Indian Jou rnal of Experimental Biology Vol. 4 1, June 2003, pp. 620-626

Xylanase production by Ganoderma lucidum on liquid and solid state fermentation

K Malarvizhi, K Murugesan & P T Kalai chelvan*

Ccntre for Advanced Studics in Botany, University of Madras, Guindy Campus, Chcnnai 600025, India

Received 6 Septe/llber 2002; revised I April 2003

C(/I/udenlw IIICidll lll , a whitc rot fungus, was cxp loited for ils potcnlial s lo produce xy lanase employ ing shake and solid-slate cu lture condili ons. DifFcrcnt culturc condilions such as pH , lempcralurc, carbon andnitrogcn requiremcnts for its growlh and production of xy lanusc wcrc opl imized. Thc cullurc media p H 6.0-7 .0 and temperalUrcs 30°-35°C signir"icuntly promoled lhc growth as wc ll as xylanasc sccret ion into thc mcdia. Xylan and pcptone we rc found to be thc su itable carbon and nitrogen sources. Among the differcllt agrowastes uscd, whea t bran was foundlo bc thc best substrate for the tcs t fungus for the production of xylanase lhan sugarca llc bagassc and ri ce bran in so lid-statc fcrmentalion.

Keywords : Fcrmcn lalion, Call0Lienl/II Ill cie/IIII/, Liquid state fermcnlalion , Solid state fcrmcntalion, Xylanase produclion.

Plant biomass is compri sed of three major polymeric const ituents such as celluloses, hemi celluloses and lignins. Hemicelluloses are a composite of different non-ce llulosic polysaccharides, in whi ch xylan being the major polysaccharide, g luca n and m<lnnan to a limited extent. In nature, xy lans ha ve L-arabino­furanosyl, acetyl, glucuronic, 4-0-mcthylgl ucuronic and p-coumaric side chains and ferulic ac id linkages l. The enzymes that degrade them are ubiquitous and diverse in nature. A consortium of at least seven different enzymes i.e., endo 1 ,4-xy lanase, -xy los idase, -glucu ronidase, L-arabinofuranosidase, acetyl esterase, the coumari c acid and ferulic acid es terases are required for the complete degradation of xylan to its monomeri c units.

Fungal xylanase can be prod uced using two main methods, so l id state cui ti vation system and su bmerged liquid systems. Most research has used submerged culture2

.3 , which all ows contro l of the degree of aeration , pH and temperature of the medium and the contro l of other environment factors required for the optimum growth of microorgani sms. However, solid state fermentation (SSF) has gai ned renewed interes t from researchers in recent years and has often been

' h d' f' I 4-6 b employed tor t e pro uctl on 0 xy anases , ecause of number of econo mic and engineerin g advantages. Xy lanase have becn isolated from basidiomycetes7

-11

but relat ively little is known from Calloderma IIICit/ulIl. Peru mal and Kalaichel van 11 reported the

"'Corrcspondcnt author: E· mail : botany 0l vsnl.coll1

production of xy lanase by C. lucidum during the degradation of li gnin in sugarcane baggase. This paper describes the results on the xy lanase production by C. lucidlllll both under liquid and solid state fermentation.

Materials and Methods M icroorgallism and inoculum - Canoderma

lucidul11 was isolated from infested Cassia marginata and identified according to FurtadolJ. The culture was mainta ined at 4°C on Potato Dextrose Agar Medium. Mycelial discs (6 mm diam.) from 7 day old cu lture of the fungus were used as inoculum for the ex peri men ts.

Media alld culture condiliol1s -- The Nutrient Salt Medium l4 was used th at contained the following composition per liter (NH4hP04 (4.0g), K2HP04 (2 .0g), KH 1P04 (O.8g), CaCl 1 (O.02g), MgS04.7H10 (0.89g), ZnS04.4 H20 (O.022g), MnS04.4 H10 (O.OOSg), Peptone (O.Sg), CuS04.SH20 (0.00 I g), FeS04.7H20 (O.OOSg), Th iam ine (0.00 1 g), Xylelll (S.Og) and pH 6.5. Erl enmeyer flasks (250 ml) co ntaining SO ml of the medium were aut oc laved at IS psi for 20 min. The medium was inocu lated with mycelial discs of actively grow ing mycel ia. Flasks were incubated for 15 days in stati c condition except for carbon sources. Cultures were harvested at every 3 day interv al. The mycelia were separated from culture broth and its dry weight was measured. The broth obtained was centrifuged at 10000 g for 10 min and the supernatant was dialyzed against cold glass-d istilled

MALARVIZHI el 01 .. XYLANASE PRODUCTION BY CANODERMA LUCIDUM 621

water and used for the xylanase assay15 and protein estimation 16.

Effect of pH and temperature on production of xylanase - To find out the optimum pH for xylanase production by G. lucidum the initi al pH of the nutrient medium was adjusted to pH 3.0 to 8.0 before inoculation, and to optimize the temperature for the max imum xylanase production the cultures were incubated at diffe rent temperatures i.e. 25°, 30°, 35°, 40° and 45°C in static conditions.

Effect of carbon sources on production of xylanase­To find out the efficacy of different carbon sources on the production of xylanase by G. lucidum, the fungus was grown in nutrient medium amended with the following carbon sources individually such as glucose, carboxy methyl cellulose, galac tose, arabinose, xylose and xy lan at 0.5 % (w/v) concentration. The cultures were maintained in stat ic as well as shaking conditions (l00 rpm) at room temperature. Duplicates were simultaneo usly maintained at the same cond iti ons.

£;ffect of Ilitrogen sources 0 11 production of xylanase ­The effect of different nitrogen sources on the production of xy lanase was tested with various nitrogen sources, viz. ammonium tartrate, sodium nitrate, peptone and yeast extract in the medium indi vidually at 0.5% (w/v) concentrations and the cultures were maintained at 30°C in stat ic condition.

Solid state f ermentation (SSF) - Three different natural lignocellulosic material s (Agro-industri al wastes), such as ri ce and wheat brans and sugarcane bagasse were used for th is study. Two hundred and fifty grams of wheat and rice brans and hundred grams of sugarcane bagasse were taken in autoclavable polypropy lene bags separately and moi stened with 80% water devoid of other mineral nutri ents and steam sterili zed at 15psi, 12 1°C for 20 min. Each bag was inocul ated with L5 plugs of myce li al discs of G. lucidum grow n on PDA medium and incubated at room temperature for 60 days. At every 15 days the after inocul ation fermented mass was mixed with phosphate buffer ( 100 mM, pH 7.0) wh ich was kept at 4°C overnight and the slurry was filtered through three layers of nylon cloth and the filtrate was centrifuged at 10000 g for 30 min. The clear filtrate obtained was used for xy lanase assay and gel electrophoresis. The so lid matters were dried in an oven at 80°C for 24 hI' and the dry we ight was es ti mated in te rms of percentage.

Delenllinatioll of protein - The protein co ntent of the supernatant was determined by th e dye binding

method of Bradford 16 with bovine serum albumin fract ion (V) (Sigma Chemicals Co. USA) as the standard.

Xylanase assay-Xy lanase act ivit/ 5 was assayed usi ng 1 ml of enzyme was preincubated with 1 ml of Oat Spelt Xylan (0.5% of oat spelt xylan in Phosphate buffer pH 6.8) at 60 °C for 4 hr. The reducing sugars were determined by using dinitrosalicylic ac id method 17 and compared with xy lose standard curves. One unit of activ ity was defined as the amount of re lease one ~lmol of product per min (IU) and is reported as U/g substrate used in the SSP.

Polyacrylamide gel electrophoresis - Native-Polyacrylamide ge l e lectrophores is of protein was performed by method of Dav is l8 . The culture fil trate was subjected to precipitation with ammonium sulphate at 50-70% (w/v) saturation. The precipitate was collected by centrifugation at 10000 g and dialyzed against sod ium acetate buffer (pH 5.0, 20 mM). The dialyzed protein sample was used to analyze protein pattern and xy lanase activity on PAGE. Detecti on of xy lanase act ivity on native PAGE was carri ed ou t by the method of Beguinel~ and Mackenzie et al. 20 using the Congo red stain .

Results Effect of pH and temperature on production of

xylanase - Figure 1 shows the effect of pH on the production of xy lanase by C. lucidum. It is obv ious that the pH had a striking effec t. Acidic pH values 3.0-5.5 did not show any marked effect on the production of xylanase. The pH nearer to the neutral ranges 6.0-7.0 favored the xy lanase production and the max imum production was observed at pH 6.5 (0. L80 U/min). The maximum xy lanase production

0.2 .. ,---- - ---

0.18

0.16

?:- 0.14 .;; "ti 0.12 '" ::: 0.1 '" ~ 0 .08

~ 0.06

0 .04

0 .02 ------._--

o +---~.--~------~----~----~ 3 6 9 12 15

Time (Days)

-3 --3.5 -+-4 --4.5 -+-5 - :- 5.5

-.-·6 ~6.5 ~7 ---'7.5-8

Fig. I - Effect of pH on xy l;.tnase production by Cal/oderlllG 11I6dlllll.

622 INDIAN J EXP BIOl, JUNE 2003

occurred between days 6-9 irrespective of the protein content and mycelial growth on ISlh day.

Of the different temperatures the maximum activity (0.180 U/min) was observed at 35°C on 61h day and similar activity was also noted in cultures grown at 30°C (Fig. 2). Increase and decrease of the temperature from 30°-35°C reduced xylanase production.

Effect of lime on rate of hydrolysis - The xylanase activity was tested with xylan as substrate, at different periods of incubation from 1-6 hI' at intervals of I hr. The amount of reducing sugar rel eased was maximum at fours of incuhation (0.11 and 0 .05 U/min) in stil l and shake flask cultures respectively (Fig. 3).

Effeet of corboll sO/.trees all production ofxylallase­The effect of different carbon sources on the growth of G. lucidul1l and xylanase prod uction was tested with various . carbon sources. Fig. 4a and b show. mycelial growth of G. I uddulII in sti II and shake cultures. Among the 6 di fferent carbon sources tested glucose promoted the growth initially (1.5 gIL) but the mycel ial growth was not progressed after 61h day whereas in xylan and xy lose supported the mycelial

0 .2

0.18 el2S· 030· ~3S· 040· 045·

:c 0.16 ..

.s; 0.14 .. U 0.12 OJ .'. ., 0.1 .'. <II OJ 0.08 .. c f.:-OJ .'. >. 0 .06 .'. x

0.04 .. 0 .02

.. .'.

a '.' ..

3 6 9 15

Time (Days)

Fig. 2 - Effecl of lelllpcralLtre or xy lanase production by CWlOdemw lucidlllll.

0.12 ,-1- --- ---- ------ - ----,

C 0.1 ] :§ 0.08 2-f 0.06 ti '" :l: 0.04 c '" ~ 0.02

. .... . Sl ill --Shaker

2 3 4 5 6

Time (Hours) Fig. 3 - Rale of hydrolysis of xylan by xy lanasc of Cal/oderllla IIICidlllll.

growth ti II IS lh day. Fig . 4c, d shows the effect of various carbon sources on the prod uction of xylanase in sti ll and shake flask cu lture respectively. It can be seen that xy lan showed maximum activity (0.229 U/min) on day 3 in shake flask culture foll owed by carboxymethy l cellulose (0.105 U/min) on day 12. In xylose amended medium xylanase activity was maximum (0.09 U/min) on day 3 in both still and shake tlask cu ltures . In galactose and arabinose amended medium there was no activity ti ll day 6 and the activity was meager (0.05 U/min) on day IS. The xylanase production was totally absent in glucose­amended medium.

Effect of nitrogen source on xylanase produclioll­The nutrient medium was supplemented with various nitrogen sources (0.5 w/v) individually with xylan as the sole carbon source. Among the different nitrogen sources tested the yeast extract promoted more mycelial growth (3 gIL) than other nitrogen sources (Fig.Sa) whereas the peptone induced the xylanase production and the maximum production (0.285 U/min) was observed on day 9 (Fig. Sb). There was no significant difference in the xylanase activity when the medium was amended with either sodium nitrate (0.172 U/min) or ammonium tartrate (0.166 U/min).

Xylanase production on solid state fermentation­The test fungus was screened fo r the production of xylanase in solid-s tate fe rmentation at 30°C for 60 days with rice and wheat brans and sugarcane bagasse as sole carbon sources without supplement of any mineral nutrients. The fungus showed extensi ve mycelial growth and enzyme production and there was a dramatic difference in enzyme production among substrates. The dry weight of the agro wastes were estimated in terms of percentage and summarized in Table I. Galloderma lucidum uti lized 22% of wheat bran, 26. 1 % rice bran and 65.8% of sugarcane bagasse on day 60.

Table 2 shows the xylanase production by the G. lucidum when grown on various lignocellulosic substrates. Xylanase production was increased with increase in the incubation period (Table 2). The maximum xylanase activity was observed in wheat bran ( 18.17 Ulg) on day 60. It was observed as 30-fold enhancement than in the xy lanase activity observed in liquid culture. Rice bran and Sugarcane bagasse showed moderate activities of 10.55 and 4.02 U/g respectively on day 60.

The culture filtrate obtained from 30 day old culture of rice bran was used for the xy lanase activity on native PAGE incorporated with oat spelt xylan

MALARVIZHI el ul.: XYLANASE PRODUCTION BY CANODERMA LUCIDUM 623

0.2% w/v. A clear lytic zone appeared after the gel was stained with Congo red. It indicated the presence of xylanase in the culture filtrate of C. lucidwn (Fig 6).

Discussion Canoderma lucidum, a polyporous and white-rot

fungus is reported to cause simultaneous decay of cellulose, lignin and hemicellulose in wood. Studies concerning the production of xylanase by C. lucidum have been made in recent years 12.21 which, formed the basis of this investigation . In this study , different parameters have been optimized for the production for xylanase by C. lucidum.

The influence of various pH and temperatures has been studied on the production of xylanase by C. lucidum. The synthesis of xylanase by the fungus depended on pH of the medium and the maximum production occurred at neutral pH range (6.0-7.0). Production of xylanase by ChaelOlniull1 cellulolticum was maximum in the medium with neutra l pH22.

Temperature also affected the production of xylanase, although the variations in temperature were less dramatic than variation in pH. Good growth of

C. lucidum and xylanase secretion occurred at 35°C on day 9. The growth and synthesis of xylanase was highly reduced with increase in temperature. Similar

o Glucose B2 Arabinose

o Cellulose o Galactose

IS Xylose 63 Xylan

2.5 ,--------------------------, Fig . 4a

~ 1 C o

05

2.5,---- ------------------,

~ 1 C o

0.5

Fig . 4b

9 Time (Days)

12 15

effects have already been reported in Aspergillus avvamori23

, where they showed the optimal production

of xylanase at 35°C. Results observed from other fungi have been similar in ' this respect, where xylanase production by Penicillium pinophilwn and several Trichoderma species have been shown to be markedly depending on temperature and pH24

.25

.

The rate of time of hydrolysi s of xylan by the culture filtrate was found to be maximun at four hours, likewise, similar effect was observed in Crptocotcus albidui6 but the rate of time on hydrolys is of xylan was found to be three hours.

Canoderma lucidum gave the best growth and xylanase sy nthesis when grown on oat spell xylan as sole carbon source followed by carboxymethyl cellulose. The production of enzyme was maximum day 9. This fact is very similar to other reports where oat spelt xylan being the best source for mycelial growth and enzyme production . The same effect has also been found 111 Bacillus circulani7 and StrelOmyces Sp2X. The xylanase secretion was completely absent in the presence of glucose such was the case for B. circulans27

. They reported that the g lucose completely suppressed the production of xylanase in B. circulans. The repress ion of xylanase in glucose-amended medium could be due to the catabolic repression, which will be answered only by studying the effect of cAMP in C. lucidum.

• Glucose 0 CMC 0 Arabinose &J Galactose (3 Xylose 0 Xylan 0.14 .r------------------~

0.12 .

. ~ 0.1 ' "il '" 0.08 . OJ

~ 0.06 c ~ 0.04 x

Fig . 4c

0.02

O ·~~~L,~~UL~~~~~~~~~~

0.25 ,----------------.---~

Z' 0.2 '> .~ 0.15 OJ If)

~ 0.1 ro 0>-X 0.05

3

Fig . 4d

6 9 12 15

Time (Days)

Fi g. 4- Effect of ca rbon sources on mycelial growth and xy lanasc production by CUllodenI/O lucidlllll. (a) M yccli al growth in stili condi­tion. (b) M ycelial growth in shaken condi ti on. (c) Xylanase production in st ill conditi on. (d) Xylan:1sc producti on in shakcn conditi on.

624 INDIAN J EXP BIOL, JUNE 2003

D-xy lose is the end product of xylanase. It was therefore unexpected that thi s compound at 33 mM concentration induced xylanase in G. lucidwn up to 3 days. This result is comparable to certain extent with those described earlier. Lianas and Hugues29 have reported that xy lose at 33 mM (5 giL ) induced the production of ~-xylanase, which was not detected in 4 mM concentration. Ganoderma lucidul11 shares a few characters with that of Clostridium stercorariun?l and Aspergillus awamor?' . These three organisms showed max imum xy lanase activity in xy lan-amended medium and none of the other so lub le carbon sources induced xylanase except xylose, galactose and arabinose. The enzyme activity was strongly inhibited by glucose. This may be due to the presence of readil y metaboli zable substrates that could have acted as catabo l ic repressor.

Nitrogen sources have a dramatic effect on the production of xy lanase by G. lucidum. Of the various organ ic and inorganic nitrogen sources tested peptone seems to be the best inducer of xy lanase. Elisashivili 9

has reported a similar observati on with respect to CerrelZa unico for.

Table 2 shows the profi le of xy lanase production by G. fucidum using various li gnoce ll ulose as substrates moistened with 80% dist illed water. Although fun ga l growth was apparently abundant on al l substrates, there were large differences in the producti on of xylanase. Wheat bran shoot out prominently without the additi on of any other supplements It was observed as 30-fo ld enhancement than in the xylanase activity observed in liquid culture

since wheat bran contains 36% wlv of hemicellu lose in which xylan is in complex form". Alam et af.32 reported similar results in Th ernlOmyces ianuginosus and Thermoascus aurantiacus. The author has reported abundant fun gal growth on all the substrates with great differences in growth and production of xyl.:mase. Similarly, G. lucidum cultivated on the agrowastes showed abundant growth and enzy me production in wheat bran th an rice bran and sugarcane

~ Y caSI exlracl Q Peptone ~ Ammoni um tartrate [) Sodium nitrate 3 .5

3 Fig . 5a

::::;-2.5 ~

1: 2 OJ '0; 1.5 ~ f7: ~ ~ r.: c:-O

0.5

0

::1 : :

'. - :

~ '7' :

3 6 9 12 15 0 .3 --. -------------- .----- ---.--- ____ _

Fig 5b 0.25

c-: ~ 0.2 u OJ

'" 0.15 '" OJ

I c '" 0.1 >-><

0.05

a - --,

3 6 9 12 15

Time (Days)

Fig. 5 - Effect of nitrogen sources on mycel ial growth and xy­lanase producti on by COllodenllo lucidul11. (a) M ycelial growth. (b) Xylanasc product ion.

Table I - Utilization of agrowastc by COllodeml(l IlIc idul11 undcr ill vitro so lid statc fermentation

I Values arc means of th rce experiments]

Incubat ion Whea t bran Ri ce bran Sugarcane bagasse peri od (days) Initia l wt Final wt Wt loss Initial wt Final wt Wt loss Initi al WI Final wt Wtl oss

(g) (g) (%) (g) (g) (%) (g) (g) (%)

IS 250 24 1.9 8.1 250 243 7 100 90.3 9.7 30 250 230.0 20 250 225 25 100 6 1.7 38.3 45 250 2 15. 1 24.9 250 206 44 100 43 .2 56.8 60 250 196. 1 53.9 250 185 65 100 34.2 65.8

Table 2 - Xylanase producti on by Cwwdenllo lucid/lin sol id slate fermentulion using agrowastes

rVal lies are means of thrcc ex periments]

Incllbalion peri od (days)

15 30 45 60

Wheat bran Prote in (gi l ) Xylanase

Ulg/min

7.89 958 9.88 12.7

11.6 16.9 15.8 IR. I

Rice bran Sugarcane bagasse Protein (gi l) Xylanase Protein (gi l) Xylanase

U/g/mi n U/glmin

5. 16 2.7 2.75 1.0 6.36 4.4 2.95 2. 1 7.88 6.9 3.5 1 3.6 9.6 1 10.5 3.74 4.0

MALARVIZHI et al.: XYLANASE PRODUCTION BY GANODERMA LUCIDUM 625

a b

Fig. 6 - Xylanase aCllvlty on na ti ve- PAGE incorporated with 0.2% oat spelt xy lan. (a) Ge l stained with Congo red fo r detection of xy lanase ac ti vi ty . (b) Gel stained with Coomass ie Blue R 250 for prote in .

bagasse (Tables 1 & 2). The 80% moisture level of agrowastes for the production of xylanase in G.lucidum was in agreement with Humicula I . 33 14 S Ibh 15 h d ' '1 anugtnosus ". u a' as reporte slim ar observation in Streptomyces sp. In contrast, Milagres and Rita" reported the higher production of xylanase by Poria medulla-pan is (brown rot) and Wolfiporia cocos (white rot) in wheat bran liquid medium. Even though there was a drastic weight loss in sugarcane bagasse, the amount of xylanase produced was comparatively less than the other two substrates (Tables 1 & 2). Thi s is because G. lucidum efficiently degrade the lignin than the other cell wall co mponents36

.

The diffe rent profiles o f xy lano lytic enzymes obtained in our ex periments with different ag rowastes are consi stent with different patterns of decay described in the literature37

.3x

.

Acknowledgement One of the authors (PTK) acknowledge the

financial ass istance by UGC, New Delhi (G rant No. F.3-100/200 \/SR II).

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626 IND IAN J EXP BIOL, JUNE 2003

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