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Chitinolytic Bacteria-Assisted Conversion of Squid Pen and Its Effect on Dyes and Pigments Adsorption.

Liang TW, Lo BC, Wang SL - Mar Drugs (2015)

Bottom Line: One chitosanase induced from squid pen powder (SPP)-containing medium by Bacillus cereus TKU034 was purified in high purification fold (441) and high yield of activity recovery (51%) by ammonium sulfate precipitation and combined column chromatography.The SDS-PAGE results showed its molecular mass to be around 43 kDa.The enzyme products revealed that the chitosanase could degrade chitosan with various degrees of polymerization, ranging from 3 to 9, as well as the chitosanase in an endolytic manner.

View Article: PubMed Central - PubMed

Affiliation: Life Science Development Center, Tamkang University, No. 151, Yingchuan Rd., Tamsui, New Taipei City 25137, Taiwan. ltw27@ms55.hinet.net.

ABSTRACT
The aim of this work was to produce chitosanase by fermenting from squid pen, and recover the fermented squid pen for dye removal by adsorption. One chitosanase induced from squid pen powder (SPP)-containing medium by Bacillus cereus TKU034 was purified in high purification fold (441) and high yield of activity recovery (51%) by ammonium sulfate precipitation and combined column chromatography. The SDS-PAGE results showed its molecular mass to be around 43 kDa. The TKU034 chitosanase used for the chitooligomers preparation was studied. The enzyme products revealed that the chitosanase could degrade chitosan with various degrees of polymerization, ranging from 3 to 9, as well as the chitosanase in an endolytic manner. Besides, the fermented SPP was recovered and displayed a better adsorption rate (up to 99.5%) for the disperse dyes (red, yellow, blue, and black) than the water-soluble food colorants, Allura Red AC (R40) and Tartrazine (Y4). The adsorbed R40 on the unfermented SPP and the fermented SPP was eluted by distilled water and 1 M NaOH to confirm the dye adsorption mechanism. The fermented SPP had a slightly higher adsorption capacity than the unfermented, and elution of the dye from the fermented SPP was easier than from the unfermented. The main dye adsorption mechanism of fermented SPP was physical adsorption, while the adsorption mechanism of unfermented SPP was chemical adsorption.

No MeSH data available.


Hydrolysis time course measurement of reducing sugar (···) and total sugar (—) with the chitosanase from B. cereus TKU034. Chitosanase activity: ●, 10 U/mL; ▲, 5 U/mL; ■, 1 U/mL.
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marinedrugs-13-04576-f004: Hydrolysis time course measurement of reducing sugar (···) and total sugar (—) with the chitosanase from B. cereus TKU034. Chitosanase activity: ●, 10 U/mL; ▲, 5 U/mL; ■, 1 U/mL.

Mentions: To evaluate the applicability of the TKU034 chitosanase for the enzymatic digestibility of chitosan into oligosaccharides, the crude enzyme from B. cereus TKU034 was used in the experiments. The production system of TKU034 chitosanase was highly efficient and fully available in our laboratory. The chitosan hydrolysis was carried out at 37 °C for six days. The reaction mixture in an Erlenmeyer conical flask (50 mL capacity) contained 0.5% of colloidal chitosan in 20 mL of phosphate buffer (pH 7, 50 mM) and 2.5 mL of concentrated enzyme preparation. Aliquots were withdrawn at different intervals and heated in a boiling water bath for 15 min to terminate the enzyme activity. After removing the undigested material by centrifugation (10,000 rpm, 20 min), the course of chitosan sample degradation was conveniently studied by measurement of the reducing sugars and total sugars [34] in the supernatant. We confirmed that the release of reducing sugars into the supernatant fraction completely ceased after six days of incubation. The amounts of the insoluble materials remaining after the digestion of TKU034 chitosanase were determined and compared with the starting amounts of the chitosan. The amount of insoluble materials remaining was the lowest at the sixth day of digestion with TKU034 chitosanase, indicating that TKU034 chitosanase has 99% digestibility for chitosan (data not shown). As shown in Figure 4, the results revealed the total sugar and the reducing sugar of the sample as a function of reaction time. The total and reducing sugars increased, and the chitosan sample recovery decreased dramatically in the early reaction stage, which can be attributed to an endo-type degradation process. The TKU034 chitosanase was added into the reaction solution after six days and continued hydrolyzing, but it did not improve the increase in total and reducing sugar levels (data not shown). Selective precipitation in 90% methanol and acetone solutions was performed to obtain low DP oligomers, as described earlier [16]. A product analysis of the 90% acetone solution precipitation by MALDI-TOF revealed that various COS had DP values up to 9 (Figure 5). The higher DP chitooligomers were precipitated as a light yellow powder in the methanol solution. The low DP oligomer fraction MALDI-TOF-MS revealed pronounced differences among the crude enzyme-generated chitooligomers, as demonstrated for chitosan depolymerization in Figure 5. The hydrolysate ions present in the mass spectra were identified as sodium adducts [M + Na]+. The MALDI-TOF analysis is limited to molecular weights higher than 500 Da because of matrix signal interference; therefore, DP < 2 oligomers could not be determined by this method. After the hydrolysis from the enzymatic reaction over six days, (GlcN)3 (m/z 536), GlcN-(GlcNAc)2 (m/z 608), (GlcN)2-(GlcNAc)2 (m/z 769), (GlcN)3-(GlcNAc)2 (m/z 930), and (GlcN)4-(GlcNAc)2 (m/z 1091) were the major products. In addition, other clear signals (m/z 685, 727, 811, 972, 1133, 1175, 1294, 1455, and 1616) were also detected (Figure 5). The differences in m/z values of these signals with those of the corresponding COSs were 42 atomic mass units, which corresponded to the mass weight of acetyl. The hydrolysates contained chitooligomers (GlcN-oligomers) and several partial N-acetylated forms. The TKU034 chitosanase reaction product is a mixture of DP 3–9 hetero-chitooligomers. These results indicate that the TKU034 chitosanase might hydrolyze chitosan in an endo-type fashion. From these results, chitosan hydrolysis by the TKU034 chitosanase, combined with a selective methanol precipitation, is a quick and simple method to obtain good chitooligosaccharide yields with DPs up to nine and low molecular weight oligomers.


Chitinolytic Bacteria-Assisted Conversion of Squid Pen and Its Effect on Dyes and Pigments Adsorption.

Liang TW, Lo BC, Wang SL - Mar Drugs (2015)

Hydrolysis time course measurement of reducing sugar (···) and total sugar (—) with the chitosanase from B. cereus TKU034. Chitosanase activity: ●, 10 U/mL; ▲, 5 U/mL; ■, 1 U/mL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556994&req=5

marinedrugs-13-04576-f004: Hydrolysis time course measurement of reducing sugar (···) and total sugar (—) with the chitosanase from B. cereus TKU034. Chitosanase activity: ●, 10 U/mL; ▲, 5 U/mL; ■, 1 U/mL.
Mentions: To evaluate the applicability of the TKU034 chitosanase for the enzymatic digestibility of chitosan into oligosaccharides, the crude enzyme from B. cereus TKU034 was used in the experiments. The production system of TKU034 chitosanase was highly efficient and fully available in our laboratory. The chitosan hydrolysis was carried out at 37 °C for six days. The reaction mixture in an Erlenmeyer conical flask (50 mL capacity) contained 0.5% of colloidal chitosan in 20 mL of phosphate buffer (pH 7, 50 mM) and 2.5 mL of concentrated enzyme preparation. Aliquots were withdrawn at different intervals and heated in a boiling water bath for 15 min to terminate the enzyme activity. After removing the undigested material by centrifugation (10,000 rpm, 20 min), the course of chitosan sample degradation was conveniently studied by measurement of the reducing sugars and total sugars [34] in the supernatant. We confirmed that the release of reducing sugars into the supernatant fraction completely ceased after six days of incubation. The amounts of the insoluble materials remaining after the digestion of TKU034 chitosanase were determined and compared with the starting amounts of the chitosan. The amount of insoluble materials remaining was the lowest at the sixth day of digestion with TKU034 chitosanase, indicating that TKU034 chitosanase has 99% digestibility for chitosan (data not shown). As shown in Figure 4, the results revealed the total sugar and the reducing sugar of the sample as a function of reaction time. The total and reducing sugars increased, and the chitosan sample recovery decreased dramatically in the early reaction stage, which can be attributed to an endo-type degradation process. The TKU034 chitosanase was added into the reaction solution after six days and continued hydrolyzing, but it did not improve the increase in total and reducing sugar levels (data not shown). Selective precipitation in 90% methanol and acetone solutions was performed to obtain low DP oligomers, as described earlier [16]. A product analysis of the 90% acetone solution precipitation by MALDI-TOF revealed that various COS had DP values up to 9 (Figure 5). The higher DP chitooligomers were precipitated as a light yellow powder in the methanol solution. The low DP oligomer fraction MALDI-TOF-MS revealed pronounced differences among the crude enzyme-generated chitooligomers, as demonstrated for chitosan depolymerization in Figure 5. The hydrolysate ions present in the mass spectra were identified as sodium adducts [M + Na]+. The MALDI-TOF analysis is limited to molecular weights higher than 500 Da because of matrix signal interference; therefore, DP < 2 oligomers could not be determined by this method. After the hydrolysis from the enzymatic reaction over six days, (GlcN)3 (m/z 536), GlcN-(GlcNAc)2 (m/z 608), (GlcN)2-(GlcNAc)2 (m/z 769), (GlcN)3-(GlcNAc)2 (m/z 930), and (GlcN)4-(GlcNAc)2 (m/z 1091) were the major products. In addition, other clear signals (m/z 685, 727, 811, 972, 1133, 1175, 1294, 1455, and 1616) were also detected (Figure 5). The differences in m/z values of these signals with those of the corresponding COSs were 42 atomic mass units, which corresponded to the mass weight of acetyl. The hydrolysates contained chitooligomers (GlcN-oligomers) and several partial N-acetylated forms. The TKU034 chitosanase reaction product is a mixture of DP 3–9 hetero-chitooligomers. These results indicate that the TKU034 chitosanase might hydrolyze chitosan in an endo-type fashion. From these results, chitosan hydrolysis by the TKU034 chitosanase, combined with a selective methanol precipitation, is a quick and simple method to obtain good chitooligosaccharide yields with DPs up to nine and low molecular weight oligomers.

Bottom Line: One chitosanase induced from squid pen powder (SPP)-containing medium by Bacillus cereus TKU034 was purified in high purification fold (441) and high yield of activity recovery (51%) by ammonium sulfate precipitation and combined column chromatography.The SDS-PAGE results showed its molecular mass to be around 43 kDa.The enzyme products revealed that the chitosanase could degrade chitosan with various degrees of polymerization, ranging from 3 to 9, as well as the chitosanase in an endolytic manner.

View Article: PubMed Central - PubMed

Affiliation: Life Science Development Center, Tamkang University, No. 151, Yingchuan Rd., Tamsui, New Taipei City 25137, Taiwan. ltw27@ms55.hinet.net.

ABSTRACT
The aim of this work was to produce chitosanase by fermenting from squid pen, and recover the fermented squid pen for dye removal by adsorption. One chitosanase induced from squid pen powder (SPP)-containing medium by Bacillus cereus TKU034 was purified in high purification fold (441) and high yield of activity recovery (51%) by ammonium sulfate precipitation and combined column chromatography. The SDS-PAGE results showed its molecular mass to be around 43 kDa. The TKU034 chitosanase used for the chitooligomers preparation was studied. The enzyme products revealed that the chitosanase could degrade chitosan with various degrees of polymerization, ranging from 3 to 9, as well as the chitosanase in an endolytic manner. Besides, the fermented SPP was recovered and displayed a better adsorption rate (up to 99.5%) for the disperse dyes (red, yellow, blue, and black) than the water-soluble food colorants, Allura Red AC (R40) and Tartrazine (Y4). The adsorbed R40 on the unfermented SPP and the fermented SPP was eluted by distilled water and 1 M NaOH to confirm the dye adsorption mechanism. The fermented SPP had a slightly higher adsorption capacity than the unfermented, and elution of the dye from the fermented SPP was easier than from the unfermented. The main dye adsorption mechanism of fermented SPP was physical adsorption, while the adsorption mechanism of unfermented SPP was chemical adsorption.

No MeSH data available.