Indian Journal of Experimental Biology Vol. 40, July 2002, pp. 802-806

Secretion of ligninperoxidase by Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus

Meera Kumari , R S S Yadav & K 0 S Yadav*

Department of Chemistry, D D U Gorakhpur University , Gorakhpur 273009, India

Received 8 May 2001; revised 12 February 2002

Secretion of ligninperoxidase [E.C.I .II . I.7 J by Penicillium cilrinUll1, Fusarium oxysporurn and AspergilluJ ierreus in liquid cu llure growth medium has been demonstrated. Enzymatic characteri sti cs like Km , pH and temperature optima using veratry l alcohol as the organi c substrate of Iigni npcroxidases from above sources have been determined. Kill va lues using veratry l alcohol as substrate for enzymes from P. cilrinulIl , F. oxysporulll and A. lerrells were 69, 64 and 60/lM respecti vely. Km values using H20 2 as the va ri able substrate were 64, 72 and 80/lM.The pH optima were 4.0, 2.3 and 2.0 respecti vely. The values of temperature optima were 30°, 25° and 22°C for the enzymes from P. cilrilllllll, F. oxysporLIm an A. lerreliS respectively.

Ligninperox idase [E.C.1.11.!.7] catalyses H20 2 de­pendent oxidati on of a variety of lignin model com­pounds'.General sequences of reactions involved in the catalys is are -

LiP+ H20 2 -----=>~LiPI + HO­

Li pr+R--->~LiPlI + R

LiPIJ + R--->~ LiP + R +H20

(I )

(2)

(3)

Ligninperoxidase compound I (LiP] ) carries both oxidi zing equivalents of H20 2, one as an oxyferryl (Fe4

+ -0) center and one as porphyrin JI cation radical whereas ligninperox idase compound II (LiPIl) carries only one ox idi zing equ ivalent. The substrate R is oxi­di zed by LiPI to an aryl cation rad ical which is COIl­

vel1ed to the final product by non enzymatic reactions.

Crystal structure of ligni nperox idase at 2A resolu­tion has been so lved2

. Basic helical fo ld and connec­ti vity defined by II helical segments wi th the heme sandwiched between distal and prox imal helices found in cytochrome c peroxidase is maintained in ligninperoxidase. The enzy me has histidine as a proximal heme ligand, wh ich is hydrogen bonded to a buried aspartic ac id side chain. The perox ide binding pocket contains di stal argi nine and hi stid ine like cyt -chrome c peroxidase. Lign inperox idase has phenyl a­lanines contac ting the distal and proximal heme su r­faces . This explai ns in part the fo rmation of a porphy­rin JI cation radical by the reaction of ligninperox idase

*Correspondent author

with perox ide. The detailed structural functional rela­tionship is not yet well understood2

.

Ligninperox idases3.6 are importan t group of en­zy mes having potentials in (i) delignifi ca ti on of li gno­cellulosic materi als? whi ch are seen as an alternati ve to depleting oil reserves, (ii ) in conversion of coa l to low molecular mass fractions8 which could be used as feedstock for the production of commodity chemicals, (iii) in biopulping and biobleaching9 in paper indus­tries, (iv) in removal of recalci trant organ ic pollut­:1 nts I0

.12; and (v) in enzymatic polymerisation '3 in in­

dustries . All these applications req uire lignolyti c en­zy mes of varying properties. Though a large number of li gnolytic microorgani sms have been reported ' 4,

the li gnolytic enzymes of a few microorgani sms have been purified and characterised3.5. '5.17.There is a sc ientific need to identify the source of novel lignolytic enzymes with properties more sui ted for above technological applications.

Keeping the above in view, studies to search of new sources of ligni nperoxidases and their enzymatic characteri stics have been initiated. Earli er, we have reported the secretion of ligninperox idase by Pleuro­tus saj or-caju l6 and Rhizopus II ig ricall.s 1 7. The enzy­matic characteristics of ligninperox idases from these sources have also been reported I6

. '7. In this communi­cation, we have reported the secretion of li gn inper­ox idase by Penicillium citrinum, Fusarium ox­ysporum and Aspergillus terr eU!i in liqu id culture me­di a. Enzymatic characteristics like Km, pH and tem­perature optima of li gninperoxidases from these sources have also been determined .

-1'

KUMAR I er af.: SECRETION OF L1GNINPEROXIDASE BY P. CITRINUM, F. OXYSPORUM & A. TERREUS 803

Materials and Methods Veratryl alcohol, which is 3,4-dimethoxy benzyl

alcohol, was from Aldrich (Wisconsin, USA). Di­methyl succinate and nitrilotriacetate were from Sigma Chemical Company (St. Louis, USA). All other chemicals were either from CDH (Delhi ) or Loba Chemie (Mumbai) and were used without fur­ther purification.

The fungal strains were isolated using pour plate technique'8 from soil samples collected from sites where waste water of Sanjai Paper and Chemical In­dustries Khalilabad, Sant Kabir Nagar, India were being discharged. The medium' 9 consisted of glucose (10g), malt extract (lOg), peptone (2g), yeast extract

,.., 0 ~

x

E '-.. ... . -

c ::1

QI

E >-N C w

1: >

u <1:

2

O.S

0.5

o

o

B

A ~~~-

2 4 6 8

c

10 Time .tdaysl_

A

Fig. I-Secretion of ligninperoxidase by (A) P. cirrinum; (B) F. oxysporum ; and (C) A. rerreus in the liquid culture medium sup­plemented with different natural lignin containing substrates. [(a) Coirdust (0); (b) Sawdust (t1); (c) Bagasse (. ); (d) Corn-cob (D); (e) Wheat straw (e ); and (f) Without substrate ( ~ )l

(2g), L-asparagine (lg), KH2P04 (2g), MgS04.7H20 (lg), thiamin-HCl (lmg) and agar (20g) dissolved in double distilled water (1 litre).

Isolated and purified microorganisms were tested for extracellular secretion of ligninperoxidase in liq­uid culture growth medium, which was a modification of liquid cu lture growth medium reported earlier' 9. The growth medium consisted of (per litre) 109, glu­cose; 1.32g, ammonium tartrate; 0.2g, KH2P04; 50mg, MgS04.7H20; 10mg, CaCI2; 101lg, thi amin ; and I ml, a solution containing (per litre) 3g, MgS04.7H20; 0 .5g, MnS04. H20; Ig, NaCI; 100mg, FeS04.7H20; 185mg, CoCh.6H20; 80mg, CaCI2; 180mg, ZnS04.7H20; 10mg, CuS04.5H20; lOmg, AIK(S04h; 10mg, H3B03; 12mg, Na2Mn04.2H20 ; and 1.5g, nitrilotriacetate. The pH of basal medi um was adjusted to 4.5 with 20mM dimethyl succinate. Growth media containing natural lignin sources like coirdust, sawdust, corncob, bagasse particles and wheat straw were separately prepared by adding 0.5g of one of the natural lignin sources to 20ml of growth medium in 100mi culture flasks which were sterilized. The sterilized growth medium was inoculated with 1 rnl of spore suspension (spore density 5x 106spores/ml) under aseptic condition and the fungal culture was grown under stationary condition at 30°C in BOD.

Activity of ligninperoxidase secreted in the liquid culture medium was assayed using veratryl alcohol as the substrate and monitoring the formation of vera­traldehyde spectrophotometrically at A=3lOnm with UVIVIS spectrophotometer Hitachi (Japan) model U-2000, which was fitted with electronic temperature control unit. Aliquot (1 ml) of the growth medium was

.!! o E

3 >

G o • ~

I~ 500

e -..... C1I

'0300 G E

::t

.... 1> 4 8 , -1

(S)(mM)

1.0 I, 2.0

(S) (mM)

12

Fig. 2-Michaelis-Menten and double reciprocal plots for ligni n­peroxidase of P. cirrinlllll using veratryl alcohol as the variable substrate.

804 INDIAN J EXP SIOL, JULY 2002

withdrawn at regular intervals of 1 day and was fil ­tered through Millipore Millex-GS filter (0.22JLM) unit. Fi ltered growth medium (0.2ml) was added to 0.8ml of the reaction mixture containing veratryl al­cohol (2.0mM), H20 2 (OAOmM) in SOmM of tartaric acid/disodium tartrate buffer (pH 2.5) contained in 1 ml cuvette maintained at 2SoC.One unit of ligninper­oxidase was defined as the amount of enzyme which converts IJLmole of veratryl alcohol to veratraldehyd under the condition specified above '9

. The enzyme unit was calculated using molar extinction coefficie t value of 9300 M· I cm· 1 for veratraldehyde. The least count of the absorbance measurement was 0.00 I absorbance unit.

Ligninperoxidases for the reported research work were prepared by growing the fungal cultures (lOx20ml volumes) in ten culture flasks (lOOml) as mentioned above. The maximum activity appeared on 5th day after inoculation of fungal spores. On fifth day, cultures in all the ten flasks were pooled, fi ltered through four layers of cheese cloth and concentrated using Amicon Concentration Cell Unit model 8200 and PM-IO membranes. Ten times concentrated en­zyme samples were stored at 4°C. The enzymes did not lose appreciable activities for one month und r these conditions.

The enzymatic characteristics of the enzymes like Km, pH and temperature optima were determined us­ing veratryl alcohol as the substrate following the re­action spectrophotometricall y at wavelength 31 On m as mentioned above. For determination of Kill> steady

~.O

0 ....

I~

S! C

>< ·e ---~

0

E

~ :l. 0

E ~I> :5 :>-

0.2

0

800

0 GOO

0

'0 80 1 _I

(S)(mM)

0.4

( Sl (mM)

( <>-)

(b)

120

Fig. 3-Michaelis-Men(en and double reciprocal plots for lignin­peroxidase of P. citrinw/l using H20 2, as the variable substrate.

state velocities of the enzyme catalysed reaction were determined at different concentrations of veratryl al­cohol and Km was calculated from the double recipro­cal plot of I / [V] vs I / [S] where [V] is the steady state velocity of the enzyme catalysed reaction and [S] is the substrate concentration. For determination of pH optimum, the steady state velocity of the enzyme cata­lysed reaction was determined at different pH values and a plot of V vs pH was drawn. The pH range from

4.0

M 0

X

c

E ""- 2.0

01

0 E

~ >

4.5 pH

Fig. 4-Variation of the activities of the lign inperoxidases of different fungi with variation of reaction pH. [(a) P. citrinulII (0); (b) F. oxysporum (6); and (c) A. terreLiS (0)]

4.0

M

~ X

c

E 2.0 --01

0

E

~ 0

>

°O~--------~2~0~------~30~--·----+'2o'------

Tempe rClt ur e t ·C )

Fig. 5-Variation of the activities of the lign inperoxidases of different fungi with variation of reaction temperature. [(a) P. ci­trinum (0); (b) F. oxysporum (6); and (c) A. terre/Is (0)

)

KUMAR I et aJ.: SECRETION OF L1GNINPEROXIDASE BY P. CITRINUM, F. OXYSPORUM & A. TERREUS 805

M a

X

c

4·0

.z. 2.0 QI

o E

=i :>

o

2 4 ,

Inhibitor (mM)

Fig. 6-lnhibition of the ac tiviti es of the ligninperoxidases from different sources by sodium azide. [(a) P. citril1um (0) ; (b) F. oxysporum (0 ) : and (c) A. terreus (L\) ]

1.5 to 4.5 was maintained using tartaric acid/disodium tartrate buffer (50mM).For determination of tempera­ture optimum, steady state velocity of the enzyme catalysed reaction was determined at different tem­peratures and a plot of V vs temperature was drawn. Inhibition by sodium azide20 was studied by measur­ing the steady state velocity of the enzyme catalysed reaction in solutions containing varying concentration of sodium azide in the range 0 to 6mM.

The fungal stains were got identified at the Micro­bial Type Culture Collection Center and Gene Bank, Institute of Microbial Technology, Chandigarh as Penicillium citrinum, Fusarium oxysporum and As­pergillus lerreus and have been deposited there with catalogue no. MTCC-3565, 3379 and 3374 respec­tively.

Results and Discussion Secretion of ligninperoxidases in the liquid culture

media by P. citrinum, F. oxysporum and A. terreus has been shown in Figure I. The liquid culture growth medium has been amended with different natural lig­nin containing substrates for induction of ligninper­oxidase. Order of induction of ligninperoxidase pro­duction in the liquid culture medium by P. citrinum was coirdust > sawdust> bagasse> corncob> wheat­straw> control, while for F. oxysporum the order was coirdust > sawdust> wheatstraw > corncob> bagasse

> control. The order of induction of ligninperoxidase production in case of A. terreus was coirdust > ba­gasse > sawdust> wheatstraw > corncob> control. In cases of all the three lignolytic fungal strains, maxi­mum induction in the production of ligninperoxidase was observed with coirdust. One point is quite clear that the production of ligninperoxidase by all the re­ported fungal strains was affected by the nature of naturallignolytic substrates and these microorganisms can be used for modifying the lignin components pre­sent in the lignolytic substrates.

Ligninperoxidase from P. citrinum followed Michaelis-Menten kinetics and the calculated Km of the enzyme for veratryl alcohol was 69p,M (Fig. 2a) which was of the same order of magnitude as Km value of 60p,M as reported for the ligninperoxidase of Phanerochaete chrysosporiuml9. Michaelis-Menten curves and double reciprocal plots for ligninperoxi­dase of F. oxysporum and A. terreus using veratryl alcohol as the substrate have also been drawn, but not shown because of similarity with Figure 2a and b re­spectively. The ligninperoxidases of F. oxysporum and A. lerreus also followed Michaelis-Menten kinet­ics and Km values for veratryl alcohol were 64 and 60p,M respectively again lying in the range of Km value reported for Phanerochaete chrysosporium l9.

Michaelis-Menten and double reciprocal plots for the ligninperoxidase of P. citrinum using hydrogen peroxide as the variable substrate has been shown in Figure 3a and b. Similar plots were obtained using ligninperoxidases of F. oxysporum and A. terreus. The calculated Km values were 64, 72 and 80p,M re­spectively for ligninperoxidases of P. citrinum, F. oxysporum and A. terreus which are also in the same range as Km value for H20 2 of ligninperoxidase of Phanerochaete chrysosporiuml9.

Figure 4 shows the variation of activities of lignin­peroxidases of P. citrinum, F. oxysporium and A. ter­reus with variation of pH of the reaction medium.The pH optima for P. citrinum, F. oxysporum and A. ter­reus were 4.0,2.3 and 2.0 respectively. These values are similar to pH optimum (3.0) of ligninperoxidase of Phanerochaete chrysosporiuml9.

Variations of activities of ligninperoxidases of P. citrinum, F. oxysporum and A. terreus with tempera­ture are shown in Figure 5. The temperature optima of these enzymes were 30°, 25° and 22°C respectively.

Ligninperoxidases from all the three reported sources were inhibited by sodium azide as shown in Figure 6. That was not unusual because azide ion is

806 INDIAN J EXP BIOL, JULY 2002

known to bind heme iron and to inhibit the activity of ligninperoxidase of Phanerochaete chrysosporiuml 9

.

Ligninperoxidases are biotechnologically important groups of enzymes and have industri al use. Three in­digenous sources of ligninperoxidase and their enzy­matic characteristics reported in the present study can be exploited for preparing ligninperox idases for in­digenous applications.

Acknowledgement The financial ass istances provided by DST through

its grant no. SP/SO/D-4S/90 and DOE through its gran t no. 19/47/90-RE are thankfully acknowledged. M.K is thankful to CSIR for granting her a SRF.

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