Synthesis of l-Tryptophan from Indole and dl-Serine by Tryptophan Synthetase Entrapped in Fibres

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.

The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.

The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.

Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 days) provided the accumulation of indole on the fibres was avoided.     

L-Malic acid production by polyacrylamide gel entrapped Pichia membranaefaciens

Summary The production of L-malic acid from fumaric acid has been achieved byPichia membraneafaciens cells entrapped in a polyacrylamide gel lattice. The reaction rate was found to be 0.15 mmoles/h/g of immobilized cells. The optimum pH for fumarase activity of immobilized cells was stable after repeated uses it increased after storing the gel pellets at 5°C. A good yield of L-malic acid production (up to 3.77 g/l) was also observed in wine added with Na fumarate.     

Degradation of 4-aminobenzenesulfonate by alginate encapsulated cells of Agrobacterium sp. PNS-1

Studies were carried out on 4-aminobenzenesulfonate (4-ABS) degradation by free and alginate entrapped cells of Agrobacterium sp. PNS-1. Degradation rate in batch reactors with free cells was marginally higher than Ca-encapsulated cells. Comparison of Ca2+ and Ba2+ as gelling agents showed that 4-ABS removal rate was significantly less with Ba-alginate entrapped cells. Specific degradation rates, using linear regression analysis and based on the initial biomass in the beads, varied from 49.7 mg/mg biomass/h to 92.0 mg/mg biomass/h for Ca-alginate encapsulated cells for different initial 4-ABS concentrations ranging from 200 to 800 mg/L. UV spectra of the aliquots drawn at different time intervals from batch reactors did not show accumulation of any intermediate during degradation. Ca-alginate immobilized cells could be repeatedly reused upto five cycles without any loss of activity. Studies with packed bed reactors, operated in a semi-continuous mode, showed that this could be used for 4-ABS degradation.     

Properties of fibre-entrapped aspartase

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) was extracted and purified from Escherichia intermedia cells. The enzyme was entrapped in cellulose triacetate porous fibres and the properties of the immobilized enzyme compared with those of the free enzyme. Similar behaviour was observed with regard to optimum pH, temperature, heat stability and kinetic constants. The stability of the entrapped enzyme was tested under operating conditions in a series of batch reactions. Good results were obtained for both the stability and the efficiency of the immobilized enzyme. The potential use of aspartase fibres for the production of l-aspartic acid is discussed.     

Effect of the structure of polymeric materials on the properties of immobilized enzymes

The relationship between the structure of triacetate cellulose fibres and films and properties of enzymes entrapped in these materials was investigated. Trypsin and penicillin amidase were entrapped in triacetate cellulose films and fibres during their formation. The effect of permeability of the films on the catalytic properties of entrapped trypsin was studied. The porous structure of triacetate cellulose fibres with entrapped penicillin amidase was studied, the fibres being produced by different methods. An increase of porosity and the specific surface of the fibre-biocatalyst reduced the Km value and the energy of activation of the hydrolytic decomposing of benzyl penicillin but increased the Vmax value.     

Arginase,an s-phase enzyme in a human cell line

Suspension cultures of ‘Chang liver’ cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S+G₂) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G₁/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4–6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in ‘Chang liver’ cells is discussed.     

Continuous deacidification of wine by immobilized Schizosaccharomyces pombe cells: evaluation of malic acid degradation rate and analytical profiles

Schizosaccharomyces pombe cells entrapped in Ca alginate gel were used under continuous fermentation conditions to evaluate the extent of malic acid degradation at different dilution rates ( D , h⁻¹) and the analytical profiles of wines obtained. Gel entrapped cells caused the deacidification of wine without affecting the analytical profiles. Maximum deacidification rate was obtained at 0.076 h⁻¹ D while higher dilution rates (up to 0.100 h⁻¹ D ) resulted in a clear reduction of activity. At D -value of 0.055 h⁻¹, the deacidification activity remained constant during the observation period of 360 h.     

Lectin-specific targeting of beta-glucocerebrosidase to different liver cells via glycosylated liposomes

Galactosylated and mannosylated liposomes were more efficient in transporting liposome-entrapped beta-glucocerebrosidase to liver compared to nonglycosylated liposomes. The enzyme entrapped to glycoside-bearing liposomes was found to be cleared at a much faster rate than that entrapped in liposomes having no sugar on their surface. Asialoorosomucoid and hydrolyzed mannan were found to inhibit both the clearance and the uptake of galactosylated and mannosylated liposomes, respectively, supporting involvement of lectin-sugar interaction. Further studies on the uptake of glucocerebrosidase by isolated liver cells revealed that the enzyme entrapped in mannosylated liposomes has much higher affinity for nonparenchymal cells whereas the assimilation of the entrapped enzyme into hepatocytes is clearly favored for liposomes having galactose on their surface.     

Slow adaptive changes in urease levels of tobacco cells cultured on urea and other nitrogen sources

Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea as the sole source of nitrogen have urease activities about four times higher than cells which have been cultured on nitrate. When cells which had always been grown on nitrate were transferred to urea, the urease activity in these cells remained at a lower level for eight transfers (40 generations), then gradually increased 4-fold during the next seven to 10 transfers. Cells with high urease activity multiplied 19% more rapidly and accumulated less urea than cells with low urease activity. These findings suggest that elevated urease accelerates urea assimilation; therefore, urea limited growth. Clones of cells with low urease activity responded in the same way as uncloned populations when transferred from nitrate to urea, indicating that high urease cells originate from low urease cells, rather than from a preexisting subpopulation of high urease cells. The urease levels in clones of cells from a population with high urease activity were three to seven times the low urease level. The observed dependence of urease activity on generations of growth on urea was matched with a model in which high urease cells originated at mitosis of low urease cells at a frequency of 8 × 10⁻⁵, then multiplied 19% more rapidly than low urease cells. This frequency is about 10³ greater than that of other biochemical variants previously isolated from XD cells. The high urease activity gradually declined in cells transferred from urea to other nitrogen sources, but rose rapidly when such cells were returned to urea, indicating the existence within the cells of some form of record of their ancestors' growth on urea. The data indicate the existence of a mechanism for generation, at unusually high frequency, of metastable variants with high urease activity. This mechanism, coupled with enrichment for the variants' progeny by virtue of their higher multiplication rate on urea, can account for the observed slow increase in urease activity of the population. It is suggested that the molecular basis of the urease increase may be gene amplification, based on animal cell models. An alternative hypothesis, namely a specific response induced in all cells by urea and manifested as a very slow adaptive increase in urease, has not been ruled out.     

Sucrose inversion by gelatin-entrapped cells of yeast (Saccharomyces cerevisiae)

Summary Sucrose hydrolysis by invertase-active yeast cells (S. cerevisiae) entrapped in gelatin was investigated using different types of miniaturized reactors. The entrapped preparations showed the highest operational stability in a continuous stirred-tank reactor. The invertase activity of the entrapped preparation was found to be almost independent of the buffer concentration so that sucrose invermay be conducted in a non-buffered medium.     

Immobilization and stability of the NAD-dependent hydrogenase from alcaligenes eutrophus and of whole cells

Summary The purified soluble NAD-dependent hydrogenase from Alcaligenes eutrophus was immobilized to porous glass beads according to the glutaraldehyde method retaining about 80% of its original activity. Entrapment of the purified hydrogenase in photo-crosslinkable prepolymers led to apparent activity yields of 10–80% dependent on the thickness of the gel film. The storage stability of entrapped hydrogenase (t/2 = 4 d) was considerably lower than that of glass-bound hydrogenase (t/2 = 150 d). During continuous production of NADH (turnover conditions), the half-life of entrapped hydrogenase was not longer than 10 h. Whole cells of A. eutrophus entrapped in a polyurethane matrix were used to produce NADH with hydrogen gas as electron donor. After 18 runs for 4h each and storage periods overnight the residual activity was still about 50%.     

Removal of flatulence-inducing sugars by using free and polyvinyl alcohol immobilized α-Galactosidase from Aspergillus oryzae

Consumption of soymilk and soybean derived foods has been hampered due to the presence of RFOs (raffinose family oligosaccharides). Soy-based foods free from RFOs have positive impact on their acceptance as protein rich food. α-Galactosidase was entrapped in PVA (polyvinyl alcohol) cross linked with boric acid. Immobilized enzyme showed shift in optimum pH of 0.4 units and the activity yield of the immobilized α-galactosidase was found to be 76%. Immobilized enzyme was used to reduce RFOs in soymilk. In batch reaction after 12 h incubation soluble and immobilized enzyme showed 92 and 83% reduction of RFOs in soymilk, respectively. In repeated batch experiments immobilized enzyme showed 64% of its hydrolyzing activity after 6th cycle. PVA-immobilized α-galactosidase in fluidized bed reactor showed highest reduction (92%) of RFOs at a flow rate of 30 mL/h. The results of this study are interesting for their use in food processing industry.     

Migration and keratinization of cells in wool follicles

Migration of cells in wool follicles of an adult Merino sheep was studied autoradiographically in skin samples taken at intervals after an intravenous injection of [3H]thymidine. Fibre and inner root sheath cells incorporated [3H]thymidine in a cone-shape region of the follicle bulb. Labelled inner sheath cells migrated out of the bulb ahead of contemporaneous cells in the fibre and remained in advance, although to a progressively lesser extent, until the inner sheath cells sloughed into the follicle lumen. Outer root sheath cells incorporated [3H]thymidine along the length of the follicle. Cells in the proximal half of the outer sheath migrated inwards and distally and sloughed into the follicle lumen before contemporaneous inner sheath cells. Other cells in the distal half of the outer sheath migrated past the level where cells from the proximal population were shed and also sloughed into the lumen. In the most distal part of the outer sheath, which formed the epidermis-like lining of the follicle canal, little migration of cells was observed during 8 days of observation. The specific activity of tritium in fibres plucked from the same sheep at intervals after the intravenous injection of [3H]thymidine was determined by scintillation counting and assessed in terms of cell migration and hardening of the fibres. The time which the specific activity of solvent-degreased fibres reached a maximum was found to give an estimate of the time for cells in the fibre to migrate to the upper limit of the keratogenous zone. When the plucked fibres were extracted with 8 M urea the times of the maximum specific activities of the urea-dispersible and urea-insoluble material provided respectively estimates of the times at which hardening of the fibres began and ended. The effects of different planes of nutrition were examined in two other Merino sheep by radioassay of fibres plucked after intravenous injections of [3H]thymidine given after equilibration period of at least 2 months on each level of feeding. A high plane of nutrition the rate of cell migration and hastened the onset of hardening of the fibres, but prolonged the hardening process. The prolongation of the hardening process was confirmed by the specific activities of fibres plucked after intravenous injections of [35S]cystine.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Conversion of alpha-ketoglutarate into L-glutamic acid with urea as ammonium source using multienzyme systems and dextran-NAD+ immobilized by microencapsulation within artificial cells in a bioreactor

Urea could be effectively converted into L-glutamic acid with semipermeable nylon-polyethylenimine artificial cells containing L-glutamic dehydrogenase (EC 1.4.1. 3), yeast alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1. 5) and soluble dextran-NAD(+). For batch conversion, the artificial cell suspension to total reaction volume ratios ranged from 1 in 5 to 1 in 60. From 22.6 to 53.4 mumol of L-glutamic acid could be produced by 0.4 mL artificial cell suspension within 2 h. The corresponding conversion ratios were 56.5-11. 1%. The L-glutamic dehydrogenase multienzyme system showed a good storage stability: 66.0% of the original activity was retained after 1 month of storage at 4 degrees C. A small bioreactor was prepared to contain 4.0 mL artificial cells. At a flow rate of SV = 1.5 h(-1), the maximum conversion rate was 49.6 mumol L-glutamic acid/p h. Thirty-eight percent of the maximum activity was retained when continuously used for four days at 22 degrees C. A kinetic analysis for the L-glutamic dehydrogenase multienzyme system was studied. The Michaelis constants are as follows: alpha-ketoglutarate is 0.838 mM; urea is 1.90 mM; dextran- NAD(+) is 0.345 mM; and ethanol is 5.31 mM.     

Properties of glucose uptake in vegetative and sporulating cells of Saccharomyces cerevisiae

The effect of digitonin, acetic acid, urea and ethanol treatment on the glucose uptake of vegetative cells and of sporulating cells (3 h after transfer to sporulation medium) was examined in Saccharomyces cerevisiae. Both glucose uptake activities decreased at a similar rate, and a slightly different rate, in treatment with various concentrations of digitonin and of acetic acid, respectively, at 25 degrees C for 10 min. The glucose uptake activity of the sporulating cells was much more stable to urea treatment than that of the vegetative cells; the activity decreased about 36% and 76% in the sporulating cells and the vegetative cells, respectively, under conditions of 2.5 M urea at 25 degrees C for 10 min. The glucose uptake activity of the vegetative cells was more stable to ethanol treatment than that of the sporulating cells; the activity decreased about 56% and 88% in the vegetative cells and the sporulating cells, respectively, in 25% ethanol at 25 degrees C for 10 min.     

Production of nikkomycin by immobilized Streptomyces cells — Physiological properties

Summary The antibiotic nikkomycin can be produced by calcium alginate immobilized mycelium of Streptomyces tendae Tü 901 in batch and continuous culture.Scanning electron micrographs show the porous structure of the matrix and the distribution of the cells in the gel.Some physiological properties of free and immobilized mycelia were compared. Immobilization does not change the relative amounts of nikkomycin compounds in the culture broth. DNA and protein content were the same in free and immobilized cells. The specific activity of fructosediphosphate aldolase dropped during fermentation and was lower for entrapped than for free cells. The specific activity of mannitol dehydrogenase increased up to the end of the fermentation and was the same for free and immobilized mycelium.In continuous culture the relative amount of mannitol consumed decreased with increasing flow rate. When the medium was supplemented with amino acids mannitol consumption increased significantly.Dedicated to Prof. Dr. L. Acker on the occasion of his 70th birthday     

LDH isoenzymes in the axial muscle of the sharks Galeus melastomus and Etmopterus spinax

The lactate dehydrogenase isoenzyme patterns have been studied in the axial muscles of the sharks Etmopterus and Galeus. Samples from red, intermediate and white muscle fibres were run separately on a polyacrylamide slab-gel. Both sharks have three isoenzymes; all three are present in the red and intermediate fibres, while the white fibres contain only the two slowest-moving isoenzymes. The red fibres of both sharks contain most of the fastest-moving isoenzyme.
The isoenzymes have a high tolerance towards urea; the slow moving isoenzyme is inhibited at about 2 m urea, the next isoenzyme at 4-6 M urea, and some activity of the fast-moving isoenzyme is still present at 10 M urea in the incubation medium. The LDH distribution in the fibre types is studied by histochemistry on frozen sections.     

Dialysis Culture of T-Strain Mycoplasmas

Using dialyzing cultures of T-strain mycoplasmas, it was possible to make some observations relevant to the growth and metabolism of these organisms which would not be possible in nondialyzing cultures due to growth inhibition of the organisms by elevated pH and increased ammonium ion concentration in media containing urea. The rate of ammonia accumulation was found to be related to the initial urea concentration in the medium and could not be accounted for by any change in the multiplication rate of the organisms. More ammonia was generated than could be accounted for by the added urea alone, suggesting that an ammonia-producing activity other than urease may be present in T-strain mycoplasmas. Titers above 10⁷ color change units per ml were achieved in dialysis cultures of a T-strain mycoplasma in the presence of urea, and such titers were maintained for approximately 60 h during dialysis culture in the absence of added urea.     

Branchial and renal handling of urea in the gulf toadfish,Opsanus beta: the effect of exogenous urea loading

The objective of this study was to determine whether the pulsatile facilitated diffusion transport mechanism (tUT) found in the gills of the gulf toadfish (Opsanus beta) and the active secretion transporter thought to be present in its kidney could be saturated when faced with elevated plasma urea concentrations. Toadfish were infused with four consecutive exogenous urea loads at a rate of 0, 150, 300 and 600 micromol kg(-1) h(-1). Initial plasma and urine urea concentrations were 8.1+/-0.9 and 12.4+/-1.5 mmol l(-1), respectively, and steadily increased with increasing infused loads of urea to a maximum of 36.8+/-2.8 mmol l(-1) in the plasma and 39.8+/-6.5 mmol l(-1) in the urine. There was only a very weak relationship (r=0.17) between pulse size (measured as branchial excretion during pulsatile excretion of urea) and plasma urea concentration (slope=9.79 micromol-N kg(-1) per mmol-N l(-1); P<0.05) suggesting that the branchial excretion mechanism was already saturated at normal plasma urea concentrations. Urine flow rate (0.15+/-0.03 ml kg(-1) h(-1)) and glomerular filtration rate (0.025+/-0.004 ml kg(-1) h(-1)) remained constant throughout the experiment despite the increased volume load. Renal urea secretion rate maintained a strong linear relationship (r=0.84) to plasma urea levels (slope=0.391 micromol-N kg(-1) h(-1) per mmol-N l(-1); P<0.001) with no observable transport maximum, suggesting that the renal secretory transport mechanism was not saturated even at plasma urea levels well above normal, in contrast to the branchial excretion mechanism.