Induction of Xylanase from Trichoderma viride by ...

Indian J Agric Biochem 25 (2), 163-166, 2012

Short Communication

Induction of Xylanase from Trichoderma viride by using Different Carbon Sources MOHD. SHAHID, ANURADHA SINGH, MUKESH SRIVASTAVA, SMITA RASTOGI* and NEELAM PATHAK* Department of Plant Pathology, CS Azad University of Agriculture and Technology, Kanpur-208 002, India *Department of Biotechnology, Integral University, Kursi Road, Lucknow - 226 026, India

Downloaded From IP - 14.139.246.34 on dated 19-Dec-2013

www.IndianJournals.com

Members Copy, Not for Commercial Sale

Trichoderma viride strain 01PP produced extra cellular xylanases when grown in liquid medium cultures containing oat spelt xylan and birch wood xylan as inducer. Trichoderma viride 01PP was grown in Vogel’s medium with different carbon sources such as glucose, maltose, carboxymethylcellulose (CMC), sucrose, corn cobs, wheat bran, oat spelt xylan and birch wood xylan at 28ºC with shaking at 150 rpm to check the induction of xylanase. Expression of xylanase was induced maximally by xylan and repressed by glucose. The pH and temperature optima of the crude xylanase were 5.5 and 28ºC, respectively. Highest xylanase activity (5.5±0.01 IU/mL) was observed at 120 hours with 1% birch wood xylan as a carbon source. T. viride did not show xylanolytic activity without the presence of any carbon source. Key words: Trichoderma viride, xylanase, Vogel’s medium

Xylan, the major component of hemicellulose, is being utilized for improving the efficiencies of a bio-based economy for paper industry. Interest in xylanases production and productivity has increased markedly in

28ºC temperature. Inoculation medium (100ml) was prepared in the same way but without agar –agar. A loopful culture of Trichoderma viride from sporulation medium was transferred aseptically into the Erlenmeyer flask,

the past decade because of applications of these enzymes in the pulp and paper industry. Research on micro-organisms that utilize xylan and on the involvement of enzyme system, is important from ecological and economic point of view (1). Among the mesophilic fungi,

containing 100ml Vogel’s medium prepared as per standard method (6). The flasks were incubated on rotary shaker at 150 rpm at 28ºC for 5 days. Liquid state cultures were harvested by centrifugation at 10,000 rpm for 20 min. at 4ºC, and the resulting supernatant was

the genera Aspergillus and Trichoderma are pre-eminent for xylanase production (2). Most of industrial xylanase producing strains are species of Aspergillus and Trichoderma (3). Trichoderma has been shown to be

used as crude enzyme preparation. Xylanase activity of this crude enzyme was measured as per method described by Khanna and Gauri using Xylose as standard (7). The unit (1U) of enzyme activity was defined as the

efficient producer of xylan degrading enzyme activity (4).

amount of enzyme required to liberate 1 m mole min-1 of xylose from the appropriate substrate under the standard conditions. Trichoderma viride was grown in Vogel’s

A fungal strain namely Trichoderma viride 01PP isolated from rhizospheric soil of pigeonpea fields of Hardoi district (U.P.) was employed for the assay of xylanase activity. Trichoderma viride 01PP was maintained at 4ºC after growing for 7 days in a culture medium containing 0.2% malt extract, 0.2% yeast extract, 2% glucose and 2% agar (MYG medium) at 28ºC (5). Sporulation medium was prepared by using Trisodium citrate (0.5g), KH2 PO4(0.5g), NH4NO3 (0.2g), (NH4)2 SO4 (0.4g), MgSO4 (0.02g), peptone (0.1g), yeast extract (0.2g), glucose (0.2g), and agar – agar (2.5g) and the volume was made upto 100ml with double distilled water at 5.5 pH and

medium with different carbon sources like glucose, maltose, CMC, sucrose, corn-cobs, wheat bran, oat splet xylan and birch wood xylan to check their effect on the expression of xylanase. The crude xylanase activity was measured in pH range of 4.0 to 8.0 with different buffers. Sodium acetate buffer was used to maintain pH of 4-5.5 and citrate phosphate buffer was used to maintain pH of 6-8. The reactions were carried out at 40, 50, 60 and 70ºC for determination of optimum temperature and xylanase assay was carried out.

The optimum pH for the growth of Trichoderma viride 01PP and xylanase production was found to be 5.5 (Fig.1) in Vogel’s medium in all subsequent experiments. Simpson (8) while working on a thermostable endo 1, 4b xylanase form a culture of Thermogota spp. noted its maximum activity at optimum pH of 5.0 and 5.5. Rose and Vanzyl (9) found highest xylanase activity at pH of 5-6 in T. reesei. However, Fadel et.al. (10) found optimal initial pH of 4.5 f or xylanase pr oduction f rom T. harzianum F-416. was 4.5. The optimal temperature 28ºC and shaking at 150 rpm observed in this study (Fig.2), was in accordance with the previous work (11, 12). Whereas, Gomes et al. (13) and Juhasz et al. (14) used optimum temperature of 31 and 30°C, respectively, for the maximum pr oduct ion of xylanase. Since

conditions play an important role in enzyme production (15) and the choice of an appropriate substrate is also of great importance for the successful production of xylanase as it not only serves as a carbon source but also produces necessary inducing compounds for the organism (16). With this view, Trichoderma viride 01PP was grown in Vogel’s medium with various carbon sources viz glucose, maltose, CMC, sucrose, corn cobs, wheat bran, oat spelt xylan and birch wood xylan and expression of xylanase was studied (Table 1). After 5 days the culture mixture was taken out of orbital shaker. Supernatant obtained after centrifugation was tested for measur ement of xylanase activity. Among the lignocellulosic materials tested as carbon source, birch wood xylan was most effective for xylanase production.

maximum xylanase activity was achieved with citrate phosphate buffers, therefore, citrate phosphate buffer was used in all subsequent experiments for xylanase activity det ermination. Carbon source along wit h culture

Wheat bran exhibited moderate activity, while the other carbon sources resulted in (Table 1) lowest activity. Since xylanase induction causes glucose repression, hence effect of different carbon sources on xlyanase expression was also studied at various time intervals. All carbon sources tested were taken at a concentration of 1% (w/ Table 1: Xylanase activity produced by Trichoderma viride grown on different carbon sources

Xylanase activity (1U)


Indian J Agric Biochem 25(2), 2012

Carbon source (1%)

Fig.1:

0.05  0.001

Maltose

0.4  0.008

Sucrose

pH Value Effect of Initial pH value on Xylanase production by T. viride.

*Xylanase activity (1U) Mean  SD

Glucose CMC

3.2  0.04 0.31  0.006

Corn cobs

2.5  0.04

Wheat bran

4.1  0.06

Oat spelt xylan

4.2  0.03

Birch wood xylan

5.5  0.01

*Values are the means of three replications

v). Fig. 3a shows the effect of glucose on xylanase expression. When maltose was used as a carbon source xylanase activity was detected upto 48 hours, after which there was a decreasing trend (Fig. 3b). In presence of CMC, xylanse activity increased upto 48 hours and reached maximum at 72 hours, after which there was a decrease (Fig. 3c). In presence of sucrose maximum xylanse activity of 0.3 1U was observed after 72 hours (Fig 3d). Maximum xylanolytic activity with 1% corn cob




164

o

Temperature ( C) Fig. 2: Effect of Initial temperature on Xylanase production by T. viride

as a substrate was achieved after 72 hours after which there was a decreasing trend and xylanase activity reached to a baseline after 120 hours (Fig. 3e). Xylanase

Induction of Xylanase from Trichoderma viride using Different Carbon Sources 165

1% Maltose Xylanase activity (1U)


1% Glucose

Time (hours)

Time (hours)

3 (a)

3 (b) 1% Sucrose



1% CMC

Time (hours)

3 (c)

3 (d) 1% Wheat bran



1% Corn cobs

Time (hours)

Time (hours)

3 (e)

3 (f) 1% Oat spelt xylaN

1% Birch wood xylan Xylanase activity (1U)


Time (hours)

Time (hours)

3 (g)

3 (h) Without any carbon source Xylanase activity (1U)




Time (hours)

Time (hours)

3 (i) Fig. (3a) Time course of xylanse production by T. viride in Vogel’s medium containing 1% glucose. (3b) Time course of xylanase production by T. viride in Vogel’s medium containing 1% maltose. (3c) Time course of xylanse production by T. viride in Vogel’s medium containing 1% carboxymethylcellulose. (3d) Time course of xylanase production by T. viride in Vogel’s medium containing 1% sucrose. (3e) Time course of xylanase production by T. viride in Vogel’s medium containing 1% corncobs. (3f) Time course of xylanase production by T. viride in Vogel’s medium containing 1% wheat bran. (3g) Time course of xylanase production by T. viride in Vogel’s medium containing 1% oat spelt xylan. (3h) Time course of xylanase production by T. viride in Vogel’s medium containing 1% birchwood xylan. (3i) Time course of xylanase production by T. viride in Vogel’s medium without any carbon source.




166

Indian J Agric Biochem 25(2), 2012

activity with wheat bran and oat spelt xylan reached the maximum after 120 hours (Fig 3f, 3g). Expression of xylanase was induced maximally by xylan and repressed by glucose. The pH and temperature optima of the crude xylanase were 5.5 and 28ºC, respectively (16). Highest xylanase activity (5.5±0.01 U/mL) was observed at 120 hours with 1% birch wood xylan as a carbon source (Fig. 3h) (17). T. viride did not show xylanolytic activity in absence of carbon source (Fig.3 i). It is concluded that among various substances used for the maximum xylanase production, birch wood xylan at a concentration of 1% proved to the best carbon source (substrate) for Trichoderma viride as it observed highest xylanase activity at a concentration of 1%.

3

Dobrev GT, Pishtiytski IG, Stanchev VS & Mircheva R (2007) Bioresour Technol, 96, 2671.

4

Silveira FQD, Melo IS & Fitho EXF (1997) Rev Miobil, 28, 152.

5

Kubicek GP & Pentilla ME (1998) Taylor and Francis Ltd, 3, 49.

6

Haltrich D, Nidetzky B, Kulbe KD, Steiner W & Zuparcic S (1996) Bioresour Technol, 58, 937.

7

Khanna S & Gauri (1993) Enzyme and Microbial Technology. 15, 995.

8

Simpson HE, Haufler UR & Daniel RM (1991) J Biochem, 277, 413.

9

Rose SH & Vanzyl WH, (2002) Appl Microbiol Biotechnol, 58, 461.

10

Fadel M (2001) Annals of Microbiol, 51, 61.

11

Franco PF, Malheiros H & Folho EXF (2004) Biotechnol Appl Biochem, 40, 255.

Acknow ledgement

12

The authors are grateful for the financial support granted by the ICAR, New Delhi.

Sexton AC, Paulsen M, Woestermiger J & Howlett BJ (2000) Gene, 248, 89.

13

Gomes I, Gomes J, Stainer W & Esterbaver H (1992) Appl Microbiol Biotechnol, 36, 701.

14

Juhasz T, Szengel Z, Reczey K, Siika M, Aho & Vikkari L (2005) Process Biochem, 40, 351.

15

Montene court BS, & Eveleigh DF (1977) Appl Environ Microbiol, 34, 777.

Received August 02, 2012; accepted November 10, 2012

References 1

Meenakshi G, Kalra KL, Sareen VK & Soni G (2008) Braz J Microbiol, 39, 3.

16

2

Poflezeli MLT, M and Rizzatti AC, Montic R, Terenzi HF, Jorge JA & Smith DS (2005) Minireview Appl Microbiol Biotechnol, 67, 577.

Zafer M, Ahmed S, Sajjad-Ur Rehman & Jamil A (2004) Pakis, Life and Social Sci, 2, 99.

17

Mohan G, Guhan Kumar P & Balakrishnan V (2011) Res In Plant Biology, 3, 15.