Ann Microbiol (2012) 62:1255–1268 DOI 10.1007/s13213-011-0371-x
ORIGINAL ARTICLE
Optimization of protease and chitinase production by Bacillus cereus SV1 on shrimp shell waste using statistical experimental design. Biochemical and molecular characterization of the chitinase Olfa Ghorbel-Bellaaj & Laila Manni & Kemel Jellouli & Noomen Hmidet & Moncef Nasri
Received: 6 June 2011 / Accepted: 3 October 2011 / Published online: 26 October 2011 # Springer-Verlag and the University of Milan 2011
Abstract The current increase in the amount of shell waste produced by the shrimp industries has led to the need to find new methods for its disposal. In this study, statistical methodologies were used for chitinase and protease coproduction optimization by Bacillus cereus SV1 in media containing shrimp shell powder. Medium composition and culture conditions were optimized using two statistical methods: Plackett–Burman design was applied to find the key ingredients and conditions for the best yield of enzymes production, and central composite design was used to optimize the levels of the five significant variables: shrimp shell powder (SSP), NH4Cl, CaCl2, K2HPO4 and speed of agitation. The medium optimization resulted in protease and chitinase activities of 8,445.8 U ml−1 and 82.8 mU ml−1, respectively. The crude chitinase exhibited maximal activity at 55°C and pH 7.0 using colloidal chitin as substrate. The metal ions Fe2+ and Mg2+ increased chitinase activity, while Hg2+ strongly inhibited this activity. The nucleotide sequencing of SV1 chitinase revealed an open reading frame containing 2,025 bp and encoding 642 amino acids. Keywords Bacillus cereus . Shrimp shells . Protease . Chitinase . Response surface methodology . Gene sequence O. Ghorbel-Bellaaj (*) : L. Manni : K. Jellouli : N. Hmidet : M. Nasri Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, B.P. 1173, 3038 Sfax, Tunisia e-mail:
[email protected]
Introduction Shrimp shell waste is an important source of bioactive molecules. The major components (on dry weight basis) of shrimp shell waste are proteins (48%), chitin (38%), and minerals (14%) (Wang et al. 2011). Chitin, a highly insoluble biopolymer, is one of the most abundant organic compounds in nature (after cellulose). Chitin is composed of linear chains of ß-1,4-linked N-acetylglucosamine residues that are highly cross-linked by hydrogen bonds. It is a major constituent of the cell walls of many fungi, insect exoskeletons, and crustacean shells (Patil et al. 2000). Degradation of chitin is essentially catalyzed by chitinases (Cohen-Kupiec and Chet 1998). These enzymes are found in bacteria, fungi, virus, and higher plants (Felse and Panda 1999; Kao et al. 2009). Potential application of the chitinolytic enzymes in biotechnology is wide. In fact, these hydrolases could find uses in various fields such as the food, agricultural, and pharmaceutical industries (Wang et al. 2006, 2011). That is why the search for new chitinolytic organisms and/or enzymes is still of interest. Many chitinases derived from different organisms have been purified and their genes analyzed. Almost all the chitinase-producing strains use chitin (or colloidal chitin) as a major carbon source (Wang et al. 2006). However, the utilization of chitinous shellfish waste not only solves environmental problems but also decreases the production costs of microbial chitinases. The production of inexpensive chitinolytic enzymes is an important element in the process (Wang et al. 2008; Wang and Yeh 2008). The
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bioconversion of chitinous materials has been proposed as a waste treatment alternative for the disposal of shellfish waste (Wang et al. 2008; Wang and Yeh 2008). To further enhance the utilization of chitin-containing marine crustacean waste, recently the bioconversion of shellfish chitin waste was investigated for the production of chitinases and/ or proteases (Wang et al. 2008). Proteases constitute one of the most important groups of industrial enzymes, accounting for more than 65% of the total industrial enzyme market (Banik and Prakash 2004). The vast diversity of proteases, in contrast to the specificity of their action, has attracted worldwide attention focused on exploiting their physiological and biotechnological applications (Shikha and Darmwal 2007). Proteases are also envisaged to have extensive applications in the development of environmentally friendly technologies, as well as in several bioremediation processes (Bhaskar et al. 2007; Roberts et al. 2007). In addition, proteases have applications in leather processing, food processing and production of protein hydrolysates (Banik and Prakash 2004). Recently, the application of proteases to the production of certain oligopeptides has received great attention as a viable alternative to a chemical approach (Manni et al. 2010a; Ding et al. 2011). In this study, shrimp shell powder was used as a substrate for the co-production of chitinase and protease by Bacillus cereus SV1. Significant variables influencing enzymes production were identified using the Plackett– Burman design. The levels of the significant variables were further optimized using central composite design (CCD). The biochemical and molecular properties of the extracellular chitinase were also determined.
Material and methods Reagents Casein, ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), acrylamide, ammonium persulfate, N,N,N′,N′-Tetramethylethylenediamine (TEMED) and chitin were purchased from Sigma (St Louis, USA). Hydrochloric acid was from Panreac Quimica (Spain). Coomassie Brilliant Blue R-250 was from BioRad Laboratories (Mexico). Trichloroacetic acid (TCA) was from Carlo Erba Reagenti (Italy). All other reagents were of analytical grade. Preparation of shrimp waste powder The shrimps (Metapeneaus monoceros) shells were procured in fresh condition from a shrimp processing plant located at Sfax city, Tunisia. Prior to use, the shrimp shells
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were washed thoroughly with tap water. The shells were then stored at −20°C until further analysis. The shrimp waste powder (SWP) was prepared in our laboratory according to the method described by Jellouli et al. (2008). Briefly, shrimp waste was washed thoroughly with distilled water and then cooked for 20 min at 100°C. The solid material obtained was dried, minced to obtain a fine powder, and then stored in glass bottles at room temperature. The chemical composition (proteins, chitin, lipids, and ash) was determined. Bacterial strain The strain used in this study was isolated from an oil sewage station from a fishing port of Sfax in Tunisia. It was identified as Bacillus cereus SV1 based on its morphological and physiological characteristics and 16S rRNA sequence analysis. The nucleotide sequence of 16S rRNA has been submitted to the GenBank database and assigned accession number EU327888 (Manni et al. 2008). Protease activity Protease activity was determined by the method described by Kembhavi et al. (1993) using casein as a substrate. A 0.5-ml aliquot of the culture supernatant, suitably diluted, was mixed with 0.5 ml of 100 mM TrisHCl buffer (pH 8.0) containing 2 mM CaCl2 and casein (1%; w/v), and incubated for 10 min at 60°C. The reaction was stopped by the addition of 0.5 ml of TCA (20%; w/v). The mixture was further incubated at room temperature for 15 min and then the acid soluble material was estimated spectrophotometrically at 280 nm after removing the precipitate by centrifugation at 13,000 rpm for 15 min. A standard curve was performed using solutions of 0– 50 mg l−1 tyrosine. One unit of protease activity was defined as the amount of enzyme which liberates 1 μg of tyrosine per minute under the experimental conditions used. Chitinase activity Colloidal chitin (0.5% in 50 mM Tris-HCl buffer pH 7.0) was used as the substrate for the measurement of chitinase activity. The mixture of enzyme solution (0.4 ml) and substrate (0.4 ml) was incubated at 55°C for 5 min. After centrifugation, the amount of reducing sugar produced in the supernatant was determined by the colorimetric assay method using dinitrosalicylic acid (DNS) reagent (Miller 1959), with N-acetylglucosamine as a reference compound. One unit of enzyme activity was defined as the amount of enzyme that catalyses the release of 1 μmol of NAG per minute.
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Table 1 Experimental variables at different levels used for the coproduction of chitinase and protease by B. cereus SV1 using Plackett– Burman Variable
SSP NH4Cl MgSO4 CaCl2 K2HPO4 KH2PO4 Temperature Speed of agitation Incubation period Inoculum size Initial pH
Unit
g l−1 g l−1 g l−1 g l−1 g l−1 g l−1 °C rpm h % -
Symbol code
X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11
Exprimental value Lower
Center
Higher
10 2 0.05 0.5 0.05 0.05 30 150 36 1 6.0
35 6 0.525 0.75 0.525 0.525 35 200 67 3.5 8.0
60 10 1 1 1 1 40 250 98 6 10.0
SSP: shrimp shell powder
Identification of the significant factors by the Plackett–Burman design The important medium components with respect to their main effects were screened by the Plackett–Burman design with a two-factorial design. It identifies the main physicochemical parameters required for maximal chitinase and protease production by screening n variables in n+1 experiments; each variable was examined at three levels (Plackett and Burman 1946). Table 1 lists the factors under investigation as well as the levels of each factor used in the Table 2 Plackett–Burman experimental design matrix
experimental design with the symbol code and actual level of the variables, whereas Table 2 presents the design matrix. Design Expert1 7.0 (Stat-Ease, Minneapolis, USA), was used to analyse the experimental Plackett– Burman design. Optimization of key ingredients by central composite design (CCD) The levels of five significant factors and the interaction effects between various medium constituents which influence the protease and chitinase production significantly were analyzed and optimized by the response surface methodology, using a CCD. The significant factors utilized were SSP, NH4Cl, CaCl2, K2HPO4 and speed of agitation. In this study, the experimental plan consisted of 43 trials and the independent variables were studied at three different levels: low, middle and high (Table 3). All the experiments were done in triplicate and the average protease and chitinase enzymes production obtained were taken as the dependent variables or responses. The data obtained from CCD on protease and chitinase production were subjected to analysis of variance (ANOVA). A second-order polynomial equation was then fitted to the data by multiple regression procedure. This resulted in an empirical model that related the response measured in the independent variables to the experiment. For a five-factor system, the model equation was: Y ¼ b0 þ
X
b i xi þ
X
bii xi 2 þ
X
bij xi xj
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
1 2 3 4 5 6 7
1 1 1 0 −1 1 0
1 −1 −1 0 1 −1 0
1 1 1 0 1 −1 0
−1 1 1 0 1 −1 0
−1 −1 1 0 −1 1 0
−1 1 −1 0 −1 −1 0
1 1 −1 0 −1 1 0
−1 1 −1 0 1 1 0
1 −1 1 0 −1 −1 0
1 −1 −1 0 1 1 0
−1 −1 1 0 1 1 0
8 9 10 11 12 13 14
−1 0 −1 −1 0 0 −1
−1 0 −1 1 0 0 −1
−1 0 1 1 0 0 −1
1 0 −1 −1 0 0 −1
−1 0 1 1 0 0 −1
1 0 1 1 0 0 −1
1 0 −1 1 0 0
−1 0 1 −1 0 0
1 0 1 −1 0 0
1 0 1 −1 0 0
1 0 −1 1 0 0
15 16 17
1 −1 1
1 1 1
−1 −1 −1
−1 1 1
−1 1 1
1 −1 1
−1 −1 1 −1
−1 1 1 −1
−1 1 1 −1
−1 −1 −1 1
−1 1 −1 −1
1258 Table 3 Experimental conditions in coded variables of the central composite design and the corresponding experimental responses
Ann Microbiol (2012) 62:1255–1268
Run
Variable
Response
X1
X2
X4
X5
X8
Protease (U ml−1)
Chitinase (mU ml−1)
1 2 3 4 5 6 7 8 9 10
1 −1 −1 −1 −1 1 −1 −1 −1 −1
1 −1 1 1 −1 1 −1 1 1 −1
−1 1 1 −1 1 −1 1 1 −1 1
1 −1 −1 1 1 1 −1 −1 1 1
−1 −1 1 1 1 −1 −1 1 1 1
8,975 5,512 8,500 6,000 5,527 9,222 4,553 8,000 6,400 4,880
42 22 14 77 30 43 18 22 65 28
11 12 13 14 15 16 17 18
0 −1 0 1 1 1 1 1
0 1 0 −1 −1 1 −1 −1
0 −1 0 −1 −1 −1 −1 −1
0 −1 0 −1 1 −1 −1
0 −1 0 −1 1 1 −1
7,864 9,629 8,494 7,460 9,050 8,320 8,200
41 27 42.5 8 75 86 8
19 20 21 22 23 24 25 26 27 28 29
1 0 −1 1 −1 1 0 0 1 1 0
1 0 1 −1 1 −1 0 0 1 1 0
−1 0 1 1 1 1 0 0 1 1 0
1 −1 0 1 1 1 1 0 0 −1 −1 0
1 1 0 −1 −1 −1 −1 0 0 −1 −1 0
9,658 8,552 8,370 4,043 6,880 5,700 7,200 8,509 8,143 5,963 5,760 8,223
68 79 37.5 18 29 20 23 39 40 43.5 43.5 40
30 31 32 33 34 35 36 37 38 39 40 41 42 43
−1 1 −1 1 1 −1 1 1 0 0 0 0 0.5 0.5
−1 −1 −1 1 −1 −1 1 0 1 0 0 0 0.5 0.5
−1 1 −1 1 1 −1 1 0 0 1 0 0 0.5 0.5
−1 −1 1 1 −1 1 1 0 0 0 1 0 0.5 0.5
1 1 −1 1 1 −1 1 0 0 0 0 1 0.5 0.5
9,076 8,580 5,120 8,190 8,000 5,760 7,300 8,436 7,781 6,821 7,141 7,781 7,534 7,720
14.5 25.5 49 10 25.5 39 17 42.5 40 27 38 43.5 31 29
where Y is the predicted response, ß0, ßi, ßii, ßij are regression coefficients of the model for the intercept, linear,
squared and interaction effect, respectively, and xi, xj are the coded independent variables.
Ann Microbiol (2012) 62:1255–1268 Table 4 Identification of significant variables for enzyme production by B. cereus SV1 using the Plackett–Burman design
Variable
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Chitinase
Protease
Effect
t ratio
P value
Effect
t ratio
P value
Intercept X1 X2 X3 X4 X5 X6 X7 X8 X9
42.44 7.75 −11.25 0.58 −6.5 9.58 −2.42 −0.5 11.83 −2.92
29.68 4.55 −6.61 0.34 −3.82 5.63 −1.42 −0.29 6.95 −1.71
