Optimization Of Extra Cellular Peroxidase Production Biology Essay

A response surface methodological analysis was used for the optimisation of excess cellular laccase production utilizing Pleurotus sp. Central composite experimental design was applied in the analysis of consequences and this process required limited figure of experiments performed while supplying possible interactions of selected constituents. Optimum values of tried variables for the maximal laccase production were ; mannitol 4.8gm/l, beef infusion 3gm/l and pH 6.2 and Cu sulfate 4 mg/l, the maximal laccase production was 90.861U/ml.

Merely 27 experiments were attained this optimal conditions for enzyme production and the coefficient of finding was 89.0 % . In the present survey, we have demonstrated that the usage of cardinal composite factorial design for high output of enzymes.Key words ABTS, Pleurotus sp. , Laccase, Response surface methodological analysis

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Lignin and cellulose are both instead stiff organic polymers ( Tuor et al. , 1995 ) , which have developed during development for building and saving intents ( Call & A ; Mucke, 1997 ) . The debasement of lignin in the pulping and bleaching procedures is indispensable for the fabrication of paper merchandises.

These compounds have to be exposed to harsh physiochemical conditions to modify or degrade their construction for use in the mush and paper industry ( Coll et al. , 1993 ) . The jobs caused by chemicals used in decoloring forced industry to see alternate, more environmental friendly methods ( Yang & A ; Eriksson, 1992 ) . Such a biological option to traditional bleaching was provided through the find of oxidative enzymes ( Poppius-Levlin et al. , 1997 ) . Laccase is a polyphenol oxidase that contains four Cus, and is able to oxidise its substrates by utilizing molecular O as an negatron acceptor.

Laccase can be found in workss, insects and bacteriums, but its major beginnings from Fungi. Fungi associated with many biological maps such as lignin debasement, remotion of potentially toxic phenols in add-on to paper industry ( Argyero Poulos, D.S. , 2001. ) . The oxidative enzymes can be used for biodecolorization and detoxification of industrial wastewater waste H2O ( Feng Xu, 2005 ) . Laccase and peroxidase enxymes are by and large produced by submersed agitation in commercial pattern ; the medium composing play a important function in the enzyme production. Research attempts have been pointed towards measuring the consequence of assorted organic and inorganic foods, metal ions, pH, temperature, aeration, and agitation on the output of enzymes.

From the conventional method the following parametric quantities were selected for the Response Surface Methodological optimisation. Mannitol, beef infusion, pH and ferric sulfate were found to be of import factors in heightening the laccase production. The conventional method of optimisation involves changing one parametric quantity at a clip and maintaining the others are invariant.

Response surface methodological analysis ( RSM ) is a simple theoretical account to analyse the consequence of assorted factors act uponing the responses by changing them at the same time. Carbon and N beginnings have been promised to play a important function in heightening the production of fungous laccase. The cardinal composite design used to analyse the interaction effects of variables on production of laccase enzymes ( Fannin et al. , 1981 ; Deshayes 1980 ; Matthews et al. , 1981 ) ( Box and Wilson 1951 ; Cochran and Cox, 1957 ; Box et al. , 1978 ; Akhnazarova and Kafarov, 1982 ; Khuri and Cornell, 1987 ; Yee and Blanch, 1993 ; Mak et al. , 1995 ) .

Conventional method of optimisation involves altering one independent variable at a clip ( vanillin, glycerin and mineral salt solution ) and keeping all the others at a invariable.

Materials and methods

Isolation and designation

Laccase-producing Fungis were screened from Dry Evergreen forest of Guindy National Park and IIT Madras, Chennai. Fungi were isolated by puting pieces of unexposed tissue of fruit organic structure on MEA ( Malt Extract Agar ) and PDB ( Potato dextrose Broth ) plates incorporating different index compounds. The home bases were incubated at 27oC for at least a hebdomad and strains were sub cultured when clear positive reactions were seeable. Production of laccase was carried out in production medium and these medium were sterilized by autoclaving at 121A°C for 15 min. The above medium ( 50ml in 250ml Erlenmeyer flasks ) was inoculated with two mycelial phonograph record ( 8mm ) and maintained at 27A°C in a inactive status. Fermented broth was filtered in filter paper at 4A°C and the supernatant was recovered and used as enzyme beginning.

The scope of trial variables are given in table 1. The concentration of Osmitrol, beef infusion, pH and ferric sulfate were varied harmonizing to the experimental design ( table 2 ) . The pH value of the medium was adjusted before sterilisation.

Enzymatic check

Extra cellular laccase activity of cell free filtrate was assayed spectrophotometrically on 9th twenty-four hours, 10 millimeter of ABTS as substrates with 100 millimeters sodium ethanoate buffer ( pH 5.0 ) , 0.1 milliliter of the civilization filtrate ( assay for 1ml of reaction mixture ) and optical density was measured at 436 nanometer. ( Mansur et al. , 1997 ) .

0.1 milliliter of distilled H2O, 0.9 milliliter of Na ethanoate buffer was kept as space.Optimization by RSMBox-Behnken design consists of a group of controlled experimental factors and mensural responses, harmonizing to one or more selected standards.

A anterior cognition to understanding the procedure and procedure variables under probe are necessary for accomplishing a more realistic theoretical account. Among the different constituents of the media, C, N, pH and metal ion were the major variables were selected to happen optimum conditions for higher enzyme production utilizing Response Surface Methodology. The scope of experimental variables was investigated in this survey ( Table 1 ) . Several experimental design have been selected the Box-Behnken design proposed by Box et al. , ( 1978 ) . For this survey, 4 factorial design were employed to suit the 2nd order multinomial theoretical account which indicates 27 experiments were required for this process.

The mid values ( zero degree ) chosen for experimental design comprised Osmitrol, beef infusion, pH and ferric sulfate. The three important independent variables X1, X2, X3 and X4 and the mathematical relationship of response Yttrium on these variables can be approximated by quadratic / ( Second grade ) multinomial equation as shown below:


WhereYttrium is the predicted response,bo is the changeless,b1, b2, B 3 & A ; b4 are the additive co-efficients,b11, b22, b33 & A ; b44 are the quadratic co-efficients andb12, b13, b23, b24 & A ; b34 are the cross-product co-efficients. The optimal values of the selected variables were obtained by work outing the arrested development equation ( Cornell 1987 ) . In that, the 27 agitation experiments were conducted in triplicate.

Consequences and treatment

Pleurous sp.

was grown in CDB at different temperatures runing from 20 and 40EsC at 5A°c interval. Maximal production of 44 U/ml was recorded at 25EsC. The temperature above and below 25EsC did non heighten the Laccase production ( fig.

2 ) . Saravanakumar and Kaviyarasan ( 2010 ) studied the optimum parametric quantity of maximal laccase enzyme production was found to be pH 8.0, temperature 25A°C, glycerin, vanillin and CuSo4, but the laccase production was decreased at 30A°C ( Thurston, 1994 ) .

In general, the Fungis were cultivated at temperatures between 25A°C and 30A°C for optimum laccase production ( Arora & A ; Gill, 2000 ; Fahreus & A ; Reinhammar, 1967 ; Indicating et al. , 2000 ; Vasconcelos et al. , 2000 ) . When the temperatures higher than 30A°C the activity of ligninolytic enzymes was reduced ( Zadrazil et al. , 1999 ) .

The pH showed a important influence on the production of excess cellular Laccase, the trial fungus produced Laccase between the pH of 5.0 to 9.0. Maximal Laccase production of 94.

3 and 94.0 U/ml was recorded at pH 5.5 and 7.5 severally, and the moderate activity was observed in pH 9.0 and declined ( fig.1 ) .

The optimum pH for laccase production was found to be at pH 6.5 in submersed civilization of Chaetomium globosum ( El-Zayat, 2008 ) . The laccase production was 25-fold higher in Botryosphaeria rhodina showed a important consequence with increased pH of 3.5 – 7.5 for both induced and noninduced civilizations ( Dekker et al. , 2007 ) .

Among the different C beginnings tested viz. Glucose, Sucrose, Mannitol, Maltose, Glycerol and Fructose at 1 % concentration, the degree of Laccase production was maximum at 161.1 U/ml, in Osmitrol amended medium. The enzyme production of 149 U/ml and 90.

9 U/ml was recorded with Fructose and Glycerol on 15th twenty-four hours of incubation. The C beginnings like glucose and malt sugar showed reduced consequence on Laccase production from Pleurotus sp. , ( fig.3 ) .

( Dekker et al. , 2007 )Fig.1Fig.2Fig. 3Churapa Teerapatsakul et al. , ( 2007 ) showed that Ganoderma sp. produced maximal laccase activity in the presence of glycerin as a C beginning in liquid medium.

In add-on, Rodriguez Couto et al. , ( 2006 ) reported glycerin as a exclusive C beginning in Trametes hirsuta, which showed a maximal laccase activity of 19,400 U/L, which was reported the highest from the fungus. Pleurotus sp. produced maximal laccase activity when beef infusion was exclusive N beginning ( 114 U/ml ) as shown in fig.4. However, most of the fungous laccases were stimulated by organic N than inorganic N beginning. Kirk and Farrell 1987, Higuchi 1990, Cullen 1997 ) reported Laccase and other ligninolytic enzyme activity was increased due to carbon and organic N.

Saravanakumar and Kaviyarasan ( 2010 ) reported maximal laccase production ( 84 U/ml ) by utilizing vanillin. Monteiro and De Carvalho ( 1998 ) reported high laccase activity with semi-continuous production in shake-flasks utilizing a low C to nitrogen ratio ( 7: 8 g.g ) . Buswell et al. , ( 1995 ) found that laccases were produced at high N concentrations, although it is by and large accepted that a high C to nitrogen ratio is required for laccase production.

Laccase was besides produced earlier when the fungus was cultivated in a substrate with a high N concentration and these alterations did non reflect differences in biomass. Heinzkill et al. , ( 1998 ) besides reported a higher output of laccase utilizing N rich media instead than N limited media normally employed for production of oxido reductase.Fig.4

Response surface methodological analysis ( RSM )

A response surface methodological analysis allowed computation of maximal production based on a few sets of experiments in which all the factors were varied within chosen scopes. This method has been successfully applied in the optimisation of medium composings ( Lee, S.L.

and W.C. Chen, 1997 ) conditions of enzymatic hydrolysis ( A.Y.M. Ma and B.

Ooraikul, 1986 ) and agitation procedures ( Rosi et al. , ( 1987 ) and Sonia ( 2005 ) . In optimising the consequence of C and N beginnings on laccase production from Pleurotus sp.

, 4 factorial designs were applied utilizing Osmitrol, peptone, pH and metal ion. Box-Behnken design aims to choose most of import variables in the system that influence over all enzyme productiveness. Each variable varies for a coveted response represented at high and low degrees. By and large calculated F values should be several times more than tabulated value, if the theoretical account is a good anticipation of experimental consequences and estimated factors effects are existent. Besides high F value and a really low chance ( P & gt ; F = 0.

0001 ) indicate that present theoretical account is in a good anticipation of experimental consequences. The F value of theoretical account implies that theoretical account is important. ( Akhnazarova and Kafarov, 1982 ; Cornell, 1987 ) . The goodness of tantrum was checked by finding coefficient ( R2 ) , and value of the finding coefficient ( R2 = 89.

5 % ) indicates that merely 10.5 % was non explained by the theoretical account. The adjusted coefficient ( Adj. R2 = 75 % ) was besides really high, which indicates a high significance of the experiment ( Box and Wilson 1951 ; Cochran and Cox 1957 ; Akhnazarova and Kafarov 1982 ; Khuri and Cornell 1987 ; Yee and Blanch 1993 ; Mak et al.

, 1995 ) . Box et al. , 1978 reported a higher value of the correlativity coefficient ( R = 98.1 % ) signifies an first-class correlativity between the independent variables ( Box and Wilson 1951 ; Cochran and Cox 1957 ; khnazarova and Kafarov 1982 ; Yee and Blanch 1993 ; Mak et al. , 1995 ) . The application of Response Surface Methodology ( Box and Wilson 1951 ; Khuri and Cornell 1987 ; Mak et al.

, 1995 ) was yielded higher enzyme production. The logarithmic values of enzyme outputs and trial variables in uncoded unit as follows:Y=50.33+b1 ( -3.44 ) +b2 ( 1.22 ) +b3 ( 16.29 ) +b4 ( -3.44 ) +b11 ( -0.

95 ) +b22 ( -14.23 ) +b33 ( -12.27 ) +b12 ( -3.44 ) ( 1.24 ) +b13 ( -3.44 ) ( 16.29 ) +b23 ( 1.

24 ) ( 16.29 ) +b14 ( -3.44 ) ( -3.44 ) +b24 ( 16.29 ) + ( -3.

44 )The enzyme concentrations expressed in logarithmic values, and X1, X2, X3 and X4 were the coded values of trial variables ( Osmitrol, beef infusion, pH and Cu sulfate severally ) . The significance of each coefficient was determined by t- value and P values ( Table 2 ) . The larger t- value and smaller the p- value indicates the significance of the theoretical account. This implies the quadratic chief effects of vanillin, glycerin and mineral salt solution of the medium are more important ( Akhnazarova and Kafarov 1982 ; Khuri and Cornell 1987 ) . The C and N beginning support the maximal enzyme activity and their several P-values were P X1 2 & gt ; 0.021, P X2 2 & lt ; 0.166 and P X3 2 & lt ; 0.022.

The Osmitrol and beef infusion have a direct relationship on the production of the enzyme. From the theoretical account each observed values ( Y degree Fahrenheit ( 0 ) was compared with the predicted values ( Y degree Fahrenheit ( P ) ( table 2 ) . Disturbance secret plan is an of import diagrammatic representation to compare consequence of all variable at a peculiar point in design infinite ; existent benefits from this secret plan were choosing axes and invariables in wire frame. Response surface secret plans showed two variables at a clip and keeping others were fixed degree, it was more helpful to understanding the interaction effects of these two factors. A circle in the square shows that response is sensitive to that factor ( fig.5,8,9,10 ) . A comparatively other showed insensitiveness to the enzyme outputs ( fig.

6,7 ) . If there are more than two factors,Table.1 Observed responses and predicted valuesS.No


Beef infusion


Ferous sulfate

Peroxidase Yield ( U/ml )

Residual value

Observed Value






























2 Model coefficients estimated by multiples additive arrested development


t- value

p- value

Changeless50.335.0820.000X1 ( C )-3.44-0.6950.500X2 ( N )1.240.

2510.806X3 ( pH )16.293.

2900.006X4 ( M )-3.44-0.6950.500X11 ( C+C )-9.22-1.2420.238X22 ( N+N )-14.

23-1.9150.080X33 ( pH+pH )-17.05-2.2950.041X44 ( M+M )-11.32-1.5250.

153X12 ( C+N-0.95-0.1110.914X13 ( C+pH )4.880.

5680.580X14 ( C+M4.300.5010.625X23 ( B+pH )-12.27-1.4310.178X24 ( B+M )-12.

05-1.4050.185X34 ( pH+M )5.230.

6090.554Table.3 Analysis of discrepancy ( ANOVA ) for the factorial designBeginning of fluctuationSum of squaresDegree of freedomMean squareF- valueProbe ( P )& gt ; FArrested development702314501.71.700.180Residual mistake1465123532Entire1055526R= 89 % ; Adjusted R2= 75 %Fig.


8Fig.9Fig.10disturbance secret plan could be used to happen consequence of variables on enzyme production. These influential factors are good picks for axes on counter secret plans and can be easy obtained by ciphering from theoretical account. The output values of different variables can besides predicated from the several response surface secret plans ( fig. 5 to 10 ) . The laccase production was preponderantly influenced by Osmitrol and peptone. Mannitol and peptone are therefore the key factors which control the biogenesis of laccase enzyme.

At lower concentration of beef infusion every bit good as higher metal ion may non do suppression of enzyme synthesis. ( Ikehata et al. , 2004 ; Sakurai et al. , 2002 ; Ragalski et al.

, 2006 ; Stajic et al. , 2006 ) . The arrested development equation solve the optimum values of trial variables in uncoded units and the values were beef extract 4.8 gm/l, Osmitrol 3gm/l, pH 6.

2 and Cu sulfate 4mg/l. Monteiro and De Carvalho ( 1998 ) reported high laccase activity with semi-continuous production in shake-flasks utilizing a low C to nitrogen ratio ( 7: 8 g.g ) . Buswell et al. , ( 1995 ) found that laccases were produced at high N concentrations, although it is by and large accepted that a high C to nitrogen ratio is required for laccase production. This theoretical account predicts maximal production of enzyme that can be obtained by utilizing the above RSM and the production was increased up to 2-3 creases.


Box- Behnken experimental design for procedure optimisation involves a survey of specific part of single factors. Output of laccase increased by supplementation of medium.

In this survey, response surface analysis and cardinal composite design were used for optimising civilization media for maximising laccase production by utilizing Pleurotus sp. in submersed agitation. The optimized agitation medium composing increased 2 to 3 crease of enzyme production.


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