (A / AH⁻). The A / AH⁻ ratio showed minimum values in winter (3.7 ± 0.2 10^− 5 AU) and increased in summer (18 ± 5 10^− 5 AU). A similar pattern was observed for lipid radical content (122 ± 29 pmol mg^− 1 fresh mass [FW] in winter and 314 ± 45 pmol mg^− 1 FW in summer), iron content (0.99 ± 0.07 and 2.7 ± 0.6 nmol mg^− 1 FW in winter and summer, respectively) and catalase activity (2.9 ± 0.2 and 7 ± 1 U mg^− 1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe–MGD–NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.     

Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L.

The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g⁻¹ DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l⁻¹ produced a 5-fold enhancement of OA accumulation (0.37 mg g⁻¹ DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l⁻¹ for 96 h or with pectin at 2 mg l⁻¹ for 48 h produced identical cellular levels of OA (0.22 mg g⁻¹ DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g⁻¹ DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.     

Co-production of S-adenosyl-l-methionine and glutathione from spent brewer’s yeast cells

Both S-adenosyl-l-methionine (AdoMet) and glutathione (GSH) are important small molecules with pharmaceutical importance. The co-production of AdoMet and GSH using abundant spent brewer’s yeast cells from the beer industry and with l-methionine supplement was successfully realized. Experimental data showed that improvement of GSH productivity was accompanied by AdoMet accumulation. AdoMet productivity of 40–45 mg g⁻¹ (DCW) was successfully achieved and an additional 13–18 mg g⁻¹ (DCW) GSH was synthesized in spent brewer’s yeast cells.     

Snail herbivory on submerged macrophytes and nutrient release: Implications for macrophyte management

Radix swinhoei (H. Adams) is a freshwater snail commonly found in shallow regions of Lake Taihu. This research estimated, based on experiments, the consumption rates of R. swinhoei on three young submerged plants (Vallisneria spiralis, Hydrilla verticillata and Potamogeton malaianus) and its rates of nutrient release. Results showed that the snails consumed V. spiralis at the highest rate (23.34 mg g⁻¹ d⁻¹), P. malaianus at a lower rate (11.97 mg g⁻¹ d⁻¹), and H. verticillata at the lowest rate (7.04 mg g⁻¹ d⁻¹). The consumption rates on V. spiralis varied significantly, with snail size, ranging from 13.63 mg g⁻¹ d⁻¹ for large-size snails to 143.42 mg g⁻¹ d⁻¹ for small-size ones.The average nutrient release rates of snails grazing on different macrophytes were 45.93 μg PO₄-P and 0.58 mg NH₄-N g⁻¹ d⁻¹. The food species had a significant effect on NH₄-N release rates but not on PO₄-P. However, the snail size had a significant effect on PO₄-P release rates and not on NH₄-N. The present study indicates that through selective grazing and nutrient release, snails may impose a significant impact on the macrophyte community, which should be considered in managing the macrophytes of a lake.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Effect of culture medium composition on Trichoderma reesei’s morphology and cellulase production

The objective of this study was to determine how fungal morphology influences the volumetric cellulase productivity of Trichoderma reesei cultured in four media with lactose and lactobionic acid as fed-batch in a 7 L stirred tank bioreactor. The use of a cellulose–yeast extract culture medium yielded the highest enzyme production with a volumetric enzyme activity of 69.8 U L⁻¹ h⁻¹, and a maximum fungal biomass of 14.7 g L⁻¹. These findings were associated with the following morphological characteristics of the fungus: total mycelia was 98% of total mean projected area, mean hyphae length of 10 mm, mean hyphae volume of 45.1 mm³, mean hyphae diameter of 7.9 μm, number of branches 9, and number of tips per hypha 29. A positive correlation was found between the total mycelia, the number of tips and the volumetric enzyme productivity, indicating the weight of these variables on the enzyme productivity.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Biochemical characterization of Rhodococcus erythropolis N′4 nitrile hydratase acting on 4-chloro-3-hydroxybutyronitrile

The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α₂β₂. The purified enzyme had a high specific activity of 860 U mg⁻¹ toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min⁻¹ mg⁻¹. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.     

Treatment of an artificial sulphide containing wastewater in subsurface horizontal flow laboratory-scale constructed wetlands

In general, treatment wetlands seem to be a potential method of tackling the sulphide problem of post-treatment of anaerobic digester effluents.Because of insufficient practical experience and lack of knowledge of sulphide removal, sulphur transformation was investigated, particularly in horizontal subsurface flow constructed wetlands (depth of 35 cm) under laboratory-scale conditions with artificial wastewater.The plants affected a clear stimulation of the sulphide and ammonia removal rates. Sulphide concentration in the range of 1.5–2.0 mg l⁻¹ was tolerated by the plants and completely removed in the planted model wetlands; sulphide concentration of >2.0 mg l⁻¹ caused instabilities in sulphide and nitrogen removal. Area-specific sulphide removal rates of up to 94 mg sulphide m⁻² d⁻¹ were achieved in the planted beds at hydraulic retention times of 2.5 d. Sulphate affected the sulphide removal. While in the unplanted control bed an almost stable removal in the range of 150–300 mg N m⁻² d⁻¹ was observed variations of hydraulic retention time, sulphide and sulphate concentrations influenced the ammonia removal rate within the planted beds in a broader range (600–1400 mg N m⁻² d⁻¹).These results showed that nitrification, sulphide oxidation, denitrification and sulphate reduction can occur simultaneously in the rhizosphere of treatment wetlands caused by dynamic redox gradients (aerobic–anaerobic) conditions.     

Factors affecting ferulic acid release from Brewer’s spent grain by Fusarium oxysporum enzymatic system

In this study, the factors affecting ferulic acid (FA) release from Brewer’s spent grain (BSG), by the crude enzyme extract of Fusarium oxysporum were investigated. In order to evaluate the importance of the multienzyme preparation on FA release, the synergistic action of feruloyl esterase (FAE, FoFaeC-12213) and xylanase (Trichoderma longibrachiatum M3) monoenzymes was studied. More than double amount of FA release (1 mg g⁻¹ dry BSG) was observed during hydrolytic reactions by the crude enzyme extract compared to hydrolysis by the monoenzymes (0.37 mg g⁻¹ dry BSG). The protease content of the crude extract and the inhibitory effect of FA as an end-product were also evaluated concerning their effect on FA release. The protease treatment prior to hydrolysis by monoenzymes enhanced FA release about 100%, while, for the first time in literature, FA in solution found to have a significant inhibitory effect on FAE activity and on total FA release.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.