Yong-Su Song

Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media

To investigate the expression patterns of chitinase isozymes on native-PAGE and SDS-PAGE gels Paenibacillus chitinolyticus MP-306 was cultured on culture media with and without chitin substrate. P. chitinolyticus MP-306 had a strong chitinolytic activity on colloidal chitin medium. Chitinase isozymes of MP-306 were expressed as six bands (CN1-CN6) on native-PAGE gels and thirteen bands (CS1-CS13) on SDS-PAGE gels after incubation in chitin medium. Three bands (CN1, CN2, and CN3) of chitinase isozymes of MP-306 on native-PAGE gels were expressed as nine bands (CS1, CS2, CS3, CS4, CS5, CS6, CS8, CS10, and CS13) of chitinase isozymes on SDS-PAGE gels. Three bands (CN4, CN5, and CN6) of chitinase isozymes of MP-306 were strongly inhibited by metal ions on native-PAGE and SDS-PAGE gels.

Detection of chitinase ChiA produced by Serratia marcescens PRC-5, using anti-PrGV-chitinase

In this study, a bacterium Serratia marcescens PRC-5 that displayed strong chitinolytic activity on 0.5% colloidal chitin-containing agar medium was isolated from soil. The chitinase activity increased rapidly with a maximum level (6.14 U/mL) on 4 days of incubation with swollen chitin (pH 5.0). Three active bands of chitinase isozymes were observed (53, 44, and 34 kDa) on SDS-PAGE gel and there pI values ranged from pI 5.4 to 5.8 on 2D gels. The chitinase from the PRC-5 strain was also able to produce GlcNAc monomers on TLC plates. The chitinase of PRC-5 inhibited the mycelial growth of Rhizoctonia solani KACC40111, which indicates that it could be used as a biocontrol agent for phytophathogens. The chitinase isozyme N1, which had a molecular weight of 62 kDa, was transferred from a native and SDS-PAGE gel onto an immunoblot and was probed using an anti-PrGV-chitinase.

Expression patterns of chitinase and chitosanase produced from Bacillus cereus in suppression of phytopathogen.

Bacillus cereus MP-310 was incubated on various culture media substrates as LB, colloidal chitin, chitosan powder, and chitosan beads to investigate the concurrent expression patterns of chitinase and chitosanase isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitinase activity increased rapidly with a maximum level after 6 days of incubation in CM-chitin medium. Major bands of chitinase isozymes were strongly expressed on SDS-PAGE in LB medium (four bands) and in colloidal chitin medium (five bands) after 6 days after incubation, and in chitosan powder medium (one band) and in chitosan beads medium (five bands) after 12 days after incubation. A major band of chitosanase isozymes was strongly expressed on SDS-PAGE in chitosan powder medium (one band) and in chitosan beads medium (one band) after 12 days of incubation.

Production of chitin and chitosan from the exoskeleton of adult two-spotted field crickets (Gryllus bimaculatus)

Chitin and chitosan were extracted from all specimens of Type I and II two‐spotted field crickets (Gryllus bimaculatus) following chemical treatment with an acid and alkali. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve demineralization and deproteinization, respectively. For chitosan extraction, 50 % NaOH (w/v) and 50 % NaOH (w/w) solutions were used to achieve deacetylation. Chitosan yielded from adult exoskeletons of G. bimaculatus in Test A of Type I was 1.76 and 8.40 % on a fresh weight (FW) and dry weight (DW) basis, respectively, after treatment with 50 % NaOH (w/v) at 95°C for 3 h. Furthermore, the chitosan yielded in Test D of Type II was 1.79 and 7.06 % on FW and DW basis, respectively, after treatment with 50 % NaOH (w/w) at 105°C for 3 h. The average yield of chitin and chitosan was 2.42 and 1.65 % on a FW basis, and 10.91 and 7.50 % on DW basis, respectively. The deacetylation (%) of chitosan extracted from adult exoskeletons in Tests A, B, C1, C2, D1, and D2 were 81.2 %, 14.5 %, 19.6 %, 90.7 %, 17.1 %, and 95.5 %, respectively. The viscosities of the chitosans extracted from adult exoskeletons in Tests A, C2, and D2 were 32.0, 21.6, and 62.4 cP (centi Poise), respectively. The molecular weight of chitosan from adult exoskeletons of G. bimaculatus was 308.3 kDa. Our results indicate that adult exoskeletons of G. bimaculatus could be used as a source of chitin and chitosan for use as functional additives in industrial animal feeds.

Expression and degradation patterns of chitinase purified from Xuehuali (Pyrus bretschneiderilia) pollen

The present study investigated the expression pattern of chitinase in Xuehuali (Pyrus bretschneiderilia) pollen, as well as its subsequent degradation. The chitinase was purified and collected using chitin affinity column chromatography with regenerated chitin. After purification, four additional chitinase isozymes (chiA, chiB, chiC, and chiD) and chitinase (Chi II) were clearly expressed on SDS-PAGE gels that contained 0.01% glycol chitin. The chitinase reaction products were examined using GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 as substrates at 2 and 24h after reaction via TLC and HPLC. The (GlcNAc)4 oligosaccharide was slightly degraded to (GlcNAc)2 after 24h of reaction with Xuehuali pollen chitinase on TLC. Meanwhile, (GlcNAc)5 was degraded to (GlcNAc)2-4, and 2300ppm (GlcNAc)6 was degraded to 246ppm (GlcNAc)2, 208ppm (GlcNAc)3, 572ppm (GlcNAc)4, and 336ppm (GlcNAc)5 on HPLC. With regard to temperature, the strongest Xuehuali pollen chitinase activity (0.69 unit/mL) was observed at 37°C after 3h of incubation, and with regard to pH, the strongest activity (0.72unit/mL) was observed at pH 3 after 3h of incubation. The main chitin oligomers degraded from (GlcNAc)6 were (GlcNAc)2 and (GlcNAc)4.

Extraction of chitin and chitosan from larval exuvium and whole body of edible mealworm, Tenebrio molitor : Chitin and chitosan from edible mealworm

The purpose of this study was to investigate the production of chitin and chitosan from both the exuvium and whole body of mealworm (Tenebrio molitor) larvae. Chitin from the exuvium and whole body of T. molitor larvae was chemically extracted with acid and alkali solutions to achieve demineralization (DM) and deproteinization (DP), respectively. The average DM (%) and DP (%) on a dry weight (DW) basis was 32.56 and 73.16% from larval exuvium, and 41.68 and 91.53% from whole body, respectively. To obtain chitosan, chitin particles from the exuvium and whole body of T. molitor larva were heated at various temperatures in different concentrations of NaOH. Average chitin yields were 18.01% and 4.92% of DW from the exuvium and whole body, respectively. The relative average yield of chitosan from whole body was 3.65% of DW. On average, over 90% of chitosan derived from whole body was deacetylated. The viscosity of chitosan from whole body was ranged from 48.0 cP to 54.0 cP. The chitin content of dry and wet byproducts from whole body were 17.32% and 16.94% respectively, compared to dry weight. The chitosan contents of byproducts on a DW basis were 14.48% in dry and 13.07% in wet byproduct. These results indicate that the exuvium and whole body of T. molitor larva may serve as a source of chitin and chitosan for use in domestic animal feed.

Enzyme activity and expression pattern of intra- and extracellular chitinase and β-1,3-glucanase of Wickerhamomyces anomalus EG2 using glycol chitin and glucan-containing high polymer complex

We investigated cell growth and activity of intra- and extracellular chitinase, β-1,3-glucanase, and chitin deacetylase with SDS-PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular β-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular β-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular β-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin.