Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification

The aim of this work was to remove nitrate-N and organic pollutants from wastewater of the dairy industry by denitrification. An artificially prepared wastewater, containing 250 mg/l nitrate-N and 1.5 g/l whey powder, was completely denitrified with removal of 90%–93% of the chemical oxygen demand (COD) of the whey powder by suspended or immobilized mixed cultures and by a suspended or immobilized pure culture that was isolated from the mixed culture inoculum. For the above COD/nitrate-N ratio of 6:1, the results indicated that the organic compounds of the wastewater served as electron donors for complete denitrification and that there was no need to add an external carbon source. In batch denitrification assays the suspended or immobilized mixed cultures proved to be more active and reacted faster than the isolated pure cultures. In continuous denitrification processes with immobilized pure or mixed cultures, the alginate beads, used for immobilization, were not stable for more than 12 days of incubation. The mixed free cultures removed the nitrate-N and COD continuously with no change of their activity for at least 15 days at an optimum hydraulic retention time of 0.27 days with a loading rate of 900 mg nitrate-N l⁻¹ day⁻¹. Received: 13 October 1997 /  Received revision: 16 December 1997 / Accepted: 19 December 1997     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Poly-β-Hydroxybutyrate Accumulation as a Measure of Unbalanced Growth of the Estuarine Detrital Microbiota

The procaryotic endogenous storage material poly-β-hydroxybutyrate (PHB) can be induced to accumulate in the estuarine detrital microbiota under conditions which suggest unbalanced growth, such as limitation of a critical factor(s) in the presence of carbon and energy sources. Changes in PHB-to-lipid phosphate ratios detected in field samples can be mimicked in the laboratory with common estuarine stresses. Acute anoxia or low pH induces conditions of no growth with depression of both the synthesis and catabolism of PHB without change in the lipid phosphate. Balanced growth induced by nutrients increases the lipid phosphate, depresses PHB synthesis, and stimulates PHB catabolism, resulting in a low ratio of PHB to lipid phosphate. Unbalanced growth induced to a small extent by high salinity or much more readily by dark upland runoff water results in rapid accumulation of PHB and slowing of PHB catabolism with little change in lipid phospate. Unbalanced growth conditions result in high PHB-to-lipid phosphate ratios in the detrital microbiota.     

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins,PhaP6 and PhaP7, in Ralstonia eutropha H16

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with “phasin 2 motifs.” To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.     

Effect of high nitrate concentration on PHB storage in sequencing batch reactor under anoxic conditions

The study investigated effect of high influent nitrate concentration on poly-beta-hydroxybutyrate, (PHB), storage in a sequencing batch reactor, (SBR), under anoxic conditions. Acetate was fed as pulse during anoxic phase, sustained with external nitrate feeding. SBR operation involved three runs at steady state with COD/N ratios of 3.84, 2.93 and 1.54 gCOD/gN, where external nitrate concentrations gradually increased from 50 mg N/l to 114 mg N/l and 226 mg N/l, in 1st, 2nd and 3rd runs, respectively. In 1st run, acetate was fully converted into PHB with the storage yield value of 0.57-0.59 gCOD/gCOD, calculated both in terms of PHB formation and NO(X) utilization, confirming storage was the sole substrate utilization mechanism. In the following runs, PHB formation was reduced and the storage yield based on PHB dropped down to 0.40 and 0.33 gCOD/gCOD with increasing influent nitrate concentrations, indicating that higher portions of acetate were diverted to simultaneous direct growth. The observations suggested that nitrite accumulation detected at low COD/N ratios was responsible for inhibition of PHB storage.     

To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha,Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure.     

The Efficacy of Hyperbaric Oxygen Therapy on Middle Cerebral Artery Occlusion in Animal Studies: A Meta-Analysis

Background

Inconsistent results have been reported for hyperbaric oxygen therapy (HBO) for acute stroke. We conducted a systematic review and meta-analysis to evaluate the benefit of HBO in animal studies of middle cerebral artery occlusion (MCAO).

Methods

A systematic search of the literature published prior to September 2015 was performed using Embase, Medline (OvidSP), Web of Science and PubMed. Keywords included “hyperoxia” OR “hyperbaric oxygen” OR “HBO” AND “isch(a)emia” OR “focal cerebral ischemia” OR “stroke” OR “infarct” OR “middle cerebral artery occlusion (MCAO).” The primary endpoints were the infarct size and/or neurological outcome score evaluated after HBO treatment in MCAO. Heterogeneity was analyzed using Cochrane Library’s RevMan 5.3.5.

Results

Fifty-one studies that met the inclusion criteria were identified among the 1198 studies examined. When compared with control group data, HBO therapy resulted in infarct size reduction or improved neurological function (32% decrease in infarct size; 95% confidence interval (CI), range 28%–37%; p < 0.00001). Mortality was 18.4% in the HBO group and 26.7% in the control group (RR 0.72, 95% CI, 0.54–0.98; p = 0.03). Subgroup analysis showed that a maximal neuro-protective effect was reached when HBO was administered immediately after MCAO with an absolute atmospheric pressure (ATA) of 2.0 (50% decrease; 95% CI, 43% -57% decrease; p < 0.0001) and more than 6 hours HBO treatment (53% decrease; 95% CI, 41% -64% decrease; p = 0.0005).

Conclusions

HBO had a neuro-protective effect and improved survival in animal models of MCAO, especially in animals given more than 6 hours of HBO and when given immediately after MCAO with 2.0 ATA.     

Kinetics of Membrane Transport during Chloroplast Development

In the course of plastid development there are changes in the permeability of the envelope membranes. An investigation of the kinetics of transport with largely uncontaminated and intact etioplast/etiochloroplast preparations from greening Avena sativa laminae demonstrates: (a) that etioplasts already possess specific translocators for the transporation of orthophosphate, dihydroxyacetone phosphate, 3-phosphoglycerate (“phosphate translocator”), and dicarboxylic acids (“dicarboxylate translocator”); (b) that changes in the rates of uptake during development are mainly due to changes in velocity for specific transport and not due to changes in the affinity for transport (K_m) or nonspecific permeation. The very low competitive inhibition of transport of orthophosphate by dihydroxyacetone phosphate and 3-phosphoglycerate, observed for developmental stages corresponding to up to 3 hours of illumination of etiolated tissue, is discussed with respect to the possibility of an early phosphate transport mechanism that is different from the phosphate translocator of more developed plastids.     

Protozoan biomass relation to nutrient and chemical oxygen demand removal in activated sludge mixed liquor

The relationship between biomass concentration to nutrient and chemical oxygen demand (COD) removal in mixed liquor supplemented with sodium acetate was investigated, using three protozoan isolates and three different initial biomass concentrations (10(1), 10(2) and 10(3) cells/mL). The study was carried out in a shaking flask environment at a shaking speed of 100 rpm for 96 h at 25 degrees C. Aliquot samples were taken periodically for the determination of phosphate, nitrate, COD and dissolved oxygen, using standard methods. The results revealed remarkable phosphate removal of 82-95% at biomass concentration of 10(3)cells/mL. A high nitrate removal of over 87% was observed at all initial biomass concentration in mixed liquor. There was an observed COD increase of over 50% in mixed liquor in at the end of 96-h incubation and this was irrespective of initial biomass concentration used for inoculation. The study shows the trend in nutrient and COD removal at different biomass concentrations of the test isolates in mixed liquor.     

Transformation of phosphorus and relevant intracellular compounds by a phosphorus-accumulating enrichment culture in the presence of both the electron acceptor and electron donor

This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors.     

Poly(3-hydroxybutyrate) synthesis in fed-batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations

Poly(3-hydroxybutyrate) (PHB) was produced by fed-batch cultures of Ralstonia eutropha with phosphate limitation under different glucose concentrations. When glucose was kept at 2.5 g l^–1, cell growth and PHB synthesis were limited due to the shortage of carbon source but a higher PHB content occurred in the cell-growth stage. This shows that a low glucose concentration is favorable for PHB accumulation in R. eutropha. PHB obtained with glucose at 9 g l^–1 is 1.6 times that obtained with 40 g l^–1. When glucose was in the range of 9 to 40 g l^–1, PHB concentration and productivity decreased significantly with the increase of glucose concentration. The highest PHB productivity was obtained with glucose at 9 g l^–1.     

Simulated acid azo dye (Acid black 210) wastewater treatment by periodic discontinuous batch mode operation under anoxic–aerobic–anoxic microenvironment conditions

Treatment of simulated acid azo dye (C.I. Acid black 210) wastewater was studied in periodic discontinuous batch mode operation employing sequencing batch reactor (SBR) with suspended growth configuration under anoxic–aerobic–anoxic microenvironment. The performance of the reactor was evaluated at two organic loading rates (0.56 kg COD/m³-day and 0.75 kg COD/m³-day) with a total cycle period of 24 h [fill phase: 30 min; react phase: 23 h; settle phase: 15 min; decant phase: 15 min] at room temperature. The performance of SBR was assessed by monitoring both COD and colour (OD-617 nm) concentrations. Periodic discontinuous batch mode operation feasibility for dye colour removal along with simultaneous substrate (COD) removal. The performance efficiency of the system was found to depend on the operating organic loading rate. Induced anoxic microenvironment during cycle operation and persistent anoxic microenvironment in the internal layer of suspended biomass may be probable reasons for dye mineralization/reduction. Rapid startup period and non-inhibited performance at higher loading rate are some of the advantages observed in the SBR operation.     

Can Serum Levels of Alkaline Phosphatase and Phosphate Predict Cardiovascular Diseases and Total Mortality in Individuals with Preserved Renal Function? A Systemic Review and Meta-Analysis

Background

It is demonstrated that elevated serum levels of alkaline phosphatase (ALP) and phosphate indicate a higher risks of cardiovascular disease (CVD) and total mortality in population with chronic kidney disease (CKD), but it remains unclear whether this association exists in people with normal or preserved renal function.

Method

Clinical trials were searched from Embase and PubMed from inception to 2013 December using the keywords “ALP”, “phosphate”, “CVD”, “mortality” and so on, and finally 24 trials with a total of 147634 patients were included in this study. Dose-response and semi-parametric meta-analyses were performed.

Results

A linear association of serum levels of ALP and phosphate with risks of coronary heart disease (CHD) events, CVD events and deaths was identified. The relative risk(RR)of ALP for CVD deaths was 1.02 (95% confidence interval [CI], 1.01–1.04). The RR of phosphate for CVD deaths and events was 1.05 (95% CI, 1.02–1.09) and 1.04 (95% CI: 1.03–1.06), respectively. A non-linear association of ALP and phosphate with total mortality was identified. Compared with the reference category of ALP and phosphate, the pooled RR of ALP for total mortality was 1.57 (95% CI, 1.27–1.95) for the high ALP group, while the RR of phosphate for total mortality was 1.33 (95% CI, 1.21–1.46) for the high phosphate group. It was observed in subgroup analysis that higher levels of serum ALP and phosphate seemed to indicate a higher mortality rate in diabetic patients and those having previous CVD. The higher total mortality rate was more obvious in the men and Asians with high ALP.

Conclusion

A non-linear relationship exists between serum levels of ALP and phosphate and risk of total mortality. There appears to be a positive association of serum levels of ALP/phosphate with total mortality in people with normal or preserved renal function, while the relationship between ALP and CVD is still ambiguous.     

Production of polyhydroxybutyrate by activated sludge performing enhanced biological phosphorus removal

In this study, polyhydroxybutyrate (PHB) – a biodegradable plastics material – was produced by activated sludge performing enhanced biological phosphorus removal (EBPR) in batch experiments under anaerobic, aerobic and anaerobic/aerobic conditions. Under anaerobic conditions, the maximum PHB content of the dry biomass was 28.8% by weight, while under aerobic or anaerobic/aerobic conditions, the maximum PHB content was about 50%. The PHB production rate with respect to the volatile suspended solids (VSS) was: (i) 70 mg/(g VSS) h under aerobic conditions that followed anaerobic conditions, (ii) 156 mg/(g VSS) h under anaerobic condition, and (iii) 200 mg/(g VSS) h under aerobic conditions with energy also supplied from polyphosphate. A side stream, with initially anaerobic conditions for PHB accumulation and phosphorus release, and then aerobic conditions for PHB accumulation, was proposed. In this side stream, biomass with a high PHB content and a high PHB production rate could be both achieved.     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Biological Ion Exchanger Resins: I. Quantitative Electrostatic Correspondence of Fixed Charge and Mobile Counter Ion

Utilizing Escherichia coli as the prototype of an ion-accumulating cell, the ion exchange isotherm is introduced as a concise method of characterizing biological ion exchange events. The ion exchange isotherm for the alkali cation exchange, K ↔ Na, is described. The total charge profile of this bacterium is compiled and compared for bacteria in the Na form and in the K form. Macromolecule fixed charge was found to provide 80% of the counter ions that pair with potassium. Therefore, in its physiological state, 80% of the cell potassium in E. coli is associated with an ion exchange site on a macromolecule. The primary cation exchange sites are found to be about equally divided between carboxylate and phosphate sites indicating that E. coli is a bifunctional resin with respect to cation exchange. During substrate-dependent cation accumulation (“active transport”), phosphate esters and organic acids were shown to accumulate. One may conclude that the role of intermediate metabolism in “active transport” is to increase the ion exchange capacity of the biological resin by the production of charged metabolites that sorb to the framework of the resin.     

In Silico Analysis of the Metabolic Potential and Niche Specialization of Candidate Phylum "Latescibacteria" (WS3)

The “Latescibacteria” (formerly WS3), member of the Fibrobacteres–Chlorobi–Bacteroidetes (FCB) superphylum, represents a ubiquitous candidate phylum found in terrestrial, aquatic, and marine ecosystems. Recently, single-cell amplified genomes (SAGs) representing the “Latescibacteria” were obtained from the anoxic monimolimnion layers of Sakinaw Lake (British Columbia, Canada), and anoxic sediments of a coastal lagoon (Etoliko lagoon, Western Greece). Here, we present a detailed in-silico analysis of the four SAGs to gain some insights on their metabolic potential and apparent ecological roles. Metabolic reconstruction suggests an anaerobic fermentative mode of metabolism, as well as the capability to degrade multiple polysaccharides and glycoproteins that represent integral components of green (Charophyta and Chlorophyta) and brown (Phaeophycaea) algae cell walls (pectin, alginate, ulvan, fucan, hydroxyproline-rich glycoproteins), storage molecules (starch and trehalose), and extracellular polymeric substances (EPSs). The analyzed SAGs also encode dedicated transporters for the uptake of produced sugars and amino acids/oligopeptides, as well as an extensive machinery for the catabolism of all transported sugars, including the production of a bacterial microcompartment (BMC) to sequester propionaldehyde, a toxic intermediate produced during fucose and rhamnose metabolism. Finally, genes for the formation of gas vesicles, flagella, type IV pili, and oxidative stress response were found, features that could aid in cellular association with algal detritus. Collectively, these results indicate that the analyzed “Latescibacteria” mediate the turnover of multiple complex organic polymers of algal origin that reach deeper anoxic/microoxic habitats in lakes and lagoons. The implications of such process on our understanding of niche specialization in microbial communities mediating organic carbon turnover in stratified water bodies are discussed.     

Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active β-galactosidase was obtained in a single step using the above described method.