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

Abstract

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.

Conversion of Squid Pen to Homogentisic Acid via Paenibacillus sp. TKU036 and the Antioxidant and Anti-Inflammatory Activities of Homogentisic Acid

“…In our previous study [3], we discovered chitosan oligomers with a low degree of polymerization that showed both antioxidant and anti-inflammatory activity. In this study, the anti-inflammatory activity of Fraction 4-4 (HGA) was estimated using an in vitro model: LPS-stimulated RAW 264.7 cells.…”

“…The mixture was vigorously shaken and incubated for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank [3]. The scavenging ability was calculated as described in our previous paper [3]. …”

“…For recycling squid pens in order to produce additional highly value-added products other than chitin or chitosan, we investigated the reutilization of this fishery processings via microbial conversion in order to produce enzymes [2,3,4,5], exopolysaccharides [6,7], chitooligomers [3], antioxidants [8,9], insecticidal materials [10], and biosorbents [11,12]. …”

“…Recently, we isolated strains of Paenibacillus species that converted squid pen to exopolysaccharides [7], chitosanase [3], and chitooligomers [3]. …”

Biosynthesis of α-Glucosidase Inhibitors by a Newly Isolated Bacterium, Paenibacillus sp. TKU042 and Its Effect on Reducing Plasma Glucose in a Mouse Model

“…The mixture was kept in the dark for 30 min before absorbance at 517 nm was measured [39]. Radical scavenging activity was calculated using the following formula:$\mathrm{Antioxidant}\mathrm{activity}\left(\%\right)=\left[\left(AB-AA\right)/AB\right]\times 100$ where AB and AA stand for absorption of the blank sample ( t = 0 min) and absorption of the tested sample solution ( t = 30 min), respectively.…”