Tzu-Wen Liang

Purification and characterization of a chitosanase from Serratia marcescens TKU011.

A chitosanase was purified from the culture supernatant of Serratia marcescens TKU011 with shrimp shell wastes as the sole carbon/nitrogen source. Zymogram analysis revealed the presence of chitosanolytic activity corresponding to one protein, which was purified by a combination of ion-exchange and gel-filtration chromatography. The molecular weight of the chitosanase was 21 kDa and 18 kDa estimated by SDS-PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase were 5, 50 degrees C, pH 4-8, and <50 degrees C, respectively. The chitosanase was inhibited completely by EDTA, Mn(2+), and Fe(2+). The results of peptide mass mapping showed that three tryptic peptides of the chitosanase were identical to a chitin-binding protein Cbp21 from S. marcescens (GenBank accession number gi58177632) with 63% sequence coverage.

Fermented and enzymatic production of chitin/chitosan oligosaccharides by extracellular chitinases from Bacillus cereus TKU027.

Two chitinases, Chi I and Chi II, were purified from the culture supernatant of Bacillus cereus TKU027 with shrimp head powder (SHP) as the sole carbon/nitrogen source. The molecular masses of Chi I and Chi II determined using SDS-PAGE were approximately 65kDa and 63kDa, respectively. Chi I toward various surfactants showed high stability, such as SDS, Tween 20, Tween 40 and Triton X-100, and these surfactants were stimulator of Chi I chitinase activity. Concomitant with the production of Chi I and Chi II, chitin oligosaccharides were also observed in the culture supernatant, including chitobiose, chitotriose, chitotetrose and chitopentose at concentrations of 0.44mg/mL, 0.08mg/mL, 0.09mg/mL and 0.43mg/mL, respectively. Chitosan with 60% deacetylation was degraded by TKU027 crude enzyme to prepare chitooligosaccharides. MALDI-TOF MS analysis of the enzymatic hydrolyzates indicated that the products were mainly chitooligosaccharides with degree of polymerization (DP) in the 4-9 range.

Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants

Two proteases (P1 and P2) and a chitinase (C1) were purified from the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source. The molecular masses of P1, P2 and C1 determined by SDS-PAGE were approximately 50 kDa, 50 kDa and 60 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of P1, P2 and C1 were (pH 10, 40 degrees C, pH 7-11, and <50 degrees C), (pH 10, 40 degrees C, pH 8-11, and <40 degrees C) and (pH 6, 50 degrees C, pH 5-8, and <50 degrees C), respectively. P1 and P2 were inhibited by Mg(2+), EDTA and C1 was inhibited completely by Cu(2+). The antioxidant activity of TKU013 culture supernatant was 72% per mL (DPPH scavenging ability). With this method, we have shown that squid pen wastes can be utilized and have revealed its hidden potential in the production of functional foods.

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

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 44kDa, 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 0.3μg/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.

Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007

BSN1, a nattokinase, was purified from the culture supernatant of Bacillus subtilis TKU007 with shrimp shell wastes as the sole carbon/nitrogen source. The BSN1 was purified to homogeneity by three-step procedure with a 515-fold increase in specific activity and 12% recovery. The molecular masses of BSN1 determined by SDS-PAGE and gel filtrations were approximately 30 kDa and 28 kDa, respectively. The results of peptide mass mapping showed that four tryptic peptides of BSN1 were identical to the nattokinase from B. subtilis (GenBank accession number gi14422313) with 37% sequence coverage. The N-terminal amino acid sequence of the first 12 amino acids of BSN1 was AQSVPYGISQIK. The optimum pH, optimum temperature, pH stability, and thermal stability of BSN1 were 8, 40 °C, pH 4-11, and less than 50°C, respectively. BSN1 was inhibited completely by PMSF, indicating that the BSN1 was a serine protease. Using this method, B. subtilis TKU007 produces a nattokinase/fibrinolytic enzyme and this enzyme may be considered as a new source for thrombolytic agents.

Production and characterization of exopolysaccharides and antioxidant from Paenibacillus sp. TKU023.

Using squid pen powder (SPP) as the sole C/N source, Paenibacillus sp. TKU023 produced exopolysaccharides (EPS) and antioxidant. With medium containing 1.5% SPP, 0.1% K(2)HPO(4), and 0.05% MgSO(4)·7H(2)O, pH 7.23, the culture was incubated at 37°C in liquid (50 mL/250 mL) for five days. The resultant culture supernatant had higher EPS productivity (4.55 g/L). The crude EPS were isolated by centrifugation, methanol precipitation and deproteinization. The characterization of the EPS demonstrated that it was mainly composed of glucose and maltose. In addition, the culture supernatant incubated for four days by using baffled base flask showed the strongest antioxidant activities and the highest total phenolic content, but maximum EPS production was found at the fifth day by using flat base flask. The production of two invaluable environmental-friendly biomaterials (EPS and antioxidant) is unprecedented. Besides, the use of SPP (waste) is green that made the whole process more valuable and attractive.

Production and purification of a fungal chitosanase and chitooligomers from Penicillium janthinellum D4 and discovery of the enzyme activators

Chitosanases have received much attention because of their wide range of applications. Although most fungal chitosanases use sugar as their major carbon source, in the present work, a chitosanase was induced from a squid pen powder (SPP)-containing Penicillium janthinellum D4 medium and purified by ammonium sulphate precipitation and combined column chromatography. The purified D4 chitosanase exhibited optimum activity at pH 7-9, 60°C and was stable at pH 7-11, 25-50°C. The D4 chitosanase that was used for chitooligomers preparation was studied. The enzyme products revealed various chitooligomers with different degrees of polymerisation (DP) from 3 to 9, as determined by a MALDI-TOF mass spectrometer, confirming the endo-type nature of the D4 chitosanase. D4 chitosanase activity was significantly inhibited by Cu(2+), Mn(2+), and EDTA. However, Fe(2+) activated or inhibited D4 chitosanases at different concentrations. The D4 chitosanase was also activated by some small synthetic boron-containing molecules with boronate ester side chains.

Conversion of squid pen by Pseudomonas aeruginosa K187 fermentation for the production of N-acetyl chitooligosaccharides and biofertilizers

Pseudomonas aeruginosa K187, a protease- and chitinase-producing bacterium, exhibited protease and chitinase activity after three and five days of incubation, respectively. The protease and chitinase were both produced by using 1% squid pen powder (SPP) (w/v) as sole carbon and nitrogen source. After fermentation, the deproteinization rate of the recovered squid pen gradually increased up to 68% on the fourth day. After five days of fermentation, the production of GlcNAc, (GlcNAc)(2), (GlcNAc)(3), (GlcNAc)(4) and (GlcNAc)(5) were 1.18mg/mL, 0.76mg/mL, 1.02mg/mL, 0.93mg/mL and 0.90mg/mL, respectively. The culture supernatant of K187 also exhibited activity of enhancing vegetable growth. For Brassica chinensis Linn treated with the fifth day culture supernatant, the total weight and total length increased up to 529% and 148%, respectively, compared to the control group. With this method, the production of protease, chitinase, N-acetyl chitooligosaccharides and biofertilizers may be useful for biological applications.0008-6215/

An Amphiprotic Novel Chitosanase from Bacillus mycoides and Its Application in the Production of Chitooligomers with Their Antioxidant and Anti-Inflammatory Evaluation

see front matter 2010 Elsevier Ltd. A doi:10.1016/j.carres.2010.01.025 * Corresponding author at present address: 151 Yin County 25137, Taiwan. Tel.: +886 2 2626 9425; fax: + E-mail address: sabulo@mail.tku.edu.tw (S.-L. Wan Pseudomonas aeruginosa K187, a proteaseand chitinase-producing bacterium, exhibited protease and chitinase activity after three and five days of incubation, respectively. The protease and chitinase were both produced by using 1% squid pen powder (SPP) (w/v) as sole carbon and nitrogen source. After fermentation, the deproteinization rate of the recovered squid pen gradually increased up to 68% on the fourth day. After five days of fermentation, the production of GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4 and (GlcNAc)5 were 1.18 mg/mL, 0.76 mg/mL, 1.02 mg/mL, 0.93 mg/mL and 0.90 mg/mL, respectively. The culture supernatant of K187 also exhibited activity of enhancing vegetable growth. For Brassica chinensis Linn treated with the fifth day culture supernatant, the total weight and total length increased up to 529% and 148%, respectively, compared to the control group. With this method, the production of protease, chitinase, N-acetyl chitooligosaccharides and biofertilizers may be useful for biological applications. 2010 Elsevier Ltd. All rights reserved.

Isolation and Identification of a Novel Antioxidant with Antitumour Activity from Serratia ureilytica Using Squid Pen as Fermentation Substrate

The objectives of this investigation were to produce a novel chitosanase for application in industries and waste treatment. The transformation of chitinous biowaste into valuable bioactive chitooligomers (COS) is one of the most exciting applications of chitosanase. An amphiprotic novel chitosanase from Bacillus mycoides TKU038 using squid pen powder (SPP)-containing medium was retrieved from a Taiwan soil sample, which was purified by column chromatography, and characterized by biochemical protocol. Extracellular chitosanase (CS038) was purified to 130-fold with a 35% yield, and its molecular mass was roughly 48 kDa. CS038 was stable over a wide range of pH values (4-10) at 50 °C and exhibited an optimal temperature of 50 °C. Interestingly, the optimum pH values were estimated as 6 and 10, whereas CS038 exhibited chitosan-degrading activity (100% and 94%, respectively). CS038 had Km and Vmax values of 0.098 mg/mL and 1.336 U/min, separately, using different concentrations of water-soluble chitosan. A combination of the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer data revealed that the chitosan oligosaccharides obtained from the hydrolysis of chitosan by CS038 comprise oligomers with multiple degrees of polymerization (DP), varying from 3-9, as well as CS038 in an endolytic fashion. The TKU038 culture supernatant and COS mixture exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities. The COS activities were dose dependent and correlated to their DP. The COS with high DP exhibited enhanced DPPH radical scavenging capability compared with COS with low DP. Furthermore, the COS exhibited inhibitory behavior on nitric oxide (NO) production in murine RAW 264.7 macrophage cells, which was induced by Escherichia coli O111 lipopolysaccharide (LPS). The COS with low DP possesses a more potent anti-inflammatory capability to decrease NO production (IC50, 76.27 ± 1.49 µg/mL) than that of COS with high DP (IC50, 82.65 ± 1.18 µg/mL). Given its effectiveness in production and purification, acidophilic and alkalophilic properties, stability over ranges of pH values, ability to generate COS, antioxidant activity, and anti-inflammatory, CS038 has potential applications in SPP waste treatment and industries for COS production as a medical prebiotic.