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Optimization of extracellular keratinase production by poultry farm isolate Scopulariopsis brevicaulis
Bioresource Technology 98 (2007) 1298–1303
Optimization of extracellular keratinase production by poultry farm isolate Scopulariopsis brevicaulis P. Anbu a
a,1,2
, S.C.B. Gopinath
a,*,1,3
, A. Hilda a, T. Lakshmipriya b, G. Annadurai
c,4
Centre for Advanced studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India b Department of Mathematics, SBK College, Aruppukottai, Tamil Nadu, India c Department of Chemical Engineering, Anna University, Chennai 600 025, Tamil Nadu, India Received 5 March 2004; received in revised form 1 May 2006; accepted 1 May 2006 Available online 1 August 2006
Abstract A Scopulariopsis brevicaulis poultry farm isolate was chosen to study factors influencing keratinase production. The parameters were optimized by factorial design. The highest enzyme production by this fungus was obtained at pH 7.5, a temperature of 30 C and a growth period of 5 weeks. The production of the enzyme was enhanced when the culture medium was supplemented with glucose (1%), sodium nitrate (2%), feather (1.5%) and CaCl2 (1 mM). According to the responses from the experimental design, the effects of each variable were calculated, and the interactions between them were determined. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9978. 2006 Elsevier Ltd. All rights reserved. Keywords: Box–Behnken; Keratinase; Keratin; Optimization; Scopulariopsis brevicaulis
1. Introduction Keratinases (E.C. 3.4.99.11) belong to the group of serine hydrolases that are capable of degrading keratin, a fibrous and insoluble structural protein extensively crosslinked with disulfide, hydrogen and hydrophobic bonds. The keratin chain of hair (hard keratin) is similar to that of the epidermis (soft keratin) as it is highly packed as an
*
Corresponding author. Tel.: +81 298 61 6085; fax: +81 298 61 6095. E-mail addresses: [email protected], gopis11@hot mail.com (S.C.B. Gopinath). 1 These authors equally contributed to this work. 2 Present address: Division of Biological Sciences, College of Natural Sciences, Chonbuk National University, Jeonju 561-756, South Korea. 3 Present address: Functional Nucleic Acids Group, Institute for Biological Resources and functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1 Higashi, Central 6, Tsukuba, Ibaraki 305-8566, Japan. 4 Present address: Graduate Institute of Environmental Engineering, National Central University, Chung-Li, Taiwan, ROC. 0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.05.047
a-helix but differs from the latter in that it contains several fold higher amounts of cysteine. In feathers, the polypeptide chain assumes a b-confirmation, which is more readily hydrolyzed than a-keratin (Ramnani and Gupta, 2004). Keratinase production by microbes is influenced by number of factors such as temperature, pH and the nature of carbon and nitrogen sources present in the medium. These factors have varied effects in different species (Singh et al., 1975). The synthesis of extracellular protease (keratinase) is governed at least in part by the concentration of individual nutrients (North, 1982). Substrate induction is a major regulatory mechanism, and keratinase biosynthesis was not completely repressed by addition of other carbon and nitrogen sources (Ignatova et al., 1999). Catabolite repression and end product inhibition appear to be the two main regulatory mechanisms governing the synthesis of extracellular keratinase. Scant information is available on the factors which control the synthesis and release of extracellular keratinases. Since the keratinase production varies from organism to organism, the study on the
P. Anbu et al. / Bioresource Technology 98 (2007) 1298–1303
nutritional and environmental factors controlling the keratinase production from this highly potent strain of fungi is warranted. Optimization is one of the most important criteria when it comes to developing any new microbial process. Factorial design and response surface analysis are important tools to determine the optimal process conditions. Factorial design of a limited set of variables is advantageous compared to the conventional method which handles a single parameter per trial, as such an approach frequently fails to locate optimal conditions due to its failure to consider the effect of possible interactions between factors (Kalil et al., 2000). Unlike for production of other biotechnologically important enzymes using microorganisms, only a limited research effort has been made towards improving the yield of keratinases to make industrial application possible. In fact, there has been no systematic statistical study to achieve optimum medium composition and process conditions (Ramnani and Gupta, 2004). The Box–Behnken design experiment was used to determine the maximum keratinase production at the most adequate pH, temperature and catalyst concentration. 2. Methods 2.1. Microorganism The fungus Scopulariopsis brevicaulis was isolated from a poultry farm soil at Namakkal, India. In brief, 20 g of soil sample was placed in 9 cm petri dishes. Autoclaved sterile chicken feather fragments (1 cm length) were scattered on the soil surface. The plates were then moistened with an antibiotic solution containing cycloheximide (0.5 mg/ml), chloramphenicol (0.05 mg/ml) and streptopenicillin (1000 I.U/ml), to prevent other microbial growth. The plates were incubated at room temperature for a period of two months and were remoistened with sterile deionized water whenever necessary. At periodic intervals (once every week) feather fragments were selected at random from each petri plate, transferred onto plates containing Sabouraud dextrose agar medium amended with cycloheximide (0.5 mg/ml), chloramphenicol (0.05 mg/ml) and incubated at room temperature for a period of 2 weeks. Colonies were examined under the microscope, identified, and their keratinase production capability was assessed by the agar plates containing keratin and the method formulated by Yu et al. (1968).
1299
ing (g/l) K2HPO4 – 1.5; MgSO4 Æ 7H2O – 0.05; CaCl2 – 0.025; FeSO4 Æ 7H2O – 0.015; ZnSO4 Æ 7H2O – 0.005; pH 7.5 were poured into 500 ml Erlenmeyer flasks. The keratin powder (1.5%) was weighed and added separately to each flask. The flasks were autoclaved for 15 min at 121 C. All flasks were inoculated with a fungal spore suspension to give a final concentration of 1 · 106 spores/ml of culture medium, and shaken at 100 rpm at 30 C. After 5 weeks of incubation, the culture filtrate was passed through the Whatman no. 42 filter then through a Millipore cellulose filter (0.45 lm) under vacuum. The filtrate was centrifuged at 4000 rpm for 5 min, and the supernatant was used as a crude enzyme solution. 2.3. Assay of keratinase activity Keratinase activity was assayed by the modified method of Yu et al. (1968). In brief, ground chicken feather (20 mg) was suspended in 3.8 ml of 100 mM Tris–HCl buffer (pH 7.8) to which 0.2 ml of the culture filtrate (enzyme source) was added. The reaction mixture was incubated at 37 C for 1 h. After incubation the assay mixture was cooled in ice cold water for 10 min and the remaining feathers were removed by filtration through a Whatman no. 42. The filtrate was subjected to filtration through a Millipore cellulose filter (0.45 lm) under vacuum. The absorbance of the mixture was measured at 280 nm. One unit of the keratinase activity was defined as the amount of enzyme that increased absorbance by 0.1 under the assay conditions used (1 KU = 0.100 corrected absorbance). The data are mean values of three parallel determinations. 2.4. Factorial design and model The production of keratinase was optimized using the Box–Behnken design (Box and Behnken, 1960; Gopinath et al., 2002, 2003a,b; Anbu et al., 2005), where keratinase production (y) is related to experimental variables by a response equation, Y ¼ f ðX 1 ; X 2 ; X 3 ; . . . ; X k Þ
ð1Þ
The true relationship between Y and Xk may be complicated and, in most cases, it is unknown; however, a second-degree quadratic polynomial can be used to represent the function in the range of interest (Annadurai and Sheeja, 1998), k X
k X
Rii X 2i þ
k1 X k X
2.2. Maintenance of culture and enzyme production
Y ¼ R0 þ
The fungus S. brevicaulis was grown on Sabouraud’s dextrose agar slants (Dextrose, 4%; peptone, 1%; agar, 2%; pH 7.6). For keratinase production, chicken feathers were washed with water and commercial sodium-salt detergents followed by defattening with chloroform:methanol (1:1), washed 2 or 3 times using glass distilled water, dried and ground. Fifty millilitres mineral salt solution contain-
where X1, X2, X3, . . ., Xk are the input variables which affect the response Y, R0, Ri, Rii and Rij (i = 1–k, j = 1–k) are the known parameters, e is the random error. A second-order model is designed such that variance of Y is constant for all points equidistant from the center of the design. The parameters and their values (in brackets) were weeks (1, 5 and 9), pH (4, 7.5 and 11), temperature (10, 30 and
i¼1
Ri X i þ
i¼1
Rij X i X j þ e
ð2Þ
i¼1;i
1300
P. Anbu et al. / Bioresource Technology 98 (2007) 1298–1303
50 C), glucose (0.1%, 1% and 1.9%), sodium nitrate (0.2%, 2% and 3.8%), feather (0.2%, 1.5% and 2.8%) and CaCl2 (0.2, 1 and 1.8 mM). These parameters were chosen as the critical variables and designed X1, X2, X3, X4, X5, X6 and X7, respectively. The low, middle and high levels of each variable (equally spaced) were designated 1, 0 and +1, respectively. A total of 62 treatments were necessary to estimate the coefficients of the model using multiple linear regressions. All data were treated with the aid of Design Expert software (Stat Ease, Inc., Statistical made Easy, Minneapolis, MN, 5.0.9.1999). 3. Results and discussion Keratin is an insoluble structural protein difficult to digest by humans and animals, however, some microorganisms are able to degrade it. Certain proteolytic enzymes, particularly keratinase, have attracted attention because they are able to degrade keratin. The mechanical stability of keratin and its resistance are due to the tight packing of the protein chain. The ability to break down keratin varies from organism to organism. Based on a previous study, S. brevicaulis is a prolific keratinase producer (Anbu et al., unpublished data). The extracellular keratinolytic enzyme was obtained from the culture filtrate of S. brevicaulis, and the yield of enzyme depended on various growth conditions. The production of keratinase by S. brevicaulis was optimized by response surface methodology with middle range parameters, as it is a powerful technique for testing multiple process variables. It includes factorial designs and regression analysis, and is suitable for multifactor experiments. The experimental conditions and the results for enzyme production, according to the factorial design, are presented in Table 1. Regression analysis of the experimental data yielded the following quadratic equation for keratinase production: Y ¼ 213:96 þ 0:06X 1 þ 0:038X 2 þ 0:20X 3 þ 1:14X 4 þ 0:26X 5 þ 0:019X 6 þ 0:063X 7 þ 43:91X 21 þ 106:25X 22 þ 96:32X 23 þ 26:74X 24 þ 32:61X 25 þ 5:99X 26 þ 39:26X 27 þ 0:0021X 1 X 2 þ 0:0066X 1 X 3 þ 0:0066X 1 X 4 þ 0:0015X 1 X 5 þ 0:011X 1 X 6 þ 0:061X 1 X 7 þ 0:026X 2 X 3 þ 0:00061X 2 X 4 þ 0:018X 2 X 5 þ 0:0012X 2 X 6 þ 0:031X 2 X 7 þ 0:14X 3 X 4 þ 0:060X 3 X 5 þ 0:0012X 3 X 6 þ 0:028X 3 X 7 þ 0:0098X 4 X 5 þ 0:21X 4 X 6 þ 0:47X 4 X 7 þ 0:19X 5 X 6 þ 0:005X 5 X 7 þ 0:080X 6 X 7
ð3Þ
An estimate of a main effect is obtained by evaluating the difference in process performance caused by a change from the low (1) to the high (+1) level of the corresponding factor (Haaland, 1989). The process performance was mea-
sured by the keratinase activity response. The calculated regression analysis gives an R2 value of 0.9978 and F-value (Fisher’s F-test) of 341.18 (Table 2), indicating that keratinase production by S. brevicaulis has a good model fit, due to the high values of R2 and F. The equation used fits the experimental data with a standard deviation (Root MSE/Dep mean) of 0.13 and 1.32 (Table 2). Values greater than 0.1000 indicate the model terms are not significant. It is evident that all the factors are important for keratinase production under experimental conditions. The maximum keratinase production was 6.2 KU/ml after 5 weeks at pH 7.5, declined thereafter. The decline in keratinase production might be due to nutrient depletion or feed-back inhibition. The statistical analysis was performed with data obtained at 5 weeks, as there was no significant increase in keratinase activity after this time. Similarly, Okafor and Ada (2000) obtained the highest keratinolytic activity after 5 weeks of incubation by Microsporum gypseum in the presence of human hair. In contrast, Ramesh (1996) measured highest enzyme production in the presence of hair by the keratinophilic fungi Microsporum nanum and Chrysosporium kertinophilum at 50 days. At extreme acidic (pH 4) and alkali (pH 11) condition, enzyme production was negligible, suggesting that extreme pH is a detrimental factor for enzyme production. Furthermore, there was no growth and no keratinase production when the incubation temperature was above 50 C. This observation differs from that of Sangali and Brandelli (2000) who obtained maximum enzyme production at pH 8.0 and 55 C. The maximum keratinase production (6.2 KU/ml) at 30 C was observed with 1% glucose as carbon source (Fig. 1a). Studies on the effect of carbon sources on keratinase production in S. brevicaulis showed that a variety of sugars (xylose, lactose, maltose, sucrose and mannitol) suppressed enzyme production when added to the medium. These results are in accordance with the findings of Singh (1998) with Malbranchea gypsea and Ramnani and Gupta (2004) with Bacillus licheniformis. Sugar-related suppression of enzyme activity appears to be common in fungi and other microorganisms. For example, catabolic repression of protease by sucrose has been shown in Neurospora crassa (Drucker, 1975), by fructose in Trichophyton rubrum (Meevootisom and Niederpruem, 1979) and by maltose in B. licheniformis (Sen and Satyanarayana, 1993). A three-dimensional plot representing maximum keratinase production (6.2 KU/ml) with carbon (1%) and nitrogen (2%) is shown in Fig. 1b. Of the several nitrogen sources (sodium nitrate, peptone, potassium nitrate, ammonium nitrate, ammonium sulphate, ammonium chloride and yeast extract) tested, maximum enzyme production was measured in the presence of 1.5–2% sodium nitrate (6.2 KU/ml) followed by peptone (6 KU/ml) and potassium nitrate (5.5 KU/ml). Sodium nitrate in amounts lower than 1–1.5% permitted the enzyme synthesis, but was inhibitory at a concentration of more than 2%. A similar beneficial effect of nitrate has been observed for many microorganisms (Johnson, 1967).
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Table 1 The Box–Behnhen design for the seven independent variables S. no.
Week
pH
Temp (C)
Glucose (%)
Sodium nitrate (%)
Feather (%)
CaCl2 (mM)
Keratinase activity (KU/ml)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57–62
5 5 5 5 5 5 5 5 1 9 1 9 1 9 1 9 5 5 5 5 5 5 5 5 1 9 1 9 1 9 1 9 5 5 5 5 5 5 5 5 1 9 1 9 1 9 1 9 5 5 5 5 5 5 5 5 5
7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 4 11 4 11 4 11 4 11 4 4 11 11 4 4 11 11 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 4 11 4 11 4 11 4 11 7.5
30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 10 50 10 50 10 50 10 50 10 10 50 50 10 10 50 50 10 10 50 50 10 10 50 50 30
0.1 1.9 0.1 1.9 0.1 1.9 0.1 1.9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.1 0.1 0.1 0.1 1.9 1.9 1.9 1.9 0.1 0.1 1.9 1.9 0.1 0.1 1.9 1.9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0.2 0.2 3.8 3.8 0.2 0.2 3.8 3.8 2 2 2 2 2 2 2 2 0.2 0.2 3.8 3.8 0.2 0.2 3.8 3.8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0.2 0.2 0.2 0.2 3.8 3.8 3.8 3.8 2 2 2 2 2 2 2 2 2
0.2 0.2 0.2 0.2 2.8 2.8 2.8 2.8 0.2 0.2 2.8 2.8 0.2 0.2 2.8 2.8 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 0.2 0.2 0.2 0.2 2.8 2.8 2.8 2.8 1.5
1 1 1 1 1 1 1 1 0.2 0.2 0.2 0.2 1.8 1.8 1.8 1.8 0.2 0.2 0.2 0.2 1.8 1.8 1.8 1.8 1 1 1 1 1 1 1 1 0.2 0.2 0.2 0.2 1.8 1.8 1.8 1.8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2.7 2.4 2.7 2.6 3.1 2.5 2.8 1.7 1.7 2.1 1.9 2.3 2.1 2.3 2.0 1.9 0.3 0.3 0.0 0.0 0.4 0.0 0.1 0.0 0.4 0.4 0.3 0.3 0.1 0.0 0.0 0.0 0.5 0.1 0.4 0.2 1.5 0.5 0.1 0.0 0.3 0.5 0.1 0.1 0.0 0.1 0.1 0.1 0.2 0.0 0.0 0.2 0.1 0.0 0.0 0.0 6.2
Keratinase production at a level of 6.2 KU/ml in the presence of 2% nitrogen and 1.5% feather is presented in Fig. 1c. Among various keratinous substrates (feather,
hair, nail, horn and hoof) tested, chicken feather (1.5%) induced maximum production of keratinase followed by hoof (6.2 and 5.1 KU/ml, respectively). Siesenop and
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Table 2 ANOVA of variable effects for optimization of keratinase production Source
Sum of squares
Degree of freedom
Mean square
F-value
Prob > F
Model Residual Lack of fit Pure error Cor total
213.96 0.47 0.47 0.000 214.43
35 26 21 0
6.11 0.018 0.022 5.0 61
341.18
<0.0001*
R2 0.9978; adj R-squared 0.9949; root MSE 0.13; dep mean 1.32 * Values of ‘‘Prob > F’’ less than 0.0500 indicate model terms are significant.
Bohm (1995) also showed that the duration and intensity of keratinase secretion were strongly influenced by various keratinous substrates. Keratinase activity is inducible with guinea pig hair in keratin salt broth by M. gypseum and with human hair by Microsporum canis was reported by Meevootisom and Niederpruem (1979) and Takiuchi et al. (1982), respectively. These results are further substantiated by Malviya et al. (1992) who showed in three fungi isolated from the grounds of a gelatin factory that keratinase production was inducible. Similarly, Trichophyton mentagrophytes var. erinacei showed highest keratinase production with wool and Aspergillus flavus with chicken feather (Muhsin and Hadi, 2002). Induction of keratinase production by the keratin substrates was also observed by Cheng et al. (1995) with B. licheniformis PWD-1 where keratinase production was induced by 1% hammer milled feather powder and sheep wool. Fig. 1d shows maximum keratinase production (6.2 KU/ml) in the presence of
1.5% feather and 1 mM CaCl2. The CaCl2 effect differs from that observed with B. licheniformis since CaCl2 showed a negative effect in this organism (Ramnani and Gupta, 2004). An empirical model was designed based on experimental data, in which the independent and dependent variables were fitted to the second-order model equation and examined in terms of the goodness of fit. ANOVA was used to evaluate the adequacy of the fitted model. The R-squared value provided a measure of how much of the variability in the observed response values could be explained by the experimental factors and their interactions. The closer the R-squared value is to 1.00, the stronger the model and the better the response predictions (Haaland, 1989). Based on the F-test, the model is predictive, since its calculated Fvalue is higher that the critical F-value and the regression coefficient (341.18) is close to unity. Thus statistical studies could prove useful for optimization of keratinases produced by other microorganisms. The condition optimized in the present study will also be useful for keratinases from other Scopulariopsis strains as they are common saprophytic fungi, with a view to industrial applications such as dehairing as proved previously for S. brevicaulis (Anbu et al., 2005). Acknowledgements The author (P.A.) thanks the Council of Scientific and Industrial Science (CSIR) for financial assistance. Part of the work was done in fulfillment of the requirements for the Ph.D. degree.
Fig. 1. (a) Response surface optimization of keratinase production with respect to temperature and glucose. All data were treated with the aid of Design Expert Software. Values were not using the whole data set, but only from treatments where the pH was 7.5 and the temperature was at 30 C. (b) Response surface optimization of keratinase production with respect to glucose and sodium nitrate. (c) Response surface optimization of keratinase production with respect to sodium nitrate and feather. (d) Response surface optimization of keratinase production with respect to feather and calcium chloride.
P. Anbu et al. / Bioresource Technology 98 (2007) 1298–1303
References Anbu, P., Gopinath, S.C.B., Hilda, A., Lakshmipriya, T., Annadurai, G., 2005. Purification and characterization of keratinase from poultry farm isolate-Scopulariopsis brevicaulis and statistical optimization of enzyme activity. Enzyme Microb. Technol. 36, 639–647. Annadurai, G., Sheeja, R.Y., 1998. Use of Box–Behnken design experiments for the adsorption of verofix red using biopolymer. Bioprocess Eng. 18, 463–466. Box, G.E.P., Behnken, D.W., 1960. Three level design for the study of quantitative variables. Technometrics 2, 455–475. Cheng, S.W., Ha, H.M., Shen, S.W., Takagi, H., Asano, M., Tsai, Y.C., 1995. Production and characterization of keratinase of a feather degrading Bacillus licheniformis PWD-1. Biosci. Biotechnol. Biochem. 59, 2239–2243. Drucker, H., 1975. Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process. J. Bacteriol. 122, 1117– 1125. Gopinath, S.C.B., Hilda, A., Laksmipriya, T., Annadurai, G., 2002. Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology. World J. Microbiol. Biotechnol. 18, 449–458. Gopinath, S.C.B., Hilda, A., Lakshmipriya, T., Annadurai, G., Anbu, P., 2003a. Statistical optimization of amylase production by Aspergillus versicolor. Asian J. Microbiol. Biotechnol. Environ. Sci. 5, 327–330. Gopinath, S.C.B., Hilda, A., Laksmipriya, T., Annadurai, G., Anbu, P., 2003b. Purification of lipase from Geotrichum candidum: conditions optimized for enzyme production using Box–Behnken design. World J. Microbiol. Biotechnol. 19, 681–689. Haaland, P.D., 1989. Experimental design in biotechnology. Marcel Dekker Inc., New York. Ignatova, Z., Gousterova, A., Spassov, G., Nedkov, P., 1999. Isolation and partial characterisation of extracellular keratinase from a wool degrading thermophilic actinomycete strain Thermoactinomyces candidus. Can. J. Microbiol. 45, 217–222. Johnson, A.G., 1967. Pilot-plant production of protease by Alternaria tenuissima. Appl. Microbiol. 15, 319–324. Kalil, S.J., Maugeri, F., Rodrigues, M.I., 2000. Response surface analysis and simulation as a tool for bioprocess design and optimization. Proc. Biochem. 35, 539–550.
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Malviya, H.K., Rajak, R.C., Hasija, S.K., 1992. Synthesis and regulation of extracellular keratinase in three fungi isolated from the grounds of a gelatin factory, Jabalpur, India. Mycopathologia 120, 1–4. Meevootisom, V., Niederpruem, D.J., 1979. Control of extracellular proteases in dermatophytes and especially Trichophyton rubrum. Sabouraudia 17, 91–106. Muhsin, T.M., Hadi, R.B., 2002. Degradation of keratin substrates by fungi isolated from sewage sludge. Mycopathologia 154, 185–189. North, M.J., 1982. Comparative biochemistry of the proteinases of eukaryotic microorganisms. Microbiol. Rev. 46, 308–340. Okafor, J.I., Ada, N., 2000. Keratinolytic activity of five human isolates of the dermatophytes. J. Common. Dis. 32, 300–305. Ramesh, V.M., 1996. Studies on keratinophilic fungi – characterization of keratinolytic potential and fungitoxic evaluation of some plant extracts. Ph.D. Thesis, University of Madras, Chennai, Tamil Nadu, India. Ramnani, P., Gupta, R., 2004. Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology. Biotechnol. Appl. Biochem. 40, 191–196. Sangali, S., Brandelli, A., 2000. Isolation and characterization of a novel feather degrading bacterial strain. Appl. Biochem. Biotechnol. 87, 17– 24. Sen, S., Satyanarayana, T., 1993. Optimization of alkaline protease production by thermophilic Bacillus licheniformis S40. Indian J. Microbiol. 33, 43–47. Siesenop, U., Bohm, K.H., 1995. Comparative studies on keratinase production in Trichophyton mentrgrophytes strains of animal origin. Mycoses 38, 205–209. Singh, C.J., 1998. Exocellular proteases of Malbranchea gypsea and their role in keratin deterioration. Mycopathologia 143, 147–150. Singh, D.P., Sing, R., Vyas, S.R., 1975. Effect of pH, temperature, nitrogen sources, glucose concentrations on acid protease production by Aspergillus niger mutant. Indian J. Microbiol. 15, 109–113. Takiuchi, I., Higuchi, D., Sei, Y., Koga, M., 1982. Isolation of an extracellular proteinase (keratinase) from Microsporum canis. Sabouraudia 20, 281–288. Yu, R.J., Harmon, S.R., Blank, F., 1968. Isolation and purification of an extracellular keratinase of Trichophyton mentagrophytes. J. Bacteriol. 96, 1435–1436.
Optimization of extracellular keratinase production by poultry farm isolate Scopulariopsis brevicaulis P. Anbu a
a,1,2
, S.C.B. Gopinath
a,*,1,3
, A. Hilda a, T. Lakshmipriya b, G. Annadurai
c,4
Centre for Advanced studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India b Department of Mathematics, SBK College, Aruppukottai, Tamil Nadu, India c Department of Chemical Engineering, Anna University, Chennai 600 025, Tamil Nadu, India Received 5 March 2004; received in revised form 1 May 2006; accepted 1 May 2006 Available online 1 August 2006
Abstract A Scopulariopsis brevicaulis poultry farm isolate was chosen to study factors influencing keratinase production. The parameters were optimized by factorial design. The highest enzyme production by this fungus was obtained at pH 7.5, a temperature of 30 C and a growth period of 5 weeks. The production of the enzyme was enhanced when the culture medium was supplemented with glucose (1%), sodium nitrate (2%), feather (1.5%) and CaCl2 (1 mM). According to the responses from the experimental design, the effects of each variable were calculated, and the interactions between them were determined. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9978. 2006 Elsevier Ltd. All rights reserved. Keywords: Box–Behnken; Keratinase; Keratin; Optimization; Scopulariopsis brevicaulis
1. Introduction Keratinases (E.C. 3.4.99.11) belong to the group of serine hydrolases that are capable of degrading keratin, a fibrous and insoluble structural protein extensively crosslinked with disulfide, hydrogen and hydrophobic bonds. The keratin chain of hair (hard keratin) is similar to that of the epidermis (soft keratin) as it is highly packed as an
*
Corresponding author. Tel.: +81 298 61 6085; fax: +81 298 61 6095. E-mail addresses: [email protected], gopis11@hot mail.com (S.C.B. Gopinath). 1 These authors equally contributed to this work. 2 Present address: Division of Biological Sciences, College of Natural Sciences, Chonbuk National University, Jeonju 561-756, South Korea. 3 Present address: Functional Nucleic Acids Group, Institute for Biological Resources and functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1 Higashi, Central 6, Tsukuba, Ibaraki 305-8566, Japan. 4 Present address: Graduate Institute of Environmental Engineering, National Central University, Chung-Li, Taiwan, ROC. 0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.05.047
a-helix but differs from the latter in that it contains several fold higher amounts of cysteine. In feathers, the polypeptide chain assumes a b-confirmation, which is more readily hydrolyzed than a-keratin (Ramnani and Gupta, 2004). Keratinase production by microbes is influenced by number of factors such as temperature, pH and the nature of carbon and nitrogen sources present in the medium. These factors have varied effects in different species (Singh et al., 1975). The synthesis of extracellular protease (keratinase) is governed at least in part by the concentration of individual nutrients (North, 1982). Substrate induction is a major regulatory mechanism, and keratinase biosynthesis was not completely repressed by addition of other carbon and nitrogen sources (Ignatova et al., 1999). Catabolite repression and end product inhibition appear to be the two main regulatory mechanisms governing the synthesis of extracellular keratinase. Scant information is available on the factors which control the synthesis and release of extracellular keratinases. Since the keratinase production varies from organism to organism, the study on the
P. Anbu et al. / Bioresource Technology 98 (2007) 1298–1303
nutritional and environmental factors controlling the keratinase production from this highly potent strain of fungi is warranted. Optimization is one of the most important criteria when it comes to developing any new microbial process. Factorial design and response surface analysis are important tools to determine the optimal process conditions. Factorial design of a limited set of variables is advantageous compared to the conventional method which handles a single parameter per trial, as such an approach frequently fails to locate optimal conditions due to its failure to consider the effect of possible interactions between factors (Kalil et al., 2000). Unlike for production of other biotechnologically important enzymes using microorganisms, only a limited research effort has been made towards improving the yield of keratinases to make industrial application possible. In fact, there has been no systematic statistical study to achieve optimum medium composition and process conditions (Ramnani and Gupta, 2004). The Box–Behnken design experiment was used to determine the maximum keratinase production at the most adequate pH, temperature and catalyst concentration. 2. Methods 2.1. Microorganism The fungus Scopulariopsis brevicaulis was isolated from a poultry farm soil at Namakkal, India. In brief, 20 g of soil sample was placed in 9 cm petri dishes. Autoclaved sterile chicken feather fragments (1 cm length) were scattered on the soil surface. The plates were then moistened with an antibiotic solution containing cycloheximide (0.5 mg/ml), chloramphenicol (0.05 mg/ml) and streptopenicillin (1000 I.U/ml), to prevent other microbial growth. The plates were incubated at room temperature for a period of two months and were remoistened with sterile deionized water whenever necessary. At periodic intervals (once every week) feather fragments were selected at random from each petri plate, transferred onto plates containing Sabouraud dextrose agar medium amended with cycloheximide (0.5 mg/ml), chloramphenicol (0.05 mg/ml) and incubated at room temperature for a period of 2 weeks. Colonies were examined under the microscope, identified, and their keratinase production capability was assessed by the agar plates containing keratin and the method formulated by Yu et al. (1968).
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ing (g/l) K2HPO4 – 1.5; MgSO4 Æ 7H2O – 0.05; CaCl2 – 0.025; FeSO4 Æ 7H2O – 0.015; ZnSO4 Æ 7H2O – 0.005; pH 7.5 were poured into 500 ml Erlenmeyer flasks. The keratin powder (1.5%) was weighed and added separately to each flask. The flasks were autoclaved for 15 min at 121 C. All flasks were inoculated with a fungal spore suspension to give a final concentration of 1 · 106 spores/ml of culture medium, and shaken at 100 rpm at 30 C. After 5 weeks of incubation, the culture filtrate was passed through the Whatman no. 42 filter then through a Millipore cellulose filter (0.45 lm) under vacuum. The filtrate was centrifuged at 4000 rpm for 5 min, and the supernatant was used as a crude enzyme solution. 2.3. Assay of keratinase activity Keratinase activity was assayed by the modified method of Yu et al. (1968). In brief, ground chicken feather (20 mg) was suspended in 3.8 ml of 100 mM Tris–HCl buffer (pH 7.8) to which 0.2 ml of the culture filtrate (enzyme source) was added. The reaction mixture was incubated at 37 C for 1 h. After incubation the assay mixture was cooled in ice cold water for 10 min and the remaining feathers were removed by filtration through a Whatman no. 42. The filtrate was subjected to filtration through a Millipore cellulose filter (0.45 lm) under vacuum. The absorbance of the mixture was measured at 280 nm. One unit of the keratinase activity was defined as the amount of enzyme that increased absorbance by 0.1 under the assay conditions used (1 KU = 0.100 corrected absorbance). The data are mean values of three parallel determinations. 2.4. Factorial design and model The production of keratinase was optimized using the Box–Behnken design (Box and Behnken, 1960; Gopinath et al., 2002, 2003a,b; Anbu et al., 2005), where keratinase production (y) is related to experimental variables by a response equation, Y ¼ f ðX 1 ; X 2 ; X 3 ; . . . ; X k Þ
ð1Þ
The true relationship between Y and Xk may be complicated and, in most cases, it is unknown; however, a second-degree quadratic polynomial can be used to represent the function in the range of interest (Annadurai and Sheeja, 1998), k X
k X
Rii X 2i þ
k1 X k X
2.2. Maintenance of culture and enzyme production
Y ¼ R0 þ
The fungus S. brevicaulis was grown on Sabouraud’s dextrose agar slants (Dextrose, 4%; peptone, 1%; agar, 2%; pH 7.6). For keratinase production, chicken feathers were washed with water and commercial sodium-salt detergents followed by defattening with chloroform:methanol (1:1), washed 2 or 3 times using glass distilled water, dried and ground. Fifty millilitres mineral salt solution contain-
where X1, X2, X3, . . ., Xk are the input variables which affect the response Y, R0, Ri, Rii and Rij (i = 1–k, j = 1–k) are the known parameters, e is the random error. A second-order model is designed such that variance of Y is constant for all points equidistant from the center of the design. The parameters and their values (in brackets) were weeks (1, 5 and 9), pH (4, 7.5 and 11), temperature (10, 30 and
i¼1
Ri X i þ
i¼1
Rij X i X j þ e
ð2Þ
i¼1;i
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50 C), glucose (0.1%, 1% and 1.9%), sodium nitrate (0.2%, 2% and 3.8%), feather (0.2%, 1.5% and 2.8%) and CaCl2 (0.2, 1 and 1.8 mM). These parameters were chosen as the critical variables and designed X1, X2, X3, X4, X5, X6 and X7, respectively. The low, middle and high levels of each variable (equally spaced) were designated 1, 0 and +1, respectively. A total of 62 treatments were necessary to estimate the coefficients of the model using multiple linear regressions. All data were treated with the aid of Design Expert software (Stat Ease, Inc., Statistical made Easy, Minneapolis, MN, 5.0.9.1999). 3. Results and discussion Keratin is an insoluble structural protein difficult to digest by humans and animals, however, some microorganisms are able to degrade it. Certain proteolytic enzymes, particularly keratinase, have attracted attention because they are able to degrade keratin. The mechanical stability of keratin and its resistance are due to the tight packing of the protein chain. The ability to break down keratin varies from organism to organism. Based on a previous study, S. brevicaulis is a prolific keratinase producer (Anbu et al., unpublished data). The extracellular keratinolytic enzyme was obtained from the culture filtrate of S. brevicaulis, and the yield of enzyme depended on various growth conditions. The production of keratinase by S. brevicaulis was optimized by response surface methodology with middle range parameters, as it is a powerful technique for testing multiple process variables. It includes factorial designs and regression analysis, and is suitable for multifactor experiments. The experimental conditions and the results for enzyme production, according to the factorial design, are presented in Table 1. Regression analysis of the experimental data yielded the following quadratic equation for keratinase production: Y ¼ 213:96 þ 0:06X 1 þ 0:038X 2 þ 0:20X 3 þ 1:14X 4 þ 0:26X 5 þ 0:019X 6 þ 0:063X 7 þ 43:91X 21 þ 106:25X 22 þ 96:32X 23 þ 26:74X 24 þ 32:61X 25 þ 5:99X 26 þ 39:26X 27 þ 0:0021X 1 X 2 þ 0:0066X 1 X 3 þ 0:0066X 1 X 4 þ 0:0015X 1 X 5 þ 0:011X 1 X 6 þ 0:061X 1 X 7 þ 0:026X 2 X 3 þ 0:00061X 2 X 4 þ 0:018X 2 X 5 þ 0:0012X 2 X 6 þ 0:031X 2 X 7 þ 0:14X 3 X 4 þ 0:060X 3 X 5 þ 0:0012X 3 X 6 þ 0:028X 3 X 7 þ 0:0098X 4 X 5 þ 0:21X 4 X 6 þ 0:47X 4 X 7 þ 0:19X 5 X 6 þ 0:005X 5 X 7 þ 0:080X 6 X 7
ð3Þ
An estimate of a main effect is obtained by evaluating the difference in process performance caused by a change from the low (1) to the high (+1) level of the corresponding factor (Haaland, 1989). The process performance was mea-
sured by the keratinase activity response. The calculated regression analysis gives an R2 value of 0.9978 and F-value (Fisher’s F-test) of 341.18 (Table 2), indicating that keratinase production by S. brevicaulis has a good model fit, due to the high values of R2 and F. The equation used fits the experimental data with a standard deviation (Root MSE/Dep mean) of 0.13 and 1.32 (Table 2). Values greater than 0.1000 indicate the model terms are not significant. It is evident that all the factors are important for keratinase production under experimental conditions. The maximum keratinase production was 6.2 KU/ml after 5 weeks at pH 7.5, declined thereafter. The decline in keratinase production might be due to nutrient depletion or feed-back inhibition. The statistical analysis was performed with data obtained at 5 weeks, as there was no significant increase in keratinase activity after this time. Similarly, Okafor and Ada (2000) obtained the highest keratinolytic activity after 5 weeks of incubation by Microsporum gypseum in the presence of human hair. In contrast, Ramesh (1996) measured highest enzyme production in the presence of hair by the keratinophilic fungi Microsporum nanum and Chrysosporium kertinophilum at 50 days. At extreme acidic (pH 4) and alkali (pH 11) condition, enzyme production was negligible, suggesting that extreme pH is a detrimental factor for enzyme production. Furthermore, there was no growth and no keratinase production when the incubation temperature was above 50 C. This observation differs from that of Sangali and Brandelli (2000) who obtained maximum enzyme production at pH 8.0 and 55 C. The maximum keratinase production (6.2 KU/ml) at 30 C was observed with 1% glucose as carbon source (Fig. 1a). Studies on the effect of carbon sources on keratinase production in S. brevicaulis showed that a variety of sugars (xylose, lactose, maltose, sucrose and mannitol) suppressed enzyme production when added to the medium. These results are in accordance with the findings of Singh (1998) with Malbranchea gypsea and Ramnani and Gupta (2004) with Bacillus licheniformis. Sugar-related suppression of enzyme activity appears to be common in fungi and other microorganisms. For example, catabolic repression of protease by sucrose has been shown in Neurospora crassa (Drucker, 1975), by fructose in Trichophyton rubrum (Meevootisom and Niederpruem, 1979) and by maltose in B. licheniformis (Sen and Satyanarayana, 1993). A three-dimensional plot representing maximum keratinase production (6.2 KU/ml) with carbon (1%) and nitrogen (2%) is shown in Fig. 1b. Of the several nitrogen sources (sodium nitrate, peptone, potassium nitrate, ammonium nitrate, ammonium sulphate, ammonium chloride and yeast extract) tested, maximum enzyme production was measured in the presence of 1.5–2% sodium nitrate (6.2 KU/ml) followed by peptone (6 KU/ml) and potassium nitrate (5.5 KU/ml). Sodium nitrate in amounts lower than 1–1.5% permitted the enzyme synthesis, but was inhibitory at a concentration of more than 2%. A similar beneficial effect of nitrate has been observed for many microorganisms (Johnson, 1967).
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Table 1 The Box–Behnhen design for the seven independent variables S. no.
Week
pH
Temp (C)
Glucose (%)
Sodium nitrate (%)
Feather (%)
CaCl2 (mM)
Keratinase activity (KU/ml)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57–62
5 5 5 5 5 5 5 5 1 9 1 9 1 9 1 9 5 5 5 5 5 5 5 5 1 9 1 9 1 9 1 9 5 5 5 5 5 5 5 5 1 9 1 9 1 9 1 9 5 5 5 5 5 5 5 5 5
7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 4 11 4 11 4 11 4 11 4 4 11 11 4 4 11 11 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 4 11 4 11 4 11 4 11 7.5
30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 10 50 10 50 10 50 10 50 10 10 50 50 10 10 50 50 10 10 50 50 10 10 50 50 30
0.1 1.9 0.1 1.9 0.1 1.9 0.1 1.9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.1 0.1 0.1 0.1 1.9 1.9 1.9 1.9 0.1 0.1 1.9 1.9 0.1 0.1 1.9 1.9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0.2 0.2 3.8 3.8 0.2 0.2 3.8 3.8 2 2 2 2 2 2 2 2 0.2 0.2 3.8 3.8 0.2 0.2 3.8 3.8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0.2 0.2 0.2 0.2 3.8 3.8 3.8 3.8 2 2 2 2 2 2 2 2 2
0.2 0.2 0.2 0.2 2.8 2.8 2.8 2.8 0.2 0.2 2.8 2.8 0.2 0.2 2.8 2.8 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 0.2 0.2 0.2 0.2 2.8 2.8 2.8 2.8 1.5
1 1 1 1 1 1 1 1 0.2 0.2 0.2 0.2 1.8 1.8 1.8 1.8 0.2 0.2 0.2 0.2 1.8 1.8 1.8 1.8 1 1 1 1 1 1 1 1 0.2 0.2 0.2 0.2 1.8 1.8 1.8 1.8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2.7 2.4 2.7 2.6 3.1 2.5 2.8 1.7 1.7 2.1 1.9 2.3 2.1 2.3 2.0 1.9 0.3 0.3 0.0 0.0 0.4 0.0 0.1 0.0 0.4 0.4 0.3 0.3 0.1 0.0 0.0 0.0 0.5 0.1 0.4 0.2 1.5 0.5 0.1 0.0 0.3 0.5 0.1 0.1 0.0 0.1 0.1 0.1 0.2 0.0 0.0 0.2 0.1 0.0 0.0 0.0 6.2
Keratinase production at a level of 6.2 KU/ml in the presence of 2% nitrogen and 1.5% feather is presented in Fig. 1c. Among various keratinous substrates (feather,
hair, nail, horn and hoof) tested, chicken feather (1.5%) induced maximum production of keratinase followed by hoof (6.2 and 5.1 KU/ml, respectively). Siesenop and
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Table 2 ANOVA of variable effects for optimization of keratinase production Source
Sum of squares
Degree of freedom
Mean square
F-value
Prob > F
Model Residual Lack of fit Pure error Cor total
213.96 0.47 0.47 0.000 214.43
35 26 21 0
6.11 0.018 0.022 5.0 61
341.18
<0.0001*
R2 0.9978; adj R-squared 0.9949; root MSE 0.13; dep mean 1.32 * Values of ‘‘Prob > F’’ less than 0.0500 indicate model terms are significant.
Bohm (1995) also showed that the duration and intensity of keratinase secretion were strongly influenced by various keratinous substrates. Keratinase activity is inducible with guinea pig hair in keratin salt broth by M. gypseum and with human hair by Microsporum canis was reported by Meevootisom and Niederpruem (1979) and Takiuchi et al. (1982), respectively. These results are further substantiated by Malviya et al. (1992) who showed in three fungi isolated from the grounds of a gelatin factory that keratinase production was inducible. Similarly, Trichophyton mentagrophytes var. erinacei showed highest keratinase production with wool and Aspergillus flavus with chicken feather (Muhsin and Hadi, 2002). Induction of keratinase production by the keratin substrates was also observed by Cheng et al. (1995) with B. licheniformis PWD-1 where keratinase production was induced by 1% hammer milled feather powder and sheep wool. Fig. 1d shows maximum keratinase production (6.2 KU/ml) in the presence of
1.5% feather and 1 mM CaCl2. The CaCl2 effect differs from that observed with B. licheniformis since CaCl2 showed a negative effect in this organism (Ramnani and Gupta, 2004). An empirical model was designed based on experimental data, in which the independent and dependent variables were fitted to the second-order model equation and examined in terms of the goodness of fit. ANOVA was used to evaluate the adequacy of the fitted model. The R-squared value provided a measure of how much of the variability in the observed response values could be explained by the experimental factors and their interactions. The closer the R-squared value is to 1.00, the stronger the model and the better the response predictions (Haaland, 1989). Based on the F-test, the model is predictive, since its calculated Fvalue is higher that the critical F-value and the regression coefficient (341.18) is close to unity. Thus statistical studies could prove useful for optimization of keratinases produced by other microorganisms. The condition optimized in the present study will also be useful for keratinases from other Scopulariopsis strains as they are common saprophytic fungi, with a view to industrial applications such as dehairing as proved previously for S. brevicaulis (Anbu et al., 2005). Acknowledgements The author (P.A.) thanks the Council of Scientific and Industrial Science (CSIR) for financial assistance. Part of the work was done in fulfillment of the requirements for the Ph.D. degree.
Fig. 1. (a) Response surface optimization of keratinase production with respect to temperature and glucose. All data were treated with the aid of Design Expert Software. Values were not using the whole data set, but only from treatments where the pH was 7.5 and the temperature was at 30 C. (b) Response surface optimization of keratinase production with respect to glucose and sodium nitrate. (c) Response surface optimization of keratinase production with respect to sodium nitrate and feather. (d) Response surface optimization of keratinase production with respect to feather and calcium chloride.
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References Anbu, P., Gopinath, S.C.B., Hilda, A., Lakshmipriya, T., Annadurai, G., 2005. Purification and characterization of keratinase from poultry farm isolate-Scopulariopsis brevicaulis and statistical optimization of enzyme activity. Enzyme Microb. Technol. 36, 639–647. Annadurai, G., Sheeja, R.Y., 1998. Use of Box–Behnken design experiments for the adsorption of verofix red using biopolymer. Bioprocess Eng. 18, 463–466. Box, G.E.P., Behnken, D.W., 1960. Three level design for the study of quantitative variables. Technometrics 2, 455–475. Cheng, S.W., Ha, H.M., Shen, S.W., Takagi, H., Asano, M., Tsai, Y.C., 1995. Production and characterization of keratinase of a feather degrading Bacillus licheniformis PWD-1. Biosci. Biotechnol. Biochem. 59, 2239–2243. Drucker, H., 1975. Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process. J. Bacteriol. 122, 1117– 1125. Gopinath, S.C.B., Hilda, A., Laksmipriya, T., Annadurai, G., 2002. Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology. World J. Microbiol. Biotechnol. 18, 449–458. Gopinath, S.C.B., Hilda, A., Lakshmipriya, T., Annadurai, G., Anbu, P., 2003a. Statistical optimization of amylase production by Aspergillus versicolor. Asian J. Microbiol. Biotechnol. Environ. Sci. 5, 327–330. Gopinath, S.C.B., Hilda, A., Laksmipriya, T., Annadurai, G., Anbu, P., 2003b. Purification of lipase from Geotrichum candidum: conditions optimized for enzyme production using Box–Behnken design. World J. Microbiol. Biotechnol. 19, 681–689. Haaland, P.D., 1989. Experimental design in biotechnology. Marcel Dekker Inc., New York. Ignatova, Z., Gousterova, A., Spassov, G., Nedkov, P., 1999. Isolation and partial characterisation of extracellular keratinase from a wool degrading thermophilic actinomycete strain Thermoactinomyces candidus. Can. J. Microbiol. 45, 217–222. Johnson, A.G., 1967. Pilot-plant production of protease by Alternaria tenuissima. Appl. Microbiol. 15, 319–324. Kalil, S.J., Maugeri, F., Rodrigues, M.I., 2000. Response surface analysis and simulation as a tool for bioprocess design and optimization. Proc. Biochem. 35, 539–550.
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Malviya, H.K., Rajak, R.C., Hasija, S.K., 1992. Synthesis and regulation of extracellular keratinase in three fungi isolated from the grounds of a gelatin factory, Jabalpur, India. Mycopathologia 120, 1–4. Meevootisom, V., Niederpruem, D.J., 1979. Control of extracellular proteases in dermatophytes and especially Trichophyton rubrum. Sabouraudia 17, 91–106. Muhsin, T.M., Hadi, R.B., 2002. Degradation of keratin substrates by fungi isolated from sewage sludge. Mycopathologia 154, 185–189. North, M.J., 1982. Comparative biochemistry of the proteinases of eukaryotic microorganisms. Microbiol. Rev. 46, 308–340. Okafor, J.I., Ada, N., 2000. Keratinolytic activity of five human isolates of the dermatophytes. J. Common. Dis. 32, 300–305. Ramesh, V.M., 1996. Studies on keratinophilic fungi – characterization of keratinolytic potential and fungitoxic evaluation of some plant extracts. Ph.D. Thesis, University of Madras, Chennai, Tamil Nadu, India. Ramnani, P., Gupta, R., 2004. Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology. Biotechnol. Appl. Biochem. 40, 191–196. Sangali, S., Brandelli, A., 2000. Isolation and characterization of a novel feather degrading bacterial strain. Appl. Biochem. Biotechnol. 87, 17– 24. Sen, S., Satyanarayana, T., 1993. Optimization of alkaline protease production by thermophilic Bacillus licheniformis S40. Indian J. Microbiol. 33, 43–47. Siesenop, U., Bohm, K.H., 1995. Comparative studies on keratinase production in Trichophyton mentrgrophytes strains of animal origin. Mycoses 38, 205–209. Singh, C.J., 1998. Exocellular proteases of Malbranchea gypsea and their role in keratin deterioration. Mycopathologia 143, 147–150. Singh, D.P., Sing, R., Vyas, S.R., 1975. Effect of pH, temperature, nitrogen sources, glucose concentrations on acid protease production by Aspergillus niger mutant. Indian J. Microbiol. 15, 109–113. Takiuchi, I., Higuchi, D., Sei, Y., Koga, M., 1982. Isolation of an extracellular proteinase (keratinase) from Microsporum canis. Sabouraudia 20, 281–288. Yu, R.J., Harmon, S.R., Blank, F., 1968. Isolation and purification of an extracellular keratinase of Trichophyton mentagrophytes. J. Bacteriol. 96, 1435–1436.