Biotechnology and Bioprocess Engineering 16: 669-678 (2011) DOI 10.1007/s12257-010-0410-7
RESEARCH PAPER
Alkaline Proteases Produced by Bacillus licheniformis RP1 Grown on Shrimp Wastes: Application in Chitin Extraction, Chicken Featherdegradation and as a Dehairing Agent Anissa Haddar, Noomen Hmidet, Olfa Ghorbel-Bellaaj, Nahed Fakhfakh-Zouari, Alya Sellami-Kamoun, and Moncef Nasri
Received: 14 November 2010 / Revised : 21 February 2011 / Accepted : 8 March 2011 © The Korean Society for Biotechnology and Bioengineering and Springer 2011
Abstract The current increase in the amount of shrimp several biotechnological processes.
wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, Bacillus licheniformis RP1 was shown to produce proteases when grown in media containing shrimp wastes powder as a sole carbon and nitrogen source, indicating that this bacteria could obtain its carbon and nitrogen requirements directly from shrimp wastes. The maximum protease production was obtained when the strain was grown in a medium containing (g/L): shrimp wastes powder 30, KCl 1.5, K2HPO4 0.5, and KH2PO4 0.5. Using casein zymography, the crude protease preparation was found to produce at least seven proteases. The proteases of B. licheniformis RP1 were tested for shrimp waste deproteinization in the preparation of chitin. The percent of protein removal after 3 h hydrolysis at 60°C and at an enzyme/ substrate (E/S) ratio of 0.5 and 5 (Unit of enzyme/mg of protein) were about 68 and 81%, respectively. Additionally, B. licheniformis RP1 showed important feather degrading activity. Complete solubilisation of whole feathers was observed after 24 h of incubation at 50°C. More interestingly, the RP1 proteolytic preparation demonstrated powerful dehairing capabilities for hair removal from skin. Collagen, which is the major leather-forming protein, was not significantly degraded. Considering its promising properties, B. licheniformis RP1 enzymatic preparation may be considered a potential candidate for future use in Anissa Haddar*, Noomen Hmidet, Olfa Ghorbel-Bellaaj, Nahed FakhfakhZouari, Alya Sellami-Kamoun, Moncef Nasri Laboratoire de Génie Enzymatique et de Microbiologie - Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, B. P. 1173-3038 Sfax, Tunisia Tel: +216-74-274-088, Fax: +216-74-275-595 E-mail:
[email protected]
Keywords: shrimp wastes, Bacillus licheniformis RP1,
enzymatic deproteinization, keratine-degradation, dehairing function
1. Introduction Proteases represent an important class of enzymes, which constitute more than 50% of the total industrial enzyme market [1]. Recently, the use of alkaline proteases has increased significantly in various industrial process, including detergents [2], feed additives, dehairing, leather preparation, decomposition of gelatin on X-ray films, and shrimp waste deproteinization for the production of chitin [3]. Growth and enzyme production by microorganisms are greatly inuenced by chemical factors such as pH, temperature, inoculum density and incubation time, and by the composition of the medium, especially the carbon and nitrogen sources [4,5]. The growth substrates constitute a major cost in the production of bioproducts through the fermentation process. In most instances the growth medium accounts for approximately, 30 ~ 40% of the production cost of industrial enzymes [6]. Thus, the development of cost-effective growth medium can significantly reduce the cost of enzyme production [7]. Protease production from species of Bacillus using various agricultural residues (such as soybean meal, rice bran, and wheat flour) or marine byproducts has been widely described in literature. Naidu and Devi [8] reported that Bacillus sp. K30 produced thermostable alkaline protease utilizing rice bran. In addition, Joo
670
and Chang [9] showed that maximum protease synthesis by alkalophilic Bacillus sp. I-312 was obtained when the bacterium was grown in a medium containing wheat flower and soybean meal as the carbon and nitrogen sources, respectively. Soybean meal was also used as a nitrogen source for protease production by Bacillus sp. L21 in low cost producing media [10]. Protease production was the highest for Bacillus mojavensis A21 [11] and Bacillus subtilis A26 strains [12] when grown on the hulled grain of wheat. Fish by-products have only been used as a fermentation substrate to a minor extent for protease production, despite its availability in large quantities and low cost. Ellouz et al. [13] have shown that protease synthesis was strongly induced when B. subtilis was grown in media containing only sardinelle heads and viscera powder. Shrimp by-products have been shown to hold great promise for use as an animal protein source and as an important source of chitin and asthaxanthin [14]. Only 65% of shrimp is edible and the remainder is discarded as inedible waste (cephalothorax and exoskeleton). Over the years, techniques have been developed to recover and exploit these by-products in valuable biopolymers such as chitin and chitosan [15,16]. These biomolecules are widely used in the food industry, pharmacy, textiles, chemical industries, etc. [17]. B. licheniformis RP1 strain, producing an alkaline proteases, was isolated from polluted water [18]. The crude extracellular protease produced by the isolate had optimal activity at 70°C and pH 10.0 ~ 11.0. More interestingly, the RP1 crude enzyme showed remarkable stability in a wide range of ingredients currently used in bleach-based detergent formulations. The present paper describes the production of alkaline proteases from B. licheniformis RP1 grown on shrimp wastes powder. The dehairing capacity, as well as the ability of RP1 proteases to accomplish the whole keratin-degradation of various keratinacious wastes and its potentiel application in the deproteinization of shrimp wastes were also investigated.
Biotechnology and Bioprocess Engineering 16: 669-678 (2011)
ly with tap water and then incubated 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 protein content was determined to be 40.83 ± 1.22% of the dry weight. The fat, ash, and carbohydrate content of the material were 6.26 ± 0.22%, 34.69 ± 0.19%, and 20 ± 1.41%, respectively [3]. To obtain M. jalapa tuber powder (MJTP), the raw material was peeled, grinded, and then dried at 80°C for at least 5 h. The dried preparation was minced again to obtain a fine powder and then stored in glass bottles at room temperature. The MJTP contained 32.6 ± 2% starch, 17.3 ± 3% proteins, 20 ± 2% ash, and low content of lipids [19]. Feather meal (FM) was obtained as described by Fakhfakh-Zouari et al. [20]. Chicken feather was washed thoroughly with tap water, dried at 105°C, and then grinded. The FM contained 71.48% proteins, 3% lipids, and 0.73% ash. Hulled grain of wheat (HGW) is a by-product of semolina factories and contains 50 ~ 60% starch, 8 ~ 12% proteins, 15% cellulose, and 5% carbohydrates.
2.3. Cultivation and media
Inocula were routinely grown in Luria-Bertani (LB) broth medium composed of (g/L): peptone, 10; yeast extract, 5; and NaCl, 5 [21]. The initial medium used for protease production (M1) was composed of the following (g/L): carbon source, 10; ammonium sulfate, 2; K2HPO4, 0.5; KH2PO4, 0.5; and KCl, 1.5; pH 8.0. Media were autoclaved at 120°C for 20 min. Cultivations were conducted in 25 mL of medium in 250 mL conical flasks maintained at 37°C. Cells were grown under agitation at 200 rpm for 24 h. The growth of the microorganism was estimated using the total plate count method on nutrient agar. This procedure involves making decimal serial dilutions of the sample in sterile physiological water. Nutrient agar plates were then incubated at 37°C for 24 h. The cultures were centrifuged and the supernatants were used to estimate proteolytic activity.
2.4. Assay of protease activity 2. Materials and Methods
2.1. Bacterial strain
The microorganism used was an alkaliphilic bacterium, which was isolated from polluted water in Sfax city (Tunisia). It was identied as B. licheniformis RP1 [18].
2.2. Preparation of complex organic substrates
Mirabilis jalapa tuber powder (MJTP), shrimp wastes
powder (SWP), and feather meal (FM) were prepared in our laboratory. To obtain SWP, raw material collected from the marine food processing industry was washed thorough-
Protease activity was measured using casein as a substrate [22]. A 0.5 mL aliquot of the culture supernatant, suitably diluted, was mixed with 0.5 mL of 100 mM glycine-NaOH buffer (pH 10.0) containing 1% (w/v) casein, and incubated for 15 min at 70°C. The reaction was stopped by the addition of 0.5 mL of trichloroacetic acid (20%, w/v). The mixture was allowed to stand at room temperature for 15 min and then centrifuged at 13,000 rpm for 15 min to remove the precipitate. A blank was treated in the same manner, except that 0.5 mL of 100 mM glycine-NaOH buffer (pH 10.0) was used instead of the crude enzyme. The absorbance of the supernatant was measured at 280
Alkaline Proteases Produced by Bacillus licheniformis RP1 Grown on Shrimp Wastes: Application in Chitin Extraction, Chicken… 671
nm. A standard curve was generated using solutions of 0 ~ 50 mg/L tyrosine. One unit of protease activity was defined as the amount of enzyme required to liberate 1 µg of tyrosine per minute under the experimental conditions used. Protease activities were determined from the mean of at least two determinations that were carried out in duplicate, and the differences between the values did not exceed 5%.
2.5. Keratinolytic activity
Keratinase activity was determined using keratin as a substrate [23]. The reaction mixture consisted of 0.5 mL of 100 mM glycine-NaOH buffer (pH 10.0) containing 0.8% (w/v) keratin from hoofs and horns (Sigma-Aldrich) and 0.5 mL of suitably diluted crude enzyme. After 1 h of incubation at 60°C, the enzyme reaction was stopped by the addition of 0.4 mL of 10% trichloroacetic acid (TCA). The samples were left at 4°C for 15 min and then centrifuged at 10,000 rpm for 15 min. The absorbance of the supernatant was measured at 280 nm and compared to the control. The control was treated in the same manner, except that TCA was added before incubation. One unit (U) of keratinolytic activity was dened as the increase of corrected absorbance at 280 nm by 0.1 under the conditions described. The data presented are the mean value of two parallel determinations.
Enzyme/Substrate ratios (Unit of enzyme/mg of protein) for various lengths of time. The reaction was then stopped by heating the solution at 90°C for 25 min to inactivate the enzyme. The shrimp waste protein hydrolysates were then centrifuged for 20 min at 5,000 rpm. The solid phase was washed and pressed manually through four layers of gauze. The protein content was analyzed to determine the protein removal rate. The press cake was packed in a plastic bag and stored at –20°C until further processing. The deproteinization percentage (%DP) was calculated using the following equation as described by Rao et al. [25]. %DP =
PO O PR R PO O
[( × )–( × ) ] × 100 ------------------------------------------------------------×
where PO and PR are the protein concentrations (%) before and after hydrolysis; while, O and R are the mass (g) of the original sample and hydrolyzed residue on a dry weight basis, respectively.
2.8. Keratin-degradation determination
The keratin-degradation ability of B. licheniformis RP1 was investigated using whole chicken feather, feather meal, human hair, and sheep wool as keratinacious materials. Chicken feathers were collected from a local slaughter2.6. Detection of protease activity by casein zymography house, rinsed to remove excess blood, and autoclaved for Protease activity staining was performed on sodium do- sterilization. They were then shaking-incubated separately decyl sulphate-polyacrylamide gel electrophoresis (SDS- in medium at 30 g/L for 48 h at 37°C. The soluble protein PAGE) according to the method described by Garcia- concentration was measured as described by Gornall [26]. Carreno et al. [24] with a slight modification. The sample The concentration of amino acids and peptides was was not heated before electrophoresis. After electro- determined colorimetrically using the ninhydrin method phoresis, the gel was submerged in 100 mM glycine- [27]. Keratin in cultures was harvested by filtration through NaOH buffer (pH 10.0) containing 2.5% Triton X-100, Whatman no. 3 filter paper, washed twice with distilled with shaking for 30 min to remove SDS. Triton X-100 was water, and dried at 105°C to a constant weight. The removed by washing the gel three times with 100 mM percentage of keratin degradation was calculated from the glycine-NaOH buffer (pH 10.0). The gel was then incubat- differences in residual keratin dry weight between the ed with 1% (w/v) casein in 100 mM glycine-NaOH buffer control (keratin without bacterial inoculation) and treated (pH 10.0) for 30 min at 50°C. Finally, the gel was stained samples. Disintegration of whole chicken feathers was with 0.25 g Coomassie, Coomassie Brilliant Blue R-250, in assessed by incubation with the RP1 proteolyttic extract 45% ethanol-10% acetic acid and destained with 5% (7,000 U casein activities) at different temperatures ranging ethanol-7.5% acetic acid. The development of clear zones from 40 to 70°C. on the blue background of the gel indicated the presence of protease activity. 2.9. Dehairing test Pieces of bovine skin with hair (5 × 5 cm) were incubated 2.7. Enzymatic deproteinization of shrimp waste with 7,000 U/mL of RP1 proteases at 25, 30, and 37°C Shrimp shell waste (15 g) were mixed with water at a ratio under shaking conditions (150 rpm). After 24 h of of 1:3 (w/v), minced and then cooked for 20 min at 90°C. incubation, the skin pieces were removed and the hair was The cooked sample was then homogenized in a Moulinex gently pulled by hand. The dehairing efficacy was assessed blender for about 2 min. The pH of the mixture was according to the depilated area of the skin at the end of the adjusted to 9.0, and then the shrimp waste proteins were process and the quality of the dehaired skin was estimated digested by the RP1 crude enzyme at 60°C using different by the naked eye after treatment. ®
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Biotechnology and Bioprocess Engineering 16: 669-678 (2011)
Effects of different carbon sources on alkaline protease production by B. licheniformis RP1 Carbon source Glucose Maltose Lactose Sucrose Starch HGW MJTP FM SWP Protease 11 ± 0.28 13 ± 0.32 52 ± 1.3 15 ± 0.37 81 ± 2.0 25 ± 0.62 96 ± 2.5 211 ± 5.5 435 ± 11 activity (U/mL) Biomass (CFU/mL×10 ) 17 20 60 16 65 22 220 125 × 10 165 × 10 Cultivations were performed for 24 h at 37°C in media that consisted of (g/L): carbon source 10, ammonium sulphate 2, KH PO 0.5, K HPO 0.5, and KCl 1.5. Values are means of three independent experiments. Table 1.
6
2
2
2
HGW: hulled grain of wheat; MJTP: 2
4
Mirabilis jalapa
4
tubers powder; FM: feather meal; and SWP: shrimp wastes powder.
Effect of SWP concentration on protease production by B. licheniformis RP1 [SWP] (g/L) 10 20 30 40 50 Protease activity (U/mL) 430 ± 11 620 ± 16 920 ± 25 910 ± 23 900 ± 22 17 52 105 130 174 Biomass (CFU/mL × 10 )
Table 2.
9
60 850 ± 21 163
70 764 ± 19 152
Cultivations were performed for 24 h at 37°C in media that consisted of (g/L): ammonium sulphate 2, KH2PO4 0.5, K2HPO4 0.5, KCl 1.5, and different concentrations of SWP. Values are means of three independent experiments. 3. Results and Discussion
3.1. Effects of different carbon sources on protease synthesis
Protease production was first tested in medium M1 containing different carbon sources at a concentration of 10 g/L. As shown in Table 1, B. licheniformis RP1 exhibited higher growth and alkaline protease production in culture media containing SWP as the carbon source (435 U/mL) followed by feather meal (211 U/mL) and MJTP (96 U/mL). However, glucose, maltose, sucrose, and HGW reduced growth and protease synthesis. Similarly, production of extracellular proteases from a haloalkaliphilic bacterium S-20-9 was previously shown to be significantly repressed in the presence of carbon sources, such as maltose, glucose, and sucrose [28]. Sanchez-Porro et al. [29] also reported that the addition of maltose, glucose or lactose to the medium repressed protease production by Pseudoalteromonas sp. strain CP76. Since SWP was the best carbon source for proteases synthesis by RP1 strain, the effect of its concentration on alkaline proteases production was tested in media consisting of (g/L): ammonium sulfate, 2; K2HPO4, 0.5; KH2PO4, 0.5; and KCl, 1.5. As shown in Table 2, the production of proteases improved in the presence of SWP at concent-
ration ranging from 20 to 70 g/L, while maximum enzyme activity (920 U/mL) was achieved with 30 g/L SWP. It also worth noting that bacterial growth increased with an increase in the SWP concentration. Several previous studies have also examined the bioconversion of shrimp wastes for the production of proteases. Manni et al. [18] reported that maximum protease activity (5,900 U/mL) was obtained when B. cereus SV1 was grown in medium containing 40 g/L SWP as a sole carbon source. In addition, the use of 60 g/L SWP was previously shown to result in a high protease production by P. aeruginosa MN7 [30]. 3.2. Effects of different nitrogen sources on protease synthesis
The results described above indicate that SWP is an excellent substrate for the growth and proteases production by B. licheniformis RP1. Microorganisms require nitrogen (both organic and inorganic forms) for metabolism to produce primarily amino acids, nucleic acids, proteins, and cell wall components. Alkaline protease production heavily depends on the availability of both carbon and nitrogen sources in the medium and has a regulatory effect on enzyme synthesis [31]. The effects of various nitrogen sources on protease synthesis in media containing 30 g/L of SWP as a carbon source was
Growth and protease production by B. licheniformis RP1 using different nitrogen sources Nitrogen Ammonium Yeast Soy Casein None Ammonium source sulphate chloride extract peptone peptone Protease activity 1,400 926 904 1,390 1,302 1,430 (U/mL) ± 35 ± 23 ± 22.5 ± 34 ± 33 ± 36 Biomass 198 108 90 245 239 266 (CFU/mL × 10 )
Table 3.
9
Casein
Gelatin
Urea
1,410 ± 35 259
1,213 ± 30 237
1104 ±28 110
Cultivations were performed for 24 h at 37°C in media that consisted of (g/L): SWP 30, nitrogen source 2, KH2PO4 0.5, K2HPO4 0.5, and KCl 1.5. Values are means of three independent experiments.
Alkaline Proteases Produced by Bacillus licheniformis RP1 Grown on Shrimp Wastes: Application in Chitin Extraction, Chicken… 673
Effects of KCl and K HPO /KH PO concentrations on protease production by B. licheniformis RP1 [KCl] (g/L) [K HPO ] and [KH PO ] (g/L) 0 0.5 1.0 1.5 2 3 0 0.5 1.0 1.5 2 Activity 740 800 1,250 1,405 1,410 1,425 796 1,410 1,413 1,425 1,420 (U/mL) ± 18 ± 20 ± 31 ± 35 ± 35 ± 35 ± 20 ± 35 ± 35 ± 36 ± 35 Biomass 90 100 150 197 200 225 100 202 210 226 225 (CFU/mL × 10 )
Table 4.
2
4
2
4
2
4
2
4
9
Cultivations were performed for 24 h at 37°C in media that consisted of (g/L): SWP 30 and different concentrations of KCl and KH2PO4/K2HPO4. Values are means of three independent experiments.
also examined (Table 3). Interestingly, a high protease activity (1,400 U/mL) was observed during culture in media not containing a nitrogen source (control). The levels of proteases synthesis in the presence of yeast extract, casein peptone or casein were the same as those obtained without the addition of a nitrogen source. Soy peptone, gelatin, and urea decreased the yield of alkaline extracellular proteases. Proteases production by RP1 in media containing SWP as the sole organic substrate indicates that it can obtain its carbon and nitrogen directly from this substrate. The addition of inorganic nitrogen sources (ammonium chloride or ammonium sulfate) decreased enzyme production. Similarly, specific repression by ammonium ion has been reported earlier for some proteases [32,33]. The results described above are some what different from those described by Manni et al. [18] who reported the stimulation of protease synthesis by the B. cereus SV1 strain when ammonium chloride was used as a nitrogen source. These results indicate that SWP is an excellent substrate for the growth and protease production by B. licheniformis RP1. Since SWP can be obtained at low cost, its use as a carbon and nitrogen source instead of commercial substrates may considerably reduce the cost of enzyme production and could promote the growth of new industrial applications.
3.3. Effects of the concentration of KCl and K2HPO4/ KH2PO4 on protease production
Previous studies have demonstrated that extracellular protease secretion by micro-organisms is substantially influenced not only by carbon and nitrogen sources, but also by salts [34,35]. The effect of adding KCl on protease synthesis was studied. The optimum concentration of KCl was determined to range from 1.5 to 3.0 g/L (Table 4). The addition of 1.5 g/L of KCl enhanced production up to 1,405 U/mL which was approximately 1.9 fold higher than that obtained using medium without KCl. Similar to our results, several previous studies have reported that KCl is necessary for protease production by B. licheniformis NH1 [36] and B. pumilus A1 [20]. The effect of phosphorus concentration was also examined (Table 4). The addition of K2HPO4 and KH2PO4 to
the medium enhanced growth and protease production. The maximum protease activity was observed when the phosphorus concentration was between 0.5 and 2.0 g/L. However, in the absence of K2HPO4 and KH2PO4, the level of proteases synthesis was only 796 U/mL. A similar enhancement of protease synthesis by phosphorus was reported by Moon and Parulekar [37] for Bacillus firmus. These results are, however, in contrast to the results reported for alkaline protease production by haloalkaliphilic Bacillus sp., in which the addition of KH2PO4 to the medium was found to repress protease synthesis [38].
3.4. Effects of metal ions on protease production
Several studies have shown that divalent cations can stimulate or inhibit enzyme formation by microorganisms. Mabrouk et al. [39], reported that Ca2+ at 0.07% markedly affected protease production by B. licheniformis ATCC 21415 and caused a 26.6% increase in activity over the control. In order to investigate the effects of metal ions on protease production by B. licheniformis RP1, these molecules were individually added at a concentration of 0.5 g/L to medium containing (g/L): SWP 30, 2; K2HPO4, 0.5; KH2PO4, 0.5; and KCl, 1.5. The addition of CaCl2, MgSO4, and NaCl had no effect on protease production (data not shown). These results indicate that the strain can obtain its salts requirements (in particular Ca2+) directly from the SWP. In fact, Manni et al. [18] reported that the mineral content in the SWP was as follows: Ca2+ (13.45%), Na+ (0.58%), Mg2+ (0.12%), and K+ (0.07%). A slight decrease in protease yield was observed with supplementation of BaCl2 and MnSO4; however, growth of the RP1 strain was inhibited and no extracellular protease activity was detected when the production medium was supplemented with ZnCl2 and CuSO4 (data not shown). Ghorbel-Frikha et al. [40] reported that CaCl2 led to a strong increase in protease synthesis by B. cereus BG1, while ZnSO4, CuSO4, and MnSO4 affected growth and protease production. Fakhfakh-Zouari et al. [20] also showed that the addition of CaCl2, CuSO2, ZnSO4, BaCl2, and MnSO4 to media containing feather meal as the corbon source decreased the production of keratinases by B. pumilus A1.
674
Time courses of protease production and growth of B. licheniformis RP1 in 100 mL of culture medium that consists of Fig. 1.
(g/L): SWP 30, KH2PO4 0.5, K2HPO4 0.5, and KCl 1.5. Protease activity was determined in culture filtrates obtained after removal of cells by centrifugation, as described in section 2. Values are the means of three independent experiments.
3.5. Effect of temperature on protease production
Temperature is a critical factor that should be controlled and the optimal temperarure will vary from one organism to another [41]. Protease production was investigated in the temperature range of 25 ~ 45°C. Maximum activity and growth were observed at 37°C. The protease activities were about 612, 483, and 443 U/mL at 30, 40, and 45°C, respectively (data not shown). Joo and Chang [8] reported that the optimum temperature for protease production by Bacillus sp. I-312 was 32°C. Temperature strongly affects the synthesis of protease, either non-specifically by influencing the rates of biochemical reactions or specifically by inducing or repressing their production [42]. Temperature was also shown to regulate protease synthesis at the mRNA transcription and probably the translation levels [43]. Temperature also influences the secretion of extracellular enzymes possibly by changing the physical properties of the cell membrane [42].
3.6. Pattern of protease production by B. licheniformis RP1
The time courses of protease production and growth of the RP1 strain in optimized medium composed of 30 g/L SWP were studied. As reported in Fig. 1, the strain grew well in the medium. The biosynthesis of protease by the strain appeared to be growth-related, since activity was detected during the early stages of the growth, which exponentially increased at the end of the exponential phase of growth and then decrease during the stationary phase. These results are in line with those of Joshi et al. [28], which showed that alkaline protease production by the S-20-9 strain started at the beginning of the stationary phase and increased during the middle of the stationary phase. However, many previ-
Biotechnology and Bioprocess Engineering 16: 669-678 (2011)
ous studies reported that protease production ends before the stationary phase [22]. Zymogram analysis was conducted to obtain more information on the diversity of the extracellular proteases secreted by B. lichenifomis RP1. Many enzymes, such as proteases and α-amylases, have proven to be renaturable after electrophoresis in the presence of SDS [44]. Seven proteases were observed in the proteolytic activity profile of the cell-free enzymatic preparation of B. licheniformis RP1 grown on SWP medium (data not shown). Similarly, Agrebi et al. [11] and Haddar et al. [45] reported the production of at least seven and six proteases by Bacillus subtilis A26 and Bacillus mojavensis A21, respectively.
3.7. Deproteinization of shrimp wastes, feather disintegration, and hide depilation with RP1 proteases
The bioconversion of shrimp wastes and keratinous residues is attracting increasing biotechnological interest since it might represent an alternative method of waste management that could be coupled with the production of valuable products. Thus, these waste materials can be converted into economically useful products. The crude protease from B. licheniformis RP1 was tested in the deproteinization of shrimp wastes in order to extract chitin. Chitin and its derivatives hold great economic value because of their versatile biological activities and agrochemical applications. In addition, the keratinolytic activity of RP1 proteases was used for whole chiken feathers disintegration to produce fertilisers and amino acids and in the pre-tanning operations of the leather industry, for eco-friendly processing of hide by reducing the use of sodium sulfide during the dehairing process.
3.7.1. Enzymatic deproteinization of shrimp wastes Chitin in the exoskeleton of shrimp shells is closely associated with proteins. Therefore, deproteinization in the chitin extraction process is crucial. Conventionally, to extract chitin from crustacean shells, chemical processing for demineralization and deproteinization has been applied by treatment with strong acids and bases to remove calcium carbonate and proteins, respectively [46]. However, the use of these chemicals may destroy the chitin. Some efforts have been directed towards reducing chemical treatments in more eco-friendly processes such as bacterial fermentation [47] and treatment by proteolytic enzymes [47,48] which have been applied for the deproteinization of crustacean wastes. Many reports have demonstrated that proteolytic microorganisms can be used for the deproteinization of marine crustacean wastes to produce chitin [49-51]. However, few studies have been reported on the use of proteolytic enzymes for the deproteinization of crustacean wastes.
Alkaline Proteases Produced by Bacillus licheniformis RP1 Grown on Shrimp Wastes: Application in Chitin Extraction, Chicken… 675
64% deproteinization was achieved by using purified microbial protease under the same condition. The fact that 100% deproteinization can not be reached indicates that the enzymes may not be accessible to some proteins protected by chitin. 3.7.2. Degradation of chicken feathers
B. licheniformis RP1 strain was also found to grow and
Fig. 2.
waste.
Effect of the E/S ratio on the deproteinization of shrimp
The RP1 strain was found to grow well and produce proteases when cultivated in media containing only shrimp waste powder, indicating that it can deproteinize crustacean wastes to obtain its carbon and nitrogen requirements. Therefore, the crude enzyme from B. licheniformis RP1 was applied for the deproteinization of shrimp waste to produce chitin. The chitinolytic activity test showed that the strain RP1 exhibited prominent clear zone around the colony on a shrimp plate indicating that it is secretes chitinolytic enzymes (data not shown). E/S ratios between 0.5 and 15 (Unit of enzyme/mg of protein) were used to compare the deproteinization efficiency. As shown in Fig. 2, the deproteinization rate at a ratio of 0.5 was 60%. The percentage of protein removal increased with increasing E/S ratio and reached about 81% with E/S = 5. Beyond a ratio of 5, no significant increase in the deproteinization rate was observed. The deproteinization activity of RP1 crude protease was better than many proteases reported in many previous studies. The percentage of protein removal from natural shrimp waste was 78% after seven days of incubation at 37°C with the culture supernatant from Pseudomonas aeruginosa K187 [47]. Bustos and Healy [52] compared the effects of microbial and enzymatic deproteinization. A maximum deproteinization value of 82% was achieved with Pseudomonas maltophilia after 6 days of incubation, but no more than
produce proteases in medium containing feather-meal, chicken feather, and sheep wool or humain hair, as the sole carbon and nitrogen source. Of the keratin substrates tested, feather-meal was the most strongly degraded (90%), followed by sheep wool (80%) and chicken feather (75%), whereas humain hair showed a relatively low degradation rate (8%). As shown in Table 5, feather-meal and sheep wool gave the maximum level of RP1 protease production, which displayed a caseinolytic activity of 380 and 330 U/ mL, respectively; while chicken feather supported only a low activity (165 U/mL). The highest keratinolytic activity (25 U/mL) was also detected in the culture medium containing 30.0 g/L feather-meal. Usually, the rate of hair keratin degradation is inferior to that of feather keratin. Keratins are classified into α-keratin (α-helix) and βkeratin (β-sheet), which is based on their secondary structural conformation. The former is the major component of hair while the latter is the main constituent of feathers. These structural features may explain the differences between hair and feather degradation by B. licheniformis RP1. In addition to the keratin-degradation, an increase in the protein level and amino acids were observed (Table 5). Higher keratin-degradation resulted in high amino acid and peptide formation. Since this strain displayed a high feather-degrading activity, the RP1 enzyme preparation was then investigated for the hydrolysis of chiken feathers. Disintegration of whole chicken feathers was assessed by incubation with the RP1 enzymatic extract (7,000 U using casein as a substrate) for 24 h at temperatures ranging from 40 to 70°C. Complete solubilisation of the chicken feathers was observed at 50 and 60°C (Fig. 3). Commercial Subtilisin Carlsberg was used under the same conditions as RP1 proteases. The data presented show that the RP1 proteolytic preparation was
Effects of keratinacious substrates on keratinolytic alkaline protease of B. licheniformis RP1 Keratinacious Caseinolytic Keratinolytic Soluble Keratinsubstrates activity activity proteins degradation (30 g/L) (U/mL) (U/mL) (mg/mL) (%) Feather meal 380 ± 10 .25 ± 0.65 4.37 90 Chicken feather 165 ± 4.0 .16 ± 0.4 3.9 75 Sheep wool 330 ± 8.25 .22 ± 0.6 4.1 80 Human hair 20 ± 0.5 1.5 ± 0.037 1 8
Table 5.
Amino-acids and peptides (mg/mL) 21.5 3 12 0.5
Cultures were conducted for 48 h at 200 rpm in media consisting of (g/L): Keratinacious substrates 30, KH2PO4 0.5, K2HPO4 0.5, and KCl 1.5.
676
Disintegration of chicken feathers using the culture supernatant of B. licheniformis RP1 at different temperatures for 24 h and by commercial Subtilisin Carlsberg after 24 h incubation at 50°C.
Biotechnology and Bioprocess Engineering 16: 669-678 (2011)
Fig. 3.
Dehairing function of the RP1 crude protease. Bovine skins were incubated for 18 or 24 h at 25, 30, and 37°C with RP1 proteases. Fig. 4.
more efficient than commercial protease after 24 h of incubation at 50°C. In fact, complete solubilisation of the chicken feathers by the RP1 crude enzymes was observed after 24 h of incubation. 3.7.3. RP1 proteases dehairing function
In the leather industry, alkaline conditions are used to facilitate the bulge of hair followed by the subsequent attack of proteases, which allows for easy hair removal [53]. Therefore, efforts have been directed towards developing novel alkaline proteases for dehairing. Some microorganisms producing extracellular enzyme with dehairing activity have been described, Streptomyces sp. isolated from soil, degraded human hair, chicken feather, silk, wool, and unhaired goatskin [54]. In addition, the potential of using strains of B. subtilis and B. amyloliquefaciens for dehairing purposes has been examined [55]. However, many proteases are not suitable for dehairing, since they have collagendegrading activity, which destroys the collagen structure of the hide. Therefore, it is essential to identify proteases displaying dehairing activity but no collagenolytic activity [56]. Thus, the poor collagenase activity of the proteolytic preparation of RP1 (data not shown) may be advantagous to the leather industry. Incubation of the RP1 enzyme preparation with bovine skin for dehairing showed that after incubation for 24 h at pH 9.5 and at 30 or 37°C, hair was removed very easily from skin (Fig. 4). No dehairing was observed at 25°C. The dehairing function during leather processing is generally carried out at a relatively high pH values ranging from 8.0 to 10.0 [57]. Thus, the RP1 enzyme could be used for dehiaring during the leather process since it displayed high activity at pH 8.0 ~ 11.0.
Similar results were obtained with Aspergillus tamarri alkaline protease on goat skin after 18 ~ 24 h at pH 9.0 ~ 11.0 and 30 ~ 37°C [57]. Alkaline proteases with high keratinolytic activity from B. pumilus, were also found to dehair bovine hair [58], cowhides [59], and goat skins [60]. The obtained results indicate that the crude enzyme preparation could also be used in leather processing. 4. Conclusion
The use of cost-effective growth medium for the production of alkaline proteases can significantly reduce the cost of protease production. In the present study, B. licheniformis RP1 was found to grow and produce proteolytic enzymes in media containing SWP as complex microbial growth substrate, suggesting that the strain can obtain its carbon and nitrogen requirements directly from SWP. The highest protease production (1,400 U/mL) was achieved at 30 g/L SWP, 1.5 g/L KCl, 0.5 g/L K2HPO4 , and 0.5 g/L KH2PO4. Since shrimp waste is a readily available substrate, powder prepared from this by- product may be a possible candidate for the cost-effective production of extracellular proteases when used as culture medium ingredient. In this study, we also demonstrated that the culture supernatant can deproteinize shrimp wastes, where 81% of the protein was removed when E/S ratio of 5 was used (Unit of enzyme/mg of protein) was used. Therefore, proteases from the RP1 strain could be used effectively for
Alkaline Proteases Produced by Bacillus licheniformis RP1 Grown on Shrimp Wastes: Application in Chitin Extraction, Chicken… 677
the deproteinization of crustacean wastes to pruduce chitin. In addition, B. licheniformis RP1 appears to be suitable for the degradation of avian feathers and feather-meal with a potential for biotechnological applications. More interestingly, the RP1 crude extract exhibited powerful dehairing function against bovine skin with minimal damage to collagen. These results indicated that RP1 proteolytic preparation offers new and promising opportunities for use in various bioprocesses, particularly by the leather and poultry processing industries.
Acknowledgement This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia.
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