An Exochitinase with N-Acetyl-β-Glucosaminidase-Like Activity from Shrimp Head Conversion by Streptomyces speibonae and Its Application in Hydrolyzing β-Chitin Powder to Produce N-Acetyl-d-Glucosamine

Marine chitinous byproducts possess significant applications in many fields. In this research, different kinds of fishery chitin-containing byproducts from shrimp (shrimp head powder (SHP) and demineralized shrimp shell powder), crab (demineralized crab shell powder), as well as squid (squid pen powder) were used to provide both carbon and nitrogen (C/N) nutrients for the production of an exochitinase via Streptomyces speibonae TKU048, a chitinolytic bacterium isolated from Taiwanese soils. S. speibonae TKU048 expressed the highest exochitinase productivity (45.668 U/mL) on 1.5% SHP-containing medium at 37 °C for 2 days. Molecular weight determination analysis basing on polyacrylamide gel electrophoresis revealed the mass of TKU048 exochitinase was approximately 21 kDa. The characterized exochitinase expressed some interesting properties, for example acidic pH optima (pH 3 and pH 5–7) and a higher temperature optimum (60 °C). Furthermore, the main hydrolysis mechanism of TKU048 exochitinase was N-acetyl-β-glucosaminidase-like activity; its most suitable substrate was β-chitin powder. The hydrolysis experiment revealed that TKU048 exochitinase was efficient in the cleavage of β-chitin powder, thereby releasing N-acetyl-d-glucosamine (GlcNAc, monomer unit of chitin structure) as the major product with 0.335 mg/mL of GlcNAc concentration and a yield of 73.64% after 96 h of incubation time. Thus, TKU048 exochitinase may have potential in GlcNAc production due to its N-acetyl-β-glucosaminidase-like activity.


Introduction
Chitin is a straight-chain polymer of N-acetyl-d-glucosamine (GlcNAc) unit with β-1,4 linkage, which is a very common polysaccharide in the world, second only to cellulose. By expressing

Screening of Exochitinase-Producing Bacterium
The isolation was conducted on the medium containing squid pen powder (SPP, 1% w/v), MgSO 4 ·7H 2 O (0.05% w/v), K 2 HPO 4 (0.1% w/v), and agar (2% w/v). Firstly, 1 g of each soil sample, obtained from arable lands in Northern Taiwan, was gently shaken with 100 mL of sterile saline for 5 min. The suspension was then serially diluted until a 10 6 -fold dilution was obtained. One hundred microliters of the final dilution were spread over a Petri dish containing isolation medium. Inoculated Petri dishes were incubated at 37 • C for 24 h to get single colonies of bacteria. The pure bacterial strains were then taken from the single colonies by streaking method. Following this, each isolated strain was transferred to liquid media (1% SPP, 0.05% MgSO 4 ·7H 2 O, and 0.1% K 2 HPO 4 ) and cultivated for 3 days at 37 • C and 150 rpm. The culture of each bacterial strains was centrifuged again at 12,000× g (Universal 320, Hettich Zentrifugen, Tuttlingen, Germany) for 10 min to collect the supernatant, which was tested for exochitinase and chitinase activities. The bacterial strain which possessed the highest exochitinase activity was named as TKU048 and selected for further experiments. DNA sequencing, as well as biochemical and morphological methods were used to verify the identity of the TKU048 strain.

Exochitinase Activity Assay
Determination of exochitinase activity was conducted following a previously described method [2]. Briefly, 50 µL of sample (containing exochitinase) was transferred to a tube containing 500 µL of sodium acetate buffer (50 mM, pH 5.8) and 100 µL of pNPg (1 mg/mL). The tube was immediately incubated for 30 min at 37 • C. Sodium carbonate-bicarbonate buffer (325 µL) was added to the reaction solution to eliminate exochitinase activity and introduce pNP coloration, which was measured by a spectrophotometer at 420 nm. The amount of exochitinase which catalyzed the hydrolysis reaction of pNPg to liberate 1 µmol of pNP in 1 min was defined as one unit (U) of enzyme activity.

Chitinase Activity Assay
Chitinase activity assay was performed by the method of Doan et al. using colloidal chitin as substrate and N-acetyl-glucosamine as reference. The amount of chitinase which catalyzed the hydrolysis of colloidal chitin to liberate 1 µmol of N-acetyl-glucosamine in 1 min, was defined as one unit (U) of enzyme activity.

Culture Conditions for Exochitinase Production
One gram of each different fishery byproduct, including deCSP, deSSP, SHP, and SPP, was added to a glass Erlenmeyer flask (250 mL) containing 100 mL of basal salt medium to provide the carbon and nitrogen (C/N) nutrients for the growth and exochitinase production of S. speibonae TKU048. The culture was started by adding 1% (v/v) of stock solution of S. speibonae TKU048 and maintained under the following conditions: 37 • C incubation temperature and 150 rpm of agitation. An aliquot of culture (1 mL) was withdrawn every 24 h for testing exochitinase activity. After finding the best source of carbon and nitrogen for enzyme production, the optimization of culture conditions was further carried out for other parameters, including amount of C/N source (0.5%-2%, w/v), incubation temperature (30-50 • C), agitation speed (0-200 rpm), and initial pH (5-8).

Isolation of TKU048 Exochitinase
S. speibonae TKU048 was cultured as described above. One liter of culture supernatant was used for isolating TKU048 exochitinase. Further isolation steps included protein concentration by (NH 4 ) 2 SO 4 (80% saturation), Macro-Prep High Q chromatography, and KW-802.5 size-exclusion chromatography. These steps have been described in detail in a previous report [2]. The molecular weight of the TKU048 exochitinase was determined by SDS-PAGE analysis [2].

Effects of Temperature and pH on Enzyme Activities
The optimal temperature of TKU048 exochitinase was investigated by incubating the mixtures of enzyme and pNPg at different temperature points (from 20 to 100 • C) for 30 min. Meanwhile, the residual activity of enzyme solutions, which were pretreated in different temperatures for 30 min, was used to explore the thermal stability of TKU048 exochitinase. The optimal pH and pH stability of TKU048 exochitinase were carried out following the method of Tran et al. [2].

Effects of Ion Metals on Enzyme Activity
A similar amount of TKU048 exochitinase solutions were incubated with each of different ion metals (FeCl 2 , CaCl 2 , BaCl 2 , NaCl, MgCl 2 , ZnCl 2 , and CuCl 2 ) and a metalloenzyme inhibitor (EDTA) at 20 • C for 30 min. The residual activity of TKU048 exochitinase was then measured according to the exochitinase activity assay, as described above.

Hydrolysis Mechanism
To investigate the hydrolysis mechanism of TKU048 exochitinase, chitin oligosaccharides with degree of polymerizations (DP) 2-6 were used as the substrates. Five hundred microliters of substrate solution (0.5 mg/mL) was mixed with 500 µL enzyme solution (2 U, approximately) in the glass tubes. The reactions were subsequently carried out at 50 • C using a water bath. After every 20 min, 100 µL of each solution was withdrawn for analysis by HPLC method (described below).

HPLC Analysis
The chitin oligosaccharides and β-chitin powder hydrolysates, which were produced from the hydrolysis reaction catalyzed by TKU048 exochitinase, were analyzed by a Hitachi Chromaster HPLC system (column, KS-802; flow rate, 0.6 mL/min; column temperature, 80 • C; mobile phase, H 2 O; ultraviolet detection wavelength, 205 nm; volume of sample, 20 µL). To detect hydrolysis products, a series of chitin oligosaccharides (DP from 1 to 6) was used as a reference.

Screening of an Exochitinase-Producing Bacterium
More than 50 chitinolytic microorganisms from arable lands in Northern Taiwan were isolated on the media containing squid pen powder [2]. For producing chitinase activity, these strains were cultivated on liquid medium containing 1% SPP and mineral salts (0.05% MgSO 4 and 0.1% K 2 HPO 4 ) for 3 days under the following conditions: initial pH 7.2, 150 rpm, and 37 • C. Among them, the exochitinase activity of strain TKU048 culture revealed the highest value (4.285 U/mL). This strain was named as Streptomyces speibonae according to the results of 16S rRNA sequences as well as morphological and biochemical studies. So far, Streptomyces along with Bacillus, Paenibacillus, Serratia, and Aspergillus are the primary microbial strains producing chitinase. However, there are only a few reports on the production of exochitinase from Streptomyces, including S. olivaceoviridis [45], S. thermocarboxydus TKU045 [2], and S. lividans pCHIO12 [46]. Additionally, to our best knowledge, there are no reports on the production of exochitinase from S. speibonae species. Therefore, the discovery of exochitinase production in S. speibonae TKU048 is of interest, especially involving the use of byproducts containing chitin as the C/N-providing source.

Optimization of Culture Conditions for Exochitinase Production
To explore the best C/N sources for TKU048 exochitinase production, four chitinous materials, SHP, SPP, deSSP, and deCSP, were added to the basal medium (0.05% MgSO 4 and 0.1% K 2 HPO 4 ) at a concentration of 1% (w/v) for cultivating S. speibonae TKU048. As shown in Figure 1, TKU048 exhibited the most exochitinase activity on SHP with 39.379 U/mL after 2 days of cultivation, while its activity was lower than 2 U/mL on other chitinous materials sources (SPP, deSSP, and deCSP). This result was different from the research of Tran et al. which showed that SPP was the most suitable for producing exochitinase by S. thermocarboxydus TKU045 (12.2 U/mL on SPP, approximately four-fold higher than 2.39 U/mL on SHP) [2]. This result suggests that the type of chitinous material has a significant effect on exochitinase production by S. speibonae TKU048, in which SHP, a common byproduct in the seafood processing industry, was demonstrated to be the best potential source. By achieving the highest exochitinase productivity in shorter cultivation times, SHP was selected as the best C/N source for cultivating S. speibonae TKU048.

Optimization of Culture Conditions for Exochitinase Production
To explore the best C/N sources for TKU048 exochitinase production, four chitinous materials, SHP, SPP, deSSP, and deCSP, were added to the basal medium (0.05% MgSO4 and 0.1% K2HPO4) at a concentration of 1% (w/v) for cultivating S. speibonae TKU048. As shown in Figure 1, TKU048 exhibited the most exochitinase activity on SHP with 39.379 U/mL after 2 days of cultivation, while its activity was lower than 2 U/mL on other chitinous materials sources (SPP, deSSP, and deCSP). This result was different from the research of Tran et al., which showed that SPP was the most suitable for producing exochitinase by S. thermocarboxydus TKU045 (12.2 U/mL on SPP, approximately fourfold higher than 2.39 U/mL on SHP) [2]. This result suggests that the type of chitinous material has a significant effect on exochitinase production by S. speibonae TKU048, in which SHP, a common byproduct in the seafood processing industry, was demonstrated to be the best potential source. By achieving the highest exochitinase productivity in shorter cultivation times, SHP was selected as the best C/N source for cultivating S. speibonae TKU048. The effects of other culture parameters on the production of TKU048 exochitinase, such as amount of SHP, incubation temperature, pH, and agitation rate, were also investigated. The results are summarized in Table 1. Following that, S. speibonae TKU048 was found to exhibit the highest exochitinase productivity on the medium containing 1.5% SHP with an initial pH of 6.0, incubation temperature of 37 °C, and shaking speed of 175 rpm. After 2 days of fermentation, the exochitinase activity of the optimized culture supernatant reached the maximum value at 45.668 U/mL. The exochitinase activity was approximately 45-fold higher than before optimization. This result indicated that the culture conditions for the production of S. speibonae TKU048 exochitinase by using only chitinous fishery byproducts to provide the source of C/N were successfully optimized. Until now, conversion of abundant and low-cost chitinous materials by microbial activity to produce bioactive compounds has received great attention . Consequently, the current results could be promising in providing a novel beneficial application of shrimp heads-a chitin-containing byproduct from fishery processing-in producing exochitinase via S. speibonae TKU048. The effects of other culture parameters on the production of TKU048 exochitinase, such as amount of SHP, incubation temperature, pH, and agitation rate, were also investigated. The results are summarized in Table 1. Following that, S. speibonae TKU048 was found to exhibit the highest exochitinase productivity on the medium containing 1.5% SHP with an initial pH of 6.0, incubation temperature of 37 • C, and shaking speed of 175 rpm. After 2 days of fermentation, the exochitinase activity of the optimized culture supernatant reached the maximum value at 45.668 U/mL. The exochitinase activity was approximately 45-fold higher than before optimization. This result indicated that the culture conditions for the production of S. speibonae TKU048 exochitinase by using only chitinous fishery byproducts to provide the source of C/N were successfully optimized. Until now, conversion of abundant and low-cost chitinous materials by microbial activity to produce bioactive compounds has received great attention . Consequently, the current results could be promising in providing a novel beneficial application of shrimp heads-a chitin-containing byproduct from fishery processing-in producing exochitinase via S. speibonae TKU048.

Isolation of Exochitinase
Since there are no previous reports of exochitinase from S. speibonae, it is necessary to investigate the characteristics of exochitinase from this species. To explore its characteristics for comparison with other reports, TKU048 exochitinase was isolated and purified by serial steps: ammonium sulfate precipitation, Macro-Prep High Q chromatography, and size exclusion chromatography on KW-802.5 column. The result is summarized in Table 2. Using ion-exchange chromatography, one peak showing exochitinase activity was found in eluted fractions 80-91 ( Figure 2). These fractions were pooled for further purification by KW-802.5 column. Finally, 0.03 mg of TKU048 exochitinase was collected. The purification showed low yield recovery (0.1%), but high purity yield (376.3-fold). It also showed the strong specific activity result of the obtained enzyme (1.92 × 10 3 U/mg), which was higher than in other reports [2,13,35,44,[47][48][49]. This result indicates that the obtained enzyme not only showed high purity but also exhibited strong exochitinase activity.

Effects of Temperature and pH on the Activity and Stability of TKU048 Activity
A range of temperature from 20-100 °C was used to investigate the influence of temperature on TKU048 exochitinase activity. Figure 4 revealed that the optimal temperature of TKU048 exochitinase was 60 °C and its stability was up to 50 °C. However, at the optimal temperature, the enzyme still retained more than 60% of its activity. Among the chitinases from Streptomyces species, TKU048 exochitinase showed good thermal stability; most of them showed an optimal and stable temperature point similar to or lower than that of the TKU048 exochitinase [2] with some exceptions, such as S. anulatus CS242 chitinase (stability temperature was 60 °C) [51] or S. thermoviolaceus OPC-520 chitinase (optimal temperature was 70-80 °C) [53]. Thermal stability can benefit the application of chitinase in industrial uses.

Effects of Temperature and pH on the Activity and Stability of TKU048 Activity
A range of temperature from 20-100 • C was used to investigate the influence of temperature on TKU048 exochitinase activity. Figure 4 revealed that the optimal temperature of TKU048 exochitinase was 60 • C and its stability was up to 50 • C. However, at the optimal temperature, the enzyme still retained more than 60% of its activity. Among the chitinases from Streptomyces species, TKU048 exochitinase showed good thermal stability; most of them showed an optimal and stable temperature point similar to or lower than that of the TKU048 exochitinase [2] with some exceptions, such as S. anulatus CS242 chitinase (stability temperature was 60 • C) [51] or S. thermoviolaceus OPC-520 chitinase (optimal temperature was 70-80 • C) [53]. Thermal stability can benefit the application of chitinase in industrial uses. The effects of pH on activity and stability of TKU048 chitinase were also studied herein. The optimal pH of TKU048 exochitinase was found at pH 5-7 (on sodium acetate and sodium phosphate buffer, respectively). However, the enzyme also exhibited another optimal pH point at pH 3 when using glycine HCl buffer with over than 80% activity, compared with its activity at pH 5. This suggests that the optimal pH of TKU048 exochitinase also depends on the buffer system. This result was different from most of the other reports of chitinase from other Streptomyces strains, which showed an optimal pH of 5-8 [37][38][39][40][41]47,48,[50][51][52]; only several strains exhibited an optimal pH at a more acidic point, such as S. thermocarboxydus TKU045 (pH 4) [2] and Streptomyces sp. (pH 2 and 6) [54]. To determine the pH stability of S. speibonae TKU048 exochitinase, the enzyme was incubated at a range of pH from 2 to 11 using different buffer systems for 30 min, as mentioned above, and its residual activity was measured after adjusting the enzyme solution to pH 5. Since retaining over 80% of initial activity in pH range from 3-8, S. speibonae TKU048 exochitinase also exhibited good pH stability, especially under acidic conditions.

Substrate Specificity
Different kinds of substrates were used to investigate the specificity activity of TKU048 exochitinase. As shown in Table 3, TKU048 exochitinase expressed the best activity at 43.887 U/mL on pNPg (by p-nitrophenol method), followed by β-chitin powder (βCP) > colloidal chitosan (CCO) > colloidal chitin (CC) > water-soluble chitosan (WSC) > cellulose (by reducing sugar method). In addition, TKU048 exochitinase did not show activity on dextran and α-chitin powder (αCP). This result indicates that TKU048 chitinase specifically acted on the β-(1→4)-linkages and could hydrolyze different types of substrates, including chitin, chitosan, and cellulose. The different activity on αCP, and βCP suggest that the crystalline structure of chitin also affected the ability of TKU048 exochitinase. In addition, it was interesting that βCP was the most suitable substrate of TKU048 exochitinase, with the exception of pNPg. Generally, chitinases possess higher activity on colloidalchitin than on powder chitin [13,35,43,44]; however, some opposing results could be found, such as for chitinase and chitosanase from B. cereus TKU030 [21]. However, some chitinases have exhibited similar results; in producing chitin oligosaccharides or GlcNAc by enzymatic method, chitin must be pretreated with a strong acid, like concentrated HCl, in order to form colloidal chitin. This chemical process has several drawbacks, such as in releasing toxic wastewater and altering chitin's structure. By showing the most activity on β-chitin powder, TKU048 exochitinase may have potential for the direct preparation of chitin oligosaccharides or GlcNAc from chitin powder.  The effects of pH on activity and stability of TKU048 chitinase were also studied herein. The optimal pH of TKU048 exochitinase was found at pH 5-7 (on sodium acetate and sodium phosphate buffer, respectively). However, the enzyme also exhibited another optimal pH point at pH 3 when using glycine HCl buffer with over than 80% activity, compared with its activity at pH 5. This suggests that the optimal pH of TKU048 exochitinase also depends on the buffer system. This result was different from most of the other reports of chitinase from other Streptomyces strains, which showed an optimal pH of 5-8 [37][38][39][40][41]47,48,[50][51][52]; only several strains exhibited an optimal pH at a more acidic point, such as S. thermocarboxydus TKU045 (pH 4) [2] and Streptomyces sp. (pH 2 and 6) [54]. To determine the pH stability of S. speibonae TKU048 exochitinase, the enzyme was incubated at a range of pH from 2 to 11 using different buffer systems for 30 min, as mentioned above, and its residual activity was measured after adjusting the enzyme solution to pH 5. Since retaining over 80% of initial activity in pH range from 3-8, S. speibonae TKU048 exochitinase also exhibited good pH stability, especially under acidic conditions.

Substrate Specificity
Different kinds of substrates were used to investigate the specificity activity of TKU048 exochitinase. As shown in Table 3, TKU048 exochitinase expressed the best activity at 43.887 U/mL on pNPg (by p-nitrophenol method), followed by β-chitin powder (βCP) > colloidal chitosan (CCO) > colloidal chitin (CC) > water-soluble chitosan (WSC) > cellulose (by reducing sugar method). In addition, TKU048 exochitinase did not show activity on dextran and α-chitin powder (αCP). This result indicates that TKU048 chitinase specifically acted on the β-(1→4)-linkages and could hydrolyze different types of substrates, including chitin, chitosan, and cellulose. The different activity on αCP, and βCP suggest that the crystalline structure of chitin also affected the ability of TKU048 exochitinase. In addition, it was interesting that βCP was the most suitable substrate of TKU048 exochitinase, with the exception of pNPg. Generally, chitinases possess higher activity on colloidalchitin than on powder chitin [13,35,43,44]; however, some opposing results could be found, such as for chitinase and chitosanase from B. cereus TKU030 [21]. However, some chitinases have exhibited similar results; in producing chitin oligosaccharides or GlcNAc by enzymatic method, chitin must be pretreated with a strong acid, like concentrated HCl, in order to form colloidal chitin. This chemical process has several drawbacks, such as in releasing toxic wastewater and altering chitin's structure. By showing the most activity on β-chitin powder, TKU048 exochitinase may have potential for the direct preparation of chitin oligosaccharides or GlcNAc from chitin powder. The effects of pH on activity and stability of TKU048 chitinase were also studied herein. The optimal pH of TKU048 exochitinase was found at pH 5-7 (on sodium acetate and sodium phosphate buffer, respectively). However, the enzyme also exhibited another optimal pH point at pH 3 when using glycine HCl buffer with over than 80% activity, compared with its activity at pH 5. This suggests that the optimal pH of TKU048 exochitinase also depends on the buffer system. This result was different from most of the other reports of chitinase from other Streptomyces strains, which showed an optimal pH of 5-8 [37][38][39][40][41]47,48,[50][51][52]; only several strains exhibited an optimal pH at a more acidic point, such as S. thermocarboxydus TKU045 (pH 4) [2] and Streptomyces sp. (pH 2 and 6) [54]. To determine the pH stability of S. speibonae TKU048 exochitinase, the enzyme was incubated at a range of pH from 2 to 11 using different buffer systems for 30 min, as mentioned above, and its residual activity was measured after adjusting the enzyme solution to pH 5. Since retaining over 80% of initial activity in pH range from 3-8, S. speibonae TKU048 exochitinase also exhibited good pH stability, especially under acidic conditions.

Substrate Specificity
Different kinds of substrates were used to investigate the specificity activity of TKU048 exochitinase. As shown in Table 3, TKU048 exochitinase expressed the best activity at 43.887 U/mL on pNPg (by p-nitrophenol method), followed by β-chitin powder (βCP) > colloidal chitosan (CCO) > colloidal chitin (CC) > water-soluble chitosan (WSC) > cellulose (by reducing sugar method). In addition, TKU048 exochitinase did not show activity on dextran and α-chitin powder (αCP). This result indicates that TKU048 chitinase specifically acted on the β-(1→4)-linkages and could hydrolyze different types of substrates, including chitin, chitosan, and cellulose. The different activity on αCP, and βCP suggest that the crystalline structure of chitin also affected the ability of TKU048 exochitinase. In addition, it was interesting that βCP was the most suitable substrate of TKU048 exochitinase, with the exception of pNPg. Generally, chitinases possess higher activity on colloidalchitin than on powder chitin [13,35,43,44]; however, some opposing results could be found, such as for chitinase and chitosanase from B. cereus TKU030 [21]. However, some chitinases have exhibited similar results; in producing chitin oligosaccharides or GlcNAc by enzymatic method, chitin must be pretreated with a strong acid, like concentrated HCl, in order to form colloidal chitin. This chemical process has several drawbacks, such as in releasing toxic wastewater and altering chitin's structure. By showing the most activity on β-chitin powder, TKU048 exochitinase may have potential for the direct preparation of chitin oligosaccharides or GlcNAc from chitin powder.

Hydrolysis Mechanism
To investigate the hydrolysis mechanism of TKU048 exochitinase, chitin oligosaccharides with degree of polymerization (DP) 2-6 were used as the substrates. As shown in Figure 6, TKU048 exochitinase could rapidly hydrolyze all chitin oligosaccharides (2-6 of DP) to release GlcNAc as the main product. This indicates that the obtained enzyme was an exochitinase. As far as we know, exochitinase was considered to separate chitobiase and N-acetyl-β-glucosaminidase. Due to its hydrolyzing abilities (GlcNAc)2 ( Figure 6A), TKU048 exochitinase could initially be classified as an N-acetyl-β-glucosaminidase. Furthermore, the low rate of (GlcNAc)2 production from the hydrolysis of chitin oligosaccharides DP 3 to DP 6 ( Figure 6C-E) revealed that the release of this dimer was not achieved by chitobiase activity. In Figure 6E, the minor (GlcNAc)3 liberated from the hydrolysis reaction of (GlcNAc)6 in the first 20 min indicates that TKU048 exochitinase did not express endochitinase or chitotriase activity. Taken together, the hydrolysis mechanism of TKU048 exochitinase was recognized as following N-acetyl-β-glucosaminidase activity, which catalyzes the Figure 5. Effect of ion metals on the activity of TKU048 exochitinase. TKU048 exochitinase was pre-incubated with each of chemicals for 30 min. The activity of TKU048 exochitinase in the absence of treatment chemicals was used as a control to estimate relative activity (%).

Hydrolysis Mechanism
To investigate the hydrolysis mechanism of TKU048 exochitinase, chitin oligosaccharides with degree of polymerization (DP) 2-6 were used as the substrates. As shown in Figure 6, TKU048 exochitinase could rapidly hydrolyze all chitin oligosaccharides (2-6 of DP) to release GlcNAc as the main product. This indicates that the obtained enzyme was an exochitinase. As far as we know, exochitinase was considered to separate chitobiase and N-acetyl-β-glucosaminidase. Due to its hydrolyzing abilities (GlcNAc) 2 ( Figure 6A), TKU048 exochitinase could initially be classified as an N-acetyl-β-glucosaminidase. Furthermore, the low rate of (GlcNAc) 2 production from the hydrolysis of chitin oligosaccharides DP 3 to DP 6 ( Figure 6C-E) revealed that the release of this dimer was not achieved by chitobiase activity. In Figure 6E, the minor (GlcNAc) 3 liberated from the hydrolysis reaction of (GlcNAc) 6 in the first 20 min indicates that TKU048 exochitinase did not express endochitinase or chitotriase activity. Taken together, the hydrolysis mechanism of TKU048 exochitinase was recognized as following N-acetyl-β-glucosaminidase activity, which catalyzes the hydrolysis reaction of chitin oligosaccharides at the end point to release GlcNAc. The hydrolysis mechanism of TKU048 exochitinase was different from the descriptions in several reports; for instance, Chitinolyticbacter meiyuanensis SYBC-H1 strain produced a chitinase (CmChi1) which possessed both exochitinase and endochitinase abilities and poor N-acetyl-β-glucosaminidase activity [43], PbChi70 produced by P. barengoltzii showed only exochitinase activity [55], PbChi74 produced by P. barengoltzii possessed two chitinolytic activities (exochitinase and N-acetyl-β-glucosaminidase) but lacked endochitinase activity [44], and exo-Chi O1 from S. olivaceoviridis was demonstrated to be an exochitinase that catalyzed chitin to release chitin oligosaccharide with DP 2 as the major product [45].
hydrolysis reaction of chitin oligosaccharides at the end point to release GlcNAc. The hydrolysis mechanism of TKU048 exochitinase was different from the descriptions in several reports; for instance, Chitinolyticbacter meiyuanensis SYBC-H1 strain produced a chitinase (CmChi1) which possessed both exochitinase and endochitinase abilities and poor N-acetyl-β-glucosaminidase activity [43], PbChi70 produced by P. barengoltzii showed only exochitinase activity [55], PbChi74 produced by P. barengoltzii possessed two chitinolytic activities (exochitinase and N-acetyl-βglucosaminidase) but lacked endochitinase activity [44], and exo-Chi O1 from S. olivaceoviridis was demonstrated to be an exochitinase that catalyzed chitin to release chitin oligosaccharide with DP 2 as the major product [45].

Evaluation of GlcNAc Production by TKU048 Exochitinase
Since β-chitin powder was demonstrated to be a suitable substrate for TKU048 exochitinase, this material was chosen for GlcNAc production. The hydrolysis reaction was performed in sodium acetate buffer (50 mM, pH 5) with 0.455 mg/mL of β-chitin powder concentration and 2 U/mL of TKU048 exochitinase (measured by p-nitrophenol reference, approximately) on an incubator (150 rpm, 50 °C). As shown in Figure 7A, the peaks indicating GlcNAc appeared at the retention time of 13.69 min, and the maximum value was observed after 96 h of incubation time. The area of GlcNAc peaks was then used to calculate GlcNAc concentration and GlcNAc production yield. It was found that GlcNAc concentration and GlcNAc yield increased over time ( Figure 7B). Finally, 0.335 mg/mL of GlcNAc could be obtained from 0.455 mg/mL of β-chitin powder with a yield of 73.64% in 96 h. Several reports show that the hydrolysis of chitin by chitinases observed the GlcNAc concentration in the range of 9.8-39.3 µg/mL [43,56]. The higher GlcNAc may have an inhibitory effect on the activity of chitinases [56]. Consequently, it indicated that TKU048 exochitinase may be suitable for GlcNAc production in a higher concentration of this product. Figure 6. HPLC analysis of the hydrolysis products from (GlcNAc) 2-6 by TKU048 exochitinase. A-E: (GlcNAc) 2 -(GlcNAc) 6 , respectively. The reaction was conducted by adding 500 µL substrate solution (0.5 mg/mL) with 500 µL enzyme solution (2 U, approximately) and incubated at 50 • C. Twenty microliters of sample was used for a single HPLC analysis.

Evaluation of GlcNAc Production by TKU048 Exochitinase
Since β-chitin powder was demonstrated to be a suitable substrate for TKU048 exochitinase, this material was chosen for GlcNAc production. The hydrolysis reaction was performed in sodium acetate buffer (50 mM, pH 5) with 0.455 mg/mL of β-chitin powder concentration and 2 U/mL of TKU048 exochitinase (measured by p-nitrophenol reference, approximately) on an incubator (150 rpm, 50 • C). As shown in Figure 7A, the peaks indicating GlcNAc appeared at the retention time of 13.69 min, and the maximum value was observed after 96 h of incubation time. The area of GlcNAc peaks was then used to calculate GlcNAc concentration and GlcNAc production yield. It was found that GlcNAc concentration and GlcNAc yield increased over time ( Figure 7B). Finally, 0.335 mg/mL of GlcNAc could be obtained from 0.455 mg/mL of β-chitin powder with a yield of 73.64% in 96 h. Several reports show that the hydrolysis of chitin by chitinases observed the GlcNAc concentration in the range of 9.8-39.3 µg/mL [43,56]. The higher GlcNAc may have an inhibitory effect on the activity of chitinases [56]. Consequently, it indicated that TKU048 exochitinase may be suitable for GlcNAc production in a higher concentration of this product.

Conclusions
One of the most important applications of chitinase is its use in hydrolyzing chitin/chitosan to produce bioactive chitooligosaccharides and GlcNAc. In the current study, exochitinase production was reported on a novel bacterial strain, S. speibonae TKU048, by using shrimp heads, a low-cost chitinous material, as the sole C/N source. S. speibonae TKU048 exochitinase was purified with high specific activity (1.92 × 10 3 U/mg) and had a molecular mass of 21 kDa. The enzyme also showed valuable properties such as thermal stability, optimal acidic pH, and degradable β-chitin powder. In addition, the hydrolysis mechanism of TKU048 was investigated, which mainly followed N-acetyl-βglucosaminidase activity. The result also indicated that TKU048 exochitinase could hydrolyze βchitin powder to release GlcNAc at high concentrations. The excellent characteristics of S. speibonae TKU048 may give it great potential in GlcNAc production.