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Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate
Bioresource Technology 98 (2007) 881–885
Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate Shikha 1, Adhyayan Sharan, Nandan S. Darmwal
¤
Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad 224 001, UP, India Received 19 July 2004; received in revised form 7 March 2006; accepted 7 March 2006 Available online 12 June 2006
Abstract An alkalophilic bacterial isolate identiWed as Bacillus pantotheneticus, isolated from saline-alkali soils of Avadh region of UP, India, was studied for the production of alkaline protease. The mutant of the isolated species showed 44% improved production over the parent strain. Organic nitrogen sources supported better protease production than the inorganic sources. The production of alkaline protease was (242 U/ml) in the medium containing molasses, which was comparable with molasses and wheat bran (285 U/ml) as carbon and nitrogen sources, respectively. Protease production was best at pH 10 and temperature 30 °C. The Km (for casein) was 11 mg/ml and V max was 380-g tyrosine/ml/min. The enzyme was stable between pH 7 and 10.7 and temperature between 30 and 60 °C with a pH and temperature optimum at 8.4 and 40 °C, respectively. The results indicated that molasses was an optimal substrate for alkaline protease production. © 2006 Elsevier Ltd. All rights reserved. Keywords: Alkaline protease; Bacillus; Molasses
1. Introduction Microorganisms are attractive sources of protease because of their biochemical diversity and the ease with which enzyme production may be increased by environmental and/or genetic manipulation. The alkaline proteases are one of the commercially important enzyme constituents in laundry detergent. A major source of alkaline protease has been Bacillus (Han and Damodara, 1998; Huang et al., 2003; Puri et al., 2002). The extracellular proteases from alkalophilic Bacillus sp. have high activities against protein at low temperature and resistance against alkali and various surfactants (Yamagata et al., 1994),
*
Corresponding author. Tel.: +91 05278 245894. E-mail address: [email protected] ( Shikha). 1 Present address: School for Environmental Sciences, Babasaheb Bhim rao Ambedkar University, Vidya Vihar, Rai bareli Road, Lucknow 226 025, UP, India. 0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.03.023
which make them ideal detergent additives. Moreover, there is a need to develop new detergent composition especially for energetic considerations (introducing new bleaching systems for washing at low temperature) since the consumer practices vary from nation to nation (Manachini and Fortina, 1998). The amount of protease produced varies greatly with strains and media used. The existence of distinct protease from diVerent strains, which catalyze the same reactions allow Xexibility in choice of fermentation conditions since these diVerent enzymes may have diVerent stabilities and diVerent pH and temperature optima (Gupta et al., 2002; Manachini and Fortina, 1998). On an industrial scale, exoenzymes such as alkaline protease are produced in complex media containing glucose and other cost intensive substrates. For potential industrial applications, hyperproductive organisms growing on economical substrates are required. To this end, we isolated a mutant of alkalophilic Bacillus pantotheneticus and investigated the optimal conditions for the production of alkaline
882
Shikha et al. / Bioresource Technology 98 (2007) 881–885
protease, which could possess potential industrial applications. 2. Methods 2.1. Organism and growth conditions The organism was isolated from saline-alkali soil of the Avadh region of Uttar Pradesh, India, employing enrichment culture technique (Boyer et al., 1973). The strain was identiWed as B. pantotheneticus at the Institute of Microbial Technology, Chandigarh, India and deposited in the MTCC (Microbial Type Collection Center, IMTECH, Chandigarh, India). It was grown at 37 °C in media containing (g/l): glucose, 10.0; peptone, 5.0; yeast extract, 5.0; KH2PO4, 1.0; MgSO4 · 7H2O, 0.2. The pH was adjusted to 10.5 by adding sterilized Na2CO3 (20% w/v) separately to the sterilized medium at 10% (v/v). The cultures were grown in 250 ml Xasks containing 50 ml media. Each Xask was inoculated with 0.5 ml of inoculum (OD 0.5) grown for 24 h, in an orbital incubating shaker (120 rev/min). The cell free supernatants (CFS) were obtained for enzyme essay, as described earlier (Mehrotra et al., 1999). 2.2. Enzyme essay The method of Anson–Hagihara was followed (Hagihara et al., 1958) using casein as the substrate. The detailed protocol was exactly as described earlier (Mehrotra et al., 1999). One unit of the alkaline protease activity (U) was taken as the amount of enzyme liberating 1 g of tyrosine/ min under the assay conditions. The estimations were based on a tyrosine standard curve.
2.4. Optimization for protease production The optimization studies included production of protease under diVerent conditions e.g., incubation temperature (30–45 °C); pH (7.0–10.7) of the growth medium; supplementation of medium with diVerent carbon sources including molasses (% composition; water, 30.5; sucrose, 29.75; dextrose, 3.25; fructose, 4; other reducing sugar, 1.0; ashes, 15.0; nitrogenous content, 4.5; nonnitrogenous content, 8.5, waxes and sterols, 1.5 and other components, 2.0) substituting for glucose (1% w/v) and various nitrogen sources for yeast extract (0.5% w/v) + peptone (0.5% w/v) at 1% (w/v). 2.5. Properties of the crude enzyme The crude enzyme (CFS) was studied as a function of substrate concentration (0.25–2.0%), thermal stability over the temperature range 30–60 °C and alkaline stability over the pH range 7.0–10.7 for 10 min. The reaction mixture (1 ml of CFS + 1 ml of 1% casein in 10 ml of carbonate– biocarbonate buVer of pH 10.5) was incubated at diVerent substrate concentration, temperature and pH. The enzyme assay was carried out after 10 min. The eVect of some metal ions (2 mM) on the activity of protease was also studied. The activity of protease was measured as described previously (Mehrotra et al., 1999). All the experiments were carried out in triplicate. 2.6. Statistical analysis Analysis of variance was carried out using software SPSS (SPSS version 9, Windows version). GLM command of the software was adopted. Means and standard error were calculated (Tables 1–3). Univariate analysis of vari-
2.3. Isolation of mutants A 1:25 dilution of an overnight culture of the isolated B. pantotheneticus was grown to O.D. 0.5 and centrifuged (5000g, 10 min). The pellet was washed twice in an equal volume of 100 mM citrate buVer (pH 5.5), resuspended in 50 ml of buVer and placed on ice for 10 min. The cells were treated with 0.2 ml of N-methyl-N⬘-nitro-N-nitrosoguanidine (MNNG, 50 g/ml) for diVerent time intervals (5, 10, 15, 30 and 45 min) to determine the MNNG survival curve (Mishra and Goel, 1999). The treated cell suspension was centrifuged and washed twice with 100 mM phosphate buVer (pH 7). The cell suspensions (0.1 ml) were spread onto solid minimal medium (pH 10.5) and incubated at 37 °C along with untreated control. After 24 h of incubation at 37 °C, colonies were observed on the media and were collected for further analysis. MNNG treatment for 10 min resulted in 99.9% mortality after 24 h of incubation at 37 °C. All possible mutants were screened for alkaline protease production (compared to the parent strain) after 24 h of growth in minimal medium (pH 10.5). An isolate exhibiting maximum protease production was selected for further study.
Table 1 EVect of carbon sources on the alkaline protease production by the mutant at pH 10.0 and temperature 30 °C Carbon (1% w/v)
Glucose Glycerol Starch Molasses Malt extract Potato Wheat Xour Rice Xour Fructose Galactose Sucrose pb
Meana enzyme activity (U/ml) 24 h
48 h
72 h
110 § 0.11 54 § 1.3 31 § 0.04 230 § 0.9 76 § 0.34 46 § 0.25 41 § 0.3 68 § .081 66 § 0.006 64 § 0.5 51 § 0.66 <0.002c
87 § 0.3 68 § 0.65 35 § 0.4 245 § 0.7 89 § 0.58 66 § 0.75 71 § 0.36 58 § 0.93 60 § 0.42 49 § 0.58 45 § 0.45 <0.002c
42 § 0.54 50 § 0.64 31 § 0.22 219 § 0.06 63 § 0.07 55 § 0.5 43 § 0.1 56 § 0.25 47 § 0.98 39 § 0.44 43 § 0.08 <0.002c
a Average of three independent experiments. Nitrogen sources used: ‘N’—yeast extract + peptone—1%. b The p values refer to the comparison of protease activity at 1% glucose with the other carbon sources. The comparison is among values within the column. c All the values in the column are statistically signiWcant.
Shikha et al. / Bioresource Technology 98 (2007) 881–885 Table 2 EVect of nitrogen sources on the alkaline protease production by the mutant at pH 10.0 and temperature 30 °C Nitrogen (1% w/v)
Meana enzyme activity (U/ml) 24 h
48 h
72 h
Peptone + yeast extract NH4Cl (NH4)2SO4 KNO3 Urea Wheat bran Mustard oil cake Coconut oil cake Peptone Yeast extract Beef extract Soybean Rice bran pb
115 § 0.5 22 § 0.35 23 § 0.22 24 § 0.02 37 § 0.15 131 § 0.54 61 § 0.84 38 § 0.94 49 § 0.22 23 § 0.12 59 § 0.06 63 § 0.38 44 § 0.01 <0.002c
85 § 0.65 25 § 0.03 28 § 0.51 28 § 0.15 60 § 0.30 155 § 0.26 100 § 0.02 57 § 0.51 58 § 0.02 35 § 0.21 60 § 0.04 82 § 0.08 55 § 0.33 <0.002c
48 § 0.20 28 § 0.05 30 § 0.84 32 § 0.05 60 § 0.09 74 § 0.21 95 § 0.54 58 § 0.45 65 § 0.22 35 § 0.35 62 § 0.51 66 § 0.25 51 § 0.31 <0.002c
883
120 Mutant strain Wild type
Alkaline protease activity (U/ml)
100
80
60
40
20
a
Average of three independent experiments. Carbon sources used: ‘C’—glucose—1%. b The p values refer to the comparison of protease activity at 1% nitrogen (peptone + yeast extract) with the other nitrogen sources. The comparison is among values within the column. c All the values in the column are statistically signiWcant.
ance was employed on the data with substrate concentration (carbon, nitrogen, combination of molasses and nitrogen sources) and protease activity. Main eVects and interaction were tested for signiWcance (Figs. 1–3). 3. Results and discussion Eighty-six (86) mutant strains were screened for their alkaline protease secretion ability. The parent strain aVorded 68 U/ml activity after 24 h incubation in shake Xask condition. However, the alkaline protease production by the mutant strain (98 U/ml) aVorded 44% improved activity
0
0
6
12
18
24
30
36
42
48
54
Time, hour's Fig. 1. Enzyme production by mutant of B. pantotheneticus in comparison with wild type p < 0.02. The p value refers to the comparison of protease activity at diVerent concentration of molasses and time periods. All the comparisons are statistically signiWcant.
(p < 0.02) over the parent strain, which was used for further study (Fig. 1). The protease described in the present paper was produced by an alkalophilic isolate identiWed as B. pantotheneticus, during the exponential phase of growth. The protease belongs to the class of serine protease as shown by its optimum pH 10 for activity and its total inhibition in the presence of phenylmethylsulfonyl Xuoride (Ward, 1983). There was no appreciable loss in the production of alkaline protease over the experimental pH and temperature after 24 h of incubation.
Table 3 EVect of diVerent combinations of molasses and nitrogen sources on alkaline protease production by the mutant Mediaa
Meanb enzyme activity (U/ml) 30 °C
Glucose Molassesc,d Molassesd Molasses + urea Molasses + wheat bran Molasses + tryptone Molasses + mustard oil cake Molasses + (NH4)2SO4 pe a
40 °C
24 h
48 h
72 h
24 h
48 h
72 h
87 § 0.11 242 § 0.36 254 § 0.23 237 § 0.55 213 § 0.19 171 § 0.23 218 § 0.5 156 § 0.54 <0.002f
115 § 0.54 240 § 0.21 245 § 0.5 212 § 0.8 285 § 0.12 205 § 0.8 243 § .22 234 § 0.56 <0.002f
48 § 0.2 194 § 0.5 246 § 0.5 218 § 0.85 236 § 0.31 160 § 0.11 196 § 0.34 140 § 0.52 <0.002f
111 § 0.22 162 § 0.31 147 § 0.52 186 § 0.21 207 § 0.55 155 § 0.02 192 § 0.03 149 § 0.50 <0.002f
76 § 0.5 144 § 0.51 137 § 0.35 126 § 0.57 172 § 0.63 150 § 0.12 180 § 0.98 129 § 1.2 <0.002f
39 § 0.23 109 § 0.2 135 § 0.56 128 § 0.13 168 § 0.8 151 § 1.56 159 § 1.01 114 § 0.03 <0.002f
Carbon and nitrogen added at 1% (w/v). Average of three independent experiments. c KH2PO4 and MgSO4 omitted. d Nitrogen omitted. e The p values refer to the comparison of protease activity at 1% glucose and nitrogen with the activity at diVerent combination of molasses and nitrogen sources. The comparison is among values within the column. f All the values in the column are statistically signiWcant. b
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30 ºC 35 ºC 125
40 ºC
Enzyme activity (U/ml)
100
75
50
25
0
9
9.5
10
10.5
pH Fig. 2. EVect of pH and temperature on alkaline protease production by the mutant strain. An average of three observations, standard error D §3.45%, p < 0.002. The p value refers to the comparison of protease activity at diVerent pH and temperature. All the comparisons are statistically signiWcant. Carbon and nitrogen sources used: ‘C’—glucose— 1%; ‘N’—yeast extract + peptone—1%.
300 24 h
Enzyme activity (U/ml)
250
48 h 72 h
200 150 100 50 0 0.25
0.5
1
1.25
1.5
Molasses (Sugar %w/v) Fig. 3. EVect of molasses on alkaline protease production by the mutant at pH 10.0 and temperature 30 °C. An average of three observations, standard error D 12.66%, p < 0.002. The p value refers to the comparison of protease activity at concentration of molasses and time period. All the comparisons are statistically signiWcant. Carbon and nitrogen sources used: ‘C’—glucose—1%; ‘N’—yeast extract + peptone—1%.
Antibiotic sensitivity test was performed. The wild type strain was found to exhibit resistance to the antibiotics Ampicillin and Penicillin G for which the mutant strain was sensitive. However, the mutant strain showed resistance to the antibiotic Cefadroxil for which the wild type was sensitive. The aforesaid results clearly demonstrated the diVerential sensitivity of the wild type and mutant strain of the B. pantotheneticus. However, no signiWcant alteration was
observed in the general morphology and generation time of the parent and mutant strains. The eVect of pH and temperature on the alkaline protease activity of the mutant is shown in Fig. 2. Results showed that the mutant grew in a pH range 9.0–10.5 and over a range of temperature (30–40 °C). However, the optimal activity (115 U/ml) was observed at pH 10 and temperature 30 °C (p < 0.002). Among the various carbon sources studied, molasses (4% reducing sugar) was found to be the best source for the protease production (Table 1). Although glucose was fairly good carbon source but there was an appreciable loss in production from 48 to 72 h of incubation. Likewise, no other carbon source was found to be comparable since alkaline protease production by mutant (245 U/ml) increased as compared to the control after 48 h of incubation at optimum pH and temperature (pH 10 and 30 °C). Owing to a substantial increase in the protease production in the presence of molasses, its eVect was studied as a function of graded concentration in sugar equivalent (Fig. 3). Molasses containing 40% sugar was (w/w) was diluted to the desired concentration (0.25–1.5% sugar w/v) and was substituted for the carbon sources in the basal medium (devoid of organic carbon and nitrogen sources). Molasses containing 1% sugar (w/v) as source for protease production was found to exhibit the highest (p < 0.02) enzyme activity (242 U/ml) over 48 h of incubation. The best carbon source for protease production was molasses, followed by glucose. Although diverse carbon sources have been employed for the production of enzyme (Ellaiah et al., 2002; Puri et al., 2002) no reports are available on the use of molasses as a substrate. Molasses is a comparatively economical carbon sources and become a cost eVective substrate in large-scale fermentation process. Among the diVerent nitrogen sources studied, wheat bran was found to be the best (p < 0.002) source for protease production by mutant, while the combination of peptone and yeast extract was comparable (Table 2). Among the oil cakes studied, mustard oil cake was found to be appreciably optimal over 48 h of incubation (100 U/ml). Addition of inorganic nitrogen sources in the medium resulted in lesser enzyme production in comparison to organic sources. Wheat bran was found to be the best nitrogen sources. It has been reported that the organic sources enhance the production of enzyme (Fujiwara and Ymamoto, 1987; Gajju et al., 1996). However, addition of inorganic sources resulted in decreased production of enzyme. Similar results have been obtained with other Bacillus sp. (Moon and Parlukar, 1991; Razak et al., 1994). Since molasses and wheat bran, studied independently, were found to be best carbon and nitrogen sources for alkaline protease production, respectively, the mutant was grown in diVerent combinations of carbon and nitrogen sources. Molasses (1% sugar w/v) as carbon sources was combined with diVerent nitrogen sources in the basal medium (Table 3). Results showed that molasses–wheat bran medium supported better production (p < 0.002) of
Shikha et al. / Bioresource Technology 98 (2007) 881–885
protease (285 U/ml), which was followed closely by molasses containing MgSO4 + KH2PO4 (254 U/ml) but devoid of any nitrogen source and crude molasses without any supplement (242 U/ml). Although, crude molasses can serve as a self-supporting medium for protease production, the role of Mg2+ and K+ salts could not be ruled out, as slightly better production was exhibited when molasses was supplemented with MgSO4 and KH2PO4 (Ellaiah et al., 2002). The overall alkaline protease production was maximum at 30 °C over 48 h of incubation whereas, at 40 °C the same was obtained over 24 h of incubation which may be attributed to partial denaturation of the enzyme with increased period of incubation. The protease activity was found to be optimum at 1.5% substrate concentration and was stable over wide temperature (30–60 °C) and pH (7–10.7) range. The optimum protease activity was observed at pH 8.4 and temperature 40 °C. The enzyme activity was enhanced in the presence of Fe2+, Ca2+ and Mg2+ whereas; Ni2+, Mn2+ and Hg2+ inhibited the enzyme activity. The enzyme was compatible with the locally available detergents such as Surf excel, Hindustan Lever Ltd., 165/166, Backbay Relamation, Mumbai; Aerial, Proctor & Gamble Home Product Limited, Mumbai; Wheel, Rohini Surfactant Limited, 236, Chauepur Kalan, Kanpur and Sipahi, Sipahi Industries, Pandu Nagar Kanpur. The protease activity increased with the increase in substrate (casein) concentration from 2.5 to 15 mg/ml, beyond this, there was no signiWcant increase in activity (p > 0.002). From a double reciprocal (Lineweaver–Burk) plot, Michaelis Menten constant (Km) for this enzyme was calculated as 11 mg/ml and its Vmax was 380 g tyrosine/ml/min. The optimum pH and temperature for in vitro enzyme was pH 8.4 and 40 °C. However, the enzyme was found to be stable over wide temperature (30–60 °C) and pH (7–11) range. Thermophilic bacilli, which grow and produce enzyme at 50–55 °C and pH 6–7 have been found to produce enzymes, which prefer alkaline pH and high temperature than the growth temperature for activity (Sonnleitner, 1983). The optimum concentration of casein for enzyme reaction was 1.0% (w/v). Besides pH and temperature stability an optimal detergent protease should also be stable in the presence of detergents. In the present case, the enzyme exhibited stability in the presence of diVerent locally available detergents. This is an important property since stable enzymes are generally not available except for a few reports (Gupta et al., 1999). The results of the present study show the potential of using molasses as a substrate in the fermentation media which would not only be an economical substrate but one of the possible ways of utilizing the distillery byproduct. Considering the overall property of diVerent alkaline proteases of microbial origin and their evaluation, the reported alkaline protease could be an ideal detergent additive with
885
regards to pH and temperature stability that might be of commercial interest. Acknowledgements Research grant from Council of ScientiWc and Industrial Research (Scheme No. 38(1008)/01/EMR-II), New Delhi to NSD and to Shikha in the form of Research Associateship is gratefully acknowledged. References Boyer, E.W., Ingle, M.B., Mercer, G.D., 1973. Bacillus alcalophilus sub sp. halodurans sub sp. nov: an alkaline amylase producing, alkalophilic microorganisms. Int. J. Syst. Bacteriol. 23, 238–242. Ellaiah, P., Srinivasulu, B., Adinarayana, K., 2002. A review on microbial alkaline proteases. J. Sci. Ind. Res. 61, 690–704. Fujiwara, N., Ymamoto, K., 1987. Production of alkaline protease in a low cost medium by alkalophilic Bacillus sp. and properties of the enzyme. J. Ferment. Technol. 65, 242–345. Gajju, H., Bhalla, T.C., Agarwal, H.O., 1996. Thermostable alkaline protease from thermophilic Bacillus coagulans PB-77. Ind. J. Microbiol. 36, 153–155. Gupta, R., Gupta, K., Saxena, R.K., Khan, S., 1999. Bleach stable, alkaline protease from Bacillus sp. Biotechnol. Lett. 21, 135–138. Gupta, R., Beg, Q.K., Khan, S., Chauhan, 2002. An overview on fermentation, downstream processing and properties of microbial alkaline protease. Appl. Microbiol. Biotechnol. 60, 381–385. Hagihara, B., Matsubara, H., Nakai, M., Okunuki, K., 1958. Crystalline bacterial proteinase of Bacillus subtilis. J. Biochem. 45, 185–194. Han, X.Q., Damodara, 1998. PuriWcation and characterization of protease Q: a detergent and urea-stable serine endopeptidase from Bacillus pumilus. J. Agric. Food Chem. 46, 3396–3603. Huang, Q., Peng, Y., Li, X., Way, H., Zhang, Y., 2003. PuriWcation and characterization of an extracellular alkaline serine protease with dehairing function for Bacillus pumilis. Curr. Microbiol. 46, 169–173. Manachini, P.L., Fortina, M.G., 1998. PuriWcation in sea water of thermostable alkaline proteases by halotolerant strain of Bacillus licheniformis. Biotechnol. Lett. 20 (6), 565–568. Mehrotra, S., Pandey, P.K., Gaur, R., Darmwal, N.S., 1999. The production of alkaline protease by a Bacillus sp. isolate. Biores. Technol. 67, 201–203. Mishra, M., Goel, R., 1999. Development of a cold resistant mutant of plant growth promoting Pseudomonas Xuorescence and its functional characterization. J. Biotechnol. 75, 71–75. Moon, S.H., Parlukar, S.J., 1991. A parametric study of protease production in batch and fed-batch cultures of Bacillus Wrmus. Biotechnol. Bioeng. 37, 467–483. Puri, S., Beg, Q.K., Gupta, R., 2002. Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Curr. Microbiol. 44, 286–290. Razak, N.A., Samad, M.Y.A., Basri, M., Yunus, W.W.Z.W., Ampon, K., Sallen, A.B., 1994. Thermostable extracellular protease of Bacillus stearothrmophilus: factors aVecting its production. World J. Microbiol. Biotechnol. 10, 260–263. Sonnleitner, B., 1983. Biotechnology of thermophilic bacteria, growth products and application. In: Fiechter, A. (Ed.), Adv. Biotechnol. Springer, Berlin, pp. 70–138. Ward, O.P., 1983. Proteinases. In: Fogaarty, W.M. (Ed.), Microbial Enzymes and Biotechnolgy. Applied Science Publ., New York, pp. 251–317. Yamagata, Y., Arakawa, K., Yamaguchi, M., Kobayashi, M., Ichishima, E., 1994. Functional changes of dextran-modiWed alkaline proteinase from alkalophilic Bacillus sp.. Enzyme Microb. Technol. 16, 99–103.
Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate Shikha 1, Adhyayan Sharan, Nandan S. Darmwal
¤
Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad 224 001, UP, India Received 19 July 2004; received in revised form 7 March 2006; accepted 7 March 2006 Available online 12 June 2006
Abstract An alkalophilic bacterial isolate identiWed as Bacillus pantotheneticus, isolated from saline-alkali soils of Avadh region of UP, India, was studied for the production of alkaline protease. The mutant of the isolated species showed 44% improved production over the parent strain. Organic nitrogen sources supported better protease production than the inorganic sources. The production of alkaline protease was (242 U/ml) in the medium containing molasses, which was comparable with molasses and wheat bran (285 U/ml) as carbon and nitrogen sources, respectively. Protease production was best at pH 10 and temperature 30 °C. The Km (for casein) was 11 mg/ml and V max was 380-g tyrosine/ml/min. The enzyme was stable between pH 7 and 10.7 and temperature between 30 and 60 °C with a pH and temperature optimum at 8.4 and 40 °C, respectively. The results indicated that molasses was an optimal substrate for alkaline protease production. © 2006 Elsevier Ltd. All rights reserved. Keywords: Alkaline protease; Bacillus; Molasses
1. Introduction Microorganisms are attractive sources of protease because of their biochemical diversity and the ease with which enzyme production may be increased by environmental and/or genetic manipulation. The alkaline proteases are one of the commercially important enzyme constituents in laundry detergent. A major source of alkaline protease has been Bacillus (Han and Damodara, 1998; Huang et al., 2003; Puri et al., 2002). The extracellular proteases from alkalophilic Bacillus sp. have high activities against protein at low temperature and resistance against alkali and various surfactants (Yamagata et al., 1994),
*
Corresponding author. Tel.: +91 05278 245894. E-mail address: [email protected] ( Shikha). 1 Present address: School for Environmental Sciences, Babasaheb Bhim rao Ambedkar University, Vidya Vihar, Rai bareli Road, Lucknow 226 025, UP, India. 0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.03.023
which make them ideal detergent additives. Moreover, there is a need to develop new detergent composition especially for energetic considerations (introducing new bleaching systems for washing at low temperature) since the consumer practices vary from nation to nation (Manachini and Fortina, 1998). The amount of protease produced varies greatly with strains and media used. The existence of distinct protease from diVerent strains, which catalyze the same reactions allow Xexibility in choice of fermentation conditions since these diVerent enzymes may have diVerent stabilities and diVerent pH and temperature optima (Gupta et al., 2002; Manachini and Fortina, 1998). On an industrial scale, exoenzymes such as alkaline protease are produced in complex media containing glucose and other cost intensive substrates. For potential industrial applications, hyperproductive organisms growing on economical substrates are required. To this end, we isolated a mutant of alkalophilic Bacillus pantotheneticus and investigated the optimal conditions for the production of alkaline
882
Shikha et al. / Bioresource Technology 98 (2007) 881–885
protease, which could possess potential industrial applications. 2. Methods 2.1. Organism and growth conditions The organism was isolated from saline-alkali soil of the Avadh region of Uttar Pradesh, India, employing enrichment culture technique (Boyer et al., 1973). The strain was identiWed as B. pantotheneticus at the Institute of Microbial Technology, Chandigarh, India and deposited in the MTCC (Microbial Type Collection Center, IMTECH, Chandigarh, India). It was grown at 37 °C in media containing (g/l): glucose, 10.0; peptone, 5.0; yeast extract, 5.0; KH2PO4, 1.0; MgSO4 · 7H2O, 0.2. The pH was adjusted to 10.5 by adding sterilized Na2CO3 (20% w/v) separately to the sterilized medium at 10% (v/v). The cultures were grown in 250 ml Xasks containing 50 ml media. Each Xask was inoculated with 0.5 ml of inoculum (OD 0.5) grown for 24 h, in an orbital incubating shaker (120 rev/min). The cell free supernatants (CFS) were obtained for enzyme essay, as described earlier (Mehrotra et al., 1999). 2.2. Enzyme essay The method of Anson–Hagihara was followed (Hagihara et al., 1958) using casein as the substrate. The detailed protocol was exactly as described earlier (Mehrotra et al., 1999). One unit of the alkaline protease activity (U) was taken as the amount of enzyme liberating 1 g of tyrosine/ min under the assay conditions. The estimations were based on a tyrosine standard curve.
2.4. Optimization for protease production The optimization studies included production of protease under diVerent conditions e.g., incubation temperature (30–45 °C); pH (7.0–10.7) of the growth medium; supplementation of medium with diVerent carbon sources including molasses (% composition; water, 30.5; sucrose, 29.75; dextrose, 3.25; fructose, 4; other reducing sugar, 1.0; ashes, 15.0; nitrogenous content, 4.5; nonnitrogenous content, 8.5, waxes and sterols, 1.5 and other components, 2.0) substituting for glucose (1% w/v) and various nitrogen sources for yeast extract (0.5% w/v) + peptone (0.5% w/v) at 1% (w/v). 2.5. Properties of the crude enzyme The crude enzyme (CFS) was studied as a function of substrate concentration (0.25–2.0%), thermal stability over the temperature range 30–60 °C and alkaline stability over the pH range 7.0–10.7 for 10 min. The reaction mixture (1 ml of CFS + 1 ml of 1% casein in 10 ml of carbonate– biocarbonate buVer of pH 10.5) was incubated at diVerent substrate concentration, temperature and pH. The enzyme assay was carried out after 10 min. The eVect of some metal ions (2 mM) on the activity of protease was also studied. The activity of protease was measured as described previously (Mehrotra et al., 1999). All the experiments were carried out in triplicate. 2.6. Statistical analysis Analysis of variance was carried out using software SPSS (SPSS version 9, Windows version). GLM command of the software was adopted. Means and standard error were calculated (Tables 1–3). Univariate analysis of vari-
2.3. Isolation of mutants A 1:25 dilution of an overnight culture of the isolated B. pantotheneticus was grown to O.D. 0.5 and centrifuged (5000g, 10 min). The pellet was washed twice in an equal volume of 100 mM citrate buVer (pH 5.5), resuspended in 50 ml of buVer and placed on ice for 10 min. The cells were treated with 0.2 ml of N-methyl-N⬘-nitro-N-nitrosoguanidine (MNNG, 50 g/ml) for diVerent time intervals (5, 10, 15, 30 and 45 min) to determine the MNNG survival curve (Mishra and Goel, 1999). The treated cell suspension was centrifuged and washed twice with 100 mM phosphate buVer (pH 7). The cell suspensions (0.1 ml) were spread onto solid minimal medium (pH 10.5) and incubated at 37 °C along with untreated control. After 24 h of incubation at 37 °C, colonies were observed on the media and were collected for further analysis. MNNG treatment for 10 min resulted in 99.9% mortality after 24 h of incubation at 37 °C. All possible mutants were screened for alkaline protease production (compared to the parent strain) after 24 h of growth in minimal medium (pH 10.5). An isolate exhibiting maximum protease production was selected for further study.
Table 1 EVect of carbon sources on the alkaline protease production by the mutant at pH 10.0 and temperature 30 °C Carbon (1% w/v)
Glucose Glycerol Starch Molasses Malt extract Potato Wheat Xour Rice Xour Fructose Galactose Sucrose pb
Meana enzyme activity (U/ml) 24 h
48 h
72 h
110 § 0.11 54 § 1.3 31 § 0.04 230 § 0.9 76 § 0.34 46 § 0.25 41 § 0.3 68 § .081 66 § 0.006 64 § 0.5 51 § 0.66 <0.002c
87 § 0.3 68 § 0.65 35 § 0.4 245 § 0.7 89 § 0.58 66 § 0.75 71 § 0.36 58 § 0.93 60 § 0.42 49 § 0.58 45 § 0.45 <0.002c
42 § 0.54 50 § 0.64 31 § 0.22 219 § 0.06 63 § 0.07 55 § 0.5 43 § 0.1 56 § 0.25 47 § 0.98 39 § 0.44 43 § 0.08 <0.002c
a Average of three independent experiments. Nitrogen sources used: ‘N’—yeast extract + peptone—1%. b The p values refer to the comparison of protease activity at 1% glucose with the other carbon sources. The comparison is among values within the column. c All the values in the column are statistically signiWcant.
Shikha et al. / Bioresource Technology 98 (2007) 881–885 Table 2 EVect of nitrogen sources on the alkaline protease production by the mutant at pH 10.0 and temperature 30 °C Nitrogen (1% w/v)
Meana enzyme activity (U/ml) 24 h
48 h
72 h
Peptone + yeast extract NH4Cl (NH4)2SO4 KNO3 Urea Wheat bran Mustard oil cake Coconut oil cake Peptone Yeast extract Beef extract Soybean Rice bran pb
115 § 0.5 22 § 0.35 23 § 0.22 24 § 0.02 37 § 0.15 131 § 0.54 61 § 0.84 38 § 0.94 49 § 0.22 23 § 0.12 59 § 0.06 63 § 0.38 44 § 0.01 <0.002c
85 § 0.65 25 § 0.03 28 § 0.51 28 § 0.15 60 § 0.30 155 § 0.26 100 § 0.02 57 § 0.51 58 § 0.02 35 § 0.21 60 § 0.04 82 § 0.08 55 § 0.33 <0.002c
48 § 0.20 28 § 0.05 30 § 0.84 32 § 0.05 60 § 0.09 74 § 0.21 95 § 0.54 58 § 0.45 65 § 0.22 35 § 0.35 62 § 0.51 66 § 0.25 51 § 0.31 <0.002c
883
120 Mutant strain Wild type
Alkaline protease activity (U/ml)
100
80
60
40
20
a
Average of three independent experiments. Carbon sources used: ‘C’—glucose—1%. b The p values refer to the comparison of protease activity at 1% nitrogen (peptone + yeast extract) with the other nitrogen sources. The comparison is among values within the column. c All the values in the column are statistically signiWcant.
ance was employed on the data with substrate concentration (carbon, nitrogen, combination of molasses and nitrogen sources) and protease activity. Main eVects and interaction were tested for signiWcance (Figs. 1–3). 3. Results and discussion Eighty-six (86) mutant strains were screened for their alkaline protease secretion ability. The parent strain aVorded 68 U/ml activity after 24 h incubation in shake Xask condition. However, the alkaline protease production by the mutant strain (98 U/ml) aVorded 44% improved activity
0
0
6
12
18
24
30
36
42
48
54
Time, hour's Fig. 1. Enzyme production by mutant of B. pantotheneticus in comparison with wild type p < 0.02. The p value refers to the comparison of protease activity at diVerent concentration of molasses and time periods. All the comparisons are statistically signiWcant.
(p < 0.02) over the parent strain, which was used for further study (Fig. 1). The protease described in the present paper was produced by an alkalophilic isolate identiWed as B. pantotheneticus, during the exponential phase of growth. The protease belongs to the class of serine protease as shown by its optimum pH 10 for activity and its total inhibition in the presence of phenylmethylsulfonyl Xuoride (Ward, 1983). There was no appreciable loss in the production of alkaline protease over the experimental pH and temperature after 24 h of incubation.
Table 3 EVect of diVerent combinations of molasses and nitrogen sources on alkaline protease production by the mutant Mediaa
Meanb enzyme activity (U/ml) 30 °C
Glucose Molassesc,d Molassesd Molasses + urea Molasses + wheat bran Molasses + tryptone Molasses + mustard oil cake Molasses + (NH4)2SO4 pe a
40 °C
24 h
48 h
72 h
24 h
48 h
72 h
87 § 0.11 242 § 0.36 254 § 0.23 237 § 0.55 213 § 0.19 171 § 0.23 218 § 0.5 156 § 0.54 <0.002f
115 § 0.54 240 § 0.21 245 § 0.5 212 § 0.8 285 § 0.12 205 § 0.8 243 § .22 234 § 0.56 <0.002f
48 § 0.2 194 § 0.5 246 § 0.5 218 § 0.85 236 § 0.31 160 § 0.11 196 § 0.34 140 § 0.52 <0.002f
111 § 0.22 162 § 0.31 147 § 0.52 186 § 0.21 207 § 0.55 155 § 0.02 192 § 0.03 149 § 0.50 <0.002f
76 § 0.5 144 § 0.51 137 § 0.35 126 § 0.57 172 § 0.63 150 § 0.12 180 § 0.98 129 § 1.2 <0.002f
39 § 0.23 109 § 0.2 135 § 0.56 128 § 0.13 168 § 0.8 151 § 1.56 159 § 1.01 114 § 0.03 <0.002f
Carbon and nitrogen added at 1% (w/v). Average of three independent experiments. c KH2PO4 and MgSO4 omitted. d Nitrogen omitted. e The p values refer to the comparison of protease activity at 1% glucose and nitrogen with the activity at diVerent combination of molasses and nitrogen sources. The comparison is among values within the column. f All the values in the column are statistically signiWcant. b
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Shikha et al. / Bioresource Technology 98 (2007) 881–885
30 ºC 35 ºC 125
40 ºC
Enzyme activity (U/ml)
100
75
50
25
0
9
9.5
10
10.5
pH Fig. 2. EVect of pH and temperature on alkaline protease production by the mutant strain. An average of three observations, standard error D §3.45%, p < 0.002. The p value refers to the comparison of protease activity at diVerent pH and temperature. All the comparisons are statistically signiWcant. Carbon and nitrogen sources used: ‘C’—glucose— 1%; ‘N’—yeast extract + peptone—1%.
300 24 h
Enzyme activity (U/ml)
250
48 h 72 h
200 150 100 50 0 0.25
0.5
1
1.25
1.5
Molasses (Sugar %w/v) Fig. 3. EVect of molasses on alkaline protease production by the mutant at pH 10.0 and temperature 30 °C. An average of three observations, standard error D 12.66%, p < 0.002. The p value refers to the comparison of protease activity at concentration of molasses and time period. All the comparisons are statistically signiWcant. Carbon and nitrogen sources used: ‘C’—glucose—1%; ‘N’—yeast extract + peptone—1%.
Antibiotic sensitivity test was performed. The wild type strain was found to exhibit resistance to the antibiotics Ampicillin and Penicillin G for which the mutant strain was sensitive. However, the mutant strain showed resistance to the antibiotic Cefadroxil for which the wild type was sensitive. The aforesaid results clearly demonstrated the diVerential sensitivity of the wild type and mutant strain of the B. pantotheneticus. However, no signiWcant alteration was
observed in the general morphology and generation time of the parent and mutant strains. The eVect of pH and temperature on the alkaline protease activity of the mutant is shown in Fig. 2. Results showed that the mutant grew in a pH range 9.0–10.5 and over a range of temperature (30–40 °C). However, the optimal activity (115 U/ml) was observed at pH 10 and temperature 30 °C (p < 0.002). Among the various carbon sources studied, molasses (4% reducing sugar) was found to be the best source for the protease production (Table 1). Although glucose was fairly good carbon source but there was an appreciable loss in production from 48 to 72 h of incubation. Likewise, no other carbon source was found to be comparable since alkaline protease production by mutant (245 U/ml) increased as compared to the control after 48 h of incubation at optimum pH and temperature (pH 10 and 30 °C). Owing to a substantial increase in the protease production in the presence of molasses, its eVect was studied as a function of graded concentration in sugar equivalent (Fig. 3). Molasses containing 40% sugar was (w/w) was diluted to the desired concentration (0.25–1.5% sugar w/v) and was substituted for the carbon sources in the basal medium (devoid of organic carbon and nitrogen sources). Molasses containing 1% sugar (w/v) as source for protease production was found to exhibit the highest (p < 0.02) enzyme activity (242 U/ml) over 48 h of incubation. The best carbon source for protease production was molasses, followed by glucose. Although diverse carbon sources have been employed for the production of enzyme (Ellaiah et al., 2002; Puri et al., 2002) no reports are available on the use of molasses as a substrate. Molasses is a comparatively economical carbon sources and become a cost eVective substrate in large-scale fermentation process. Among the diVerent nitrogen sources studied, wheat bran was found to be the best (p < 0.002) source for protease production by mutant, while the combination of peptone and yeast extract was comparable (Table 2). Among the oil cakes studied, mustard oil cake was found to be appreciably optimal over 48 h of incubation (100 U/ml). Addition of inorganic nitrogen sources in the medium resulted in lesser enzyme production in comparison to organic sources. Wheat bran was found to be the best nitrogen sources. It has been reported that the organic sources enhance the production of enzyme (Fujiwara and Ymamoto, 1987; Gajju et al., 1996). However, addition of inorganic sources resulted in decreased production of enzyme. Similar results have been obtained with other Bacillus sp. (Moon and Parlukar, 1991; Razak et al., 1994). Since molasses and wheat bran, studied independently, were found to be best carbon and nitrogen sources for alkaline protease production, respectively, the mutant was grown in diVerent combinations of carbon and nitrogen sources. Molasses (1% sugar w/v) as carbon sources was combined with diVerent nitrogen sources in the basal medium (Table 3). Results showed that molasses–wheat bran medium supported better production (p < 0.002) of
Shikha et al. / Bioresource Technology 98 (2007) 881–885
protease (285 U/ml), which was followed closely by molasses containing MgSO4 + KH2PO4 (254 U/ml) but devoid of any nitrogen source and crude molasses without any supplement (242 U/ml). Although, crude molasses can serve as a self-supporting medium for protease production, the role of Mg2+ and K+ salts could not be ruled out, as slightly better production was exhibited when molasses was supplemented with MgSO4 and KH2PO4 (Ellaiah et al., 2002). The overall alkaline protease production was maximum at 30 °C over 48 h of incubation whereas, at 40 °C the same was obtained over 24 h of incubation which may be attributed to partial denaturation of the enzyme with increased period of incubation. The protease activity was found to be optimum at 1.5% substrate concentration and was stable over wide temperature (30–60 °C) and pH (7–10.7) range. The optimum protease activity was observed at pH 8.4 and temperature 40 °C. The enzyme activity was enhanced in the presence of Fe2+, Ca2+ and Mg2+ whereas; Ni2+, Mn2+ and Hg2+ inhibited the enzyme activity. The enzyme was compatible with the locally available detergents such as Surf excel, Hindustan Lever Ltd., 165/166, Backbay Relamation, Mumbai; Aerial, Proctor & Gamble Home Product Limited, Mumbai; Wheel, Rohini Surfactant Limited, 236, Chauepur Kalan, Kanpur and Sipahi, Sipahi Industries, Pandu Nagar Kanpur. The protease activity increased with the increase in substrate (casein) concentration from 2.5 to 15 mg/ml, beyond this, there was no signiWcant increase in activity (p > 0.002). From a double reciprocal (Lineweaver–Burk) plot, Michaelis Menten constant (Km) for this enzyme was calculated as 11 mg/ml and its Vmax was 380 g tyrosine/ml/min. The optimum pH and temperature for in vitro enzyme was pH 8.4 and 40 °C. However, the enzyme was found to be stable over wide temperature (30–60 °C) and pH (7–11) range. Thermophilic bacilli, which grow and produce enzyme at 50–55 °C and pH 6–7 have been found to produce enzymes, which prefer alkaline pH and high temperature than the growth temperature for activity (Sonnleitner, 1983). The optimum concentration of casein for enzyme reaction was 1.0% (w/v). Besides pH and temperature stability an optimal detergent protease should also be stable in the presence of detergents. In the present case, the enzyme exhibited stability in the presence of diVerent locally available detergents. This is an important property since stable enzymes are generally not available except for a few reports (Gupta et al., 1999). The results of the present study show the potential of using molasses as a substrate in the fermentation media which would not only be an economical substrate but one of the possible ways of utilizing the distillery byproduct. Considering the overall property of diVerent alkaline proteases of microbial origin and their evaluation, the reported alkaline protease could be an ideal detergent additive with
885
regards to pH and temperature stability that might be of commercial interest. Acknowledgements Research grant from Council of ScientiWc and Industrial Research (Scheme No. 38(1008)/01/EMR-II), New Delhi to NSD and to Shikha in the form of Research Associateship is gratefully acknowledged. References Boyer, E.W., Ingle, M.B., Mercer, G.D., 1973. Bacillus alcalophilus sub sp. halodurans sub sp. nov: an alkaline amylase producing, alkalophilic microorganisms. Int. J. Syst. Bacteriol. 23, 238–242. Ellaiah, P., Srinivasulu, B., Adinarayana, K., 2002. A review on microbial alkaline proteases. J. Sci. Ind. Res. 61, 690–704. Fujiwara, N., Ymamoto, K., 1987. Production of alkaline protease in a low cost medium by alkalophilic Bacillus sp. and properties of the enzyme. J. Ferment. Technol. 65, 242–345. Gajju, H., Bhalla, T.C., Agarwal, H.O., 1996. Thermostable alkaline protease from thermophilic Bacillus coagulans PB-77. Ind. J. Microbiol. 36, 153–155. Gupta, R., Gupta, K., Saxena, R.K., Khan, S., 1999. Bleach stable, alkaline protease from Bacillus sp. Biotechnol. Lett. 21, 135–138. Gupta, R., Beg, Q.K., Khan, S., Chauhan, 2002. An overview on fermentation, downstream processing and properties of microbial alkaline protease. Appl. Microbiol. Biotechnol. 60, 381–385. Hagihara, B., Matsubara, H., Nakai, M., Okunuki, K., 1958. Crystalline bacterial proteinase of Bacillus subtilis. J. Biochem. 45, 185–194. Han, X.Q., Damodara, 1998. PuriWcation and characterization of protease Q: a detergent and urea-stable serine endopeptidase from Bacillus pumilus. J. Agric. Food Chem. 46, 3396–3603. Huang, Q., Peng, Y., Li, X., Way, H., Zhang, Y., 2003. PuriWcation and characterization of an extracellular alkaline serine protease with dehairing function for Bacillus pumilis. Curr. Microbiol. 46, 169–173. Manachini, P.L., Fortina, M.G., 1998. PuriWcation in sea water of thermostable alkaline proteases by halotolerant strain of Bacillus licheniformis. Biotechnol. Lett. 20 (6), 565–568. Mehrotra, S., Pandey, P.K., Gaur, R., Darmwal, N.S., 1999. The production of alkaline protease by a Bacillus sp. isolate. Biores. Technol. 67, 201–203. Mishra, M., Goel, R., 1999. Development of a cold resistant mutant of plant growth promoting Pseudomonas Xuorescence and its functional characterization. J. Biotechnol. 75, 71–75. Moon, S.H., Parlukar, S.J., 1991. A parametric study of protease production in batch and fed-batch cultures of Bacillus Wrmus. Biotechnol. Bioeng. 37, 467–483. Puri, S., Beg, Q.K., Gupta, R., 2002. Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Curr. Microbiol. 44, 286–290. Razak, N.A., Samad, M.Y.A., Basri, M., Yunus, W.W.Z.W., Ampon, K., Sallen, A.B., 1994. Thermostable extracellular protease of Bacillus stearothrmophilus: factors aVecting its production. World J. Microbiol. Biotechnol. 10, 260–263. Sonnleitner, B., 1983. Biotechnology of thermophilic bacteria, growth products and application. In: Fiechter, A. (Ed.), Adv. Biotechnol. Springer, Berlin, pp. 70–138. Ward, O.P., 1983. Proteinases. In: Fogaarty, W.M. (Ed.), Microbial Enzymes and Biotechnolgy. Applied Science Publ., New York, pp. 251–317. Yamagata, Y., Arakawa, K., Yamaguchi, M., Kobayashi, M., Ichishima, E., 1994. Functional changes of dextran-modiWed alkaline proteinase from alkalophilic Bacillus sp.. Enzyme Microb. Technol. 16, 99–103.