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

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.

Production and Characterization of Antioxidant Properties of Exopolysaccharide(s) from Peanibacillus mucilaginosus TKU032.

Natural polysaccharides have received much attention due to their wide range of applications. Although most microbial exopolysaccharides (EPSs) use sugars as the major carbon source, such as glucose or sucrose, in this study, EPSs were induced from a squid pen powder (SPP)-containing medium by Paenibacillus mucilaginosus TKU032, a bacterial strain isolated from Taiwanese soil. Under the optimal culture conditions, the maximum EPS yield (14.8 g/L) was obtained. MALDI-TOF MS analysis of an EPS fraction purified by gel filtration revealed two mass peaks with molecular weights of ∼1.05 × 104 and ∼1.35 × 104 Da, respectively. The analysis of the hydrolysates of TKU032 EPS with cellulase, pectinase or α-amylase indicated that the glycosidic bond of TKU032 EPS is most likely an α-1,4 glycosidic bond and the hydrolysates are similar to those of starch. In addition, the purified EPS demonstrated strong antioxidant abilities.

Application of Chitinous Materials in Production and Purification of a Poly(l-lactic acid) Depolymerase from Pseudomonas tamsuii TKU015.

The management of fishery residues and plastics is considered to be a vital strategy for conserving resources and maintaining the quality of the environment. Poly(l-lactic acid) (PLA) is a commercially promising, renewable, and biodegradable plastic. In this study, a PLA depolymerase was produced in a squid pen powder (SPP) and recycled plastic waste (PLA powder)-containing medium by Pseudomonas tamsuii TKU015, a bacterial strain isolated from Taiwanese soil. This PLA depolymerase had a molecular weight of 58 kDa and was purified to homogeneity from the supernatant of a TKU015 culture. The optimum pH of TKU015 PLA depolymerase is 10, and the optimal temperature of the enzyme is 60 °C. In addition to PLA, TKU015 PLA depolymerase degraded fibrinogen and tributyrin, but did not hydrolyze casein, triolein, and poly(ß-hydroxybutyrate). Taken together, these data demonstrate that P. tamsuii TKU015 produces a PLA depolymerase to utilize SPP and polylactide as carbon/nitrogen sources.

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

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.

Recent advances in exopolysaccharides from Paenibacillus spp.: production, isolation, structure, and bioactivities.

This review provides a comprehensive summary of the most recent developments of various aspects (i.e., production, purification, structure, and bioactivity) of the exopolysaccharides (EPSs) from Paenibacillus spp. For the production, in particular, squid pen waste was first utilized successfully to produce a high yield of inexpensive EPSs from Paenibacillus sp. TKU023 and P. macerans TKU029. In addition, this technology for EPS production is prevailing because it is more environmentally friendly. The Paenibacillus spp. EPSs reported from various references constitute a structurally diverse class of biological macromolecules with different applications in the broad fields of pharmacy, cosmetics and bioremediation. The EPS produced by P. macerans TKU029 can increase in vivo skin hydration and may be a new source of natural moisturizers with potential value in cosmetics. However, the relationships between the structures and activities of these EPSs in many studies are not well established. The contents and data in this review will serve as useful references for further investigation, production, structure and application of Paenibacillus spp. EPSs in various fields.

Squid pen chitin chitooligomers as food colorants absorbers.

One of the most promising applications of chitosanase is the conversion of chitinous biowaste into bioactive chitooligomers (COS). TKU033 chitosanase was induced from squid pen powder (SPP)-containing Bacillus cereus TKU033 medium and purified by ammonium sulfate precipitation and column chromatography. The enzyme was relatively more thermostable in the presence of the substrate and had an activity of 93% at 50 °C in a pH 5 buffer solution for 60 min. Furthermore, the enzyme used for the COS preparation was also studied. The enzyme products revealed various mixtures of COS that with different degrees of polymerization (DP), ranging from three to nine. In the culture medium, the fermented SPP was recovered, and it displayed a better adsorption rate (up to 96%) for the disperse dyes than the water-soluble food colorants, Allura Red AC (R40) and Tartrazne (Y4). Fourier transform-infrared spectroscopic (FT-IR) analysis proved that the adsorption of the dyes onto fermented SPP was a physical adsorption. Results also showed that fermented SPP was a favorable adsorber and could be employed as low-cost alternative for dye removal in wastewater treatment.

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.

Purification of chitinase/chitosanase from Bacillus cereus and discovery of an enzyme inhibitor.

A chitinase and a chitosanase were induced from a squid pen powder (SPP)-containing medium of Bacillus cereus TKU030 and purified by precipitation with ammonium sulphate and combined column chromatography. The purified chitinase and chitosanase exhibited optimum activity at 60 °C, pH 5-6 and 40 °C, pH 4, respectively. The chitinase and chitosanase were stable at 25-60 °C, pH 4-7 and 25-50 °C, pH 3-7, respectively. The chitinase and chitosanase showed the highest activity toward ß-chitin and 60% DD chitosan, respectively. The chitinase was significantly inhibited by Mn(2+) and EDTA but activated by Cu(2+), Fe(2+) and Ca(2+). The chitosanase was significantly inhibited by Cu(2+), Fe(2+), Zn(2+), Mn(2+) and EDTA. The chitinase showed high stability in the presence of various surfactants, such as SDS, Tween 20, Tween 40 and Triton X-100. In contrast, these surfactants were inhibitors of the chitosanase. The chitinase and chitosanase were also inhibited by TKUPSP017, a small synthetic boron-containing molecule with a BF3K side-chain. However, TKUPSP017 enhanced the growth of B. cereus TKU030 in SPP-containing medium.

Exopolysaccharides and antimicrobial biosurfactants produced by Paenibacillus macerans TKU029.

Paenibacillus macerans TKU029 can produce exopolysaccharides (EPSs; 3.46 g/L) and a biosurfactant (1.78 g/L) in a medium with 2 % (w/v) squid pen powder as the sole carbon/nitrogen source. The biosurfactant can reduce the surface tension of water from 72.30 to 35.34 mN/m at a concentration of 2.76 g/L and reach an emulsification index of 56 % after a 24-h reaction with machine oil. This biosurfactant is stable at 121 °C for 20 min, over a pH range from 3 to 11, and in <5 % salt solutions. It also shows significant antimicrobial activity, which remains active after treatment at 121 °C and at pH values from 4 to 10, against Escherichia coli BCRC13086, Staphylococcus aureus BCRC10780, Fusarium oxysporum BCRC32121 and Aspergillus fumigatus BCRC30099. Furthermore, human skin shows from 37.3 to 44.3 % hydration after being treated with TKU029 EPSs for 180 min. These results imply that EPSs and the biosurfactant from this strain have potential in cosmetics, for removal of oil contamination, and as antimicrobial agents.

Purification of a thermostable chitinase from Bacillus cereus by chitin affinity and its application in microbial community changes in soil.

A thermostable chitinase was purified by chitin affinity from the culture supernatant of Bacillus cereus TKU028 with shrimp head powder (SHP) as the sole carbon/nitrogen source. TKU028 chitinase was purified using a one-step affinity adsorbent system, and the molecular mass of TKU028 chitinase (approximately 40 kDa) was then determined using SDS-PAGE. The enzyme was stable for 60 min at temperatures below 60 °C and stable over a broad pH range of 4-9 for 60 min. In addition, the temporal changes of a bacterial community in mangrove river sediment of the Tamsui River with added SHP were also analysed by PCR-denaturing gradient gel electrophoresis to investigate the effects of B. cereus TKU028 on the degradation of SHP. The 6-week incubation sample of SHP and B. cereus TKU028-amended mangrove river sediment displayed the highest amount of biomass, reducing sugar and total sugar, and some variance of bacterial community composition existed in the soils.

Enhancement of prodigiosin production by Serratia marcescens TKU011 and its insecticidal activity relative to food colorants.

Prodigiosin (PG) has been reported to have various biological activities. With the aim of increasing Serratia marcescens TKU011 PG production on squid pen powder (SPP)-containing medium, the effects of phosphate and ferrous ion supplementation, autoclave treatment, and aeration were studied. Autoclave treatment showed positive results for PG productivity (2.48 mg/mL), which increased 2.5-fold when the organism was incubated in 50 mL of 40-min autoclaved medium in a baffle-based flask (250 mL) containing 1.5% SPP at 30 °C for 1 day and then at 25 °C for 2 additional days. Furthermore, the use of pigments including PG and the food colorants Allura Red AC (R40) and Tartrazine (Y4) as insecticides was also investigated. The lethal concentrations causing 50% Drosophila larval mortality (LC50) of PG, Y4, and R40 using a 5-d exposure period were 230, 449, and 30000 ppm, respectively. The results indicated that the biopigment PG and the food colorant Y4 were potentially toxic to Drosophila larvae.

Applied development of crude enzyme from Bacillus cereus in prebiotics and microbial community changes in soil.

The chitosanase and chitinase activity were revealed in the culture supernatant of Bacillus cereus TKU027 with shrimp head powder (SHP) as the sole carbon/nitrogen source. The chitosan with 60% degree of deacetylation (DD) was depolymerized by TKU027 crude enzyme. The low DP oligomers stimulated the growth of Lactobacillus paracasei BCRC12193 and Lactobacillus kefir BCRC14011 in a MRS broth supplemented with low DP oligomers for 12 h. Conversely, the high DP oligomers (0.1%) had potent inhibitory effects against L. paracasei BCRC12193 and L. kefir BCRC14011 for 48 h. Besides, the study also investigated the effects of B. cereus TKU027 on degradation of SHP and the survival conditions of bacteria in mangrove river sediment of Tamsui River. The 5 weeks-incubation sample of SHP and B. cereus TKU027-amended mangrove river sediment showed the highest amounts of biomass, reducing sugar and total sugar, and some variance of bacterial community compositions existed in the soils.

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.

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.

Utilisation of chitinous materials in pigment adsorption.

The effect of adding the cells of four lactobacilli to a squid pen powder (SPP)-containing medium on prodigiosin (PG) production by Serratia marcescens TKU011 is examined. The best increase in PG productivity was shown by strain TKU012. Among the samples of strain TKU012 and the chitinous materials of cicada casting powder (CCP), shrimp shell powder (SSP), squid pen powder (SPP), α-chitin, and ß-chitin, TKU012 cells displayed the best adsorption rate (84%) for PG, followed by CCP, SSP, SPP, α-chitin, and ß-chitin. As for the water-soluble food colourants, Allura Red AC (R40) and Tartrazne (Y4), SPP and SSP had better adsorptive powers than pure chitin preparations, strain TKU012, and CCP. Treatment with organic solvents, hot alkali, or proteases (papain, bromelain) diminished the adsorption rates of the biosorbents.

A novel compound with antioxidant activity produced by Serratia ureilytica TKU013.

The secondary metabolites from the cultured supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source were isolated and ascertained the mechanism of biological activity. The EtOAc layer, which has high DPPH scavenging activity, was applied to silica gel column chromatography with a gradient of CH(2)Cl(2)/MeOH solvent system, to yield A-H and MeOH fractions. The DPPH scavenging activity and cytotoxic activities against Doay and HEp-2 cell lines of these fractions were examined. The active fractions were further applied to purification by RP-HPLC, to obtain seven compounds, including a novel compound, serlyticin-A (1), together with six known compounds, serranticin (2), serratamolide A (3), thymine (4), (4-hydroxyphenyl)acetic acid (5), methyl p-hydroxybenzoate (6), and uracil (7). Their structures were determined by physical and extensive spectral analyses such as 1D and 2D NMR data, as well as comparison with literature values. Furthermore, the major secondary metabolites of EtOAc extract of the cultured supernatant were examined by the fingerprinting data of the HPLC system.

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.

Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, Acinetobacter calcoaceticus TKU024.

Two chitosanases (CHSA1 and CHSA2) were purified from the culture supernatant of Acinetobacter calcoaceticus TKU024 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHSA1 and CHSA2 determined by SDS-PAGE were approximately 27 and 66 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSA1 and CHSA2 were (pH 6, 50°C, pH 4-10, <90°C) and (pH 7, 60°C, pH 6-11, <70°C), respectively. CHSA1 and CHSA2 had broad pH and thermal stability. CHSA1 and CHSA2 were both inhibited by EDTA and were inhibited completely by 5 mM Mn(2+). CHSA1 and CHSA2 degraded chitosan with DD ranging from 60 to 98%, and also degraded some chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU024 culture supernatant (1.5% SPP) incubated for 5 days has the most reducing sugars (0.63 mg/ml). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.

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.

Isolation and identification of a novel antioxidant with antitumour activity from Serratia ureilytica using squid pen as fermentation substrate.

The antioxidant activity of the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source was assessed by three methods, and the phenolic contents were assayed. The supernatant with the highest antioxidant activity was further purified by liquid-liquid partition, revealing the ethyl acetate extract exhibited the strongest antioxidant activity and the highest total phenolic content. Eight fractions were retrieved from silica gel column chromatography of this extract, designated F1-F8. F4 was found to possess the strong antioxidative activity and the highest total phenolic content and also exhibited strong cytotoxic activities against two different tumoural cell lines. A new compound (Serranticin) with antioxidant and antitumor activity was obtained from F4. The structure of Serranticin is analogous to that of siderophores (hexacoordinated catecholamine), which are iron chelators. As such, Serranticin has the potential for use as a deferration agent in various iron overload diseases.