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Location of glucose oxidase during production by Aspergillus niger

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Location of glucose oxidase during production by Aspergillus niger
  Appl Microbiol Biotechnol (2006) 70: 72  –  77DOI 10.1007/s00253-005-0031-9 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS K. G. Clarke .M. Johnstone-Robertson .B. Price .S. T. L. Harrison Location of glucose oxidase during productionby   Aspergillus niger  Received: 20 December 2004 / Revised: 24 May 2005 / Accepted: 26 May 2005 / Published online: 17 August 2005 # Springer-Verlag 2005 Abstract  The production of the enzyme glucose oxidase by  Aspergillus niger   is well documented. However, itsdistribution within the fungal culture is less well defined.Since the enzyme location impacts significantly on enzymerecovery, this study quantifies the enzyme distribution between the extracellular fluid, cell wall, cytoplasm andslime mucilage fractions in an  A. niger   NRRL-3. The cul-ture was separated into the individual fractions and theglucose oxidase activity was determined in each. The ex-tracellular fluid contained 38% of the total activity. Theremaining 62% was associated with the mycelia and wasdistributed between the cell wall, cytoplasm and slimemucilage in the proportions of 34, 12 and 16%, respec-tively. Intracellular cytoplasmic and cell wall sites wereconfirmed using immunocytochemical labelling of the my-celia. In the non-viable cell, the mycelial-associated en-zyme was distributed between these sites, whereas in theviable cell, it was predominantly associated with the cellwall. The distribution of the enzyme activity indicates that recovery from the solids would result in a 38% loss,whereas recovery from the extracellular fluid would result in a 62% loss. The results also suggest, however, that this62% loss could be reduced to around 34% by disintegratingthe solids prior to separation due to the contribution of theenzyme in the cytoplasm and slime mucilage. This wasconfirmed by independently establishing the percentageactivity in the liquid and solid portions of a disintegratedculture as 62 and 38%, respectively. Introduction Glucose oxidase is a commercially important enzymewhich has application in the pharmaceutical industry as a biosensor in diagnostic kits (Richter  1983) and in the foodindustry for the removal of oxygen or glucose to prevent rancidity or discolouration, respectively (Lu et al. 1996).Glucose oxidase is a fungal enzyme predominantly pro-duced by  Aspergillus  and  Penicillium  sp. (Bucke 1983). Insubmerged culture,  Penicillium  exhibits a filamentousmorphology, which tends to result in non-Newtonian be-haviour and, consequently, mass transfer limitations at highmycelial concentrations.  Aspergillus , on the other hand,grows with a pelleted morphology which results in a lessviscous broth. As a result, the  Aspergillus  process has adistinct advantage over the  Penicillium  process in that, provided the pellet is maintained below a critical diameter,the production of glucose oxidase is likely to be kineticallycontrolled. Of major concern in the  Aspergillus  process,however, is the uncertainty of the distribution of the en-zyme within the fungal culture. A knowledge of the par-titioning of the enzyme between the intracellular andextracellular locations is essential for the judicious designof an efficient enzyme recovery programme, since it im- pacts directly on the programme complexity and, conse-quently, has a considerable influence on the overall enzyme production cost.Enzyme recovery is usually initiated with separation of the solids and liquids after which the enzyme is recoveredfrom either the liquid or solid stream. It is generally con-sidered preferable to have a predominantly extracellular location because this allows the enzyme to be recovereddirectly from the liquid stream. This facilitates ease of re- K. G. Clarke .M. Johnstone-Robertson .S. T. L. HarrisonDepartment of Chemical Engineering,University of Cape Town,Private Bag,Rondebosch, 7701, South AfricaB. PriceElectron Microscope Unit,University of Cape Town,Private Bag,Rondebosch, 7701, South Africa  Present address: K. G. Clarke ( * )Department of Process Engineering,University of Stellenbosch,Private Bag X1,Stellenbosch, 7602, South Africae-mail: +27-21-8084421Fax: +27-21-8082059  covery because it precludes the need for an extra celldisruption step and further limits the number of proteinsand metabolites from which the enzyme must be purified.Under conditions where the enzyme is not predominantlyextracellular, a predominantly intracellular location wouldthen be preferred over a distribution between the locations,since processing of both liquid and solid streams would not  be practicable. Although recovery of an intracellular en-zyme realises the disadvantages mentioned above, theenzyme could be readily concentrated from a concentrationof the cells before cell disruption and separation from thesolids.The literature findings relating to the partitioning of glucose oxidase in the  Aspergillus  broth are controversial both with regard to the distribution between extra and in-tracellular locations and, in the case of an intracellular location, with regard to the identification of the exact in-tracellular site. The early literature assumed glucose oxi-dase to be an intracellular enzyme (Pazur  1966) and initialstudies concentrated on identifying the exact intracellular location. Van Dijken and Veenhuis (1980), using cyto-chemical labelling, observed glucose oxidase in cytoplas-mic microbodies, identified as peroxisomes. Subsequently,however, Witteveen et al. (1992), employing immunocyto-chemical labelling found the bulk of the glucose oxidase to be located in the cell wall and attributed the earlier ob-servation of glucose oxidase in peroxisomes to an artefact of the method used. A cell wall location was further sup- ported by the observation that while glucose oxidase was present in the lysates of intact mycelia, it was absent in thelysates of protoplasts (Witteveen et al. 1992).Mischak et al. (1985), who examined the distribution between both intra- and extracellular locations, on thecontrary, reported a predominantly extracellular location.These authors observed that 75% of the total glucoseoxidase was present in the culture fluid, only 17% of whichcould be accounted for by autolysis. Since the extracellular location was observed in a Mn ++ deficient medium only,Witteveen et al. (1992) suggested that the extracellular location might be a consequence of a an altered cell wallcomposition resulting from Mn ++ deficiency, thus allowinga cell-wall-localised glucose oxidase to enter the culturefluid and, therefore, making this finding compatible withtheir result of a cell wall locality. The alteration of cell wallcomposition had previously been associated with Mn ++ deficiency during production of citric acid by  A. niger  (Kisser et al. 1980). However, Mn ++ has also been ob-served to induce slime mucilage around the cell (Kisser et al. 1980; Mischak et al. 1985), which traps glucose oxi- dase, making it appear intracellular through diffusionallimitations (Mischak et al. 1985), suggesting that it is alsolikely that the extracellular location in a Mn ++ deficient medium is due to the absence of enzyme entrapment andnot a result of a cell wall locality. An extracellular locationof glucose oxidase is also suggested by the glycoproteinstructure of the enzyme (Pazur et al. 1965).In view of the importance of the enzyme location onenzyme recovery, this paper elucidates the enzyme distri- bution in an  A. niger   NRRL-3 culture under process con-ditions which favour glucose oxidase production. The percentage enzyme activity associated with the extracellu-lar fluid and that associated with the mycelial fractions,namely, the cytoplasm, the cell wall and the slime muci-lage surrounding the cell, is quantified. The enzyme in themycelial fractions is further assessed through immuno-cytochemical labelling of glucose oxidase. Finally, the rel-evance of the data with respect to the implications for downstream processing is discussed. Materials and methods Microorganism and culture maintenance  Aspergillus niger   NRRL-3 was used throughout this study.The fungus was maintained on malt extract agar (MEA).MEA contained the following per litre: malt extract 20 g;glucose 20 g; peptone 1 g; and agar 20 g.Inoculum development Spores from an MEA plate were suspended in 1% peptoneto 5×10 7 spores/ml. A 3-l conical flask was charged with600 ml of medium containing the following per litre: 100 gglucose, 3 g NaNO 3 , 2 g yeast extract, 1 g KH 2 PO 4 , 0.5 gMgSO 4 ·7H 2 O, 0.5 g KCl and 0.01 g FeSO 4 ·7H 2 O(Hellmuth et al. 1995). The pH was adjusted to 6.2 with0.18 ml of 5 M NaOH. Subsequently, the broth was in-oculated with 30 ml of the spore suspension to give a finalconcentration of 2.4×10 6 spores/ml. The flask was in-cubated at 30°C and 120 rpm until a final pH of 5.0 had been reached (about 17  –  19 h) when it was used as theinoculum for the bioreactor at 10% (v/v).Process conditionsThe process was carried out in a 6-l working volumeChemap bioreactor containing the medium of Hellmuth et al. (1995). Dual flat-bladed turbines providing agitation at 500 rpm and an aeration rate of 0.8 vvm maintained theoxygen level above 30% of the saturation level. Dissolvedoxygen was monitored by an oxygen probe (Mettler Toledo). The pH was controlled to 5.5 by the automaticaddition of 5 M NaOH and the temperature was controlledto 30°C.AnalysesCell dry weight was determined by filtering 20 ml of sample through a 0.45- μ  m Millipore filter. The filter wasdried at 80°C, cooled in a desiccator and weighed to four decimal places prior to use. After filtering, the filter plusmycelia were dried and weighed as before. The cell dryweight was calculated from the difference in the twoweights. 73  Glucose concentration was determined as reducing sugar in the supernatant using the standard dinitrosalycylic acidmethod (Miller  1959). The supernatant was obtained byvacuum filtration of the broth through a 0.45- μ  m Milliporefilter.Gluconic acid concentration was estimated from theweight of the NaOH consumed during pH control by as-suming a direct relationship between the base consumedand the amount of gluconic acid produced, demonstratedfor the cultivation of   A. niger   NRRL-3 (Johnson, personalcommunication).Glucose oxidase activity was determined by the dissolv-ed oxygen method, where the enzyme activity is related tothe oxygen consumption in the presence of excess glucose(Miura et al. 1970). One unit (U) of glucose oxidase ac-tivity is defined as the amount of enzyme that catalyses theoxidation of 1  μ  mol of glucose in 1 min at pH 5.5 and25°C.The analysis of the glucose oxidase activity was carriedout in triplicate for each sample. The standard deviation of the triplicate samples varied between 0 and 8% with anaverage value of 3%.Separation of culture fractionsThe distribution of glucose oxidase activity was deter-mined by separation of the culture into the extracellular fraction and fractions associated with the mycelia (slimemucilage, cell wall and cytoplasm) according to the pro-cedure outlined below and shown in Fig. 1, followed byanalysis of the activity in each of the fractions. The totalglucose oxidase activity was determined from analysis of the activity in the entire culture after disruption of the my-celia with a French Press (two passes at 20 MPa).The supernatant was separated by vacuum filtration of the culture through a 0.45- μ  m Millipore filter followed bywashing of the mycelia on the filter. The supernatant andwash water were combined for analysis of the activity inthe extracellular fluid.The washed mycelia were suspended in phosphate buff-er at pH 5.5 and disrupted with a French Press as before.The disrupted suspension was filtered through a 0.45- μ  mMillipore filter to separate the solids, or cell wall frag-ments, from the liquid containing the cytoplasm and slimemucilage. The cell wall fragments were resuspended in phosphate buffer pH 5.5 for analysis of the activity in thecell wall. The slime mucilage was separated from the cy-toplasm via ethanol precipitation, followed by recoveryof the precipitate after centrifugation (12,000×  g  , 10 min,4°C). The activity of the cytoplasm was determined fromthe liquid and the precipitate was dissolved in 0.1 M NaOHto obtain the activity in the slime mucilage.Some activity was invariably lost during the separationof the culture. The activity that was lost due to separationwas calculated using a mass balance and the data werecorrected to that which would be attained, assuming com- plete recovery in eachof the separation steps. Thecorrecteddata were used in the calculation of the percentage activityin each fraction.Immunocytochemical labellingPrimary rabbit polyclonal antibodies, immunoglobin G(IgG), were raised against a commercial glucose oxidaseobtained from Seravac (Epping, Cape Town, South Africa).Pre-immune serum was collected prior to immunisationwith glucose oxidase to check if the animals expressed pre-existing anti-fungal antibodies. An enzyme-linked immu-nosorbent assay (ELISA) was performed (Harlow and Lane1988) on the rabbit serum to identify the fractions with thehighest titre. IgG was purified from the serum by fractional precipitation with PEG 6,000 and the concentration wasdetermined by the absorbance at 280 nm ( ɛ 2801 mg/ml =1.64).To prepare cell extracts, fungal cells were ruptured by aFrench Press, followed by incubation with 1% Triton X-100. The extract was clarified by centrifugation (15,000×  g  ,15 min, 4°C), and the supernatant was transferred to a cleancontainer. Proteins were precipitated by the addition of trichloroacetic acid (TCA) to a final concentration of 5%.The suspension was centrifuged and the supernatant wasremoved. The pellet was washed with acetone to removeresidual TCA and redissolved in a minimal volume of  phosphate-buffered saline (PBS). Pure, commercial glu-cose oxidase and cell extracts were separated by reducing12% sodium dodecyl sulphate  –   polyacrylamide gel elec-trophoresis (SDS-PAGE) (20 V, 90 min, 4°C), transferredonto nitrocellulose (20 V, overnight) and stained withPonceau S. The membrane was probed with purified IgG to  A. niger culture  Disrupt TOTAL ACTIVITYIntact mycelia CYTO -PLASMIC ACTIVITY SLIME MUCILAGEACTIVITYCELL WALL ACTIVITYCytoplasmSlime mucilageEXTRA-CELLULAR ACTIVITY  Filter PrecipitateCentrifuge Disrupt Disrupted mycelia  Filter Fig. 1  Procedure for separation of glucose oxidase into individualfractions74  determine specificity and to identify any cross-reactivitywith other   A. niger   proteins. For immunogold transmissionelectron microscopy, mycelial pellets were fixed with 2% paraformaldehyde and 2% glutaraldehyde in PBS (pH 7.4),dehydrated in a graded ethanol series and embedded in LR White resin.Ultra-thin sections were collected onto nickel grids and blocked with tris-buffered saline containing 1% fish skingelatin, 0.8% bovine serum albumin (BSA) and 20 mMglycine for an hour. The sections were washed, probed withIgG (ranging from 2.5 to 10  μ  g/ml at a final volume of 5 ml, 2 h) and detected with 10 nm gold-conjugated goat anti-rabbit IgG (1/200 dilution, 5  μ  l, 1 h). The sectionswere contrasted with uranyl acetate/lead citrate and ex-amined on a transmission electron microscope at 80 kV. Results and discussion Enzyme distribution in  A. niger   culturesThe distribution of glucose oxidase activity in the cellsuspension was examined at the end of duplicate  A. niger   batch cultures when the total enzyme activity was highest.The batch cultures were complete by 25 h as indicated bynegligible residual reducing sugar with no further increasein enzyme activity or concentration of gluconic acid or mycelia (Fig. 2). The extracellular fluid and the myceliawere separated, and the mycelia were further divided intodiscrete fractions of slime mucilage, cytoplasm and cellwall, as described above. The enzyme activities in each of the extracellular fluid, slime mucilage, cytoplasm and cellwall were quantified (Table 1).The enzyme activity was found to be predominantly as-sociated with the mycelial fraction, with only 38% present in the extracellular fluid. This is contrary to the results of Mischak et al. (1985), who report a predominantly ex-tracellular location (75%). These authors conducted their studies in a Mn ++ deficient medium in which slime pro-duction was negligible suggesting that under conditionswhere slime production is significant, the finding of anintracellular location may be erroneously attributed to en-zyme entrapment in the slime. In our study, however, only16% of the mycelial-associated fraction was due toentrapment in the slime mucilage, whereas almost half of the total activity could be attributed to a truly intracellular location, namely, either the cell wall or the cytoplasm.Both cell wall and cytoplasm fractions contained appre-ciable activity with 34 and 12%, respectively. A pre-dominantly cell wall location has been reported previously(Witteveen et al. 1992) as has a predominantly cytoplasmiclocation (Van Dijken and Veenhuis 1980). The analyses of the activities in the mycelial fractions presented here sug-gest that, on the contrary, the enzyme is located in multiplesites.To examine this paradox, the cell wall and cytoplasmicsites were examined using immunocytochemical labellingof the glucose oxidase in the mycelia. SDS-PAGE and aWestern Blot confirmed that the antibodies were specificfor glucose oxidase and did not cross-react with other   A.niger   proteins (Fig. 3).Electron micrographs of sections of   A. niger   mycelia at the end of duplicate batch cultures were examined. Thesemicrographs show glucose oxidase in both the cell wall andcytoplasm (Fig. 4) in both viable and non-viable cells. Theviable and non-viable cells were distinguished by thedramatic morphological differences in the section in Fig. 4avs the section in Fig. 4 b. Specifically, the non-viable cellsin Fig. 4 b, in contrast to the viable cells in Fig. 4a, share the common characteristics: none exhibits intact nuclei or nu-clear membranes, no mitochondria are present (suggestingthat all oxidative phosphorylation has ceased) and thereappears to be extensive detachment of cytoplasm from the periphery of the cell wall.The labelling in these micrographs was confirmed to bespecific to glucose oxidase since the labelling using the pre-immune serum was statistically non-significant (95%confidence interval). The electron micrographs, in addi- 05101520250 5 10 15 20 25 30 Time (h)    G   l  u  c  o  n   i  c  a  c   i   d   (   M   )    C  e   l   l   d  r  y  w  e   i  g   h   t   (  g   /   l   ) 0204060801001201400 5 10 15 20 25 30 Time (h)    R  e  s   i   d  u  a   l  s  u  g  a  r   (  g   /   l   ) 0123456    T  o   t  a   l  a  c   t   i  v   i   t  y   (   U   /  m   l   ) Fig. 2  Batch profile of   A. niger   NRRL-3 at pH 5.5.  □ , cell dryweight; ⋄ , gluconic acid;  ♦ , residual sugar; ▪ , total activity Table 1  The percentage of glucose oxidase activity in theindividual fractions of the culture harvested at early stationary phaseLocation Percentage glucose oxidase activityBatch 1 Batch 2 AverageExtracellular fluid 42 33 38Slime mucilage 10 21 16Cell wall 32 37 34Cytoplasm 16 9 12Fluid+slime+cytoplasm 68 63 6675  tion, showed a dependence of the location of enzyme on thecell viability. Although the enzyme was present in both theviable and non-viable cells, significant cytoplasmic en-zyme was only observed in the non-viable cells. In theviable cells, the enzyme was predominantly associatedwith the cell wall with negligible amounts observed in thecytoplasm. These results indicate a statistical variation inthe cell population, with respect to the cell viability of theindividual cells, with a corresponding variation in the in-tracellular site of the enzyme. This suggests that theconflicting intracellular sites reported in the literature may,at least in part, be a consequence of cells of different vi-ability in the various studies exhibiting correspondinglydifferent enzyme sites. When considering the viable cellsalone, the results presented here are in agreement with thecell wall location reported by Witteveen et al. (1992).An association of the enzyme with the cell wall issupported by the model of Chang and Trevithick (1974),which is based on the relative sizes of the enzyme and the pores in the cell wall. Measurements of the average poresize of the cell wall indicate that these are too small to allow passage of the enzyme. However, this average value islargely dependent on the pore size of the lateral walls in thehyphae. Further, it is known that the apical tips are more porous than the lateral walls. Consequently, it is postulatedthat enzyme secretion is likely to occur solely through theapical tips and is trapped in the cell wall as the porousapical wall transforms into the non-porous lateral wall.Impact of enzyme location on downstream recoveryThe results on enzyme location reported above havesignificant consequences for the efficacy of downstreamenzyme recovery. Since it is generally only economicallyviable to purify the enzyme from either the liquid or thesolid, a choice has to be made as to whether the liquidstream or the solids portion is to be processed after solidsseparation. An activity which is predominantly associatedwith the mycelia (whether it be truly intracellular and/or trapped in the slime mucilage) implies that processing of the liquid stream after solids separation would lead to anunacceptable amount being discarded with the solids, inthis case 62% of the total activity. This loss is likely to beeven greater since the maximum enzyme recovery would be reduced due to losses during the purification operations.On the other hand, recovery of the enzyme from the solidstream would result in the loss of at least a 38% of the totalactivity. Although this represents a smaller fraction, it isstill significant, and in addition, the processing of an in-tracellular enzyme necessitates the fractionation of the en-zyme from a more complex mix of metabolites. Therefore,under conditions where the enzyme is partitioned betweenthe extracellular fluid and mycelia, processing of either of the liquid or solid streams alone is unsuitable.These results suggest an alternative downstream pro-cessing option, namely, disruption of the mycelia beforeseparation, followed by processing of the liquid stream(Fig. 5). In this manner, part of the enzyme associated withthe mycelia would be released into the liquid stream,thereby concentrating the activity in this stream. Under these conditions, the activity in the liquid stream wouldreach 66% through the contributions from the activity inthe cytoplasm and the slime mucilage to that already pres-ent in the extracellular fluid, with the remaining activity being discarded with the cell wall fragments. These resultshave been verified in a separate experiment in which theactivity in the liquid portion of a disrupted culture was Fig. 4  Immunocytochemical labelling of glucose oxidase in  A.niger   NRRL-3 Fig. 3  SDS-PAGE ( a  –  c ) and Western Blot ( d  and  e ). ( a ) Molecular marker (98, 66, 39, 21 6/4 Da). ( b ) 5  μ  g glucose oxidasecommercial. ( c ) 20  μ  l TCA homogenate. ( d ) 5  μ  g glucose oxidasecommercial. ( e ) 20  μ  l TCA homogenate76
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