Effect of tree leaf as supplementation on nutrient digestion and rumen fermentation pattern in sheep grazing semi-arid range of India – II

A study was conducted to evaluate the effects of supplementing with different tree leaves on nutrient digestion, rumen fermentation and blood parameters of sheep. Thirty adult Malpura rams (39.0 ± 0.56 kg) were divided into five groups of six each. They were grazed as a single flock on a semi-arid rangeland and after the end of routine grazing period (08:00–17:00 h), first group (G1), which was not provided with any supplementation, served as control group. Second group (G2) was supplemented with 200 g of a conventional concentrate mixture per head per day, whereas third, fourth and fifth groups (G3, G4 and G5) were supplemented with approximately 200 g dry matter (DM) per day freshly cut foliage from Ailanthes excelsa, Azardirachta indica and Bauhinia racemosa, respectively. Protein content (g kg⁻¹ DM) in A. excelsa, A. indica and B. racemosa foliage was 197, 128 and 132, respectively. A. indica and B. racemosa foliages also contained 123.2 and 211.2 g kg⁻¹ DM condensed tannin (CT) with protein precipitating capacity (PPC) of 16.5 and 46.5 g kg⁻¹ DM. None of the tree leaves contained hydrolysable tannin (HT). Dry matter intake (DMI, g day⁻¹) was 591, 766, 865, 974 and 939 in G1, G2, G3, G4 and G5, respectively. Digestible crude protein (DCP) and metabolisable energy (ME) intakes in supplemented groups G2–G5 were higher (P < 0.05) compared to control (G1). Supplementation improved digestibility of all nutrients in all groups. Rumen fermentation study indicated lower (P < 0.05) ammonia and total N in the rumen liquor collected from G5 sheep compared to the other supplemented groups. Although haemoglobin (Hb, g dl⁻¹) levels showed small changes among groups, blood urea nitrogen (BUN, mg dl⁻¹) was lowest in G5 compared to the other groups. Initial BW were similar among the groups. After 60 days of experimental feeding, all animals maintained their BW, except sheep in the control group (G1), which lost BW. Results indicate that for adult sheep grazing on a semi-arid range, supplementation with a concentrate mixture could be replaced by tree leaves like A. excelsa, A. indica and B. racemosa, during the lean season to maintain their BW. In addition, supplementing with tree leaves containing condensed tannin has advantages in terms of N utilization.     

Organ-specific distribution of dietary alkaloids in the marine opisthobranch Tylodina perversa

Specimens of the opisthobranch Tylodina perversa that were observed while feeding on the sponge Aplysina aerophoba were transferred to seawater tanks along with their prey and kept under controlled conditions. After one week the opisthobranchs were anaesthetized, dissected and studied for sequestered sponge-derived brominated alkaloids. All parts of T. perversa analyzed including feces, mucus and egg masses that had been produced during captivity contained alkaloids derived from A. aerophoba. The highest total alkaloid concentration (24.6 mg g⁻¹ dry wt) was found in mantles of T. perversa (compared to 51.2 mg g⁻¹ dry wt of total alkaloids in A. aerophoba). Hepatopancreas, egg masses and mucus (respective total alkaloid concentrations ranging from 20.4 to 12.5 mg g⁻¹ dry wt) were also rich in alkaloids. Whereas in A. aerophoba the isoxazoline alkaloids aerophobin-2 and isofistularin-3 were present in almost equal concentrations, aerophobin-2 constituted by far the major alkaloid (amounting to approximately 70% of all identified alkaloids) in mantles, mucus and egg masses of T. perversa, indicating selective sequestration by the opisthobranchs. Mantles as well as mucus also contained appreciable concentrations (approximately 20% of all identified compounds) of the brominated alkaloid aerothionin; this is not detected in A. aerophoba. It is possible that aerothionin originates from a previous encounter of T. perversa with the sponge A. cavernicola, the latter being closely related to A. aerophoba. The enrichment of aerophobin-2 (and of aerothionin) in mantles, mucus and egg masses that are vulnerable and exposed (mantles and egg masses) to predators and/or pathogens argues for defensive functions of the respective alkaloids even though this hypothesis still needs to be experimentally corroborated.     

The response of an emergent sedge Bolboschoenus medianus to salinity and nutrients

The potential for nutrient load (30, 100 and 350 g N m⁻² per year) to alter plant performance under saline conditions (control, 4.5, 9 and 13 dS m⁻¹) was examined in the sedge Bolboschoenus medianus. Relative growth rates (RGR) across nutrient loadings ranged from 30.2 to 41.8 mg g⁻¹ per day in controls and were reduced to 20.9–28.5 mg g⁻¹ per day by salinities of 13 dS m⁻¹. Whilst higher nutrient loads generally increased RGR, the response was smaller at higher salinities. Responses to salinity and nutrient load were specific. Nutrient load increased the RGR via increases in the leaf area ratio (LAR). The LAR ranged from 1.9 to 2.1 m² kg⁻¹ across salinity treatments at 30 g N m⁻² per year, and increased to 2.5–2.8 m² kg⁻¹ at 350 g N m⁻² per year. Salinity reduced the RGR via a reduction in the net assimilation rate (NAR). The NAR in control plants ranged from 14.7 to 16 g m⁻² per day across nutrient loadings and decreased to 11–12 g m⁻² per day at 13 dS m⁻¹. Carbon isotope discrimination of leaves decreased by 2–3‰ in response to 13 dS m⁻¹ at the lower nutrient loadings. A prominent response of B. medianus to salinity was a change in biomass allocation from culms to tubers. In contrast, the response to nutrient load was characterised by a shift in biomass allocation from roots to leaves.     

Cadmium biosorption on Spirulina platensis biomass

Dry biomass of Spirulina platensis re-hydrated for 48 h was employed as a biosorbent in tests of cadmium(II) removal from water. Various concentrations of biomass (from 1 to 4 g l⁻¹) and metal (from 100 to 800 mg l⁻¹) were tested. Low biomass levels (X_o  2 g l⁻¹) ensured metal removal up to 98% only at Cd₀= 100 and 200 mg l⁻¹, while X_o  2.0 g l⁻¹ were needed at Cd₀ = 400 mg l⁻¹ to achieve satisfactory results. Whereas X_o = 4.0 g l⁻¹ was effective to remove up to Cd₀ = 500 mg l⁻¹, a further increase in metal concentration (Cd₀ = 600 and 800 mg l⁻¹) led to progressive worsening of the system performance. At a given biomass levels, the kinetics of the process was better at low Cd²⁺ concentrations, while, raising the adsorbent level from 1.0 to 2.0 g l⁻¹ and then to 4.0 g l⁻¹, the rate constant of biosorption increased by about one order of magnitude in both cases and the adsorption capacity of the system progressively decreased from 357 to 149 mg g⁻¹.     

Purification and immobilization of recombinant variants of Brevundimonas diminuta glutaryl-7-aminocephalosporanic acid acylase expressed in Escherichia coli cells

Cyclotides are naturally occurring mini-proteins that have a diverse range of therapeutically useful biological activities. Although a choice of approaches is available for cyclotides synthesis; most studies have involved the use of peptides extracted from plants. In order to facilitate the screening for structure-activity studies or to exploit them in drug development, a convenient and reliable route for the biosynthesis of cyclotides is of vital importance.

Callus, suspension cultures and hydroponic plants of Oldenlandia affinis were established and have been evaluated for effective cyclotides production processes. The specific accumulation of kalata B1 was affected by cell differentiation as well as agitation; highest accumulation of 2.7 mg g⁻¹ dry weight was detected in agitated hydroponic plant cultures resulting in a productivity of 1.4 mg kalata B1 l⁻¹ day⁻¹.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Halophila ovalis (R. Br.) Hook. f. from a submarine hot spring in southern Japan

Colonies of the seagrass Halophila ovalis are found growing adjacent to coral Acropora sp. and Seriatopora hystrix in a submarine hot spring (at 15.7 m depth, 28.6°C) at the north coast of Taketomi Island, near the southern tip of Japan. Halophila plants grow in sea water containing sulphide 930 μg S ml⁻¹ and on the substratum with fine precipitates of the submarine hot spring which have sulphide content up to 5400 μg S g⁻¹ DW. The accumulated sulphide concentration reaches as high as 8400 μg S g⁻¹ DW in under ground tissues and 5700 μg S g⁻¹ DW in above-ground tissues, respectively. It is suggested that, not the sulphide concentration but light and possibly water temperature are the limiting factors for the Halophila colonization in the submarine hot spring.     

Phenol inhibition of biological nutrient removal in a four-step sequencing batch reactor

Nutrient removal from synthetic wastewater was investigated using a four-step sequencing batch reactor (SBR) at different phenol (C₆H₅OH) concentrations in order to determine the inhibition effects of phenol on biological nutrient removal. The nutrient removal process consisted of anaerobic, oxic, anoxic, and oxic phases with hydraulic residence times (HRT) of 1 h/3 h/1 h/1 h and a settling phase of 3/4 h. Solids retention time (SRT) was kept constant at 10 days in all experiments. Initial phenol concentrations were varied between 0 and 600 mg l⁻¹ at seven different levels. The effects of phenol on COD, NH₄-N, and PO₄-P removals and effluent nutrient levels were investigated. Phenol was almost completely degraded up to 400 mg l⁻¹ phenol concentration resulting in almost negligible inhibition effects on COD, NH₄-N, and PO₄-P removals. Nutrient removals were adversely affected by phenol at concentrations above 400 mg l⁻¹. Above 95% COD, 90% NH₄-N and 65% PO₄-P removal was obtained for phenol concentrations below 400 mg l⁻¹. The sludge volume index (SVI) was almost constant around 45 ml g⁻¹ for phenol concentrations below 400 mg l⁻¹ but increased to 90 ml g⁻¹ at a phenol level of 600 mg l⁻¹.     

Removal of lead (Pb(2+)) by the cyanobacterium Gloeocapsa sp

Pb²⁺ removal ability of the viable-freshwater cyanobacterium Gloeocapsa sp. was studied in batch experiments. Gloeocapsa sp. was cultured in the Medium 18 with pH adjusted to 3, 4, 5, 6 and 7. Growth was subsequently determined based on the increase of chlorophyll-a content. Gloeocapsa sp. was able to grow at all pH levels tested, except at pH 3. Removal of Pb²⁺ was then further studied under pH 4. The results showed that Pb²⁺ concentration in the range of 0–20 mg L⁻¹ was not inhibitory to Gloeocapsa sp. growth but reduced its Pb²⁺ removal efficiency (by 4.5% when Pb²⁺ concentration increased from 2.5 to 20 mg L⁻¹). Pb²⁺ removal characteristics followed the Langmuir adsorption isotherm with the maximum removal capacity (q_max) of 232.56 mg g⁻¹. Adsorption of Pb²⁺ by this cyanobacterium followed the second order rate reaction and intraparticle diffusion was likely the rate-determining step. The initial rate of Pb²⁺adsorption during intraparticle diffusion was slower under light than under dark conditions, indicating that light probably slowed down the initial rate of intraparticle diffusion through the repulsion effects on cell membrane.     

Enhanced production of free trans-astaxanthin by oxidative stress in the cultures of the green microalga Chlorococcum sp

Free trans-astaxanthin accumulated in the alga Chlorococcum sp. was markedly enhanced from 3.664 mg g⁻¹ cell dry weight to 5.724 mg g⁻¹ cell dry weight when the culture was supplemented with hydrogen peroxide (0.1 mM) under mixotrophic conditions of growth. After saponification, a total of 7.086 mg astaxanthin per g cell dry weight was achieved. Similarly, in heterotrophic cultures, the total astaxanthin content was increased from 1.034 mg g⁻¹ cell dry weight without H₂O₂ to 1.782 mg g⁻¹ cell dry weight with 0.1mM H₂O₂. Results indicate that hydrogen peroxide effectively induces the formation of free trans-astaxanthin in Chlorococcum sp.     

Effect of leaf age on CAM activity in Littorella uniflora

In this study the effect of ontogenetic drift on crassulacean acid metabolism (CAM) was investigated in the aquatic CAM-isoetid Littorella uniflora. The results of this study strengthen the general hypothesis of CAM being a carbon-conserving mechanism in aquatic plants, because high-CAM capacity (45–183 μequiv. g⁻¹ FW) was present in all leaves of L. uniflora irrespective of age. Since possession of CAM in aquatic plants allows CO₂ uptake throughout the light/dark cycle, presence of CAM in all leaves influences the carbon balance of L. uniflora positively. On average for all lakes, different leaf classes accounted for 11–36% of the total dark CO₂ uptake by the individual plant.

The capacity for both CAM and photosynthesis declined with increasing leaf age, and was in the oldest leaves only 25–53% of the capacity in the youngest. The photosynthetic capacity was estimated to be sufficiently high to ensure refixation of the CO₂ released from malate during decarboxylation in the daytime. In line with this, a linear coupling between CAM capacity and photosynthetic capacity was found. Parallel to the change in photosynthetic capacity, an age-related change in total ribulose-bisphosphate carboxylase/oxygenase (rubisco) activity from 732 μmol C g⁻¹ DW h⁻¹ in the youngest leaves to 346 μmol C g⁻¹ DW h⁻¹ in the oldest was observed. In contrast, no significant change in phosphoenolpyruvate carboxylase (PEPcase) activity with leaf age was observed (means ranged between 46 and 156 μmol C g⁻¹ DW h⁻¹).     

Transformation of Saussurea involucrata by Agrobacterium rhizogenes: Hairy root induction and syringin production

Agrobacterium rhizogenes-mediated genetic transformation of Saussurea involucrata was investigated. Four bacterial strains, A4, LBA 9402, R1000 and R1601 and three explant types, leaf blade, petiole and root, were examined. Over 100 hairy root lines were successfully established with strains R1601, R1000 and LBA9402, but none with A4. The highest transformation efficiency of 67% was achieved by using strain R1601 with root explants. One hairy root line isolated from this combination, HR1601-1, produced up to 43.5 ± 1.13 mg syringin g⁻¹ dw, which is about 50-fold higher than that in the wild type plants.Two other lines, HR1000-1 and HRLBA9402-1, isolated from R1000- and LBA9402-transformed roots, respectively, also displayed high capacity of syringin production, being 32.5 ± 3.08 and 39.7 ± 1.37 mg syringin g⁻¹ dw. These three lines were characterized in detail. Polymerase chain reaction analyses confirmed these root lines were of A. rhizogenes origin.     

Effect of feeding tree leaves as supplements on the nutrient digestion and rumen fermentation pattern in sheep grazing on semi-arid range of India – I

A study was carried out to determine the effect of feeding different tree leaves as supplements on nutrient digestion, rumen fermentation and blood parameters of sheep grazing on a semi-arid rangeland. Thirty adult Malpura rams of uniform body weight (39.0 ± 0.75) were divided into five groups of six each. They were grazed as a single flock from 08.00 to 17.00 h on a semi-arid rangeland. After the end of the grazing period, the first group (G1), which was not provided with any supplementation, served as the control. The second group (G2) was supplemented with 200 g of a concentrate mixture per head per day, whereas the third, fourth and fifth groups (G3–G5) were provided with approximately 200 g DM d⁻¹ of freshly cut foliage from Prosopis cineraria, Acacia nilotica and Albezia lebbek. The foliage from P. cineraria contained 133.4 g kg⁻¹ DM condensed tannin (CT) with protein precipitating capacity (PPC) of 66 g kg⁻¹ DM, whereas A. nilotica contained 18.9 g kg⁻¹ DM hydrolysable tannin (HT) with PPC of 11.5 g kg⁻¹ DM. However, A. lebbek did not contain any tannin. The protein contents were 119, 139 and 194 g kg⁻¹ DM, respectively. The DMI (g d⁻¹) was 688, 916, 1024, 1003, 999 in G1, G2, G3, G4 and G5, respectively. Digestible crude protein (DCP) and metabolizable energy (ME) intakes in supplemented groups G2–G5 were higher (P < 0.05) than in the control (G1). Supplementation improved the DM digestibility in all groups, whereas CP digestibility was lower (P < 0.05) in G3 compared to G2, G4 and G5. Rumen fermentation study conducted 6 h after supplementation revealed that total N, ammonia N, and total VFA levels were lower (P < 0.05) in G3 compared to the other supplemented groups. Although the haemoglobin (Hb) levels were similar among groups, blood urea N (BUN) was lowest in G3 compared to the other groups. The initial body weights were similar among groups (mean 39 kg). After 60 days of experimental feeding, all groups maintained their body weight, except the control group (G1), which lost body weight. It was observed, that supplementation with tree leaves containing CT like P. cineraria helps in better rumen fermentation pattern by preventing excessive loss of nitrogen. It was concluded that maximum nutritional benefits of tree leaves could be harvested, if used as supplement rather than as a sole feed.     

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum and Spirulina platensis under phosphate limitation

Nostoc muscorum and Spirulina platensis were grown under phosphate deficiency in order to investigate the role of internal phosphate pool and activity of alkaline phosphatase on poly-β-hydroxybutyrate (PHB) accumulation. PHB accumulation in N. muscorum increased to 22.7% of dry weight (dw) after 4 day of phosphate deficiency, while the internal phosphate pool reduced to the level of 0.02 μM mg dw⁻¹ at a maximum APase activity of 2.57 nM PNP mg dw⁻¹ h⁻¹. In contrary, S. platensis depicted maxima of 1.39 nM PNP mg dw⁻¹ h⁻¹ on day 30 of incubation, which was about 2 fold lower than the observed value of N. muscorum. PHB content in S. platensis remained low even after prolonged phosphate starvation, and a rise only up to 3.5% of dw was recorded on day 60 of phosphate deficiency. Supplementation of NADPH exogenously to S. platensis cultures grown under phosphate deficiency favoured PHB accumulation in 10, 20 and 30 days old cultures, but not in the cultures grown under phosphate deficiency for 60 days. The possible role of phosphate limitation on PHB accumulation is discussed.     

Maackiain in Cicer bijugum Rech. f. associated with resistance to Botrytis grey mould

Resistance in Cicer bijugum Rech. f. a wild relative of chickpea, to Botrytis grey mould (BGM), caused by Botrytis cinerea Pers., was shown to be associated with high concentrations of maackiain when compared to three susceptible species. The two BGM resistant accessions of C. bijugum contained between 200 and 300 μg maackiain g⁻¹ of foliage whereas the BGM susceptible species C. arietinum, C. echinospermum and C. reticulatum contained less than 70 μg g⁻¹. Furthermore, the concentration of maackiain increased to more than 400 μg g⁻¹ in the resistant wild species after being inoculated with the pathogen whereas no significant increase was recorded in the susceptible species. The germination of spores of Botrytis cinerea, treated with maackiain, was inhibited in a dose dependent manner; less than 10% of spores germinated when treated with 500 μg ml⁻¹. The data indicate that maackiain may be an important component in BGM resistance in the wild chickpea C. bijugum and that the resistance is enhanced in the presence of the pathogen.     

Polyamine and methyl jasmonate-influenced enhancement of betalaine production in hairy root cultures of Beta vulgaris grown in a bubble column reactor and studies on efflux of pigments

Hairy root cultures of red beet (Beta vulgaris) were grown in 3 l bubble column reactor for studying growth and pigment production under the influence of polyamines (PA) and elicitor treatment. Earlier studies with shake flask cultures had shown that combined feeding of spermidine (spd) and putrescine (put) (each 0.75 mM) significantly enhanced betalaine productivity in hairy root cultures of red beet. The present study has been focused on betalaine production in 3 l bubble column bioreactor where the growth pattern and betalaine synthesis under the influence of similar levels of polyamines were followed. A combination of spermidine and putrescine fed to the roots each at levels of 0.75 mM efficiently increased growth and pigment production resulting in 1.23-fold higher biomass (39.2 g FW l⁻¹) and 1.27-fold higher betalaine content (32.9 mg g⁻¹ DW) than control. Treatments with various levels of elicitor-methyl jasmonate (MJ), though progressively retarded biomass, at 40 μM level resulted in a significant increase in betalaine content resulting in 36.13 mg g⁻¹ DW which was 1.4-fold higher than the control. Further higher concentrations of methyl jasmonate treatments supported high as well as rapid accumulation of betalaines, the overall betalaine productivity was hampered mainly because of the inhibitory action on biomass. Pigment release studies with cetyl trimethyl ammonium bromide (CTAB) resulted in optimization of concentration for better efflux of betalaines without showing any inhibitory effect on hairy root viability. These studies on product enhancement and on-line extraction of pigment are useful for developing a bioreactor system for betalaine production using B. vulgaris hairy root cultures. In particular the use of elicitors and efflux studies provide an insight for integrating unit operations and developing a process for continuous operation and higher production of phytochemicals.     

Metal accumulation by Halodule wrightii populations

Metal concentrations and population parameters of the seagrass Halodule wrightii were determined at three locations at Rio de Janeiro State, Brazil. The possible increase of metal availability in one of these areas, Sepetiba Bay, as a result of dredging of contaminated bottom sediments which ocurred, was evaluated by analyses of Al, Cd, Cr, Cu, Fe, Ni, Pb and Zn in root, rhizome and shoots. In addition, analyses were carried out in H. wrightii populations from non-contaminated areas located at northwestern (Cabo Frio) and southeastern (Angra do Reis) regions of Rio de Janeiro State. Concurrently, abundance and density data of the seagrass populations were obtained. It was found that concentration from Sepetiba Bay samples up to 1.6 ± 0.4 μg g⁻¹ of Cd, 12 ± 1.0 μg g⁻¹ of Cr, 27 ± 2.4 μg g⁻¹ of Pb, 291 ± 47 μg g⁻¹ of Mn, 128 ± 23 μg g⁻¹ of Zn were significantly higher than that from two other collection sites. An increase in Cd and Zn concentration was observed in H. wrightii from Sepetiba Bay indicating that metal mobilization from contaminated sediments through dredging activities were, at least in part, transferred to the biotic compartment via accumulation by the seagrass. The populations of seagrass within the region demonstrated quite substantial changes in biomass data but not in shoot or rhizome density during the study. Such changes in biomass are to be expected, as these dynamics are typical of the small, isolated monospecific populations of H. wrightii along the Rio de Janeiro coast.     

Aerobic chromate reduction by chromium-resistant bacteria isolated from serpentine soil

A group of 34 chromium-resistant bacteria were isolated from naturally occurring chromium percolated serpentine soil of Andaman (India). These isolates displayed different degrees of chromate reduction under aerobic conditions. One of the 34 isolates identified as Bacillus sphaericus was tolerant to 800 mg l⁻¹ Cr(VI) and reduced >80% Cr(VI) during growth. In Vogel Bonner broth, B. sphaericus cells (10¹⁰ cells ml⁻¹) reduced 62% of 20 mg l⁻¹ of Cr(VI) in 48 h with concomitant discoloring of yellow medium to white one. Reduction of chromate was pronounced by the addition of glucose and yeast extract as electron donors. In the presence of 4.0 g l⁻¹ of glucose, 20 mg l⁻¹ of Cr(VI) was reduced to 2.45 mg l⁻¹ after 96 h of incubation. Optimum pH and temperature for reduction were 6.0 and 25 °C, respectively. Increase in cell density and initial Cr(VI) concentration increased chromate reduction but was inhibited by metal ions like, Ni²⁺, Co²⁺, Cd²⁺ and Pb²⁺. Experiments with cell-free extracts indicated that the soluble fraction of the cell was responsible for aerobic reduction of Cr(VI) by this organism.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).