Thermal characterization of hyperproduced glucose

oxidase from Aspergillus niger BCG-5 mutant strainM. Anjum Zia', Khalil-ur-Rahman', M. Khalid Saeed*2 and Fozia Anjum3

'Department of Chemistry (Biochemistry), University of Agriculture, Faisalabad, Pakistan.2Food and Biotechnology Research Center, PCSIR Laboratories Complex, Lahore, Pakistan

3National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.Corresponding Author: M. Khalid Saeed

E-mail: ksaeed95ghotmail.com

Abstract-Introduction: Diabetes mellitus is a metabolic problemand is prevalent in many parts of the world. In developedcountries, the incidence rate is 5% and an equal number is liableto develop the disease. One fundamental aspect of diabetes is anabnormality of glucose metabolism as due to insufficient action ofinsulin, owing either to its absence or to resistance in action.Blood glucose level in diabetes becomes so elevated that theglucose "spills over" into urine, providing a convenientdiagnostic test for the disease. Three enzymes mutarotase,glucose oxidase and peroxidase are involved in the process ofdetermination of glucose level. Glucose oxidase (GOD, ,I-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) is an importantenzyme, which catalyzes the oxidation of ,I-D-glucose to D-glucono-1,5-lactone and hydrogen peroxide and finally togluconic acid using molecular oxygen as electron acceptor.Methods: Aspergillus niger spore suspension of 48 hours Vogel'sbroth was exposed to gamma irradiation and 80 k Rad. dose wasselected as optimized dose to induce the mutation. For selectionof resistance to catabolite repression, 2-deoxy-D-glucose was usedat 1 mg mL-'. An intracellular glucose oxidase was produced fromthe mycelium extract of a gamma rays mutated strain ofAspergillus niger BCG-5. Furthermore, enzyme was purified andsubjected to kinetic/thermodynamic characterization. Results:The enzyme was purified to a yield of 61.91%, 255.23 foldpurification and specific activity of 3910.06 U mg-' throughammonium sulfate precipitation, anion exchange and gelfiltration chromatography. The enzyme showed high affinity forD-glucose, with a Km value of 28 mM and Vmax 60 U mL-' with amolecular weight of 132 kDa. The enzyme exhibited optimumcatalytic activity at pH 5. Temperature optimum for glucoseoxidase catalyzed D-glucose oxidation was 400C.The enzymeshowed a high thermostability having a half-life 12.16 minutes,enthalpy of denaturation 0.171 kJ mol-' and free energy ofdenaturation 77.64 kJ mol-'. Conclusion: Glucose oxidase,produced/purified from the mycelium extract ofAspergillus nigerBCG-5 exhibited high catalytic properties and thermostability.Breakthrough work to be presented: These characteristicssuggest the use of glucose oxidase from mutant Aspergillus nigerBCG-5 as a valuable analytical tool and in the design ofbiosensors for clinical, biochemical and diagnostic assays.Key words: Glucose oxidase; Mutagenesis; Aspergillus niger;Thermal stability.

I. INTRODUCTION

Diabetes mellitus is a metabolic problem and is prevalent inmany parts of the world. In developed countries, the incidence

rate is more than 500 and an equal number is liable to developthe disease. One fundamental aspect of diabetes is anabnormality of glucose metabolism as due to insufficientaction of insulin, owing either to its absence or to resistance inaction [1]. Blood glucose level in diabetes becomes soelevated that the glucose "spills over" into urine, providing aconvenient diagnostic test for the disease [2].

The success of Aspergillus niger for industrial productionof biotechnological products is largely due to the metabolicversatility of this strain. The industrial importance of A. nigeris not only limited on its more than many products but also onthe development and commercialization of the new productswhich are derived by modern molecular biology techniques[3].

Glucose oxidase also known as f-D-glucose: oxygen-oxidoreductase (EC 1. 1.3.4), is of commercial interest,produces D-glucono- 1 ,5-lactone and a reduced acceptor.Glucose oxidase is FAD dependent glycoprotein catalyzingthe oxidation of f-D-glucose to glucono- 1 ,5-lactone. Itremoves hydrogen from glucose and reduces itself, which isthen re-oxidized by molecular oxygen. The developedhydrogen peroxide is decomposed by peroxidase [4].

The importance of glucose oxidase comes from its wideapplications in many fields in crude/purified form. Inpharmaceutical and clinical/medical biochemistry, it is usedfor quantitative determination of glucose in biological fluids.Glucose "dip-sticks" became available for screening ofblood/urine glucose by coupling the reaction to peroxidase anda chromogen [5]. Glucose oxidase use in diagnostic assaysaccounts for more than 8% of the total yearly budget of theenzymatic kit market worldwide, about US$ 61 million [6].

Traditionally, strain development requires painstaking,lengthy and tedious procedures to identify superior isolatesamong a mutagen-treated population. Several attempts havebeen made to improve glucose oxidase production by strainselection using classical screening and mutagenesistechniques. Special environmental conditions, toxic to themajority of the cell type (wild type) but less toxic or non-toxicto a desired minority of cells (mutant), have often beenapplied to enrich a cell population to obtain desired mutants(Direct selection). The greatest advantage of this screeningmethod is its simplicity that does not require any profoundunderstanding of the molecular biology and physiology of the

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microorganisms being manipulated [7]. The presentinvestigations were carried out to produce the mutant ofAspergillus niger for the hyperproduction of glucose oxidaseand its purification and characterization as well.

II. MATERIALS AND METHODS

A. OrganismPure culture of Aspergillus niger NFCCP was obtained

from National Fungal Culture Collection of Pakistan(NFCCP), Department of Plant Pathology, University ofAgriculture, Faisalabad, Pakistan. It was maintained on potatodextrose agar (PDA) slants at 4oC. Inoculum was prepared bytransferring the spores from 6 days old slant culture [8].

B. Mutagenesis ofA. nigerAspergillus niger spore suspension (1X107 spores mL-1) in

36 hours Vogel's broth was transferred in McCartney vialsand exposed to gamma radiation (Co60 irradiator) at NuclearInstitute of Agriculture and Biology (NIAB), Faisalabad,Pakistan. Seven different doses i.e. 20, 40, 60, 80, 100, 120and 140 k Rad. of gamma radiation (1.25 k Rad hour'1) wereselected in this regard [7].

C. Selection ofMutantIn order to restrict the formation of fungal colonies, triton

X- 100 (2% vlv) was used as colony restrictor in PDA medium[9]. Non-irradiated spores were also plated as control andplaced in an incubator, (30oC) for 8 days. More than 1000colonies were screened while a few mutants were isolated onPDA plates to study their enzyme activity [9] & [10].For screening of resistance to catabolite repression, 2-deoxy-D-glucose was used at 1 mg mL-1 [7] & [9]. The mutant sporeswere allowed to grow in PDA at 30OC for 8 days. The coloniesthat appeared as background growth were picked andsubjected to the preliminary glucose oxidase identification.

Glucose oxidase positive strain was identified on agar platecontaining 0.1 g L-1 o-dianisidine and 6000 U mL-1 ofhorseradish peroxidase. If glucose oxidase is formed, thenenzymatic reaction will occur giving rise a brown color [3] &[9]. The strains showing the greatest diffusion areas (mm)were further studied as being scratched, homogenized inbuffer, filtered and then the reaction for glucose oxidaseactivity by enzyme assay was determinedspectrophotometerically.

D. Production ofGlucose OxidaseThe selected mutant BCG-5 (gamma rays at 80 k Rad.) and

parent strain (as control) were used for growth in submergedfermentation at pre-determined conditions, in order to analyzethe glucose oxidase activity [11]. Corn steep liquor 2% (wlv)was used as an economical substrate along with urea 0.3,KH2PO4 0.6, CaCO3 0.04 and glucose 400 to achieve higherglucose oxidase yield using submerged fermentation. pH ofthe media was adjusted to 5. Then, 500 inoculum was addedaseptically in each flask for incubation in shaker at 30OC and

120 rpm for 36 hours. All experiments and analysis werecarried out in triplicates.

After growth upto 36 hours, the culture was filtered,separated mycelia washed twice with distilled water andsuspended in 0.1 M potassium phosphate buffer (pH 6). Themycelia were disrupted by a glass cell homogenizer for 10minutes, and the resulting suspension was subjected tocentrifugation at speed 10,000 rpm for 15 minutes at -10oC, inorder to disrupt/remove the cell membranes [7].

E. AnalyticalBiuret method [12] was applied for the determination of

protein using standard curve of bovine serum albumin.Glucose oxidase activity was determined with the help of acoupled o-dianisidine-peroxidase reaction as described inreference [5].

F. Purification ofGlucose OxidaseCrude enzyme (100 mL) was subjected to ammonium

sulfate precipitation at 60 to 85% saturation [13]. Ionexchange chromatography was carried out by self-packedcolumn (2x15 cm) of DEAE-cellulose [14]. An amount of 1mL desalted sample was poured on the column and 100fractions of 2 mL each were collected by pH gradient mode. Acolumn of sephadex G-150 (lx50 cm) was prepared by themethod of [14] and the sample (recovered from ion exchange)was allowed to penetrate into packed column. A total of 60fractions of 2 mL each were collected at a constant drop rate.

G. Molecular Mass DeterminationStandard proteins along with enzyme of 1 mg mL-1 each,

were loaded on sephadex G- 150 column and eluted with 0.IMphosphate buffer, pH 6 [14]. The flow rate was adjusted to 0.5mL min-' and 1 mL of size fractions was collected.

H pH and Temperature OptimaThe effect ofpH on hyperproduced glucose oxidase activity

was determined by assaying the enzyme as mentioned beforewith the difference that the activity was determined atdifferent pH ranging from 3-7. Glucose oxidase was assayedat different temperatures ranging from 20-80oC at pH 5 [14] &[15].

I. Determination of Kinetic ParametersGlucose oxidase from Aspergillus niger BCG-5 was

assayed in the reaction mixtures containing variable amountsof glucose ranging from 4-20% w/v. The data wereplotted according to Lineweaver-Burk to determine the valuesof kinetic constants (Vmax,Km) [1 6,1 7].

J. Irreversible Thermal DenaturationIrreversible thermal denaturation of glucose oxidase was

determined by incubating enzyme in phosphate buffer (pH 5)at different temperatures (20-80oC). Time course aliquots werewithdrawn, cooled in ice for 30 minutes and then assayed forenzyme activity at 40OC [17]. This procedure was repeated atfive different temperatures (45, 50, 55, 60 and 65oC). The data

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* 0 0 0

was fitted to first-order plots and analyzed as described by[17] & [18].

K. Thermodynamic ofirreversible thermal inactivationThe thermodynamic parameters for thermostability were

calculated by rearranging the Eyring's absolute rate equationderived from the transition state theory as described by[19].

Kd = (kb/h) e (-H /RT).e (AS */R) (1)

Where,h = Planck's constant= 6.63 x 10-` Jskb= Boltzman's constant (R/N) = 1.38 x 10-2 JK-lR= Gas constant = 8.3 14 JK-' mol-N= Avogadro's No. = 6.02 x 1023 mol-T= Absolute temperatureAH* =Ea - RT (2)AG* = - RT ln(Kd,h/kb. T) (3)AS*= (AH* - AG*)/T (4)

L. Statistical AnalysisAll the experiments were carried out in triplicates and the

data obtained was analyzed through MINITAB- 14 software.

III. RESULTS AND DISCUSSION

Although several organisms have been reported to produceglucose oxidase but Aspergillus niger is still the mainorganism used for industrial production. Improvement andevaluation of new glucose oxidase overproducing strains isvery important in improving the efficiency of the industrialprocess. Several attempts have been made to improve glucoseoxidase production in A. niger by strain selection usingrandom/classical screening and mutagenesis techniques,optimizing of cultivation conditions and genetic engineering[9].

A. Mutagenesis for Enhanced Production of GlucoseOxidase

In order to restrict the fungal colonies to small size onselection medium triton X-100, was used. Initially a kill curvewas prepared using gamma radiation as a mutagen. It wasfound that a dosage of 80 k Rad. produced 88.53% killing(9x102 CFU mL-1) as 3 log kill ofthe fungal spores.A number of specific selection schemes have been adapted

to improve the biosynthetic capacity of production strains.Literature reported that resistance to toxic glucose analoguei.e. 2-deoxy-D-glucose has been used as a criterion to selectthe mutants showing increased rates of glucose oxidase [7].The mutants resistant to 2-deoxy-D-glucose were obtained bymutagenic activation and passage in the medium withgradually increasing concentrations of this non-metabolizableagent [20]. Selective isolation medium was utilized to isolate2-deoxy-D-glucose resistant mutants and was prepared by theaddition of 2-deoxy-D-glucose at the level of 1.5 g L` asdiscussed in reference [9].

In order to produce depressed mutants for enzymeproduction, 2-deoxy-D-glucose was used at lmg mL-1

concentration. Colonies were selected based on largeclearance zones than wild type microorganism. Moreover,various colonies showing best results were subjected toenzyme diffusion zone analysis to select the best one.On plate media, enzyme diffusion zone analysis is the

specific procedure to screen the specific mutant based onenzymatic reaction. Glucose oxidase positive strain wasidentified on agar plate containing 0.1 g L` o-dianisidine and6000 U mL-' of horseradish peroxidase enzyme. The size andintensity of zone color is an index of the formation of glucoseoxidase. These results indicated that mutant BCG-5 obtainedat 80 k Rad. dose of gamma rays produced 14 mm enzymediffusion zone, with 582.78% increased activity as comparedto wild type 2 mm. [21 ] found the distribution ofthe irradiatedcolonies according to the size of halos of glucose oxidasediffusion into the agar plates and reported that 54 coloniesobtained diffusion halo larger than 7 mm in diameter ascompared to parent of 3 mm. According to reference [9] therewas a good correlation between the diameter of the zonesformed on screening medium and glucose oxidase activitiesmeasured using the spectrophotometric assay in the range of0-10 mm. Furthermore, mutant derived Aspergillus nigerBCG-5 obtained at 80 k Rad. dosage was used in thefermentation processes. However, another test was alsoemployed on the colonies obtained by zone analysis. Thelarger and darker zone producing strains were scratched,dissolved into buffer, filtered, homogenized and then thereaction for glucose oxidase activity was determinedspectrophotometerically.

B. Production and Purification ofGlucose OxidaseAs compared to parent strain, a mutant derived organism

can have a new genotype [22]. The mutant derived organismwas grown on corn steep liquor with pre-determinedconditions and it obtained 172.87 U mL-' of activity. While theenzyme produced from parent strain obtained with activity of38.17 U mL-'. So, the enzyme was purified from Aspergillusniger BCG-5 strain that showed about 4.5 fold increasedactivity. The specific activity of crude extract was 15.31 U mg-I, subjected to ammonium sulfate precipitation that resultedinto 18.92 U mg-' specific activity (Table I). Authors ofreference [14] subjected the P. funiculosum 433 glucoseoxidase to 80% saturation and found 18 U mg-' specificactivity with 94% yield and 1.6 fold purification.

The ion exchange capacity of a resin is a quantitativemeasure of its ability to take up exchangeable ions. Thisproperty and exchange efficiency reflects the accessibility ofthe inorganic groups to the exchanging ions [23]. Purificationof the enzyme by anion exchange column resulted into 46.19fold with 69.55% recovery. These findings showed a finecorrelation with the previous literature. P. funiculosum 433glucose oxidase applied to DEAE-cellulose column, resultedthe decrease in protein contents, increased specific activity(1400 U mg-'), 127 fold purification and 56.2% yield [14].[24] obtained 9900 recovery and 7 fold enrichment of Botrytiscinerea glucose oxidase when treated with DEAE-sepharosecolumn. It was reported that after DEAE-sephadex treatment

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of P. chrysosporium glucose oxidase, 60% of the enzyme wasrecovered with 5.4 fold purification [25].

Sephadex G-150, is a cross-linked polymer with pores ofselected size, used for gel filtration chromatography. Largerproteins migrate faster than smaller ones in sephadex columnsbecause they take a more direct route through the column. Thesmaller proteins enter the pores and are slowed by the morelabyrinthine path they through the column [25]. The 55thfraction of ion exchange treatment was applied to sephadex G-150 column for gel filtration chromatography, which is amethod of separation according to the differences in molecularsize. It was observed that enzyme achieved 3910.06 U mg-' ofspecific activity with 255.23 fold purification and 61.91%enzyme yield (Table I). According to [14], 3730 U mg-'specific activity, 339 fold purification and 56% yield of P.funiculosum 433 extracellular glucose oxidase was obtainedthrough sephadex G-150. Moreover, according to reference[24], 36% recovery and 36 fold enrichment ofthe enzyme wasobserved after gel filtration chromatography.

C. Molecular MassThe native molecular mass of glucose oxidase from

Aspergillus niger BCG-5, has been determined on sephadexG- 150 column, which was found to be 132 kDa. According toresults in reference [6], it was found that molecular mass of A.niger glucose oxidase is of 160 kDa and this finding was alsosupported by [26].

D. pH and Temperature OptimaGlucose oxidase from Aspergillus niger BCG-5 was active

within the pH range of 4-7. Optimum activity was observed atpH 5. Our results favorably compare to those of [27] whoreported that glucose oxidase worked in the pH range of 4-7with optimum pH of 5.5. It was showed by [28] that maximumglucose oxidase activity from A. niger was obtained at pH 5.5.Optimum temperature of the enzyme under investigation

was found to be 40OC respectively. According to previous

TABLE ISUMMARY OF PURIFICATION OF GLUCOSE OXIDASE FROM ASPERGILLUS niger BCG-5

Fractions Activity Protein(U mL-1) contents

(mg mL-)

Crudeextract

Ammoniumsulfate ppt.

Ionexchange

Gelfiltration

172.87 11.29

134.71

SpecificActivity(U mg-)

Fold %purification yield

15.31 1 100

7.12 18.92 1.24 77.9

120.23 0.17 707.23 46.19

107.03 0.0273 3910.06 255.23

69.5

61.9

literature, temperature dependence of recombinant enzymewas similar to parent one from P. amagasakiense with a broadtemperature optimum at 28-40oC and thereafter activitydecreased rapidly above 40oC. It was further reported thatglucose oxidase from P. funiculosum 433 displayed a widerange of temperature as 25-50oC [17], [29] & [30].

E. Kinetic parametersThe Km and Vmax values as determined from Lineweaver-

Burk plots were 28 mM and 60 U mL'1 (Fig. 1). In reference[25] it obtained the apparent Km value for glucose to be 38mM for P. chrysosporium glucose oxidase. [31] calculated thevalue of Km 18 with Vmax of 200.1 for A. niger glucose oxidaseand these findings are in agreement to my results. So, suchhigh substrate affinity and specificity, with its stabilitytowards pH, makes Aspergillus niger BCG-5 glucose oxidasefit for application in biosensors and for industrial applications.

F. Thermal Denaturation StudiesThermostability is the ability of enzyme to resist against

thermal unfolding in the absence of substrates, whilethermophilicity is the capability of enzymes to work atelevated temperatures in the presence of substrate. Thethermal denaturation of enzymes is accompanied by thedisruption of non-covalent linkages, including hydrophobicinteractions with concomitant increase in the enthalpy ofactivation. The enzyme from A. niger BCG-5 was thermallystable at 65oC with half life of 12.16 minutes (Fig. 2). Theenzyme had a range of 0.161-0.171 kJ mol'1 enthalpy ofdenaturation (AH*) at 45-65oC. The value of free energy ofthermal denaturation (AG*) for glucose oxidase was 72.94 kJmol'1 at 45oC that was increased to 77.64 kJ mol'1 at 65oC.Here negative values were obtained when entropy ofinactivation (AS*) was calculated at each temperature.Glucose oxidase from A. niger BCG-5 showed a AS* value of-229.53 J mol 'K'1 at 65oC (Table II). The thermal inactivationof glucose oxidase was studied in the temperature range from28-60oC for periods from 0-7 hours by [32] and resulted thatthe inactivation rate increased with increase in temperature.

TABLE IITHERMODYNAMIC PARAMETERS FOR IRREVERSIBLE THERMAL INACTIVATION OF

GLUCOSE OXIDASE FROM ASPERGILLUS niger BCG-5

Temp Kd t112 AH* AG*(K) (mi -'.) (min.) (kJ mol') (kJ mol')

318 0.045 15.40 0.161 72.94

323 0.047 14.74 0164 74.32

328 0.049 14.14 0.166 75.43

333 0.054 12.83 0.169 76.54

AS*(J mol'K-1)

-228.86

-229.93

-229.80

-229.68

338 0.057 12.16 0.171 77.64

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-229.53

* 0 0 0

1/ S (MM)

Figure. 1. Effect of substrate on mutant derived glucose oxidase activity.

5.00

A 45Cc4. 20

A 500C

3340

0 550C

2. 60

1. 80 D- 650C

1.00

lime (min)

Figure. 2. Irreversible thermal denaturation of mutant derived glucoseoxidase.

Hence, it is concluded that glucose oxidase of A. nigerBCG-5 was thermally stable and could be used for analyticaland other industrial applications. A high value for free energy

of thernal denaturation at 65oC indicates that the glucoseoxidase exhibited the resistance against thermal unfolding athigher temperatures.

REFERENCES

[1] R.K. Murray, D.K. Granner, P.A. Mayes, and V.W. Rodwell. "Harper'sBiochemistry;" 25th ed. New York: Appleton and Lange, 2000, pp. 4,149, 610-611.

[2] D. Voet, J.G Voet, and C.W. Pratt, "Fundamentals of Biochemistry,"New York: John Wiley and Sons, 1999, pp. 687-689.

[3] A.H. El-Enshasy, "Optimization of glucose oxidase production andexcretion by recombinant Aspergillus niger," Ph.D. Thesis,Biochemical Engineering Dept., Gesellschaft fMr BiotechnologischeForschung mbH (GBF), Braunschweig, Germany, 1998.

[4] A. Crueger and W. Crueger, "Glucose transforming enzymes," inMicrobial Enzymes and Biotechnology, 2nd ed., W.M. Forgarty andC.T. Kelly, Eds. New York: Elsevier, 1990, pp. 177-227.

[5] C.C. Worthington, "Worthington Enzyme Manual: Enzymes andrelated biochemicals," New York: Worthington Biochem. Coop., 1988,pp. 155-158.

[6] L.F.P. Ferreira, M.E. Taqueda, A. Converti, M. Vitolo, and A. PessoaJr., "Purification of glucose oxidase from Aspergillus niger by liquid-

liquid cationic reversed micelles extraction," Biotechnol. Prog., vol.21, pp. 868-874, 2005.

[7] A. Gromada, and J. Fiedurek, "Selective isolation of Aspergillus nigermutants with enhanced glucose oxidase production," J Appl.Microbiol., vol. 82, pp. 648-652, 1997.

[8] I. Haq, S. Khurshid, S. Ali, H. Ashraf, M.A. Qadeer, and M.I. Rajoka,"Mutation ofAsperigllus niger for hyper-production of citric acid fromblack strap molasses," WorldJ. Microbiol. Biotechnol., vol. 17, pp. 35-37, 2001.

[9] A.A. Khattab, and W.A. Bazaraa, "Screening, mutagenesis andprotoplast fusion of Aspergillus niger for the enhancement ofextracellular glucose oxidase production," J Ind. Microbiol.Biotechnol., vol. 32, pp. 289-294, 2005.

[10] M. Petruccioli, U. F. Federici, U. C. Bucke, and T. Keshavarz,"Enhancement of glucose oxidase production by Penicillium variabileP16," Enzyme Microbial Technol., vol. 24, pp. 397-401, 1999.

[11] J. Fiedurek, A. Gromada, and J. Pielecki, "Simultaneous production ofcatalase, glucose oxidase and gluconic acid by Aspergillus nigermutant," Acta Microbiol. Pol., vol. 47, pp. 355-364, 1998.

[12] A.G. Gornall, C.J. Bardwill, and M.M. David, "Determination of serumproteins by means of biuret reagent," J Biol. Chem., vol. 177, pp. 751-766, 1949.

[13] K.S. Shin, H.D. Youn, Y.H. Han, S.O. Kang, and Y.C. Hah,"Purification and characterization of D-glucose oxidase from white-rotfungus Pleurotus ostreatus," Eur. J Biochem., vol. 215, pp. 747-752,1993.

[14] M.V. Sukhacheva, E. Davydova, and A. I. Netrusov, "Production ofPenicillium funiculosum 433 glucose oxidase and its properties," Appl.Biochem. Microbiol., vol. 40, pp. 25-29, 2004. (Translated fromPrikladnaya Biokhimiya i Mikrobiologiya, Vol. 40, pp. 32-36, 2004).

[15] M.H. Rashid, and K.S. Siddiqui, "Thermodynamic and kinetic study ofstability of the native and chemically modified f-glucosidase fromAspergillus niger," Process Biochem., vol. 33, pp. 109-115, 1998.

[16] K.S. Siddiqui, M.J. Azhar, M.H. Rashid, and M.I. Rajoka, "Stabilityand identification of active site residues of carboxymethylcellulasefrom Aspergillus niger and Cellulomonas biazotea," FoliaMicrobiologica., vol. 42, pp. 312-318, 1997.

[17] S. Witt, M. Singh, and H.M. Kalisz, "Structural and kinetic propertiesof nonglycosylated recombinant Penicillium amagasakiense glucoseoxidase expressed in Escherichia Coli," Appl. Environ. Microbiol., vol.64, pp. 1405-1411, 1998.

[18] 0. Munch, and D. Tritsch, "Irreversible thermoinactivation ofglycoamylase from Aspergillus niger and thermostabilization bychemical modification of carboxyl groups," Biochem. Biophys. Acta.,vol. 1041, pp. 11 1-1 16, 1990.

[19] K.S. Siddiqui, A.M. Shamsi, M.A. Anwar, M.H. Rashid, and M.I.Rajoka, "Partial and complete alternation of surface changes ofcarboxymethyl cellulase by chemical modification: thermostabilizationin water-miscible organic solvent," Enzyme Microbial. Technol., vol.24, pp. 599-608, 1999.

[20] J. Fiedurek, A. Paszczynski, G. Ginalska, and Z. Ilczuk, "Selection ofamylolytically active Aspergillus niger mutants to 2-deoxy-D-glucose,"Zentralbl. Mikrobiol., vol. 142, pp. 407-412, 1987.

[21] M. Petruccioli, P. Piccioni, F. Federici, and M. Polsinelli, "Glucoseoxidase overproducing mutants of Penicillium variabile (P16)," FEMSMicrobiol. Lett., vol. 128, pp. 107-111, 1995.

[22] M. Petruccioli, P. Piccioni, M. Fenice, and F. Federici, "Glucoseoxidase, catalase and gluconic acid production by immobilizedmycelium of Penicillum variabile," Lett. Biotechnol., vol. 16, pp. 939-942, 1997.

[23] A. Braithwaite, and F.J. Smith, "Chromatographic methods," 5th ed.,London: Champan and Hall, 1996, pp. 449-457.

[24] S. Liu, S. Oeljeklaus, B. Gerhardt, and B. Tudzynski, "Purification andcharacterization of glucose oxidase of Botrytis cinerea," Physiol. Mol.Plant Path., vol. 53, pp. 123-132, 1998.

[25] R.L. Kelley, and C.A. Reddy, "Purification and characterization ofglucose oxidase from ligninolytic cultures of Phanerochaetechrysosporium," J Bacteriol., vol. 166, pp. 269-274, 1986.

[26] H, Tsuge, 0. Natusaki, and K. Ohashi, "Preparation, properties andmolecular features of glucose oxidase from Aspergillus niger," JBiochem. Japan., vol. 78, pp. 835-845, 1975.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

* a a a

[27] M.K. Weibel, and H.J. Bright, "The glucose oxidase mechanism:Interpretation of the pH dependence," J Biol. Chem., vol. 246, pp.2734-2744, 1971.

[28] R. Luque, M. Orejas, N.I. Perotti, D. Ramon, and M.E. Lucca, "pHcontrol of the production of recombinant glucose oxidase in Aspergillusnidulans," J Appl. Microbiol., vol. 97, pp. 332-337, 2004.

[29] A.N. Eremin, D.I. Metelitsa, Z.F. Shishko, R.V. Mikhailova, M.I.Yasenko, and A.G. Lobanok, "Thermal stability of glucose oxidasefrom Penicillium adametzii," Appl. Biochem. Microbiol., vol. 37, pp.578-586, 2001. (Translated from Prikladnaya Biokhimiya iMikrobiologiya., vol. 37, pp. 678-686, 2001).

[30] H.M. Kalisz, H.J. Hecht, D. Schomburg, and R.D. Schmid, "Effects ofcarbohydrate depletion on the structure, stability and activity of glucoseoxidase from Aspergillus niger," Biochim. Biophys. Acta., vol. 1080,pp. 138-142, 1991.

[31] J. Miron, M.P. Gonzalez, L. Pastrana, and M.A. Murado, "Diauxicproduction of glucose oxidase by Aspergillus niger in submergedculture: A dynamic model," Enzyme Microbial. Technol., vol. 31, pp.6 15-620, 2002.

[32] T. Godjevargova, N. Vasileva, and Z. Letskovska, "Study of thethermal stability of glucose oxidase in the presence of water-solublepolymers," J Appl. Polymer Sci., vol. 90, pp. 1393-1397, 2003.

[33] J. Georis, F.L. Esteves, J.L. Brasseur, V. Bougnet, B. Devreese, F.Giannotta, B. Granier, and J.M. Frere, "An additional aromaticinteraction improves the thermostability and thermophilicity of amesophilic family 11 xylanase: structural basis and molecular study,"Protein Sci., vol. 9, pp. 466-475, 2000.

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