Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation

Carbohydrate Research 362 (2012) 38–46

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Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation Tzu-Wen Liang a,b, Jia-Lin Hsieh b, San-Lang Wang a,b,⇑ a b

Life Science Development Center, Tamkang University, New Taipei City 25137, Taiwan Department of Chemistry, Tamkang University, New Taipei City 25137, Taiwan

a r t i c l e

i n f o

Article history: Received 14 June 2012 Received in revised form 9 August 2012 Accepted 9 August 2012 Available online 4 September 2012 Keywords: Protease Chitosanase N-Acetyl chitooligosaccharides Shrimp heads Bacillus cereus

a b s t r a c t A protease- and chitosanase-producing strain was isolated and identified as Bacillus cereus TKU022. The protease and chitosanase were both produced using 1.5% (w/v) shrimp head powder (SHP) as the sole carbon/nitrogen source, and these enzymes were purified from the culture supernatant. The molecular masses of the TKU022 protease and chitosanase determined using SDS–PAGE were approximately 45 and 44 kDa, respectively. The high stability of the TKU022 protease toward surfactants, an optimal pH of 10 and an optimal temperature of 50–60 °C suggest that this high-alkaline protease has potential applications for various industrial processes. Concomitant with the production of the TKU022 chitosanase, N-acetyl chitooligosaccharides were also observed in the culture supernatant, including (GlcNAc)2, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 at concentrations of 201.5, 12.4, 0.5, and 0.3 lg/mL, respectively, as determined using an HPLC analysis. The chitin oligosaccharides products were also characterized using a MALDI-TOF mass spectrometer. A combination of the HPLC and MALDI-TOF MS results showed that the chitin oligosaccharides of the TKU022 culture supernatant comprise oligomers with degree of polymerization (DP) from 2 to 6. Using this method, the production of a protease, a chitosanase, and chitin oligosaccharides may be useful for various industrial and biological applications. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Proteases constitute one of the most important groups of industrial enzymes. Among the various proteases, bacterial proteases are the most significant compared with proteases of animal, plant, or fungal origin, and bacteria of the genus Bacillus produce most of the commercial proteases used today1 because the alkaline proteases of Bacillus spp. exhibit high proteolytic activity and stability under alkaline conditions.2 The characterization of alkaline proteases is interesting for bioengineering and biotechnological applications. The major application of these proteases is in the detergent industry because the pH of laundry detergents is generally in the range of 9.0–12.0. In addition, proteases have applications in leather processing, food processing, and the production of protein hydrolysates.1 Chitosanases have been found in abundance in a variety of bacteria.3–5 Most of the chitosanase-producing strains use colloidal chitosan or chitosan as a major carbon source.4,5 However, preparation of chitin/chitosan involves the demineralization and deproteinization of shellfish waste using strong acids or bases.6 The

⇑ Corresponding author at present address: No. 151, Yingchuan Rd., Tamsui Dist., New Taipei City 25137, Taiwan. Tel.: +886 2 2626 9425; fax: +886 2 2620 9924. E-mail address: [email protected] (S.-L. Wang). 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2012.08.004

utilization of shrimp head wastes not only solves environmental problems but also decreases the production cost of microbial chitosanases. Among these published protease- and/or chitosanaseproducing strains, few have been found to utilize marine wastes as a carbon/nitrogen source. The production of inexpensive proteases and chitosanases is an important element in the process. Recent studies on chitosan/chitin have attracted interest for converting these species into oligosaccharides because the oligosaccharides not only are water-soluble but also possess versatile functional properties such as antitumor activity and antimicrobial activity.6–8 Traditionally, chitin oligosaccharides were processed using chemical methods in industries. Many problems exist in these chemical processes, such as the production of a large amount of short-chain oligosaccharides, low yields of oligosaccharides, the high cost of separation, and environmental pollution. Alternatively, with its advantages in environmental compatibility, low cost, and reproducibility, enzyme hydrolysis has become more popular in recent years.8 However, in our work, the chitin oligomers were produced directly by B. cereus TKU022 fermentation. With this method, the production of chitin oligomers will omit the procedure for purifying enzymes and help decrease the cost of chitin oligomer production more effectively. In summary, in the present study, shrimp head wastes can be utilized for the production of enzymes (both protease and chitosanase) and N-acetyl chitooligosaccharides (GlcNAc-oligomers) by B. cereus TKU022 fermentation. The

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2. Results and discussions 2.1. Isolation and identification of a protease/chitosanaseproducing strain The microorganisms were isolated from soils using the procedure described above. Among more than 100 strains, isolated in the laboratory and screened for protease and chitosanase activity, TKU022 strain was selected. The TKU022 strain that showed the highest protease and chitosanase activity was isolated, maintained on nutrient agar, and used throughout the study. TKU022 is a gram-positive and endospore-forming bacillus that contains catalase but not oxidase and that grows in both aerobic and anaerobic environments. According to the result of a 16S rDNA partial base sequence (approximately 1.5 kbp) analysis, strain TKU022 is most closely related to Bacillus cereus and Bacillus thuringiensis. According to the API identification, TKU022 was most closely related to B. cereus, with a 98.2% similarity. Therefore, the isolate was identified as B. cereus. 2.2. Production of extracellular protease and chitosanase by B. cereus TKU022 In our preliminary experiments, the protease, chitosanase, and chitinase activities were measured in the culture supernatant of strain TKU022 during various cultivation times. The culture supernatants exhibited protease and chitosanase activities, but the results were negative for the chitinase assay in the supernatants. To further investigate the production of protease and chitosanase by strain TKU022 during 6 days of cultivation in the production medium, the 50 mL of basal medium containing 1.5% SHP was the most suitable for the production of protease and chitosanase at 37 °C (data not shown). After a 6-h lag phase, exponential growth was observed for 3 days, and stationary phase was achieved after 3 days. Interestingly, strain TKU022 secreted extracellular chitosanase at the beginning of the exponential phase and achieved maximal production of up to 0.6 U/mL at this phase (2 days; Fig. 1). Thereafter, the chitosanase activity decreased

7.5

pH

7

6

Cell growth (OD660)

0.6 8

6.0 0.5 4.5

0.4

0.3

3.0

0.2 5

1.5 0.1

4

0.6

0.7

0.0

0.0 0

1

2

3

4

5

6

Chitosanase activty (U/mL)

9

remarkably with the appearance of protease activity (Fig. 1). This secretion pattern of the protease is similar to those reported in previous studies in which the majority of proteases were not produced or were only produced at a low rate in the exponential growth phase and normally reached the maximal production in the stationary phase.2,11 Therefore, the protease production was closely related to the cell growth. This result indicated that the production of protease is cell growth dependent, and B. cereus TKU022 is a promising protease producer. To investigate the reason why the chitosanase activity decreased with the first appearance of protease, B. cereus TKU022 was incubated under the optimized conditions for the production of protease and chitosanase. B. cereus TKU022 was incubated for 2 days (for chitosanase production) and for 5 days (for protease production). Using the culture supernatants of B. cereus TKU022, we assayed the enzyme activities of protease and chitosanase. Equal volumes of protease and chitosanase preparations were mixed and reacted at 37 °C for 0, 10, 20, 30, and 60 min. The results are shown in Fig. 2. After 60 min of reaction, the chitosanase in the absence of protease retained 96% of its original activity, but the chitosanase in the presence of protease retained only 38% of its original activity. It is conjectured that the reason for the obvious decrease in chitosanase activity might be that the chitosanase was hydrolyzed by the protease and therefore lost its activity. Similar to this study, B. subtilis IMR-NK1 is also a protease- and chitosanase-producing strain.3 Because the recovery yield was lower for the purified chitosanase, the authors of this previous study conjectured that the hydrolysis by the protease caused the decrease in the activity of the chitosanase. Comparison of productivity by activity was often seen in review papers concerning enzymes. The maximum values of TKU022 protease activity (0.5 U/mL) and chitosanase activity (0.6 U/mL) were higher than those of some other reports in the same reaction system and the same definition of enzyme activity, such as Bacillus sp. TKU004 protease (0.067 U/mL),12 Bacillus sp. TKU004 chitosanase (0.16 U/mL),13 and B. subtilis TKU007 chitosanase (0.03 U/mL).14 However, it is indeed very difficult to make comparison because the conditions used for assaying were usually different in the literature. In addition, for comparison to be meaningful, the reaction system and definition of unit for enzyme activity need to be the same. We could not make comparison of enzyme activity of our

0.5

0.4

0.3

0.2

Protease activty (U/mL)

above technologies facilitate the potential use of this process in industrial applications and functional foods.

0.1

0.0

7

Cultivation time (day) Figure 1. Time courses of the protease and chitosanase production in a culture of B. cereus TKU022 on shrimp head-containing media: (N) protease activity (U/mL); (d) chitosanase activity (U/mL); (h) cell growth; (s) pH.

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120

4

3

2

1

M

(kDa)

5

M (kDa)

97.4

100

97.4

Relative activity (%)

66.2

66.2

80

45

45

60

29

29

20.1

20.1

14.4

14.4

40

20

0 0

10

20

30

40

50

60

70

Reaction time (min) Figure 2. Inhibitory effects of the TKU022 protease on the TKU022 chitosanase: (d) the chitosanase with protease; (s) the chitosanase without protease.

study with those reported in the references, unless the reaction system and definition of unit are the same. In fact, the main purpose of this study was to screen some protease- and chitosanase-producing strains which are able to utilize chitinous materials as the sole carbon/nitrogen source. It was expected that protease and chitosanase are produced by the microorganism and at the same time the enzymes will induce the production of N-acetyl chitooligosaccharides from SHP. 2.3. Isolation and purification The purification of the B. cereus TKU022 protease and chitosanase from the culture supernatant (930 mL) was described in the Section 4.1. As shown in Table 1, the purification steps were combined to give an overall purification of approximately 332-fold and 273-fold for protease and chitosanase, respectively. The overall activity yield of the purified protease and chitosanase was 3% and 11%, respectively, with a specific protease activity of 3 U/mg and a specific chitosanase activity of 7 U/mg. The final amount of B. cereus TKU022 protease and chitosanase obtained was 1 and 5 mg, respectively. During the enzyme purification processes, the recovery of the protease and chitosanase activities decreased gradually. This result may be due to the removal of some fractions containing protease and chitosanase with lower specific activity during the column chromatography. It is also possible that the protease and chitosanase were unstable over a series of purification steps. The purified protease and chitosanase were confirmed to be homogeneous using SDS–PAGE with molecular weights of approximately 45 and 44 kDa, respectively (Fig. 3).

Figure 3. SDS–PAGE analysis of the purified protease and chitosanase produced by B. cereus TKU022. Lanes: M, molecular markers; (1) crude enzyme; (2) the adsorbed chitosanase fractions after DEAE-Sepharose CL-6B chromatography; (3) the adsorbed chitosanase fractions purified using Macro-Prep DEAE chromatography; (4) the purified chitosanase after Sephacryl S-100 chromatography; (5) the purified protease. The molecular mass markers used for the 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 a-lactalbumin (14.4 kDa).

The molecular mass of the B. cereus TKU022 protease (45 kDa) was obviously different from most of the other B. cereus metalloproteases, such as the 28 kDa B. cereus (isolated from slaughterhouse waste) metalloprotease,15 B. cereus KCTC3674 metalloproteases (36 and 38 kDa),16 and the 29 kDa B. cereus (isolated from a patient) metalloprotease.17 The 45.6 kDa B. cereus metalloprotease18 was the only B. cereus protease with a molecular mass similar to that of B. cereus TKU022. The molecular mass of the B. cereus TKU022 chitosanase (44 kDa) was similar to those of some Bacillus chitosanases, such as B. subtilis IMR-NK1 (41 kDa),3 B. cereus S1 (45 kDa),4 Bacillus sp. KCTC0377BP (45 kDa),19 Bacillus sp. 739 (46 kDa),20 and Bacillus sp. P16 (45 kDa).21 The molecular mass of the B. cereus TKU022 chitosanase was obviously different from those of most of the other Bacillus chitosanases, such as Bacillus sp. MET1299 (52 kDa),22 B. subtilis GM9804 (27 kDa),23 B. subtilis KH-1 (28 kDa),24 and Bacillus sp. DAU101 (27 kDa).25 2.4. Effect of pH and temperature The pH activity profiles of the protease and chitosanase exhibited maximum values at pH 10 and pH 7, respectively (Fig. 4a). The pH optimum of the protease was higher than those of B. cereus metalloprotease (pH 7),18 B. proteolyticus CFR3001 alkaline protease (pH 8),26 B. pantotheneticus alkalophilic protease (pH 8.4),27 and B. cereus MCM B-326 protease (pH 9).28 The pH stability

Table 1 Purification of protease and chitosanase from B. cereus TKU022 Step

Supernatant (NH4)2SO4 ppt DEAE-Sepharose Phenyl Sepharose Macro-prep DEAE Sephacryl S-100

n2bo

Total activity (U)

Specific activity (U/mg)

Purification fold

Yield (%)

Protease

Chitosanase

Protease

Chitosanase

Protease

Chitosanase

Protease

Chitosanase

Protease

Chitosanase

12,767 1352 180 18 1

12,767 1352 497 — 43 5

93 20 16 5 — 3

304 209 206 — 124 31

0.007 0.015 0.08 0.2 3.0

0.024 0.2 0.4 — 3.0 7.0

1 2 12 32 — 332

1 6.5 18 — 121 273

100 21 17 5 — 3

100 69 67 — 41 11

B. cereus TKU022 was grown in 50 mL of liquid medium in an Erlenmeyer flask (250 mL) containing 1.5% SHP, 0.1% K2HPO4, and 0.05% MgSO47H2O in a shaking incubator for 2 days at 37 °C.

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120

(a)

Table 2 Substrate specificities of the protease from B. cereus TKU022 Substrates

100

Relative activity (%)

Relative activity (%)

Method A Casein Azocasein Albumin Azoalbumin Gelatin Hemoglobin Fibrin Elastin Myoglobin

80

60

40

a

Method Bb

100 100 60 78 0 5 0 207 47

a The activities of these substrates were measured using the method of Todd, as described in Section 2. b The activities of these substrates were determined by measuring the absorbance at 440 nm, as described in the protocol of Sigma Co.

20

0 0

4

5

6

7

8

9

10

11

pH Table 3 Substrate specificities of the chitosanase from B. cereus TKU022

120

(b)

Relative activity (%)

100 80 60 40

Substrate

Relative activity (%)

Chitosan (atype; 98% DD) Chitosan (btype; 98% DD) Chitosan (95% DD) Chitosan (80% DD) Chitosan (60% DD) Colloidal chitin Chitin (atype) Chitin (btype) CMC Glycol chitosan

29 20 35 25 100 0 0 0 0 40

20

more suitable for washing at lower temperatures. For applications in detergents for a lower temperature washing, the TKU022 protease has the characteristic.

0

0

25

30

37

40

50

60

70

80

90

Temperature (ºC) Figure 4. Effect of pH (a) and temperature (b) on the activity (solid line) and stability (dashed line) of the TKU022 protease and chitosanase. (s) protease; (d) chitosanase. For the activity, the maximum activity obtained at optimum pH and temperature was considered as 100% activity. For the stability, the original activity before preincubation was taken as 100% (protease: 0.1 U/mL; chitosanase: 0.23 U/ mL).

profiles of the protease and chitosanase activities were determined by measuring the residual activity at pH 7 after incubation at various pH values at 37 °C for 60 min. The protease and chitosanase activities were stable at pH 7-10 (Fig. 4a). The effect of temperature on the activities of the protease and chitosanase was studied. The optimum temperatures for the TKU022 protease and chitosanase were 50–60 °C and 60 °C, respectively (Fig. 4b). To examine the thermal stability of the TKU022 protease and chitosanase, enzyme solutions in 50 mM phosphate buffer (pH 7) were allowed to stand for 60 min at various temperatures, and then the residual activity was measured. TKU022 protease maintained its initial activity from 25 to 40 °C and had 85% of its activity at 50 °C, but it was completely inactivated at 70 °C. TKU022 chitosanase also maintained its initial activity from 25 to 40 °C and had 62% of its activity at 50 °C (Fig. 4b). The increasing use of synthetic fibers which cannot tolerate temperatures above 50 °C has changed the washing habits during the past 20 years toward the use of lower washing temperatures. Furthermore, the energy crisis has focused interest on washing at ambient temperatures for the purpose of saving energy.29–33 It is very important to screen for new proteolytic enzymes which are

2.5. Substrate specificity The activity of the purified protease toward various substrates is summarized in Table 2. The enzyme showed especially high activity toward casein and elastin but no activity toward gelatin and fibrin. This result is in contrast to that of the fibrinolytic enzyme from a mutant of B. subtilis IMR-NK1.34 Peng et al.35 have measured the ratio of fibrinolytic activity to caseinolytic activity

Table 4 Effects of various chemicals and surfactants on the activities of the protease and chitosanase from B. cereus TKU022 Chemicals

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

Concentration (mM)

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

Relative activity (%) Protease

Chitosanase

100 96 22 57 65 104 105 103 39 97/91 93/96/105 98/94/94 93/96/97

100 125 61 68 33 100 53 53 0 88/80 107/112/112 70/72/73 102/113/139

The purified enzyme was preincubated with the various reagents at 25 °C for 30 min, and the residual protease and chitosanase activities were determined as described in the text. One hundred percent was assigned to the activity determined in the absence of reagents.

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Figure 5. A typical HPLC profile of the chitin oligosaccharides obtained from the culture supernatant containing 1.5% SHP using B. cereus TKU022 fermentation for 2 days. (a) standards; (b) TKU022 culture supernatant.

(F/C) of various Bacillus proteases. The F/C ratios (%) of Bacillus amyloliquefaciens DC-4 (a serine protease), B. subtilis (a neutral protease), Bacillus pumilus (an alkaline protease), and Bacillus licheniformis (an alkaline protease) were 1.92, 0.92, 0.75, and 0.39, respectively.35 In contrast to the above Bacillus proteases, the F/C ratio of TKU022 protease was 0%, as calculated from the data in Table 2. For the substrate specificity of TKU022 chitosanase, chitin, and chitosan with a degree of deacetylation (DD) ranging from 60% to 98% were used as substrates, as summarized in Table 3. The enzyme could hydrolyze chitosan, but it exhibited no activity with chitin and CMC. The chitosanases from Bacillus sp. PI-7S36 and Bacillus sp. CK437 were most active with approximately 100% deacetylated chitosan. The chitosanase from Bacillus sp. P1621 was most active with approximately 80% deacetylated chitosan. The most susceptible substrate for the TKU022 chitosanase was

60% deacetylated water-soluble chitosan, suggesting that the TKU022 chitosanase has specificity to the linkages of GlcN-GlcN and GlcNAc-GlcN and/or GlcN-GlcNAc.21 2.6. Effects of various inhibitors and metal ions To further characterize the B. cereus TKU022 protease and chitosanase, we next examined the effects of some known enzyme inhibitors and divalent metals on their activities. The results are summarized in Table 4. The protease activity was not affected by some metal ions, but there was a 43%, 35%, and 61% reduction in protease activity in the presence of 5 mM Mg2+, Cu2+, and Mn2+, respectively. However, the chitosanase activity was inhibited completely by 5 mM Mn2+. An inhibitor of serine proteases (phenylmethylsulfonyl fluoride [PMSF]) had no significant effect on the activities of the purified TKU022 protease and chitosanase.

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Intens. [a.u.]

DP=3 x104

608.259

4

3

2

DP=4 811.433

1

DP=3 DP=5

650.314

1014.556

0 600

650

700

750

800

850

900

950

1000

1050 m/z

Figure 6. MALDI-TOF MS spectrum of the 90% methanol-soluble/90% acetone-insoluble fraction of the chitin oligosaccharides obtained from the TKU022 culture supernatant.

2.7. Effect of various surfactants Enzymes are usually inactivated by the addition of surfactants to the reaction solution. The effects of different surfactants on the stabilities of the purified TKU022 protease and chitosanase were also studied. The enzyme activity of a sample without any surfactants (control) was defined as 100%. A high stability was observed for the TKU022 protease and chitosanase toward various surfactants (Table 4). Upon incubation with 2% Tween 20, the TKU022 protease exhibited enhanced residual activities (105%). Upon incubation with Tween 20 and Triton X-100, the TKU022 chitosanase exhibited enhanced residual activities of 102-139%. Even in the presence of 2% Tween 40 and Triton X-100, the activity of the TKU022 protease retained 94% and 97%, respectively, of its original activity. In the presence of 2% Tween 40, the TKU022 chitosanase retained 73% of its original activity. Ionic surfactants have been reported to destabilize most alkaline proteases.2 However, in the presence of the anionic surfactant SDS (1 mM), the TKU022 protease and chitosanase retained 91% and 80% of their original activities, respectively (Table 4). 2.8. Production of chitin oligosaccharides in liquid phase fermentation In the culture supernatant, the reducing sugar content increased dramatically on the second day, and the protein concentration increased quickly on the third day. However, the relative weight of the recovered SHP decreased with an increase in cultivation time (data not shown). The chitin oligosaccharides in the supernatant were recovered and lyophilized using the method described above. The HPLC chromatograms of the chitin oligosaccharides obtained from the culture supernatant are shown in Fig. 5. The (GlcNAc)n (n = 2,4,5,6) species were produced from SHP by B. cereus TKU022 fermentation on the second day. The amounts of (GlcNAc)2, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 produced were 201.5, 12.4, 0.5, and 0.3 lg/mL, respectively, after 2 days of fermentation. Recently, the production of chitin oligomers by enzymatic hydrolysis has been reported, with GlcNAc produced as a major chitinolytic

product. These enzymes were derived from Serratia marcescens,38 Bacillus thuringiensis subsp. Pakistani,39 Aeromonas hydrophila H-2330,40 Trichoderma viride and Acremonium cellulolyticus,41 Aeromonas sp. GJ-18,42 and Paenibacillus illinoisensis KJA-424.43 However, in our work, the chitin oligomers were produced by B. cereus TKU022 fermentation. There was no production of GlcNAc by B. cereus TKU022 fermentation. Our method and results were obviously different from the above reports. In this study, this result indicates that the microbial strains used would be affected (induced) by chitin and protein contained in the shellfish chitin wastes, thus exhibiting protease and chitosanase activities simultaneously. Additionally, the chitin oligosaccharides in the culture supernatant could be recovered for biological applications. 2.9. Analysis of TKU022 chitin oligosaccharides using MALDITOF MS To obtain the low-DP oligomers in the culture supernatant, a selective precipitation in 90% methanol and acetone solutions was used, as described earlier.6 From the MALDI-TOF analysis of the precipitation in the 90% acetone solution, it appeared that chitin oligosaccharides with DP up to 5 were obtained (Fig. 6). The higher DP chitooligomers were precipitated from the methanol solution as a light yellow powder. This culture supernatant contained (GlcNAc)3 and GlcN-(GlcNAc)24 (Table 5). During B. cereus TKU022 fermentation, both the N-acetyl linkage between residues and the O-glycosidic linkage can be hydrolyzed. GlcNAc- and GlcN(GlcNAc)24 ions present in the mass spectra were identified as Table 5 Assigned ion composition of the MALDI-TOF MS spectra of the chitin oligosaccharides produced by B. cereus TKU022 fermentation and isolated by 90% methanol and acetone m/z

Types

Ion composition

608 650 811 1014

[M+Na]+ [M+Na]+ [M+Na]+ [M+Na]+

GlcN-(GlcNAc)2 (GlcNAc)3 GlcN-(GlcNAc)3 GlcN-(GlcNAc)4

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sodium adducts, [M+Na]+, corresponding to the following series of the oligosaccharides: GlcN-(GlcNAc)2, (GlcNAc)3, GlcN-(GlcNAc)3, and GlcN-(GlcNAc)4 (Table 5). Because the MALDI-TOF analysis is limited to molecular weights higher than 500 Da due to interference of the matrix signals, the DP <2 oligomers could not be determined using this method. The oligosaccharides were also analyzed using HPLC (Fig. 5). Collectively, the data presented in Fig. 5 and Fig. 6 show that the oligosaccharides of the TKU022 culture supernatant comprise oligomers with DPs from 2 to 6. From these results, the TKU022 culture supernatant combined with a selective methanol/acetone precipitation appears to be a quick and simple method to yield the oligosaccharides with low molecular weight oligomers. 3. Conclusions In contrast to other reported protease- and/or chitosanase-producing strains of Bacillus sp., this research aimed for the microbial reclamation of shrimp processing wastes. Shrimp heads were used as the sole carbon/nitrogen source to screen the protease- and chitosanase-producing strain. Consequently, although the screened TKU022 belongs to a Bacillus sp. the same as the reported proteaseand/or chitosanase-producing strains, some properties of the enzymes produced were different. B. cereus TKU022 used 1.5% (w/v) SHP as the sole carbon/nitrogen source for protease/chitosanase production. The medium for TKU022 is obviously much simpler and cheaper. Additionally, using this method, the chitin oligosaccharides were also produced on the second day. These results may be useful for biological applications in relation to enzyme and bioactive materials production. 4. Experimental 4.1. Materials The shrimp head powder (SHP) used in these experiments was prepared as described previously.7 The shrimp heads were purchased from Shin-Ma Frozen Food Co. (I-Lan, Taiwan). During the preparation of the SHP, the shrimp heads were washed thoroughly with tap water and then dried. The dried materials obtained were milled to powders for use as the carbon source for protease and chitosanase production. The DEAE-Sepharose CL-6B, phenyl sepharose, and Sephacryl S-100 were purchased from GE healthcare UK Ltd. (Little Chalfont, Buckinghamshire, England). The weakbase anion-exchanger Macro-prep DEAE was obtained from BioRad (Hercules, CA, USA). The standard proteins (Geneaid, Taiwan) used for the calibration of the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) 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 a-lactalbumin (14.4 kDa). All other reagents used were of the highest grade available. 4.2. Isolation and screening of protease- and chitosanaseproducing strains The microorganisms isolated from soils collected at different locations in northern Taiwan were screened on agar plates containing 1% SHP, 0.1% K2HPO4, 0.05% MgSO47H2O, and 1.5% agar powder (pH 7). The plates were incubated at 30 °C for 2 days. The organisms obtained from this screening were subcultured in liquid media (containing 1% SHP, 0.1% K2HPO4, and 0.05% MgSO47H2O) in shaking flasks at 30 °C on a rotary shaker (150 rpm, Yih Der LM-570R). After incubation for 2 days, the culture broth was centrifuged (4 °C and 12,000g for 20 min, Kubota 5922), and the

supernatants were collected for the measurement of protease and chitosanase activity using the procedure described below. The TKU022 strain that showed the highest protease and chitosanase activity was isolated, maintained on SHP agar and used throughout the study. 4.3. Identification of strain TKU022 The bacterial strain TKU022 was identified on the basis of morphological, physiological, and biochemical parameters as well as on the basis of a 16S rDNA-based sequence analysis after PCR amplification with primers. The 16S rDNA gene of the isolate was amplified using the upstream primer P1: 50 -ACGGCTACCTTGTTACG ACT-30 and the downstream primer P2: 50 -CCCACTGCCTCCCGTAAG GAGT-30 . The following PCR program was used: 1 cycle of 94 °C for 5 min; 30 cycles of 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 1.5 min; and a cycle of 72 °C for 5 min. The nucleotide bases of the DNA sequence obtained were compiled and compared with sequences in the GenBank databases using the BLAST program. Further identification of strain TKU022 was performed using the analytical profile index (API). Strain TKU022 grew on nutrient agar plates. The surface bacterial growth was resuspended by gentle mechanical agitation in 2 mL of sterile distilled water. This bacterial suspension was used to inoculate 50 CHB API strips (ATB system, bioMérieux SA, Marcy-I’Etoile, France) following the manufacturer’s instruction. The strips were incubated at 30 °C and observed after 16, 24, 40, and 48 h and compared to the API identification index and database. 4.4. Purification of the enzymes 4.4.1. Production of protease and chitosanase For the production of protease and chitosanase, B. cereus TKU022 was grown in 50 mL of liquid medium in an Erlenmeyer flask (250 mL) containing 1.5% SHP, 0.1% K2HPO4, and 0.05% MgSO4 7H2O (pH 7). One milliliter of the seed culture (absorbance at 660 nm 1.0) was transferred into 50 mL of the same medium and grown in an orbital shaking incubator for 2 days at 37 °C and pH 7.2 (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 using chromatography. 4.4.2. DEAE-Sepharose CL-6B chromatography For the culture supernatant (930 mL), ammonium sulfate was slowly added to the supernatant to 80% saturation and the mixture was stored at 4 °C overnight. The precipitate 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 (60 mL) was loaded onto a DEAE-Sepharose CL-6B column (5 cm  30 cm) equilibrated with 50 mM sodium phosphate buffer (pH 7). The protease was washed from the column with the same buffer, and the chitosanase was eluted with a linear gradient of 0– 1 M NaCl in the same buffer. The fractions of the two peaks containing the protease and the chitosanase activity were independently pooled and concentrated using ammonium sulfate precipitation. The resultant precipitates were collected by centrifugation and dissolved in 5 mL of 50 mM sodium phosphate buffer (pH 7). 4.4.3. Phenyl sepharose chromatography The enzyme solution that was obtained (the unadsorbed protease fractions from the DEAE–Sepharose CL-6B column) was then chromatographed on a column of phenyl sepharose

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(1.3  20 cm), which had been equilibrated with 50 mM sodium phosphate buffer (pH 7) containing 1 M (NH4)2SO4. The protease was eluted using a linear gradient of 1–0 M (NH4)2SO4 in the same buffer. The protease fractions were collected, and the enzyme activity was measured. The fractions with confirmed enzyme activity were pooled, dialyzed overnight at 4 °C against 50 mM sodium phosphate buffer pH 7, and lyophilized. 4.4.4. Macro-prep DEAE chromatography The enzyme solution that was obtained (the adsorbed chitosanase fractions from the DEAE-Sepharose CL-6B column) was then chromatographed on a column of Macro-prep DEAE (12.6  40 mm), which had been equilibrated with 50 mM sodium phosphate buffer (pH 7). The chitosanase was eluted using a linear gradient of 0–1 M NaCl in the same buffer. The fractions containing the chitosanase activity were pooled and concentrated using ammonium sulfate precipitation. The resultant precipitate was collected using centrifugation and dissolved in 50 mM sodium phosphate buffer (pH 7). 4.4.5. Sephacryl S-100 chromatography The two resultant enzyme solutions were independently loaded onto a Sephacryl S-100 gel filtration column (2.5  120 cm), which had been equilibrated with 50 mM sodium phosphate buffer (pH 7), and eluted with the same buffer. One peak exhibiting protease activity and another peak exhibiting chitosanase activity for each enzyme solution were obtained, and the pooled fractions for each enzyme solution were used as a purified preparation. 4.5. Protein determination The protein content was determined using the Bradford method with a 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.7 4.6. Measurement of enzyme activity To measure the protease activity, a diluted enzyme solution (0.2 mL) was mixed with 1.25 mL of 1.25% casein in a pH 7 phosphate buffer and incubated for 30 min at 37 °C. The reaction was terminated by adding 5 mL of 0.19 M trichloroacetic acid (TCA). The reaction mixture was centrifuged, and the soluble peptide in the supernatant fraction was measured using the method of Todd with tyrosine as the reference compound. One unit of protease activity was defined as the amount of enzyme required to release 1 lmol tyrosine/min.8 The 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 the reaction mixture at 100 °C for 15 min. The amount of reducing sugar produced was measured using the method of Imoto and Yagishita9 with glucosamine as a reference compound. One unit of enzyme activity was defined as the amount of enzyme that released 1 lmol reducing sugars/min.8 The specific activity was expressed as units per mg protein (U/mg protein) of the enzyme extract. 4.7. Determination of molecular mass The molecular masses of the purified protease and chitosanase were determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).10 SDS–PAGE was performed on a 5% (w/v) stacking and a 12.5% (w/v) resolving gel, and protein

45

bands were visualized by staining with Coomassie Brilliant Blue R-250. Before electrophoresis, the proteins were exposed overnight to 10 mM phosphate buffer (pH 7) containing b-mercaptoethanol. The molecular masses of the protease and chitosanase in the native form were determined using a gel filtration method. The sample and standard proteins were applied to a Sephacryl S-100 column (2.5  120 cm) equilibrated with 50 mM phosphate buffer (pH 7). Bovine serum albumin (molecular mass: 67 kDa), Bacillus sp. a-amylase (50 kDa) and hen egg white lysozyme (14 kDa) were used as molecular mass markers.7 4.8. Effect of pH and temperature on enzyme activities The optimum pHs of TKU022 protease and chitosanase were studied by assaying the samples at different pH values. The pH stability of TKU022 protease and chitosanase was determined by measuring the residual activity at pH 7 as described above after the sample had been dialyzed against a 50 mM buffer solution of various pH values (pH 3–11) in seamless cellulose tubing (Sankyo). The buffer systems used were glycineHCl (50 mM, pH 3), acetate (50 mM, pH 4–5), phosphate (50 mM, pH 6–8), and Na2CO3–NaHCO3 (50 mM, pH 9–11). To determine the optimum temperatures for TKU022 protease and chitosanase, the activity values of the samples were measured at various temperatures (25–90 °C). The thermal stability of TKU022 protease and chitosanase was studied by incubating the samples at various temperatures for 60 min. The residual activity was measured as described above. 4.9. Effect of various chemicals and surfactants on enzyme activities The effects of metal ions (5 mM) were investigated using Mg2+, Cu , Fe2+, Ca2+, Zn2+, Mn2+, and Ba2+. The effects of enzyme inhibitors were studied using phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA). The effects of surfactants were also studied using SDS, Tween 20, Tween 40, and Triton X-100. The enzyme was pre-incubated with various chemicals and surfactants for 30 min at 25 °C, and the residual protease and chitosanase activities were then tested. 2+

4.10. Preparation of the GlcNAc-oligomers using B. cereus TKU022 fermentation The culture supernatant was concentrated to approximately 1/5 of the original volume with a rotary evaporator under diminished pressure, and this was followed by the addition of methanol with a final methanol concentration of 90% (v/v). Yellow agglomerates formed in the solution. The agglomerates were concentrated using a rotary evaporator under diminished pressure and were collected after drying under vacuum. The supernatant was concentrated to approximately 1/10 of the original volume using a rotary evaporator under diminished pressure. Then, the supernatant was precipitated by adding acetone with a final acetone concentration of 90% (v/v). The precipitates were collected after drying under vacuum. 4.11. HPLC analysis The HPLC analysis of TKU022 GlcNAc-oligomers was performed using a Hitachi L-7000 apparatus (column, Nucleosil 5 NH2 4.6  250 mm; mobile phase, acetonitrile/water = 70/30, v/v; flow rate = 1.0 mL/min; detection, RI). After fermentation and filtration, the sample was analyzed to measure the amount of (GlcNAc)n (n = 1–6) in the culture supernatant using HPLC. The amounts of (GlcNAc)n (n = 1–6) were estimated using the calibration curve of standard 3.75 mg/mL (GlcNAc)n (n = 1–6). The yield of (GlcNAc)n (n = 1–6) was calculated using the following equation.

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The concentration of sample (mg/mL) = 3.75  the area of sample/the area of standard. 4.12. MALDI-TOF MS analysis An aliquot of 1 lL of the sample solution (2 mg/mL) was mixed on the target with 1 lL of a solution of 2,5-dihydroxybenzoic acid as a matrix (15 mg/mL) in 30% aqueous ethanol. Positive ion MALDI mass spectra were acquired using a MALDI-TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser emitting at 337 nm operating in linear mode. Each mass spectrum was the accumulated data of approximately 30–50 laser shots. An external 3-points calibration was used for the mass assignment. Acknowledgements This work was supported in part by a grant from the National Science Council, Taiwan (NSC99-2313-B-032-001-MY3). References 1. Banik, R. M.; Prakash, M. Microbiol. Res. 2004, 159, 135–140. 2. Deng, A.; Wu, J.; Zhang, Y.; Zhang, G.; Wen, T. Bioresour. Technol. 2010, 101, 7100–7106. 3. Chiang, C. L.; Chang, C. T.; Sung, H. Y. Enzyme Microb. Technol. 2003, 32, 260– 267. 4. Kurakake, M.; Yo-u, S.; Nakagawa, K.; Sugihara, M.; Komaki, T. Curr. Microbiol. 2000, 40, 6–9. 5. Gao, X. A.; Ju, W. T.; Jung, W. J.; Park, R. D. Carbohydr. Polym. 2008, 72, 513–520. 6. Liang, T. W.; Chen, Y. J.; Yen, Y. H.; Wang, S. L. Process Biochem. 2007, 42, 527– 534. 7. Wang, S. L.; Lin, T. Y.; Yen, Y. H.; Liao, H. F.; Chen, Y. J. Carbohydr. Res. 2006, 341, 2507–2515. 8. Wang, S. L.; Lin, H. T.; Liang, T. W.; Chen, Y. J.; Yen, Y. H.; Guo, S. P. Bioresour. Technol. 2008, 99, 4386–4393. 9. Imoto, T.; Yagishita, K. Agric. Biol. Chem. 1971, 35, 1154–1156. 10. Laemmli, U. K. Nature 1970, 227, 680–685. 11. Chu, W. H. J. Ind. Microbiol. Biotechnol. 2007, 34, 241–245. 12. Wang, S. L.; Kao, T. Y.; Wang, C. L.; Yen, Y. H.; Chern, K. M.; Chen, Y. H. Enzyme Microb. Technol. 2006, 39, 724–731. 13. Wang, S. L.; Wu, P. C.; Liang, T. W. Carbohydr. Res. 2009, 344, 979–984. 14. Wang, S. L.; Yeh, P. Y. Process Biochem. 2008, 43, 132–138. 15. Doddapaneni, K. K.; Tatineni, R.; Vellanki, R. N.; Rachcha, S.; Anabrolu, N.; Narakuti, V.; Mangamoori, N. Microbiol. Res. 2009, 164, 383–390.

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