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Protein Expression and Purification 21, 24–29 (2001)doi:10.1006/prep.2000.1344, available online at http://www.idealibrary.com on

b-Galactosidase of Penicillium chrysogenum: Production,urification, and Characterization of the Enzyme

Zoltan Nagy,* Tunde Kiss,† Attila Szentirmai,* and Sandor Biro*,‡,1

*Faculty of Natural Sciences, Department of Microbiology and Biotechnology, and †Department of Biochemistry, Universityof Debrecen, Egyetem ter 1, H-4010 Debrecen, Hungary; and ‡Health and Medical Sciences Center, Medical School,

epartment of Human Genetics, University of Debrecen, Nagyerdei korut 98, 4012 Debrecen, Hungary

Received April 3, 2000, and in revised form September 11, 2000

Intracellular b-galactosidase from Penicillium chry-sogenum NCAIM 00237 was purified by procedures in-cluding precipitation with ammonium sulfate, ion-ex-change chromatography on DEAE–Sephadex, affinitychromatography, and chromatofocusing. These stepsresulted a purification of 66-fold, a yield of about 8%,and a specific activity of 5.84 U mg21 protein. Somenzyme characteristics were determined using o-ni-rophenyl-b-D-galactopyranoside as substrate. The pHnd temperature optimum of the activity were about.0 and 30°C respectively. The Km and pI values were

1.81 mM and 4.6. b-Galactosidase of P. chrysogenum ismultimeric enzyme of about 270 kDa composed ofonomers with a molecular mass of 66 kDa. © 2001

Academic Press

Key Words: Penicillium chrysogenum; b-galactosidase;urification; characterization; subunit structure.

The enzyme b-D-galactoside-galactohydrolase (EC3.2.1.23) commonly known as b-galactosidase or lac-tase is involved in lactose utilization by hydrolyzinglactose into glucose and galactose in many species. It iswidely distributed in nature and many studies havebeen reported on the physiology and regulation of theenzyme from different sources, including bacteria,yeasts, molds, plants, and animals. b-Galactosidasehas recently evoked considerable interest because of itsapplication in the food industry, nutrition, and medi-cine (1). In industrial penicillin fermentations by thefilamentous fungus Penicillium chrysogenum lactose isused as carbon source, because unlike glucose or othereasily metabolizable sugars it does not exert suppres-

1 To whom correspondence should be addressed at Faculty of Nat-ural Sciences, Department of Microbiology and Biotechnology, Uni-versity of Debrecen, Egyetem ter 1, H-4010 Debrecen, Hungary. Fax:

136 52 454400. E-mail: sbiro@jaguar.dote.hu.

24

sion on penicillin biosynthesis (2–4). Therefore, lactoseutilization has a considerably important role in peni-cillin biosynthesis. In our department different aspectsof penicillin biosynthesis have been studied (5, 6). Re-cently we have shown that production of the enzyme isinduced by lactose and repressed by glucose. In order tostudy the mechanism of carbon catabolite repressionand the regulation of the b-galactosidase biosynthesisat the molecular level in P. chrysogenum we plan toclone its cognate gene. This study presents the resultsof the purification of intracellular b-galactosidase fromP. chrysogenum NCAIM 00237 and also the determi-nation of some properties of the enzyme. The purifiedenzyme will be sequenced and the sequence informa-tion will be used in cloning.

MATERIALS AND METHODS

Culture Conditions

P. chrysogenum NCAIM 00237 was grown in a me-dium containing 2% lactose (sterilized separately),0.4% peptone, 0.4% yeast extract, 0.2% KH2PO4, 0.8%Na2HPO4 3 12H2O, 0.025% MgSO4 3 6H2O.

The fungus was grown in 500-ml Erlenmeyer flasks(each containing 200 ml of the medium) in New Bruns-wick incubator shaker at 25°C and 200 rpm for 65 h.

Dry Weight Determination

For determination of the dry weight of fungal myce-lium, 5 ml culture broth was filtered through pre-weighed filter papers; the mycelium retained by thefilter papers was dried to a constant weight.

Enzyme Assay

The activity of b-galactosidase was assayed at 30°C

after 30 min of incubation of the enzyme samples with

1046-5928/01 $35.00Copyright © 2001 by Academic Press

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25b-GALACTOSIDASE OF Penicillium chrysogenum

o-nitrophenyl-b-D-galactopyranoside (ONPG, Sigma)2

as chromogenic substrate. The 1-ml reaction mixturecontained 500 ml sample and ONPG at 3 mM finalconcentration in 0.1 M phosphate buffer, pH 7.0. Thereactions were started by the addition of ONPG andterminated either on ice or by 2 ml 0.1 M sodiumcarbonate. The enzyme activity was followed spectro-photometrically at 410 nm and was expressed as spe-cific activity (U mg21 protein): 1 U is equivalent to 1mmol o-nitrophenol produced min21.

Protein was determined by the modified Lowryethod (7) with BSA as a standard.

nzyme Purification

After 65 h of growth, the culture was filtered throughglass filter paper and the mycelium was washed withistilled water to remove medium components. Theashed mycelium was suspended in 15 ml 0.1 M phos-hate buffer, pH 7.0, and was frozen and kept at 220°Cntil further processing. Cells were broken by X-pressType X25, AB Biox, Goteborg, Sweden).

In order to inhibit proteinases, PMSF (Sigma) wasdded to the melted suspension at a 3 mM final con-entration and the cell-free crude extracts were centri-uged at 10,000 rpm for 10 min at 4°C.

Redundant proteins from the supernatant were pre-ipitated at 60% and the enzyme at 100% saturation bymmonium sulfate.The pellet was suspended in 2 ml 0.01 M phosphate–

itrate buffer, pH 7.0, and was dialyzed against 5000l of the same buffer O/N at 4°C.An aliquot of the enzyme was applied to a DEAE–

ephadex A-50 column (3 cm 3 10 cm) preequilibratedwith 0.01 M phosphate buffer, pH 7.0. The proteinswere eluted at a flow rate of 1 ml min21 with a 200-mlinear NaCl gradient (0.05 to 0.2 M) in the same buffer.he fractions were examined for b-galactosidase activ-

ity and those exhibiting high enzyme activity werepooled and concentrated by ultrafiltration using Kwik-Spin macro ultrafiltration units (10,000 MWCO;Pierce, U.S.A.) at 5000g and 4°C.

The enzyme solution was applied to an affinity col-umn (p-aminobenzyl 1-thio-b-D-galactopyranoside,1.5 3 3 cm, Sigma) that had been equilibrated with0.01 M phosphate–citrate buffer, pH 7.0. The columnwas washed with 40 ml of the buffer and the enzymewas eluted with 0.1 M phosphate buffer, pH 7.0. Frac-tions with high b-galactosidase activity were pooledand concentrated by ultrafiltration as above.

Pooled fractions were applied to a PBE 94 (1 3 16cm, Pharmacia Biotech, Uppsala, Sweden) columnequlibrated with 25 mM imidazole/HCl buffer, pH 7.0,as a final purification step and to determine the iso-electric point of the enzyme. The pH gradient was

2 Abbreviation used: ONPG, o-nitrophenyl-b-D-galactopyranoside.

developed by eightfold diluted polybuffer 74 (pH 4.0) ata flow rate of 0.66 ml min21. Active fractions wereconcentrated by ultrafiltration.

Gel Electrophoresis

SDS–PAGE was performed in 10% (w/v) gels by themethod of Laemmli (8). Low-range SDS–PAGE molec-ular weight standards purchased from Bio-Rad wereused as molecular mass standards (phosphorylase b97.4 kDa, serum albumin 66.2 kDa, ovalbumin 45 kDa,carbonic anhydrase 31 kDa, trypsin inhibitor 21.5 kDa,lysozyme 14.4 kDa), and the proteins were detected bystaining with Coomassie brilliant blue.

Native gradient gel electrophoresis according to themodified method of Laemmli was also used to followthe steps of purification and detect enzyme activity inthe gel. Activity was detected by staining with X-galaccording to McGrew and Green (9). Before stainingwith X-gal, the gel was equilibrated with 0.1 M phos-phate buffer, pH 7.0 for 1 h.

Effect of pH on Enzyme Activity

The optimum pH of the enzyme was determined byincubating the enzyme in buffers of different pH (0.1 Mphosphate–citrate in the range of pH 3–4.5 and 0.1 Mphosphate in the range of pH 4.5–8) for 30 min withONPG as described in the enzyme assay.

Effect of Temperature on Enzyme Activity

The optimum temperature for the enzyme activitywas measured by incubating the enzyme at differenttemperatures in 0.1 M phosphate–citrate buffer, pH4.0, for 30 min and the activity was measured as de-scribed previously.

Molecular Weight Determination

The molecular weight (Mw) of the enzyme was deter-mined on a Sephacryl S-300 column with dimensions of1.5 3 57 cm. The elution was carried out with 0.1 Mphosphate buffer (pH 7.0) containing 0.2 M NaCl and 3mM NaN3, with a velocity of 0.87 ml min21 at 4°C. Asmolecular weight marker proteins equine myoglobin(Mw 17.8 kDa), bovine albumin (Mw 67 kDa), bovinecatalase (Mw 240 kDa), and ferritin (Mw 450 kDa), allpurchased from Serva, were used.

Kinetic Parameters Determinations

The kinetic parameters—Michaelis constants (Km)and maximal velocities (Vmax)—were determined re-garding the synthetic substrate ONPG. A linear re-gression program was used to analyze and obtain Line-weaver–Burk plots and Km and Vmax were calculated.

This investigation was carried out by ONPG in the

26 NAGY ET AL.

concentration range of 1–8 mM at the optimum pH andtemperature.

RESULTS AND DISCUSSION

Production and Purification of b-Galactosidaseof P. chrysogenum

Our preliminary experiments showed that produc-tion of b-galactosidase in P. chrysogenum is subject toglucose repression and substrate induction (Z. Nagy,unpublished results). Therefore, throughout thesestudies we used a lactose-containing medium. A typicalgrowth curve, the pH values during fermentation, andthe intra- and extracellular enzyme activities areshown in Fig. 1. The majority of the enzyme remainedcell bound. The low activity in the culture fluid isprobably due to cell lysis at later stages of growth. Forenzyme purification cell extracts from the 60- to 72-hperiod seemed to be optimal where growth reaches the

FIG. 1. b-Galactosidase biosynthesis during growth of Penicillium cE, extracellular b-galactosidase activity; ■, dry weight; h, pH.

TAB

StepTotal protein

(mg)

Crude extract 162.04Ammonium sulfate precipitation 7.49DEAE–Sephadex chromatography

and ultrafiltration 0.56Affinity chromatography and

ultrafiltration 0.42

Chromatofocusing and ultrafiltration 0.2

stationary phase, enzyme production is near to maxi-mum and cell lysis is negligible. In our experiments weused 65-h cultures.

Table 1 shows the results of the different steps of atypical purification procedure of b-galactosidase of P.chrysogenum. The crude cell extract was subjected topartial purification by precipitating the enzyme withammonium sulfate in the saturation range of60–100%. This step decreased the protein content byabout 95% and consequently the specific activity in-creased about 10-fold.

The precipitated enzyme was subjected to DEAE–Sephadex A-50 ion-exchange chromatography. This re-sulted only an additional fourfold purification butturned out to be critical because omitting this step wecould not remove two major copurifying high molecularweight proteins (see Fig. 2, lane 3).

Then the enzyme solution was applied to an affinity

sogenum in shaken culture. F, intracellular b-galactosidase activity;

1

al activity(U)

Specific activity(U/mg)

Purification(fold)

Yield(%)

14.31 0.08 1 1006.72 0.89 10 47

2.12 3.74 42 15

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27b-GALACTOSIDASE OF Penicillium chrysogenum

column (p-aminobenzyl 1-thio-b-D-galactopyranoside,Sigma) which had been successfully used for rapidpurification of b-galactosidase of Aspergillus nidulans(10), Escherichia coli (11), and Bacillus megatherium12). In our case this step resulted only a slight furtherurification probably due to the relative purity of thepplied sample.As the final purification step chromatofocusing was

sed that resulted an additional 1.5-fold purification.

FIG. 2. SDS–PAGE (A) and native-gradient PAGE detecting theenzyme activity using X-gal as substrate (B) of samples of the dif-ferent purification steps. Lane 1, molecular weight markers; lane 2,crude cell extract; lane 3, ammonium sulfate precipitate; lane 4,DEAE ion-exchange chromatography; lane 5, affinity chromatogra-phy; lane 6, chromatofocusing.

he isoelectric point of the protein proved to be 4.6.

These purification steps resulted an overall 66-foldurification and an 8% yield.The purification steps were monitored by acrylamide

el electrophoresis. Fig. 2A shows SDS–PAGE stainedy Coomassie brilliant blue and Fig. 2B shows nativeradient gel stained by X-gal for enzyme activity. Inig. 2B, lane 2, an additional very faint band is visiblet the bottom of the gel (indicated by an arrow). Thiseflects that there are two different enzymes with X-al-hydrolyzing activity in the crude extract. The pu-ified enzyme shows a single band on Coomassie-tained gel and a single X-gal-hydrolyzing signal;herefore, we think that the protein is reasonably pure.

ffect of pH on Enzyme Activity

The pH activity profile of the purified b-galactosi-dase is presented in Fig. 3A. The enzyme exhibited a

FIG. 3. The effect of pH (A) and the temperature (B) on the activity

of b-galactosidase of Penicillium chrysogenum.

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28 NAGY ET AL.

broad pH optimum of 4–5 where it exerted more than95% of its maximum activity. The enzyme activity de-creased sharply below pH 4 showing only a 15% activ-ity at pH 3. The decrease of the activity in the higherpH range was not so sharp since at pH 7 the enzymeretained more than 30% of its activity.

Effect of Temperature on Enzyme Activity

The optimum temperature range of b-galactosidasectivity is 27–37°C. The activity increased with risingemperature until the optimum of 30°C was reached.bove 37°C the activity decreased rapidly, due to the

hermal inactivation of the enzyme.

olecular Weight Determination

Pure enzyme preparations were loaded to aephacryl S300 column previously calibrated witharker proteins in the range of 17.8 to 450 kDa. Theolecular mass of the protein proved to be 270 kDa.ince SDS–PAGE showed that the molecular mass ofhe protein was 66 kDa, therefore, we concluded thathe active form of the enzyme consisted of four identi-al subunits.

Multimeric nature is common amongst microbialb-galactosidases. The enzyme has a homotetramericform in E. coli (13), Bacillus macerans (13), and Lac-tobacillus helveticus (14). In some of the studied fungithe enzymes are monomeric (15, 16); in others theenzymes are multimeric (10, 17–19). The enzyme ismainly extracellular (15–17, 20) but intracellularforms are also known (10, 19).

Kinetic Parameters Determinations

In order to determine the kinetic constants we used

FIG. 4. Kinetic parameters determination of the b-galactosidase byineweaver–Burk plot using ONPG as substrate.

ONPG as substrate in the concentration range of 1–8

mM. The determination is based on the linear Line-weaver–Burk plotting (Fig. 4). The calculated Km valueis 1.81 mM which is very similar to the data obtainedfor Aspergillus oryzae (15) and A. nidulans (10) using

NPG and p-nitrophenyl-b-D-galactopyranoside assubstrates, respectively. The calculated Vmax value is40 nkat mg21. There are no comparable data for theVmax value available.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Education (OM-FKFP0102/1999). Z.N. was the recipient of a short-term TEMPUS PHAREJEP student mobility.

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29b-GALACTOSIDASE OF Penicillium chrysogenum

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