• Journal of Microbiology and Biotechnology
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• v.20 no.7
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• pp.1114-1120
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• 2010

A chitosan-degrading fungus, BSF114, was isolated from soil. The culture preparation showed strong chitosanolytic enzyme activity at an optimum pH of 4.0 and optimum temperature of $60^{\circ}C$ after 36-40 h fermentation. The rapid decrease in the viscosity of the chitosan solution early in the reaction suggested an endo-type cleavage of the polymeric chitosan chains. To identify the isolated fungus, molecular biological and morphological methods were used. The fungal internal transcribed spacer (ITS) region 1 was amplified, sequenced, and then compared with related sequences in the GenBank database using BLAST. The phylogenetic relationships were then analyzed, and the results showed that the fungus belongs to Aspergillus fumigatus. Morphological observations were also used to confirm the above conclusion. The chitooligosaccharides (COS) obtained through hydrolyzing the colloidal chitosan showed that A. fumigatus BSF114 is suitable for degrading chitosan and producing chitooligosaccharides on a large scale. High concentrations of the COS (1,000 and 500 ${\mu}g/ml$) significantly proliferated mice marrow cells.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.686-692
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• 2004

To develop the enzyme-linked immunosorbent assay (ELISA) for the analysis of N-acetylehitooligosaccharides (NACOS) and chitooligosaccharides (COS), specific antibodies (Abs) were produced, and their properties were compared. N-acetylehitohexaose (NACOS6), chitohexaose (COS6), and COS mixture (COSM) conjugated to bovine serum albumin (BSA) were used to immunize rabbits. By the use of specific Abs and NACOS6-horseradish peroxidase (HRP), COS6-HRP, and COSM-HRP conjugates, competitive direct ELISA (cdELISA) was developed. The detection limits of NACOS6 by the anti-NACOS6 Ab and COS6 by the anti-COS6 and the anti-COSM Abs in the cdELISAs were about 0.2, 2, ana 2 ng/ml (ppb), respectively. In the cdELISA, the anti-NACOS6 Ab was found to recognize NACOS3-NACOS6, but not N-acetyl-D-glucosamine (GlcNAc), NACOS2, and COSs; the anti-COS6 Ab recognized COS2-COS6 and COSM, but not glucosamine (GlcN) and NACOSs. The recognition pattern of the anti-COSM Ab was almost the same as that of the anti- COS6 Ab, except that the former recognized COS2 and COS3 slightly better than the latter.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.877-880
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• 2004

In this study, we determined the content of chitooligosaccharides (COS) in Korean commercial salt-fermented shrimps by competitive direct enzyme-linked immunosorbent assays (cdELISAs), using anti-COS mixture (COSM) antibody and COSM horseradish peroxidase (HRP) conjugate. When COS6 was spiked into salt-fermented shrimps at the level of $10-300\mu{g/g,}$ the average recovery was $120\pm19%$ ($mean\pmS.D.$). The COS contents of the 92 samples of Korean commercial salt-fermented shrimps collected during February 2000 and August 2002 were $36.3\pm20.7\mug$ COS6 equivalent/g (expressed as "$\mug/g$" hereafter). Among the samples, the COS contents of yuk-jeot ( $40.3 \pm 22.5 \mug/g, n=27$) and buksaewoojeot ($40.2 \pm 21.6 \mug/g, n=5$) were higher than the others. The COS contents of salt-fermented shrimps produced at Gwangcheon ($47.1 \pm 20.7 \mug/g, n=18$) and Gomso ($44.1 \pm 21.8 \mug/g, n=6$) areas were higher than those produced at the other areas. This is the first report to determine COS of salt-fermented shrimps by cdELISA.

• Journal of Applied Biological Chemistry
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• v.55 no.1
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• pp.13-17
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• 2012

In order to enzymatically produce chitooligosaccharide using the crude enzyme preparation from Bacillus cereus D-11, we first studied the optimal reaction conditions. It was found that the optimal temperature for hydrolysis of chitosan was $55^{\circ}C$. The ratio of enzyme/substrate should not be lower than 0.13 U/mg in the reaction mixture. The enzyme activity was stable below $50^{\circ}C$. The products of enzymatic reaction were analyzed by both thin layer chromatography and high performance liquid chromatography. Under the appropriate condition, chitosan was hydrolyzed using the enzyme preparation. The resulting chitooligosaccharides were purified and separated by Dowex ($H^+$) ion exchange chromatography. From 4 g soluble chitosan, 0.95 g $(GlcN)_2$, 1.43 g $(GlcN)_3$, and 1.18 g $(GlcN)_4$ were recovered.

• The Korean Journal of Food And Nutrition
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• v.12 no.6
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• pp.597-601
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• 1999

Chitosan을 Bacillus pumilus BN-262 유래의 chito-sanase로 처리한 경우에는 trimet, tetramer, pentamer 가 전체 올리고당 중 64,3%에 달하는 비교적 저분자의 chitooligosaccharides로 구성괸 chitooligosacch-aride I을 얻을수 있었다 그러나 Trichoderma viride 유래의 cellulase로 처리한 경우에는 중합도 7이상의 것이 전체 올리고당 중 49.3% 에 달하는 상대적으로 분자량이 큰 chitooligosaccharides로 구성된 chitoolig-osaccharide II을 얻을수 있었다. 따라서 생리적 기능성이 높은 hexamer 이상의 chitooligosaccharides를 얻기 위해서는 chitosanase의 처리조건을 달리하여 분해가 덜 일어나도록 하거나 cellulase와 같은 효소를 처리함으로써 chitosan의 부분적인 분해를 유도하는 것이 필요하고 판단되었다. Chitin과 chitooligosac-charides의 응집성에 대하여 조사하였다 고분자의 chitosan은 2가 음이온을 함유하는 무기화합물 및 고분자의 유기화합물과 반응하여 응집이 일어났다. 분해가 많이 일어난 chitooligosaccharide I 은 무기물질과는 침전하지 않았으나 고분자화학불을 함유하는 유기화합물과는 반응하여 침전이 일어났다 분해가 적게 일어난 chitooligosaccharide II 는 2가 음이온을 함유하는 무기화합물 및 고분자의 유기화합물과 반응하여 침전이 일어났다.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2000.05a
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• pp.86-87
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• 2000

Chitosan (deacetylated form of chitin) possesses strong antibacterial activities such as antimicrobial effect, antifungal effect and the induction of plant defense response. Chitosan itself, however, has high molecular weight and viscosity as well as water-insolubility, These natures may restrict applications in various fields, especially in in vivo system. While the hydrolysates of chitosan, chitooligosaccharides (COS) are not only lower in the molecula. weight and viscosity, but also water-soluble. Thus, they would be expected more efficient absorption in vivo. Besides several documents have been reported antibacterial activities of COS against microorganisms (Kendra et al., 1989; Uchida et al., 1989). (omitted)

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.960-968
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• 2003

A bacterial isolate showing a strong endochitosanase activity was isolated from soil and then characterized. The isolate was identified and designated as Bacillus cereus P16, based on morphological and biochemical properties, assimilation tests, cellular fatty acids pattern, along with 16S rRNA gene sequence. The optimized medium for producing extracellular chitosanase in a batch culture contained 1% tryptone, 0.5% chitosan, and 1% NaCl (pH 7.0). Powder chitosan and tryptone served the best as carbon and nitrogen sources, respectively, for the chitosanase production. Chitosanase activity was the highest when culture was completed at $37^{\circ}C$ among various temperatures ($20-42^{\circ}C$) tested in a shaking incubator (200 rpm). The levels of chitosanase activity in the culture fluid were 2.0 U/ml and 3.8 U/ml, respectively, when incubated in a flask for 60 h and in a jar fermenter for 24 h. The culture supernatant showed a strong liquefying activity on the soluble chitosan. The viscosity of 1% chitosan solution, that was incubated with the culture supernatant, was rapidly decreased, suggesting the secretion of endochitosanolytic enzymes by P16. The culture fluid revealed six endo-type chitosanase isozymes, two major (38 and 45 kD), and four minor (54, 65, 82, and 96 kD) forms by staining profile. The crude enzymes were very stable, and full activity was maintained for 4 weeks at $4^{\circ}C\;or\;-20^{\circ}C$ in the culture supernatant, suggesting a highly desirable stability rate for making an industrial application of the crude enzymes. The supernatant also cleaved the insoluble chitosan powder, but the hydrolysis rate was much lower. The enzymic degradation products of chitosan contained $(GlcN)_n$ (n=2-8). The concentration of chitosan in the reaction mixture of the crude enzyme affected the chitooligosaccharides composition of the hydrolysis products. When the higher concentration of chitosan was used, the higher degree of polymerized chitooligosaccharides were produced. By comparison with other commercial chitosanase preparations, P16 was indeed found to be a valuable enzyme source for industrial production of chitooligosaccharides from chitosan.

• Applied Biological Chemistry
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• v.37 no.1
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• pp.43-48
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• 1994

A basic inducible protein, ICG, containing chitinase and ${\beta}-1,3-glucanase$ activity concomittantly was purified from cell suspension culture media of rice after the treatment of chitooligosaccharides. The isolated ICG enzyme gave a single band on native and SDS polyacrylamide gel electrophoresis and its molecular weight was estimated to be 52.53 kd. The optimal temperature and optimal pH of both enzyme activities in ICG were $60^{\circ}C$, pH 6.0 for chitinase activity and $37^{\circ}C$, pH 4.0 for ${\beta}-1,3-glucanase$ activity. $K_M$ and $V_{max}$ values for chitinase were 0.474 mM. 2.997 nM/min., and those for ${\beta}-1,3-glucanase$ were 1.004 mM 0.739 nM/min. respectively. TLC analysis of the chitooligosaccharide hydrolysates with ICG enzyme indicated that ICG acts as endochitinase.

• Proceedings of the Korean Aquaculture Society Conference
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• 2006.05a
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• pp.83-84
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• 2006

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.643-643
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• 2003