International Journal of Biotechnology and Bioengineering Research. ISSN 2231-1238, Volume 4, Number 6 (2013), pp. 521-530 © Research India Publications http://www.ripublication.com/ ijbbr.htm

Bioprocess Parameter Optimization for Laccase Production in Solid State Fermentation

G. Mathur 1, R. Nigam1, A. Jaiswal1 and C. Kumar1

1Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sec-62, Noida, INDIA.

E-mail: 1garima.mathur@jiit.ac.in, 1www.jiit.ac.in

Abstract Laccase (EC 1.10.3.2), copper containing enzyme, catalyzes the oxidation of various aromatic compounds specifically phenols and anilines while concomitantly reducing molecular oxygen to water. Promising industrial and environmental applications of laccase include textile-dye decolourization, pulp bleaching, food industry, bioremediation of soils and water, polymer synthesis, the development of biosensors and biofuel cells, pharmaceuticals & cosmetic industry. Solid state fermentation holds tremendous potential for large scale production of enzymes. In current study an attempt has been made to compare laccase production by indigenously isolated Trichoderma longibracheatum (ITCC 8996.13) in submerged and solid state fermentation. Ten-fold increase in enzyme activity was observed when a comparison between submerged fermentation and solid state fermentation was done. It was observed that maximum laccase activity of 0.06 U/ml was obtained on 3rd day of incubation in synthetic medium using submerged fermentation. 80 %RH, pH 7.0 and 30°C temperature resulted in maximum laccase activity of 1.1 U/gds in solid state fermentation using wheat bran as substrate after 5 days of incubation, whereas enzyme activity of 10.73 U/gds was obtained using rice bran as substrate under similar conditions Process parameters such as relative humidity, temperature, pH, incubation time and nitrogen source were optimised to increase the laccase production by using design of experiment approach. It was found that under high relative humidity (89%), neutral pH, 30ºC temperature 3 days incubation time and no additional nitrogen supplement wheat bran as a substrate gives maximum enzyme activity of 10.02U/gds. While high

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relative humidity (96%), neutral pH, 37ºC temperature and no additional nitrogen supplement give best enzyme activity of 45.24U/gds using rice bran as substrate. The statistical analysis shows that time of incubation is the most significant parameter using wheat bran as a substrate while, time of incubation and interaction between nitrogen suppliment annd temperature are the most significant parameters using rice bran as substarte. Proposed mathematical model based on multiple regression was in complete agreement with experimental observation. Thus, the indigenous isolate seems to be a potential laccase producer using SSF.

1. Introduction Laccase (EC 1.10.3.2) is multiple copper containing enzymes that catalyzes the oxidation of various aromatic compounds specifically phenols and anilines while concomitantly reducing molecular oxygen to water. Laccase can also oxidize non phenolic compounds in presence of appropriate mediators (Brijwani et al,2010). Laccase activity has been found in plants, some insects and few bacteria. However, most biotechnologically useful laccase (i.e. those with high redox potentials) is of fungi origin. Most fungal laccase are monomeric proteins. However, quite a few exhibit a homodimeric structure, the enzyme being composed of two identical subunits. These enzymes are copper-containing glycoproteins with molecular weight between 60 and 80 kDa and with carbohydrate content between 15% and 20%. Laccase finds widespread applications in food industry, pulp and paper industry, textile industry, pharmaceutical industry, nano-biotechnology, soil bioremediation, biodegradation of environmental phenolic pollutants and other related applications (Arora et al, 2010).

Submerged fermentation (SmF) techniques have been successfully used for enzyme production by enzyme manufacturers worldwide. However in the last decade there has been an increasing trend towards the use of the solid-state fermentation (SSF) technique for the production of a large number of enzymes. SSF is a microbial process occurring mostly on the surface of solid materials, which can absorb water, in the presence or absence of soluble nutrients. SSF, in comparison with SmF, provides higher volumetric productivities, is less prone to problems with substrate inhibition and yields enzymes with higher temperature or pH stability. SSF has marked advantages over SmF in terms of productivity, concentration of the product and effluent generation. The present study was undertaken with an objective of isolation and screening of potential laccase-producers and laccase production in submerged and solid-state fermentation. Further, Statistical approach was used for media optimization in order to obtain maximum laccase production (Mishra et al, 2008).

Bioprocess Parameter Optimization for Laccase Production in Solid State 523

2. Materials and Method 2.1 Material The reagent grade chemicals and potato dextrose agar (PDA) were procured from Hi-Media, Mumbai. Guaiacol, CBB G-250 were purchased commercially from SIGMA, USA and used for analytical purpose. The substrates for solid state fermentation, Wheat Bran and Rice Bran were procured locally from the market.

Isolated laccase producing culture Trichoderma longibracheatum (ITCC 8996.13) was used in the present study for laccase production. The organism was maintained on PDA plates. The strains were sub-cultured periodically and fresh cultures (7 days at 30 ± 2 ◦C) were prepared and used for each experiment as inoculum.

2.2 Fungal Isolation and Culture Maintenance Soil and rotting wood samples were collected from different places. Fungal isolates were obtained from these samples using serial dilution method and point inoculation method on PDA agar plates. Pure cultures were maintained on PDA slants and were screened for laccase production. 2.3 Screening for Laccase Production Laccase production by fungal isolates was screened using composite selective media plates having composition as 0.3% peptone, 1% glucose, 0.06% KH2PO4, 0.0005% FeSO4, and 0.005% MnSO4, 0.05% MgSO4, 0.0001% ZnSO4, 2% agar and 0.02% guaiacol with pH adjusted to 6. Laccase activity was visualized on the plates as reddish brown zones in medium. (Sivakumar et al, 2010) 2.4. Laccase Production in Submerged Fermentation Laccase production by Trichoderma longibracheatum was carried out using four different production media Medium1, Medium 2, Medium 3, and PDB as a control medium. Fungal spore suspension from actively growing slants was used as inoculum to inoculate 100 ml of each production medium. The cultures were cultivated in the production media at 30°C and 120 rpm for 14 days. Sampling at regular intervals was done for estimation of fungal growth and laccase activity.

Table 1: Different media for submerged fermentation.

Media Composition (g/L) Reference Medium 1 Maltose(30.0),Yeast Extract(6.0), Polypeptone(2.0),

K2HPO4(0.5), K2HPO4(0.2),MnSO4(0.2),MgSO4,PH 5.0

(Luke et al,2001)

Medium 2 Glucose(10.0), (NH4)2SO4(3.3), CuSO4.5H2O(2.0), K2HPO4(0.4),

K2HPO4(0.6), MnSO4.H2O(0.5), MgSO4.7H2O(0.5), FeSO4.7H2O(0.05),

ZnSO4.7H2O(0.007) PH 4.5

(Diaz et al,2001)

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Medium 3 Starch(20), Yeast Extract(2.5), H3PO4(1.0),Na2HPO4(0.05), MgSO4(0.5),

CaCl2(0.01),FeSO4(0.01),MnSO4 (0.001), ZnSO4 (0.001),CuSO4(0.002) PH 4.5

(Sivakumar et al,2010)

2.5 Solid State Fermentation 5 gm. of Substrate i.e. wheat bran and rice bran is autoclaved in petri plates. . Buffer solutions of pH 5.0 (10 mM Sodium-acetate buffer) and pH 10.0 (10 mM Carbonate bicarbonate buffer) were used as moistening medium and to maintain desirable pH. The enzyme was harvested by adding 20ml water and the fermented slurry was filtered through muslin cloth. The resulting extract was used for the assay of enzyme activities was determined using hygrometer. Agar plugs (0.8 mm in diameter) cut from actively growing fungal mycelium were used as inoculum. The contents of the petriplates were mixed thoroughly and incubated at 30 ±C in static condition for different time intervals (5 and 10 days). 2.6 Assay for Laccase Activity The laccase activity was measured by monitoring the oxidation of 1ml of 10mM Guaiacol buffered with 3 ml 100mM Sodium Acetate buffer at 470 nm.[10] Enzyme activity is measured in U/ml which is defined as the amount of enzyme catalyzing the production of one micromole of colored product per min per ml.

(1)

Where, Vt = final volume of reaction mixture (ml) = 5.00 Vs = sample volume (ml) = 1 € = extinction co-efficient of guaiacol = 6,740 /M/cm 4 = derived from unit definition & principle 2.7 Plackett-Burman Design PlacketteBurman design was applied to study the significant variables responsible for laccase production. Each variable was tested at two levels, high level (+) and a low levels (-),and three variables were screened by conducting 8 experiments as given in Table 2. The variables which were significant at 68% confidence interval from the regression analysis were considered to have greater impact on laccase production. The predicted response can be obtained from the model fitting techniques by applying multiple regression analysis using Design of Experiment.

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Table 4: Fit statistics for Y1 Wheat Bran Rice Bran

The overall linear regression equation showing the empirical relationship between

laccase activity (Y1 ) and three test variables in coded units is represented by Eq. (2) using wheat bran as substarte and by Eq. (3) using rice bran as substrate.

Y1 = 0.472258 ‐ 0.156697*X1 (1) Y1 = 0.558288 ‐ 0.216249*X1 ‐ 0.007144*X2 + 0.017841*X3 ‐ 0.020328*X1*X2 + .032283*X1*X3 + 0.14223*X2*X3 (2)

The pareto analysis of the effect of three indicates that time of incubation is the

most significant parameter for laccase production using wheat bran as a substrate while time of incubation and interaction between temperature and nitrogen source are most significant parameters using rice bran as a substrate.

Figure 5: Pareto chart showing the effect of three parameters on laccase production.

Bioprocess Parameter Optimization for Laccase Production in Solid State 529

4. Conclusion Of all the fungus isolates obtained Trichoderma longibracheatum (ITCC 8996.13) was found to be the best laccase producer and was selected for the optimization of laccase production. The effect of various process parameters such as relative humidity, temperature, pH, incubation time and nitrogen source on laccase production using solid-state fermentation was studied and optimised to increase laccase production. High relative humidity, neutral pH, 30ºC temperature 3 days incubation time and no additional nitrogen supplement gives maximum enzyme activity using wheat bran as substrate for SSF. While High relative humidity, neutral pH, 37ºC temperature and no additional nitrogen supplement give best enzyme activity using rice bran as substrate. A comparison between rice bran and wheat bran as substrate shows that rice bran is a better substrate for laccase production. References

[1] A Jhadav, K K Vamsi, Y Khairnar, A Boraste, N Gupta, S Trivedi, P Patil ,G

Gupta, M Gupta, A K Mujapara, B Joshi and D Mishra (2009), Int. J. of Micr. Res.,1, pp-09-12.

[2] A K Luke and S G Burton (2011), Article in regular journal, Enzyme Microb. Tech, 29, pp. 348–356.

[3] A Mishra, S Kumar and S Kumar (2008), Article in regular journal, J Sci Ind Res India, 67, pp. 1098-1107.

[4] D S Arora and R K Sharma (2010), Article in regular journal, Appl. Biochem. and Biotechnol,160, pp.1760-1788

[5] K Brijwani, A Rigdon and P V Vadlani (2010), Article in regular journal, Enzyme Research, 10, pp.1- 10.

[6] R Sivakumar, R Rajendran, C Balakumar and M Tamilvendan (2010), Article in regular journal, Int. J. Eng. Sci.Technol, 2, 12, pp. 7133-7141.

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