Using Statisticss Based Experimental Design


Production of amylase under submerged agitation Bacillus sp. was investigated utilizing wheat bran, soybean repast and CaCO3 ( WSC ) medium. Response surface methodological analysis ( RSM ) was used to measure the consequence of the chief variables, i.e. , pH ( 11.35 ) , temperature ( 35.16oC ) and inoculum size ( 2.95 % ) on amylase production by using a full factorial cardinal composite design ( CCD ) . The common interaction between these variables resulted into 4.64 fold addition in amylase activity as compared to the non-optimized environmental factors in the basal medium.

Key words

Amylase, Bacillus sp. , cardinal composite design, response surface methodological analysis

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Amylases are the hydrolytic enzymes that cleave the ?- 1-4 glucosidal linkage of complex polyoses. Amylases are obtained from assorted beginnings like works, animate being, bacterial and fungous. Amylase production was achieved by many research workers utilizing Bacillus sp. ( Yuguo et al. , 2000 ; Young et al. , 2001 ; Dharani, 2004 ; Zambare, 2010a ) Amylase has many applications in nutrient, fabric, paper and mush, pharmaceuticals, baking and drinks, detergent and leather industries ( Pandey et al. , 2000 ; Reddy et al. , 2003 ; Kar et al. , 2010 ) . Industrially of import enzymes including amylases have traditionally been obtained from submerged civilizations because of easy handling, greater control of environmental and nutritionary factors. The most often used operation in biotechnology is to better the agitation conditions for maximising cell denseness, high degree of coveted metabolic merchandise or enzyme degrees in microbic system. This attack is clip devouring and besides ignores the combined interactions between physical every bit good as nutritionary factors.

In contrast, RSM includes factorial design and arrested development analysis which helps in measuring the effectual factors and their interaction and to happen out the optimal conditions of variables for a desirable response ( Tunga and Banerjee, 1999 ; Coninck et al. , 2000 ; Reddy et al. , 2003 ; Kunamneni et al. , 2005 ; Gangadharan et al. , 2008 ) . Recently, a figure of statistical experimental designs with response surface methodological analysis have been employed for optimising enzyme production from micro-organisms ( Koteswara et al. , et al. , 2006 ; Thys et al. , 2006 ; Zambare, 2010b ; Mohandas et al. , 2010 ) . However, 3D and counter secret plans for response surfaces can supply a good manner for visualising the parametric quantity interaction. Therefore, statistical technique is frequently used for foretelling optimal procedure conditions for microbic enzyme production. It is good known that extracellular enzyme production in micro-organism is greatly influenced by nutritionary factors like C beginnings, N beginnings and mineral salts ( De et al. , 2001 ; Adinarayana and Elliaiah, 2002 ; Chauhan and Gupta, 2004 ) . Enhancement in extracellular amylase production from Bacillus sp. by environmental factor optimisation has non been attempted so far. Therefore, sing the many industrial applications of amylase, we report here the optimisation of extracellular amylase production from Bacillus sp. as a consequence of the synergistic effects of three variables ( i.e. pH, temperature and inoculant size ) utilizing response surface methodological analysis.

Materials and Methods


Bacillus sp. isolated from dirt showed true potency in extracellular amylase secernment. It was maintained on 2 % Nutrient agar angles at 40C and besides as a glycerin stocks at -200C.

Chemicals and media

Chemicals and media were all of analytical class and purchased from Sigma ( St Louis, MO, USA ) .

Production medium

Production medium incorporating wheat bran ( 1 % ) , soybean repast ( 1 % ) and CaCO3 ( 0.3 % ) , was used for the growing and amylase production by Bacillus sp.

Inoculum readying

Seed inoculant was prepared by turning the isolate on Nutrient agar in Roux bottle at 30 & A ; deg ; C for 24h. The cells were suspended in saline and cell denseness was measured spectrophotometrically ( Shimatzu UV-2501 Personal computer, Japan ) at 600 nanometers.

Experimental design and optimisation by RSM

In the RSM the synergistic effects of three variables, i.e. pH, temperature and inoculant size was studied for amylase production. Each factor in the CCD was studied at three different degrees ( -1, 0, +1 ) . The minimal and maximal scopes of variables were investigated with regard to their values in existent and coded signifier ( Table 1 ) . To optimise the conditions for amylase production, Design-Expert 8.0 CCD-RSM package ( State-Ease, Minneapolis, MN, U.S.A. ) was used. A 23 factorial CCD proposed by Box et al. , ( 1978 ) with three factors taking to a sum of 20 sets per experiment was formulated to optimise the procedure parametric quantities. This experiment included 8 factorial design, 6 star and 6 cardinal points. All the variables i.e. pH, temperature and inoculant size were taken at a cardinal coded value and considered as nothing. The conditions of these environmental factors for the production medium were varied harmonizing to the experimental design ( Table 2 ) . All the experiments were carried out in extras. The experiments were conducted in 250 milliliters Erlenmeyer flasks incorporating 100 milliliter of sterilized WSC medium of different pH 10-12, inoculated with the newly prepared 1-5 % ( 2 x 108 cells/ml ) inoculant ( as discussed earlier ) and incubated for 12 hour at 30-400C under agitating civilization status ( 150 revolutions per minute ) . After agitation, the cell-free supernatant was obtained by centrifugation at 10,000 revolutions per minute and used for amylase activity.

Using RSM, the relationship among the variables, i.e. pH, temperature and inoculant size were expressed mathematically in the signifier of a multinomial theoretical account, which gave the response as a map of relevant variables. The present work was based on the CCD to obtain the experimental information, which would suit in an empirical, full second-order multinomial theoretical account stand foring the response surfaces over a comparatively wide scope of parametric quantities as show in Eq. ( 1 ) .

… … … … … … … … … ( 1 )

where, Y was the predicted response ( amylase production ) used as a dependent variable ; xi ( one = 1, 2 and 3 ) were the input forecasters or commanding variables ; and a0, ai ( one = 1, 2, 3 ) and aij ( one = 1, 2, 3 ; J = I, . . . , 3 ) were the theoretical account coefficient parametric quantities. The coefficient parametric quantities were estimated by multiple additive arrested development analysis utilizing the least-squares method. A second-order multinomial equation was so fitted to the informations by least-squares optimisation technique. This resulted in an empirical theoretical account that related the response measured to the independent variables of the experiment.

Assay of amylase

The amylase activity in the cell free supernatant ( CFS ) was measured by incubating 0.5 milliliter of CFS with 0.5 milliliters of 2 % ( w/v ) amylum at 370C in 2 milliliter phosphate buffer ( 0.1 M, pH 6.0 ) . The cut downing sugars released were measured by 3,5-dinitrosalicylic acid method ( Miller, 1959 ) . A separate space was set for each sample to rectify the non-enzymatic release of sugars. One unit of amylase was defined as the sum of enzyme that released 1 µg of cut downing sugar as maltose per minute under the standard check conditions.

Consequences and Discussion

RSM had non merely been used for optimisation of medium constituents in the agitation procedure ( Puri et al. , 2002 ) but besides for analyzing the combined effects of civilization parametric quantities ( Dutta et al. , 2004 ; Nawani and Kapadnis, 2005 ) . A submersed civilization was used for the production of extracellular amylase from Bacillus sp. Preliminary experiments on amylase production from the above strain indicated that the most of import environmental factors were pH, temperature and inoculant size. Hence these three factors were considered as the independent variables and their consequence on amylase production was studied utilizing a CCD of RSM. The consequences of CCD experiments for analyzing the effects of three independent variables, viz. , pH, temperature and inoculant size, on amylase production are presented in Table 3 along with the predicted and ascertained responses. The standard divergences on the ascertained responses are besides presented in Table 3. The coefficients of the theoretical account were determined of least-squares optimisation by the Gauss-Newton technique ( Table 4 ) . The overall 2nd order multinomial equation for amylase production is given in Eq. ( 2 ) .

Amylase activity ( Y ) = 511.77 + 35.77X1 – 7.75X2 – 42.17X3 + 21.25X12 – 12.19X23 + 0.31X13 – 21.83X11 -184.33X22 – 219.75X33… … … … … … … … … ( 2 )

where, X1-pH, X2-temperature in degree Celsius and X3-inoculum size in % . The larger the magnitudes of F- value, smaller is the p- value, the more important value is the corresponding coefficient ( Akhnazarova and Kafarov, 1982 ; Rubinder et al. , 2002 ) . The consequences of the 2nd order response surface theoretical account adjustment in the signifier of analysis of discrepancy ( ANOVA ) are in Table. 5. The fisher F-test with a really low chance value demonstrated a really high significance for the arrested development theoretical account ( Olivera et al. , 2004 ; Zambare, 2010b ) . The adjustment of the theoretical account was checked by the finding coefficient ( R2 ) . In this instance, the value of the finding coefficient ( R2= 0.818 ) indicates that merely 18.2 % of the entire fluctuations are non explained by the theoretical account. The value of the adjusted finding coefficient ( Adj. R2= 0.655 ) is besides high, which indicates a higher significance ( p value & A ; lt ; 0.01 ) of the theoretical account ( Adinarayana and Elliaiah, 2002 ; Olivera et al. , 2004 ) . Adequate preciseness measures the signal to resound ratio. An equal preciseness value ( 5.74 ) was greater than 4 which indicates equal signal. At the same clip a comparatively lower value of the coefficient of fluctuation ( CV=42.70 ) indicates improved preciseness and dependability of the conducted experiments ( Adinarayana and Elliaiah, 2002 ) .

The 3-D counter secret plans for response surfaces matching to the combined effects of pH-temperature ( Fig. 1 ) , pH-inoculum ( Fig. 2 ) and temperature-inoculum ( Fig. 3 ) were plotted. The response surfaces obtained were proposing that Bacillus sp. secreted amylase in more alkalic status at moderate temperature and inoculant size. Thus the optimal operating conditions obtained from the RSM theoretical account were pH ( 11.35 ) , temperature ( 35.160C ) and inoculant ( 2.95 % ) with predicted amylase activity of 515.30 U/ml. After optimisation, 4.64 fold amylase activity ( 515.30 U/ml ) was enhanced when compared with non-optimized environmental factors ( pH 10, temperature 30oC, inoculant size 1 % ) in basal medium ( 110.83 U/ml ) . Therefore, RSM could be a really powerful and flexible tool for patterning the agitation procedure due to disciplinary action originating from methodological analysis and the associated appraisal process. The application of decently designed theoretical accounts with multi-factor analysis allows procedure and biochemical applied scientists to plan scale up schemes for increasing enzyme production.


The consequence obtained in the present survey indicated that Bacillus sp. could be a possible strain for amylase production in submersed agitation utilizing wheat bran like easy available C substrates. The RSM allowed the optimisation of procedure parametric quantities such as pH ( 11.35 ) , temperature ( 35.160C ) and inoculum size ( 2.95 % ) for achieving higher output of amylase.


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