Conversion and degradation of shellfish wastes by Bacillus cereus TKU018 fermentation for the production of chitosanases and bioactive materials

Biochemical Engineering Journal 48 (2009) 111–117

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Conversion and degradation of shellfish wastes by Bacillus cereus TKU018 fermentation for the production of chitosanases and bioactive materials San-Lang Wang ∗ , Tz-Rung Chen, Tzu-Wen Liang, Pei-Chen Wu Graduate Institute of Life Sciences, Tamkang University, 151 Ying-Chuan Road, Tamsui 251, Taiwan

a r t i c l e

i n f o

Article history: Received 3 June 2009 Received in revised form 24 August 2009 Accepted 27 August 2009

Keywords: Chitosanase Antioxidant Shrimp shell wastes Squid pen wastes Bacillus cereus Reducing sugar

a b s t r a c t Two chitosanases (CHSB1 and CHSB2) were purified from the culture supernatant of Bacillus cereus TKU018 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of CHSB1 and CHSB2 determined by SDS–PAGE were approximately 44 kDa and 22 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSB1 and CHSB2 were (pH 5, 60 ◦ C; pH 5–7, <40 ◦ C) and (pH 7, 50 ◦ C; pH 4–7, <50 ◦ C), respectively. CHSB1 and CHSB2 were both inhibited by EDTA and CHSB1 was inhibited completely by 5 mM Zn2+ . CHSB1 and CHSB2 degraded chitosan with DD ranging from 60% to 95%, but did not degrade chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU018 culture supernatant (1.5% SPP) incubated for 3–4 days has 75% relative antioxidant activity (DPPH scavenging ability). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Chitosan, a d-glucosamine polymer, is a totally or partially deacetylated derivative of chitin. It is usually obtained by the artificial deacetylation of chitin in the presence of alkali [1]. However, the application of the natural polysaccharides in medicine and food industry is limited since its high molecular weight results in low solubility in acid-free aqueous media. Therefore, recent studies have been focusing on converting chitin and chitosan to oligosaccharides because the oligosaccharides not only are water-soluble but also possess versatile functional properties such as antitumor activity and antimicrobial activity [1–3]. Traditionally, chitosan oligosaccharides were processed by chemical methods in industries. There are many problems existing in chemical processes, such as a large amount of short-chain oligosaccharides produced, low yields of oligosaccharides, high cost in separation, and also environmental pollution. Alternatively, with its advantages in environmental compatibility, low cost, and reproducibility, the use of chitosanase for the hydrolysis of chitin and chitosan has become popular in recent years [4,5]. Chitosanases have been found in abundance in a variety of bacteria, including Bacillus spp. [6,7]. Almost all of the chitosanaseproducing strains used colloidal chitosan or chitosan as a major

∗ Corresponding author. Tel.: +886 2 2626 9425; fax: +886 2 8631 1015. E-mail address: [email protected] (S.-L. Wang). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.08.013

carbon source, such as Bacillus cereus D-11 [7], B. cereus S1 [8] and Aspergillus sp. CJ22-326 [9]. However, preparation of chitin/chitosan involves demineralization and deproteinization of shellfish waste by the use of strong acids or bases [1,10]. The utilization of shrimp shell and squid pen wastes not only solves environmental problems but also decreases the production cost of microbial chitosanases. Among these published chitosanaseproducing strains, few have been found to utilize marine wastes as carbon/nitrogen source. The production of inexpensive chitosanase is an important element in the process. The purpose of this study was to isolate chitosanase-producing bacteria, then to purify and characterize the chitosanases from the bacteria, B. cereus TKU018, and to compare with chitosanases isolated from other bacterial sources. 2. Materials and methods 2.1. Materials The shrimp shell powder (SSP) and squid pen powder (SPP) used in these experiments were prepared as described earlier [1]. SSP and SPP were purchased from Shin-Ma Frozen Food Co. (I-Lan, Taiwan). DEAE-Sepharose CL-6B, Phenyl Sepharose and Sephacryl S-100 were purchased from GE Healthcare UK Ltd. (Little Chalfont, Buckinghamshire, England). Weak-base anion-exchanger Macroprep DEAE was from Bio-Rad (Hercules, CA, USA). All other reagents used were of the highest grade available.

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Fig. 1. Elution profile of TKU018 chitosanases on DEAE-Sepharose CL-6B: () absorbance at 280 nm; (䊉) chitosanase activity (mU/mL).

Fig. 2. Elution profile of TKU018 chitosanase (CHSB1) on Phenyl Sepharose 6 Fast Flow: () absorbance at 280 nm; (䊉) chitosanase activity (mU/mL).

2.2. Isolation and screening of chitosanase-producing strains

matographed on a column of Phenyl Sepharose (1.3 cm × 20 cm), which had been equilibrated with 50 mM sodium phosphate buffer (pH 7) containing 1 M (NH4 )2 SO4 . The chitosanase was eluted with a linear gradient of 1–0 M (NH4 )2 SO4 in the same buffer. As shown in Fig. 2, the chitosanase fractions were collected and the enzyme activity was measured. Fractions with confirmed enzyme activity were pooled, dialyzed overnight at 4 ◦ C against 50 mM sodium phosphate buffer pH 7, and lyophilized.

Microorganisms isolated from soils collected at different locations in northern Taiwan were screened on agar plates containing 1% SSP, 0.1% K2 HPO4 , and 0.05% MgSO4 ·7H2 O, and 1.5% agar powder (pH 7). The plates were incubated at 30 ◦ C for 2 days. Those organisms obtained from the screening were subcultured in liquid media (containing 1% SSP, 0.1% K2 HPO4 , and 0.05% MgSO4 ·7H2 O) in shaking flasks at 30 ◦ C and 150 rpm. After incubation for 2 days, the culture broth was centrifuged (4 ◦ C and 12,000 × g for 20 min) and the supernatants were collected for measurement of chitosanase activity using the procedure described below. The strain TKU018 that showed the highest chitosanase activity was isolated, maintained on nutrient agar, and used throughout the study. 2.3. Purification of the chitosanases 2.3.1. Production of chitosanases For the production of chitosanases, B. cereus TKU018 was grown in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) containing 0.5% SPP, 0.1% K2 HPO4 , 0.05% MgSO4 ·7H2 O (pH 7). One milliliter of the seed culture was transferred into 100 mL of the same medium and grown in an orbital shaking incubator for 3 days at 30 ◦ C and pH 7 (the pH after being autoclaved was 7.5). After incubation, the culture broth was centrifuged (4 ◦ C and 12,000 ×g for 20 min), and the supernatant was used for further purification by chromatography. 2.3.2. DEAE-Sepharose CL-6B chromatography To the culture supernatant (900 mL), ammonium sulfate was added (608 g/L). The resultant mixture was kept at 4 ◦ C overnight and the precipitate formed was collected by centrifugation at 4 ◦ C for 20 min at 12,000 × g. The precipitate was then dissolved in a small amount of 50 mM sodium phosphate buffer (pH 7), and dialyzed against the buffer. The resultant dialysate (50 mL) was loaded onto a DEAE-Sepharose CL-6B column (5 cm × 30 cm) equilibrated with 50 mM sodium phosphate buffer (pH 7). As shown in Fig. 1, one chitosanase (CHSB1) was washed from the column with the same buffer and another chitosanase (CHSB2) was eluted with a linear gradient of 0–1 M NaCl in the same buffer. The fractions of the two peaks containing the chitosanase activity were respectively pooled and concentrated by ammonium sulfate precipitation. The resultant precipitates were collected by centrifugation and dissolved in 5 mL of 50 mM sodium phosphate buffer (pH 7). 2.3.3. Phenyl Sepharose chromatography The obtained enzyme solution (the unadsorbed chitosanase fractions from DEAE-Sepharose CL-6B column) was then chro-

2.3.4. Macro-prep DEAE chromatography The obtained enzyme solution (the adsorbed chitosanase fractions from DEAE-Sepharose CL-6B column) was then chromatographed on a column of Macro-prep DEAE (12.6 mm × 40 mm), which had been equilibrated with 50 mM sodium phosphate buffer (pH 7). As shown in Fig. 3, the chitosanase was eluted with a linear gradient of 0–1 M NaCl in the same buffer. The fractions containing the chitosanase activity (Fig. 3) were pooled and concentrated by ammonium sulfate precipitation. The resultant precipitate was collected by centrifugation and dissolved in 50 mM sodium phosphate buffer (pH 7). 2.3.5. Sephacryl S-100 chromatography These two resultant enzyme solutions were respectively loaded onto a Sephacryl S-100 gel filtration column (2.5 cm × 120 cm), which had been equilibrated with 50 mM sodium phosphate buffer (pH 7), then eluted with the same buffer. One peak exhibiting chitosanase activity for each enzyme solution was obtained and pooled fractions for each enzyme solution were used as a purified preparation.

Fig. 3. Elution profile of TKU018 chitosanase (CHSB2) on Macro-prep DEAE: () absorbance at 280 nm; (䊉) chitosanase activity (mU/mL).

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2.4. Protein determination Protein content was determined by the method of Bradford using Bio-Rad dye reagent concentrate and bovine serum albumin as the standard. After column chromatography, the protein concentration was estimated by measuring the absorbance at 280 nm [1]. 2.5. Measurement of enzyme activity Chitosanase activity of the enzyme was measured by incubating 0.2 mL of the enzyme solution with 1 mL of 0.3% (w/v) water-soluble chitosan (Kiotec Co., Hsinchu, Taiwan; with 60% deacetylation) in 50 mM phosphate buffer, pH 7 at 37 ◦ C for 30 min. The reaction was stopped by heating it at 100 ◦ C for 15 min. The amount of reducing sugar produced was measured by the method of Imoto and Yagishita [11] with glucosamine as a reference compound. One unit of enzyme activity was defined as the amount of enzyme which released 1 ␮mol of reducing sugars per min [12]. 2.6. Determination of molecular mass The molecular masses of the purified chitosanases (CHSB1 and CHSB2) were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [13] using 12.5% acrylamide and 2.67% methylene bis acrylamide in 0.375 M Tris–HCl buffer (pH 8.8) with 0.1% (w/v) SDS. Before electrophoresis, proteins were exposed overnight to 10 mM phosphate buffer (pH 7) containing ␤-mercaptoethanol. The electrode buffer was 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS (pH 8.3). Electrophoresis was performed at a constant current of 70 mA through the stacking gel and 110 mA through the resolving gel. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 in methanol–acetic acid–water (5:1:5, v/v), and decolorized in 7% acetic acid. The molecular masses of CHSB1 and CHSB2 in the native form were determined by a gel filtration method. The sample and standard proteins were applied to a Sephacryl S-100 column (2.5 cm × 120 cm, Amersham Pharmacia) equilibrated with 50 mM phosphate buffer (pH 7). Bovine serum albumin (molecular mass, 67 kDa), Bacillus sp. ␣-amylase (50 kDa), and hen egg white lysozyme (14 kDa) were used as molecular mass markers [12]. 2.7. Scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) TKU018 culture supernatant (150 ␮L) was mixed with 37.5 ␮L of methanolic solution containing 0.75 mM DPPH (Sigma Chemical Co., St. Louis, MO) radicals. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank [14]. The scavenging ability was calculated as follows: Scavenging ability (%) = [Ac − (A − As )]/Ac × 100, where Ac is the absorbance of the control DPPH solution, A is the absorbance of sample with DPPH solution, and As is the absorbance of sample. 3. Results and discussions 3.1. Identification of the strain TKU018 TKU018 is a gram-positive and endospore-forming bacillus, with catalase but without oxidase, which grows in both aerobic and anaerobic environments. According to the result of 16S rDNA partial base sequence (500 bp), TKU018 was most closely aligned to B. cereus with 99% similarity. According to the results of MIDI microbial fatty acids identification system and VITEK identification

Fig. 4. Time courses of chitosanase production in a culture of B. cereus TKU018 on shrimp shell containing media: (䊉) chitosanase activity (mU/mL); () cell growth; () pH. Data are presented as means ± SD of triplicates.

system, TKU018 was most close to B. cereus with both more than 99% similarity. The physical, biochemical, and molecular biological characteristics of strain TKU018 were most close to B. cereus with more than 99% similarity. The identification of strain TKU018 was carried out by the Bioresource Collection and Research Center, Taiwan. 3.2. Culture conditions and enzyme production In our preliminary experiments, we found 100 mL of basal medium (0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O, pH 7) containing 1% SSP was better for the production of chitosanase by strain TKU018 at 30 ◦ C. To study the effect of carbon/nitrogen sources on the production of chitosanase, growth was carried out in 100 mL of basal medium (0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O, pH 7) containing additional carbon/nitrogen sources of 0.5–2% (w/v) SSP or SPP, respectively. The result showed that 0.5% SSP was more suitable as an inducer for chitosanase production than others (data not shown). Therefore, in the following experiments, 100 mL of basal medium (0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O, pH 7) containing additional carbon/nitrogen source of 0.5% (w/v) SSP was used. To study the time course of cultivation, 100 mL of the media (0.5% SSP contained basal medium, pH 7) was used, and the relationship between incubation time (1–4 days), chitosanase activity was investigated. As shown in Fig. 4, maximum activity of chitosanase (22 mU/mL) was found at the third day and then decreased gradually. 3.3. Isolation and purification The purification of the TKU018 chitosanases from the culture supernatant (900 mL) was described under Section 2. First, the supernatant was submitted to ion exchange chromatography (Fig. 1) showing two peaks, CHSB1 and CHSB2 that both display chitosanase activity. After DEAE-Sepharose CL-6B chromatography, one protein peak containing the chitosanase activity (CHSB1) was washed from the DEAE-Sepharose CL-6B column, and another peak with chitosanase activity (CHSB2) was eluted from the column. By further purification, the purified CHSB1 and CHSB2 were obtained. As shown in Table 1, the purification steps were combined to give an overall purification of about 14.82-fold for CHSB1 and 11.44fold for CHSB2. The overall activity yields of the purified CHSB1 and CHSB2 were 3% and 4% respectively, with specific chitosanase activities of 54.55 mU/mg (CHSB1) and 42.11 mU/mg (CHSB2). The final amounts of TKU018 chitosanases obtained were 11 mg (CHSB1) and 19 mg (CHSB2). CHSB1 and CHSB2 were also confirmed to be homo-

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Table 1 Purification of chitosanases (CHSB1 and CHSB2) from B. cereus TKU018. Step

Total protein (mg)

Total activity (U)

Culture supernatant (NH4 )2 SO4 ppt

4896 1070

18 7

3.68 6.54

1 1.78

100 39

42 306

1 3

23.81 9.80

6.47 2.66

6 17

DEAE-Sepharose CHSB1 CHSB2

Specific activity (mU/mg)

Purification fold

Yield (%)

Phenyl Sepharose CHSB1

13

0.7

53.85

14.63

4

Macro-prep DEAE CHSB2

21

0.8

38.10

10.35

4

Sephacryl S-100 CHSB1 CHSB2

11 19

0.6 0.8

54.55 42.11

14.82 11.44

3 4

geneous by SDS–PAGE (Fig. 5). The molecular masses of CHSB1 and CHSB2 were determined by SDS–PAGE and gel filtration were approximately 44/45 kDa and 22/20 kDa, respectively. The molecular mass of CHSB1 (44 kDa) was similar to that of most Bacillus chitosanases described below. However, the molecular mass of CHSB2 (22 kDa) was obviously different from most of the other Bacillus chitosanases, such as, those of B. subtilis IMR-NK1 (41 kDa) [6], B. cereus S1 (45 kDa) [8], B. ehimensis EAG1 (31 kDa) [15], B. megaterium P1 (43, 39.5 kDa) [16], Bacillus sp. KCTC0377BP (45 kDa) [17], Bacillus sp. MET1299 (52 kDa) [18], Bacillus sp. 739

Fig. 5. SDS–PAGE analysis of the purified chitosanases (CHSB1 and CHSB2) produced by strain TKU018. Lanes—M, molecular markers; 1, crude enzyme; 2, CHSB1; 3, CHSB2. The molecular mass markers used for calibration were phosphorylase b (molecular mass, 97.4 kDa), albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20.1 kDa), and ␣-lactabumin (14.4 kDa).

(46 kDa) [19], Bacillus sp. P16 (45 kDa) [20], Bacillus sp. 7-M (41 kDa) [21], B. subtilis GM9804 (27 kDa) [22], B. subtilis KH-1 (28 kDa) [23], and Bacillis sp. DAU101 (27 kDa) [5]. The chitosanase of B. megaterium P1 (22 kDa) [16] was the only Bacillus chitosanase that had molecular mass similar to that of B. cereus TKU018 chitosanase (CHSB2). 3.4. Effect of pH and temperature on the enzyme activity The effect of pH on the catalytic activity was studied by using soluble chitosan as a substrate under the standard assay conditions. The pH activity profile of CHSB1 and CHSB2 was with maximum values at pH 5 and pH 7, respectively. Compared with the other Bacillus chitosanases, similar optimal pHs were obtained—pH 6 for Bacillus sp. S65 [4]; pH 7.5 for Bacillus sp. DAU101 [5]; pH 4 for B. subtilis IMR-NK1 [6]; pH 6 for B. cereus D-11 [7]; pH 6 for B. cereus S1 [8]; between pH 4.5 and 6.5 for B. megaterium P1 [16]; pH 5.5 for Bacillus sp. MET1299 [18]; and pH 5.5 for Bacillus sp. P16 [20]. The pH stability profile of the chitosanase activity was determined by the measurement of the residual activity at pH 7 after incubation at various pH values at 37 ◦ C for 60 min. The chitosanase activity of CHSB1 and CHSB2 was stable at pH 5–7 and pH 4–7, respectively (Fig. 6A). Similar pH stability was obtained between pH 5.5 and 6.5 for Bacillus sp. S65 [4]. The pH stability profile of CHSB1 and CHSB2 was different from most of the other Bacillus chitosanases, such as, between pH 5 and 9 for B. subtilis IMR-NK1 [6]; between pH 6 and 10 for B. cereus D-11 [7]; between pH 6 and 11 for B. cereus S1 [8]; and between pH 4.5 and 10 for Bacillus sp. P16 [20]. The effect of temperature on the activity of chitosanase was studied with soluble chitosan as a substrate. The optimum temperature of CHSB1 and CHSB2 was 60 ◦ C and 50 ◦ C, respectively (Fig. 6B). Compared with CHSB1, similar optimal temperatures were obtained 65 ◦ C for Bacillus sp. S65 [4]; 60 ◦ C for B. cereus D11 [7]; 60 ◦ C for B. cereus S1 [8]; 60 ◦ C for Bacillus sp. MET1299 [18]; 60 ◦ C for Bacillus sp. P16 [20]. Compared with CHSB2, similar optimal temperatures were obtained 50 ◦ C for Bacillus sp. DAU101 [5]; 45 ◦ C for B. subtilis IMR-NK1 [6]; 50 ◦ C for B. megaterium P1 [16]. To examine the heat stability of the chitosanases, the enzyme solution in 50 mM phosphate buffer (pH 7) was allowed to stand for 60 min at various temperatures, and then the residual activity was measured. CHSB1 maintained its initial activity at less than 40 ◦ C but was inactivated at higher than 50 ◦ C (Fig. 6B). CHSB2 maintained 70% of its initial activity at less than 50 ◦ C but was inactivated at higher than 60 ◦ C (Fig. 6B). Similar thermostability was obtained below 40 ◦ C for Bacillus sp. S65 [4]; between 30 ◦ C and 40 ◦ C for B. subtilis IMR-NK1 [6]; below 50 ◦ C for B. cereus D-11 [7]; below 60 ◦ C for B. cereus S1 [8]; below 50 ◦ C for Bacillus sp. P16 [20].

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Table 3 Effects of various chemicals and surfactants on chitosanase activity of CHSB1 and CHSB2 purified from B. cereus TKU018. Chemicals

None PMSF EDTA Mg2+ Cu2+ Fe2+ Ca2+ Zn2+ Mn2+ Ba2+ SDS Tween 20 Tween 40 Triton X-100

Concentration (mM)

0 5 5 5 5 5 5 5 5 5 0.5/1/2 0.5/1/2 (%) 0.5/1/2 (%) 0.5/1/2 (%)

Relative activity (%) CHSB1

CHSB2

100 64 45 71 70 32 39 0 37 51 0/0/0 100/92/109 101/153/155 80/90/88

100 92 29 73 61 26 79 44 44 38 44/40/40 105/88/80 105/87/80 86/85/93

Purified enzyme was preincubated with the various reagents at 37 ◦ C for 30 min and residual chitosanase activity were determined as described in the text. One hundred percent was assigned to the activity in absence of reagents.

Fig. 6. Effect of pH (A) and temperature (B) on the activity (solid line) and stability (dashed line) of CHSB1 and CHSB2. () CHSB1; () CHSB2. Data are presented as means ± SD of triplicates.

3.5. Substrate specificity For the substrate specificity of CHSB1 and CHSB2, chitin, and chitosans with degree of deacetylation (DD) ranging from 60% to 95% were used as substrates, as summarized in Table 2. These enzymes could hydrolyze soluble chitosans, but exhibited no activity on glycol chitosan and chitin. The chitosanases from Amycolatopsis sp. CsO-2 [24], Nocardioides sp. K-01 [25], Bacillus circulans MHK1 [26], Bacillus sp. PI-7S [27], and Bacillus sp. CK4 [28] were most active on approximately 100% deacetylated chitosan. The chitosanases from Acinetobacter sp. CHB101 [29] and Bacillus sp. P16 Table 2 Substrate specificity of CHSB1 and CHSB2 purified from B. cereus TKU018. Substrate

Soluble chitosan (95% DD) Soluble chitosan (85% DD) Soluble chitosan (82% DD) Soluble chitosan (73% DD) Soluble chitosan (60% DD) Colloidal chitin Chitin (␣-type) Chitin (␤-type) CMC Glycol chitosan

Relative activity (%) CHSB1

CHSB2

74 15 12 29 100 0 0 0 53 0

10 5 18 40 100 0 0 0 0 0

[20] were most active on approximately 80% deacetylated chitosan. The chitosanase from Penicillium islandium [30] was less active in hydrolyzing chitosan that less than 40% or more than 70% DD. The most susceptible substrates for TKU018 chitosanases (CHSB1 and CHSB2) were both 60% deacetylated chitosan, suggesting that CHSB1 and CHSB2 have specificity to the linkages of GlcN–GlcN and GlcNAc–GlcN and/or GlcN–GlcNAc, and the N-acetylglucosamine residues are important in the recognition and reaction mechanism of the substrate by these enzymes [20]. Surprisingly, CHSB1 showed a hydrolysis activity for carboxymethylcellulose (CMC) comparable to soluble chitosan (DD 73%) by about 53% relative to soluble chitosan (DD 60%), though most chitosanases do not show the reactivity for CMC [24,26,27,30]. Taking into account the differential behavior of CHSB1 in attacking ␤-1,4 linked polysaccharides of different chemical structures, it could be speculated that this enzyme possesses different catalytic sites for attacking the glycosidic linkages of those two polysaccharides. It seems that CHSB1 does not recognized so severely amino group of C2 position in glucosamine residue when enzyme–substrate complex was formed. This finding could be associated with the property that some cellulose can hydrolyze chitosan. 3.6. Effects of various chemicals on the enzyme activity Metal ions had different impacts on chitosanases. It was reported that Ca2+ triggered the refolding of chitosanase from B. subtilis GM9804 [22]. Therefore, we hypothesized that different metal ions might affect CHSB1 and CHSB2 activity through influencing the structure of the protein. EDTA was an activator of Bacillus sp. R-4 chitosanase [31]. But to CHSB1 and CHSB2, it was an inhibitor. Therefore, CHSB1 and CHSB2 are metalloenzymes, the presence of metal ions non-covalently bound to the enzyme is an important means to influence its catalytic activity. To further characterize CHSB1 and CHSB2, we next examined the effects of some known enzyme inhibitors and divalent metals on their activities. The effects of various chemicals on the enzyme activity were investigated by preincubating the enzyme with chemicals in 50 mM phosphate buffer (pH 7) for 30 min at 37 ◦ C and then measuring the residual chitosanase activity by using soluble chitosan (DD 60%) as substrate. The results showed that CHSB1 was completely inactivated by Zn2+ and significantly inactivated by Ca2+ , Mn2+ and Fe2+ at 5 mM concentration (Table 3). CHSB2 was significantly inactivated by Mn2+ , Zn2+ , Ba2+ , and Fe2+ at 5 mM concentration (Table 3). However, the other ions little affected these enzymes activities.

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for optimums, the highest reducing sugar content (at the 3rd day) showed later than the optimal chitosanase production (at the 2nd day). Conjecturing from above results, those reducing sugars in the culture supernatant might be originated from the hydrolyzates (chitooligosaccharides) from hydrolysis of squid pen chitin by chitosanase. 3.9. Antioxidant activity of culture supernatant from SPP fermented by strain TKU018

Fig. 7. Time courses of reducing sugar and antioxidant in a culture of B. cereus TKU018 on squid pen containing media: () reducing sugar content (mg/mL); () antioxidant activity (%); (䊉) chitosanase activity (mU/mL). Data are presented as means ± SD of triplicates.

Moreover, CHSB1 and CHSB2 were still active when the metal ions were removed. This result was similar to those for other chitosanolytic enzymes and suggested the tested metal ions were not essential for the catalytic action of the enzymes. 3.7. Effect of various surfactants on the enzyme activity Enzymes are usually inactivated by the addition of surfactants to the reaction solution because the structure of the enzymes might be influenced. Moreover, certain surfactants affected the chitooligosaccharides synthesis by chitosanase [32]. Therefore, the effect of different surfactants (2%, v/v) on stability of CHSB1 and CHSB2 was also studied. CHSB1 and CHSB2 were incubated with surfactants (0.5–2%, v/v) at 37 ◦ C for 30 min and the remaining enzymatic activity was determined under normal assaying conditions. The chitosanase activity of the sample without any surfactants (control) was taken as 100%. It was found that in the presence of 2% nonionic surfactants of Tween 20, Tween 40, or Triton X-100, the activities of CHSB1 and CHSB2 retained more than 80% of its original activity. At the presence of 0.5–2 mM SDS (anionic surfactant), the activity of CHSB1 was completely inhibited and CHSB2 retained about 40% of its original activity (Table 3). These differences between both enzymes might be related to the dissimilarity of the ratio of their hydrophobic and hydrophilic amino acids. Surfactants such as Tween 20 and Tween 40 had stimulatory effect on CHSB1 and CHSB2 chitosanase activity which may be due to change in the conformation of these enzymes thus increasing the substrate accessibility. The CHSB1 chitosanase activity was highly stimulated (about 1.5-fold higher) in the presence of Tween 40 (2%). 3.8. The production of reducing sugar in liquid phase fermentation

It has been reported that chitin, chitosan and peptide have antioxidative [33–36] and anticarcinogenic [3,10] properties. For example, the antioxidative peptides could be obtained by hydrolyzing shrimp (Acetes chinensis) with crude protease from Bacillus sp. SM98011 [36]. To increase the utilization of the chitin/proteincontaining marine wastes, we incubated B. cereus TKU018 for 1–5 days with various concentrations (0.5–2%) of SSP and SPP under the optimal culture conditions described above (100 mL, 30 ◦ C) and analyzed the antioxidant activity and enzyme activity of the culture supernatants. The antioxidant activity assayed was the scavenging ability on DPPH. It was found that TKU018 culture supernatant (1.5% SPP) incubated for 3–4 days has the highest antioxidant activity, the DPPH scavenging ability of TKU018 culture supernatant was about 75% relative activity (Fig. 7). To analyze the antioxidant activity of the culture medium at the 0 day, we heated these marine wastes in an autoclave (121 ◦ C for 15 min), the relative antioxidant activities were 15–20% and 20–30% in the SSP and SPP supernatants, respectively. However, as shown in Fig. 7, the antioxidant activities increased significantly and reached up to approximately 75% relative activity after being fermented by TKU018. It is assumed that even though the autoclave treatment (121 ◦ C for 15 min) degrades SPP and produces some of the antioxidant materials, most of the antioxidant materials are produced by strain TKU018. Comparing the difference in culture time (Fig. 7), it was found that the optimal antioxidant activities of TKU018 showed later than the chitosanase production. The chitooligosaccharides in the supernatant were recovered by the method previously described [10] and the antioxidant activity was measured. It was found that the antioxidant activity of the chitooligosaccharides in the supernatant was approximately 65% relative activity (data not shown). These results demonstrated that antioxidative oligosaccharides might be hydrolyzed by the enzyme and present in the culture supernatant. It is consistent with the observation at the increase in the reducing sugar content. The increase in the reducing sugar content might be due to SPP was hydrolyzed to oligosaccharides by TKU018 chitosanases. The third day culture supernatant showed both high reducing sugar content and high antioxidant activity. The antioxidant materials may contain oligosaccharides (or a little oligopeptides) that are electron donors and are able to react with free radicals to terminate the radical chain reaction. 4. Conclusion

To increase the utilization of the chitin/protein-containing marine wastes, we incubated B. cereus TKU018 for 1–5 days with various concentrations (0.5–2%) of SSP and SPP under the optimal culture conditions described above (100 mL, 30 ◦ C) and analyzed the reducing sugar and enzyme activity of the culture supernatants. As shown in Fig. 7, it was found that TKU018 culture supernatant (1.5% SPP) incubated for 3 days has the most reducing sugars (0.58 mg/mL) than others. Since the medium based on the SPP, reducing sugars were mainly chitooligosaccharides. The reducing sugar content increased during initial 3 days of fermentation and reached constant values thereafter. Besides, the amount of SPP in the culture media decreased with an increase in reducing sugar content (data not shown). Comparing the difference in culture time

This research used SSP as the sole carbon/nitrogen sources to produce chitosanases. This is different from most other chitosanase-producing strains which require chitosan as carbon/nitrogen source. In this study, we have succeeded in developing the efficient production procedure of chitosanases by B. cereus TKU018 using the cheap medium based on shrimp shell. Although the productivity is significantly improved by the optimization of cultivation conditions, further studies on the improvement of the productivity of Bacillus sp. strains by mutation or genetic engineering approaches are important to develop the industrial production process of chitosanases. In addition, we also have purified and characterized the chitosanases, and found

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that the culture supernatant has reducing sugars and antioxidant activity as well. Our data provide a useful example of utilizing SSP and SPP biowaste materials as valuable functional ingredients. The literature survey found that protein hydrolysates from marine biology are known to possess strong antioxidative properties [36,37]. It is expected that this bioactive material rich liquor will have beneficial biological functions due to the inherent protein and chitin hydrolysis and other bioactive materials production occurring during fermentation. The findings of the current report appear useful for further research aiming to isolate and identify the specific compounds responsible for the antioxidant activity of TKU018 fermented supernatant. Acknowledgement This work was supported in part by a grant of the National Science Council, Taiwan (NSC96-2313-B-032-002-MY3). References [1] S.L. Wang, T.Y. Lin, Y.H. Yen, et al., Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase, Carbohydr. Res. 341 (2006) 2507–2515. [2] K. Suzuki, T. Mikami, Y. Okawa, et al., Antitumor effect of hexa-Nacetylchitohexaose and chitohexaose, Carbohydr. Res. 151 (1986) 403–408. [3] S.L. Wang, H.T. Lin, T.W. Liang, et al., Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials, Biores. Technol. 99 (2008) 4386–4393. [4] C. Su, D. Wang, L. Yao, et al., Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65, J. Agric. Food Chem. 54 (2006) 4208–4214. [5] Y.S. Lee, J.S. Yoo, S.Y. Chung, et al., Cloning, purification and characterization of chitosanase from Bacillus sp. DAU101, Appl. Microbiol. Biotechnol. 73 (2006) 113–121. [6] C.L. Chiang, C.T. Chang, H.Y. Sung, Purification and properties of chitosanase from a mutant of Bacillus subtilis IMR-NK1, Enzyme Microb. Technol. 32 (2003) 260–267. [7] X.A. Gao, W.T. Ju, W.J. Jung, et al., Purification and characterization of chitosanase from Bacillus cereus D-11, Carbohydr. Polym. 72 (2008) 513–520. [8] M. Kurakake, S. You, K. Nakagawa, et al., Properties of chitosanase from Bacillus cereus S1, Curr. Microbiol. 40 (2000) 6–9. [9] X. Chen, W. Xia, X. Yu, Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326, Food Res. Int. 38 (2005) 315–322. [10] T.W. Liang, Y.J. Chen, Y.H. Yen, et al., The antitumor activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656, Process Biochem. 42 (2007) 527–534. [11] T. Imoto, K. Yagishita, A simple activity measurement of lysozyme, Agric. Biol. Chem. 35 (1971) 1154–1156. [12] S.L. Wang, J.H. Peng, T.W. Liang, et al., Purification and characterization of a chitosanase from Serratia marcescens TKU011, Carbohydr. Res. 343 (2008) 1316–1323. [13] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [14] K. Shimada, K. Fujikawa, K. Yahara, et al., Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion, J. Agric. Food Chem. 40 (1992) 945–948.

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[15] K. Akiyama, T. Fujita, K. Kuroshima, et al., Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1, J. Biosci. Bioeng. 87 (1999) 383– 385. [16] A. Pelletier, J. Sygusch, Purification and characterization of three chitosanases activities from Bacillus megaterium P1, Appl. Environ. Microbiol. 56 (1990) 844–848. [17] Y.J. Choi, E.J. Kim, Z. Piao, et al., Purification and characterization of chitosanase from Bacillus sp. strain KCTC0377BP and its application for the production of chitosan oligosaccharides, Appl. Environ. Microbiol. 70 (2004) 4522– 4531. [18] P.I. Kim, T.H. Kang, K.J. Chung, et al., Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene, FEMS Microbiol. Lett. 240 (2004) 31–39. [19] G.E. Aktuganov, A.V. Shirokov, A.I. Melent’ev, Isolation and characterization of chitosanase from Bacillus sp. 739 strain, Prikl Biokhim Mikrobiol. 39 (2003) 536–541. [20] Y.Y. Jo, K.J. Jo, Y.L. Jin, et al., Characterization and kinetics of 45 kDa chitosanase from Bacillus sp. P16, Biosci. Biotechnol. Biochem. 67 (2003) 1875–1882. [21] M. Izume, S. Nagae, H. Kawagishi, et al., Action pattern of Bacillus sp. 7-M chitosanase on partially N-acetylated chitosan, Biosci. Biotechnol. Biochem. 56 (1992) 448–456. [22] A. Colomer-Pallas, Y. Pereira, M.F. Petit-Glatron, et al., Calcium triggers the refolding of Bacillus subtilis chitosanase, Biochem. J. 369 (2003) 731–738. [23] C.A. Omumasaba, N. Yoshida, Y. Sekiguchi, et al., Purification and some properties of a novel chitosanase from Bacillus subtilis KH1, J. Gen. Appl. Microbiol. 46 (2000) 19–27. [24] S. Okajima, A. Ando, H. Shinoyama, et al., Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2, J. Ferment. Bioeng. 77 (1994) 617–620. [25] S. Okajima, T. Kinouchi, Y. Mikami, et al., Purification and some properties of a chitosanase of Nocardioides sp, J. Gen. Appl. Microbiol. 41 (1995) 351–357. [26] M. Yabuki, A. Uchiyama, K. Suzuki, et al., Purification and properties of chitosanases from Bacillus circulans MH-K1, J. Gen. Appl. Microbiol. 34 (1988) 255–270. [27] H. Seino, K. Tsukuda, Y. Shiimasue, Properties and action pattern of a chitosanase from Bacillus sp. PI-7S, Agric. Boil. Chem. 55 (1991) 2421–2423. [28] H.G. Yoon, H.Y. Kim, H.K. Kim, et al., Thermostable chitosanase from Bacillus sp. Strain CK4: its purification, characterization, and reaction patterns, Biosci. Biotechnol. Biochem. 65 (2001) 802–809. [29] M. Shimosaka, M. Nogawa, X.Y. Wang, et al., Production of two chitosanases from a chitosan-assimilating bacterium, Acinetobacter sp. strain CHB101, Appl. Environ. Microbiol. 61 (1995) 438–442. [30] D.M. Fenton, D.E. Eveleigh, Purification and the mode of action of a chitosanase from Penicillum islandicum, J. Gen. Microbiol. 126 (1981) 151–165. [31] D. Somashekar, R. Joseph, Chitosanases—properties and applications: a review, Bioresour. Technol. 55 (1996) 35–45. [32] Y.C. Hsiao, Y.W. Lin, C.K. Su, et al., High degree polymerized chitooligosaccharides synthesis by chitosanase in the bulk aqueous system and reversed micellar microreactors, Process Biochem. 43 (2008) 76–82. [33] J.E. Pinero Estrada, P. Bermejo Besco‘s, A.M. Villar del Fresno, Antioxidant activity of different fractions of Spriulina platensis protean extract, IL FARMACO 56 (2001) 497–500. [34] H.Y. Lin, C.C. Chou, Antioxidant activities of water-soluble disaccharide chitosan derivatives, Food Res. Int. 37 (2004) 883–889. [35] R. Xing, H. Yu, S. Liu, et al., Antioxidative activity of differently regioselective chitosan sulfates in vitro, Bioorg. Med. Chem. 13 (2005) 1387–1392. [36] H. He, X. Chen, C. Sun, et al., Preparation and functional evaluation of oligopeptide-enriched hydrolysate from shrimp (Acetes chinensis) treated with crude protease from Bacillus sp. SM98011, Biores. Technol. 97 (2006) 385– 390. [37] S. Kim, E. Mendis, Bioactive compounds from marine processing byproducts—a review, Food Res. Int. 39 (2006) 383–393.